Functional dissection of DNA supercoiling factor: EF-hand domains and C-terminal HDEF motif are essential for its activity. [ 10231391 ]

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TITLE:Functional dissection of DNA supercoiling factor: EF-hand domains and C- terminal HDEF motif are essential for its activity 

[2-s2.0-0033065870] Refers [2-s2.0-0033065870] LEVEL: 0
Background: DNA supercoiling factor (SCF) was first identified in the silkworm as a protein capable of generating negative supercoils into a relaxed DNA in conjunction with eukaryotic topoisomerase II. Drosophila melanogaster SCF localizes to puffs on polytene chromosomes, implicating its role in gene expression. The factor is a Ca2+-binding protein with four EF- hand domains and possesses a tetrapeptide sequence HDEF at its C-terminus. Results: To clarify the roles of the domains of SCF, we carried out a functional dissection of the factor. A glutamic acid to glutamine substitution at the end of the loop in EF-hand domain II or III reduced both the Ca2+ binding and supercoiling activities; simultaneous substitutions at both sites abolished these activities. During native polyacrylamide gel electrophoresis, SCF migrated more rapidly in the presence of Ca2+ than in the presence of Mg2+ or EGTA. SCF binds directly to topoisomerase II. Deletion of the C-terminal HDEF sequence destroyed the binding and supercoiling activity. Conclusions: Two regions of SCF play critical roles in the supercoiling activity. The C-terminal HDEF is essential for the factor binding to topoisomerase II. The EF-hand domains II and III are functional for the Ca2+ binding that induces a mobility change in the factor upon gel electrophoresis.

TITLE:The CREC family, a novel family of multiple EF-hand, low-affinity Ca2+-binding proteins localised to the secretory pathway of mammalian cells 

[2-s2.0-0033978227] Refers [2-s2.0-0033065870] LEVEL: 1
The CREC family consists of a number of recently discovered multiple (up to seven) EF-hand proteins that localise to the secretory pathway of mammalian cells. At present, the family includes reticulocalbin, ERC-55/TCBP-49/E6BP, Cab45, calumenin and crocalbin/CBP-50. Similar proteins are found in quite diverse invertebrate organisms such as DCB-45 and SCF in Drosophila melanogaster, SCF in Bombyx mori, CCB-39 in Caenorhabditis elegans and Pfs40/PfERC in Plasmodium falciparum. The Ca2+ affinity is rather low with dissociation constants around 10-4-10-3 M. The proteins may participate in Ca2+-regulated activities. Recent evidence has been obtained that some CREC family members are involved in pathological activities such as malignant cell transformation, mediation of the toxic effects of snake venom toxins and putative participation in amyloid formation. Copyright (C) 2000 Federation of European Biochemical Societies.

TITLE:When the DREAM is gone: From basic science to future prospectives in pain management and beyond 

[2-s2.0-0037399166] Refers [2-s2.0-0033065870] LEVEL: 1
DREAM (downstream regulatory element antagonistic modulator) was identified as a novel calcium-binding protein with pleiotropic functions in vitro that are as varied as that of a transcription factor, a binding partner for presenilins, and a modulator of potassium channels. This review will discuss the findings that have implicated DREAM in its various roles. As a transcriptional repressor, DREAM may control the expression of the endogenous opioid gene prodynorphin amongst others, and itself is exquisitely regulated by second messenger molecules, protein kinases and other transcription factors. Recent genetic evidence has revealed a physiological role for DREAM in pain modulation. The interplay between DREAM and prodynorphin is discussed in light of our current understanding of this Janus-like opioid gene. The potential for the involvement of DREAM in other processes beyond pain modulation is considered at the end of this review.

TITLE:ICRF-193, a catalytic inhibitor of DNA topoisomerase II, inhibits re-entry into the cell division cycle from quiescent state in mammalian cells 

[2-s2.0-0036201868] Refers [2-s2.0-0033065870] LEVEL: 1
Background: To describe the requirement of DNA topoisomerase II (topo II) during transition from the quiescent state (G0 phase) to the cell division cycle in mammalian cells, we examined the influence of ICRF-193, a catalytic inhibitor of topo II, on re-entry into the cell division cycle of quiescent cells in response to appropriate growth stimuli. Results: The re-entry into the S phase of cultured cell lines arrested at the quiescent (G0) phase by serum-starvation was sensitive to 10 ?M ICRF-193. DNA syntheses induced by lipopolysaccharide in murine spleen cells or by release from contact-inhibition were also inhibited by ICRF-193. The cell lines with a high-level of resistance toward ICRF-193 due to a point mutation in the topo II? gene entered into the S phase from quiescence in the presence of ICRF-193. The drug did not inhibit entry into the S phase in cultured cells released from arrest at the metaphase or G1 phase. Conclusion: There is an ICRF-193-sensitive step during re-entry of quiescent mammalian cells into the cell division cycle upon growth stimulation and the drug targets topo II? during the process.

TITLE:Superoxide production by plant homologues of the gp91phox NADPH oxidase. Modulation of activity by calcium and by tobacco mosaic virus infection 

[2-s2.0-0034954408] Refers [2-s2.0-0033978227] LEVEL: 2
Genes encoding homologs of the gp91phox subunit of the plasma membrane NADPH oxidase complex have been identified in plants and are hypothesized to be a source of reactive oxygen species during defense responses. However, the direct involvement of the gene products in superoxide (O2-) production has yet to be shown. A novel activity gel assay based on protein fractionation in native or sodium dodecyl sulfate (SDS)-denaturing polyacrylamide gels was developed. In native polyacrylamide gel electrophoresis, one or two major O2--producing formazan bands were detected in tomato (Lycopersicum esculentum Mill. cv Moneymaker) and tobacco (Nicotiana tabacum var. Samsun, NN) plasma membranes, respectively. Denaturing fractionation of tomato and tobacco plasma membrane in SDS-polyacrylamide gel electrophoresis, followed by regeneration of the in-gel activity, revealed NADPH-dependent O2--producing formazan bands of 106-, 103-, and 80- to 75-kD molecular masses. The SDS and native activity bands were dependent on NADPH and completely inhibited by diphenylene iodonium or CuZn- O2- dismutase, indicating that the formazan precipitates were due to reduction by O2- radicals catalyzed by an NADPH-dependent flavin containing enzyme. The source of the plasma membrane activity bands was confirmed by their cross-reaction with antibody prepared from the C terminus of the tomato gp91phox homolog. Membrane extracts as well as the in-gel NADPH oxidase activities were stimulated in the presence of Ca2+. In addition, the relative activity of the gp91phoxhomolog was enhanced in the plasma membrane of tobacco mosaic virus-infected leaves. Thus, in contrast to the mammalian gp91phox, the plant homolog can produce O2- in the absence of additional cytosolic components and is stimulated directly by Ca2+.

TITLE:Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons 

[2-s2.0-12944314765] Refers [2-s2.0-0033978227] LEVEL: 2
The endoplasmic reticulum (ER) is the largest single intracellular organelle, which is present in all types of nerve cells. The ER is an interconnected, internally continuous system of tubules and cisterns, which extends from the nuclear envelope to axons and presynaptic terminals, as well as to dendrites and dendritic spines. Ca2+ release channels and Ca 2+ pumps residing in the ER membrane provide for its excitability. Regulated ER Ca2+ release controls many neuronal functions, from plasmalemmal excitability to synaptic plasticity. Enzymatic cascades dependent on the Ca2+ concentration in the ER lumen integrate rapid Ca 2+ signaling with long-lasting adaptive responses through modifications in protein synthesis and processing. Disruptions of ER Ca 2+ homeostasis are critically involved in various forms of neuropathology.

TITLE:Identification and validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometry 

[2-s2.0-0036280779] Refers [2-s2.0-0033978227] LEVEL: 2
Despite improvements in treatment of patients with head and neck squamous cell carcinoma (HNSCC) over the last two decades, the survival rate of these patients has not increased significantly. One of the major factors in the poor outcome of the disease is regional metastasis. To better understand the mechanisms of this process at the protein level, we performed two-dimensional electrophoresis (2-DE) and mass spectrometry using SELDI ProteinChip technology to identify proteins differentially expressed in two HNSCC cell lines, UMSCC10A and UMSCC10B, from the same patient. UMSCC10A was derived from the primary tumor and UMSCC10B from a metastatic lymph node. The differentially expressed proteins were excised from the gels. Following in-gel digestion by trypsin, mass profiles of the peptides were generated. Proteins were identified by submitting the peptide mass profiles to a public available NCBInr databases ( Two membrane-associated proteins, annexin I and annexin II, and glycolytic protein enolase-? were found to be upregulated, and calumenin precursor down-regulated, in metastatic cell line UMSCC10B. The identity of these proteins was confirmed by analyzing additional peptide mass fingerprints obtained by endoproteinase lysine-C digestion. The results were also validated by Western blotting analysis. Our results showed that enolase-?, annexin-I and annexin-II might be important molecules in head and neck cancer invasion and metastasis. The results also suggest an important complementary role for proteomics in identification of molecular abnormalities important in cancer development and progression.

TITLE:Rapidly exchanging Ca2+ stores in neurons: Molecular, structural and functional properties 

[2-s2.0-0034742490] Refers [2-s2.0-0033978227] LEVEL: 2
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TITLE:Proteomic analysis of early melanosomes: Identification of novel melanosomal proteins 

[2-s2.0-12244267707] Refers [2-s2.0-0033978227] LEVEL: 2
Melanin is a heterogeneous biopolymer produced only by specific cells termed melanocytes, which synthesize and deposit the pigment in specialized membrane-bound organelles known as melanosomes. Although melanosomes have been suspected of being closely related to lysosomes and platelets, the total number of melanosomal proteins is still unknown. Thus far, six melanosome-specific proteins have been identified, and the challenge is to characterize the complete proteome of the melanosome to further understand its mechanism of biogenesis. In this report, we used mass spectrometry and subcellular fractionation to identify protein components of early melanosomes. Using this approach, we have identified all 6 of the known melanosome-specific proteins, 56 proteins that are shared with other organelles, and confirmed the presence of 6 novel melanosomal proteins using western blotting and by immunohistochemistry.

TITLE:A chemical genetic screen for direct v-Src substrates reveals ordered assembly of a retrograde signaling pathway 

[2-s2.0-0036008093] Refers [2-s2.0-0033978227] LEVEL: 2
Using an ATP analog that is a specific substrate for an analog-specific allele of v-Src, we identified several novel cytoskeletal substrates that control actin assembly processes. A screen for less abundant v-Src substrates revealed the scaffolding protein Dok-1 as a direct substrate of v-Src. Further studies suggest that v-Src phosphorylation sites on Dok-1 are critical for its binding to RasGAP and Csk, negative regulators of Src signaling. This results in the downregulation of growth-promoting signals of the Src family kinases and the Ras pathway. Identification of the direct substrates of v-Src leads to a model for the precise order of assembly of a retrograde signaling pathway in v-Src-transformed cells and has provided new insight into the balance between those signals that promote cell transformation mediated by v-Src catalyzed tyrosine phosphorylation and those that inhibit it.

TITLE:The Golgi PMR1 P-type ATPase of Caenorhabditis elegans: Identification of the gene and demonstration of calcium and manganese transport 

[2-s2.0-0035815727] Refers [2-s2.0-0033978227] LEVEL: 2
In recent years, it has been well established that the Ca2+ concentration in the lumen of intracellular organelles is a key determinant of cell function. Despite the fact that essential functions of the Golgi apparatus depend on the Ca2+ and Mn2+ concentration in its lumen, little is known on the transport system responsible for ion accumulation. The Golgi ion pump PMR1 has been functionally studied only in yeast. In humans, mutations in the orthologous gene ATP2C1 cause Hailey-Hailey disease. We report here the identification of the PMR1 homologue in the model organism Caenorhabditis elegans and after ectopic expression the direct study of its ion transport in permeabilized COS-1 cells. The C. elegans genome is predicted to contain a single PMR1 orthologue on chromosome I. We found evidence for alternative splicing in the 5?-untranslated region, but no indication for the generation of different protein isoforms. C. elegans PMR1 overexpressed in COS-1 cells transports Ca2+ and Mn2+ with high affinity into the Golgi apparatus in a thapsigargin-insensitive manner. Part of the accumulated Ca2+ can be released by inositol 1,4,5-trisphosphate, in agreement with the idea that the Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitire Ca2+ store.

TITLE:The endoplasmic reticulum as an integrating signalling organelle: From neuronal signalling to neuronal death 

[2-s2.0-0037025272] Refers [2-s2.0-0033978227] LEVEL: 2
The endoplasmic reticulum is one of the largest intracellular organelles represented by continuous network of cisternae and tubules, which occupies the substantial part of neuronal somatas and extends into finest neuronal processes. The endoplasmic reticulum controls protein synthesis as well as their post-translational processing, and generates variety of nucleus-targeted signals through Ca2+-binding chaperones. The normal functioning of the endoplasmic reticulum signalling cascades requires high concentrations of free calcium ions within the endoplasmic reticulum lumen ([Ca2+]L), and severe alterations in [Ca2+]L trigger endoplasmic reticulum stress response, manifested by either unfolded protein response (UPR) or endoplasmic reticulum overload response (EOR). At the same time, the endoplasmic reticulum is critically involved in fast neuronal signalling, by producing local or global cytosolic calcium signals via Ca2+-induced Ca2+ release (CICR) or inositol-1,4,5-trisphosphate-induced Ca2+ release (IICR). Both CICR and IICR are important for synaptic transmission and synaptic plasticity. Several special techniques allowing real-time [Ca2+]L monitoring were developed recently. Video-imaging of [Ca2+]L in neurones demonstrates that physiological signalling triggers minor decreases in overall intraluminal Ca2+ concentration due to strong activation of Ca2+ uptake, which prevents severe [Ca2+]L alterations. The endoplasmic reticulum lumen also serves as a "tunnel" which allows rapid transport of Ca2+ ions within highly polarised nerve cells. Fluctuations of intraluminal free Ca2+ concentration represent a universal mechanism, which integrates physiological cellular signalling with protein synthesis and processing. In pathological conditions, fluctuations in [Ca2+]L may initiate either adaptive or fatal stress responses. © 2002 Elsevier Science B.V. All rights reserved.

TITLE:Identification of differentially expressed proteins in human glioblastoma cell lines and tumors 

[2-s2.0-0038326942] Refers [2-s2.0-0033978227] LEVEL: 2
An in-frame deletion of 801 bp in exons 2-7 (type III mutation) of the epidermal growth factor receptor (EGFR) is detected at high incidence in primary glioblastoma tumors. A proteomic approach was used to generate differential protein expression maps of fetal human astrocytes (FHA), human glioblastoma cell lines U87MG and U87MG expressing type III EGFR deletion (U87MG?EGFR) that confers high malignancy to tumor cells. Two-dimensional gel electrophoresis followed by in-gel digestion of separated spots and protein identification by LC-MS and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified 23 proteins expressed at higher levels or exclusively in FHA and 29 proteins expressed at higher levels or exclusively in U87MG cells. Three proteins, ubiquitin, cystatin B, and tissue transglutaminase (TTG), were upregulated in U87MG?EGFR relative to U87MG. Four proteins highly expressed by U87MG cells, Hsp27, major vault protein, TTG, and cystatin B, were analyzed by Western blot, ELISA, or RT-PCR in cell extracts and in tissue samples of glioblastoma multiforme (GBM; grade IV), low-grade astrocytomas (grades I and II), and nonmalignant brain lesions. All four proteins were highly expressed in GBM tissues compared to nonmalignant brain. These proteins may be used as diagnostic or functional (e.g., multiple drug resistance, invasiveness) markers for glioblastoma tumors. © 2003 Wiley-Liss, Inc.

TITLE:PMR1/SPCA Ca2+ pumps and the role of the Golgi apparatus as a Ca2+ store 

[2-s2.0-0038519661] Refers [2-s2.0-0033978227] LEVEL: 2
Besides the well-known sarco/endoplasmic-reticulum Ca2+-transport ATPases (SERCA), animal cells contain a much less characterized P-type Ca2+-transport ATPase: the PMR1/SPCA Ca2+/Mn2+-transport ATPase. SPCA is mainly targeted to the Golgi apparatus. Phylogenetic analysis indicates that it might be more closely related to a putative ancestral Ca2+ pump than SERCA. SPCA supplies the Golgi apparatus, and possibly other more distal compartments of the secretory pathway, with the Ca2+ and Mn2+ necessary for the production and processing of secretory proteins. In the lactating mammary gland, SPCA appears to be the primary pump responsible for supplementing the milk with high (60-100 mM) Ca2+. It could also play a role in detoxification of cells overloaded with Mn2+. Mutations in the human gene encoding the SPCA pump (ATP2C1) result in Hailey-Hailey disease, a keratinocyte disorder characterized by incomplete cell adhesion. Recent observations show that the Golgi apparatus can function as a Ca2+ store, which can be involved in setting up cytosolic Ca2+ oscillations.

TITLE:Versatility of the endoplasmic reticulum protein folding factory 

[2-s2.0-23744457478] Refers [2-s2.0-0033978227] LEVEL: 2
The endoplasmic reticulum (ER) is dedicated to import, folding and assembly of all proteins that travel along or reside in the secretory pathway of eukaryotic cells. Folding in the ER is special. For instance, newly synthesized proteins are N-glycosylated and by default form disulfide bonds in the ER, but not elsewhere in the cell. In this review, we discuss which features distinguish the ER as an efficient folding factory, how the ER monitors its output and how it disposes of folding failures. Copyright © Taylor & Francis Inc.

TITLE:The inhibitory effect of calumenin on the vitamin K-dependent Ī³-carboxylation system: Characterization of the system in normal and warfarin-resistant rats 

[2-s2.0-2942627237] Refers [2-s2.0-0033978227] LEVEL: 2
The vitamin K-dependent ?-carboxylation system is responsible for post-translational modification of vitamin K-dependent proteins, converting them to Gla-containing proteins. The system consists of integral membrane proteins located in the endoplasmic reticulum membrane and includes the ?-carboxylase and the warfarin-sensitive enzyme vitamin K1 2,3-epoxide reductase (VKOR), which provides ?-carboxylase with reduced vitamin K1 cofactor. In this work, an in vitro ?-carboxylation system was designed and used to understand how VKOR and ?-carboxylase work together as a system and to identify factors that can regulate the activity of the system. Results are presented that demonstrate that the endoplasmic reticulum chaperone protein calumenin is associated with ?-carboxylase and inhibits its activity. Silencing of the calumenin gene with siRNA resulted in a 5-fold increase in ?-carboxylase activity. The results provide the first identification of a protein that can regulate the activity of the ?-carboxylation system. The propeptides of vitamin K-dependent proteins stimulate ?-carboxylase activity. Here we show that the factor X and prothrombin propeptides do not increase reduced vitamin K 1 cofactor production by VKOR in the system where VKOR is the rate-limiting step for ?-carboxylation (Wallin, R., Sane, D. C., and Hutson, S. M. (2002) Thromb. Res. 108, 221-226). These findings put calumenin in a central position concerning regulation of ?-carboxylation of vitamin K-dependent proteins. Reduced vitamin K1 cofactor transfer between VKOR and ?-carboxylase is shown to be significantly impaired in the in vitro ?-carboxylation system prepared from warfarin-resistant rats. Furthermore, the sequence of the 18-kDa subunit 1 of the VKOR enzyme complex (Rost, S., Fregin, A., Ivaskeviclus, V., Conzelmann, E., Hortnagel, K., Pelz, H-J., Lappegard K., Seifried, E., Scharrer, I., Tuddenham, E. G. D., Muller, C. R., Storm, T. M., and Oldennburg, J. (2004) Nature 427, 537-541) was found to be identical in the two rat strains. This finding supports the notion that different forms of genetic warfarin resistance exist.

TITLE:Dose-dependent and independent temporal patterns of gene responses to ionizing radiation in normal and tumor cells and tumor xenografts 

[2-s2.0-0035940408] Refers [2-s2.0-0033978227] LEVEL: 2
U87 cells derived from human malignant gliomas and growtharrested human embryonic lung (HEL) fibroblasts were examined with respect to their response to ionizing radiation by profiling their RNAs. In the first series of experiments, cells grown in vitro were harvested and the RNAs were extracted 5 h after exposure to 1, 3, or 10 Gy. In the second series of experiments the U87 tumors were implanted in nude mice and subjected to the same doses of irradiation. The xenografts were harvested at 1, 5, or 24 h after irradiation and subjected to the same analyses. We observed and report on (i) cell-type common and cell-type specific responses, (ii) genes induced at low levels of irradiation but not at higher doses, (iii) temporal patterns of gene response in U87 xenografts that varied depending on radiation dose and temporal patterns of response that were similar at all doses tested, (iv) significantly higher up-regulation of cells in xenografts than in In vitro cultures, and (v) genes highly up-regulated by radiation. The responding genes could be grouped into nine functional clusters. The representation of the nine clusters was to some extent dependent on dose and time after irradiation. The results suggest that clinical outcome of ionizing radiation treatment may benefit significantly by taking into account both cell-type and radiation-dose specificity of cellular responses.

TITLE:Gene profile of replicative senescence is different from progeria or elderly donor 

[2-s2.0-0034805332] Refers [2-s2.0-0033978227] LEVEL: 2
In vitro cellular senescence of human diploid fibroblast has been a good model for aging research, which shows similar phenotypes to in vivo aging. Gene expression profiling would provide an insight to understand the mechanism of senescence. Using cDNA microarray containing 384 known genes, we compared the expression profiles of three different types of aging models: replicative senescence, fibroblasts from progeria or from elderly donor. Although all of them showed senescence phenotypes, distinct sets of genes were altered in each group. Pairwise plots or cluster analysis of activation fold of gene expression revealed closer relationships between fibroblasts from progeria or from old individual, but not between replicative senescence fibroblasts and either models. Differential expression pattern of several genes were confirmed by RT-PCR. We suggest that the replicative senescence model might behave differently to other types of aging models due to the distinct gene expression. © 2001 Academic Press.

TITLE:Characterization of the differential protein expression associated with thermoresistance in human gastric carcinoma cell lines 

[2-s2.0-0034849978] Refers [2-s2.0-0033978227] LEVEL: 2
Resistance to chemotherapeutic agents is one of the major problems faced during palliative therapy of tumor cells. Thus, chemotherapy is frequently combined with other modes of therapy such as radiation therapy and/or hyperthermia. Tumor cells respond to heat stress with development of thermotolerance and the interactions between chemo- and thermoresistance phenomena are not clearly understood. In this paper, we analyze the differential protein expression in vitro in human stomach cancer cells, their chemoresistant and thermoresistant counterparts using proteomics. The immediate aim was to identify sets of proteins that may lead to the development of thermoresistance. Based on these results, we aim to develop functional tests and methods for the modulation of thermoresistance and chemoresistance phenomena that may assist the therapy of inoperable cancers.

TITLE:The Ca2+/Mn2+ pumps in the Golgi apparatus 

[2-s2.0-16644383141] Refers [2-s2.0-0033978227] LEVEL: 2
Recent evidence highlights the functional importance of the Golgi apparatus as an agonist-sensitive intracellular Ca2+ store. Besides Ca 2+-release channels and Ca2+-binding proteins, the Golgi complex contains Ca2+-uptake mechanisms consisting of the well-known sarco/endoplasmic reticulum Ca2+-transport ATPases (SERCA) and the much less characterized secretory-pathway Ca2+-transport ATPases (SPCA). SPCA supplies the Golgi compartments and, possibly, the more distal compartments of the secretory pathway with both Ca2+ and Mn 2+ and, therefore, plays an important role in the cytosolic and intra-Golgi Ca2+ and Mn2+ homeostasis. Mutations in the human gene encoding the SPCA1 pump (ATP2C1) resulting in Hailey-Hailey disease, an autosomal dominant skin disorder, are discussed. © 2004 Elsevier B.V. All rights reserved.

TITLE:Study of therapy resistance in cancer cells with functional proteome analysis 

[2-s2.0-0036235429] Refers [2-s2.0-0033978227] LEVEL: 2
Different types of cancer are naturally resistant to many anticancer drugs. Additionally, these tumours develop acquired drug resistance, which includes the classical multidrug resistance (MDR) accompanied by the synthesis of P-glycoprotein, a member of the superfamily of ATP-binding cassette (ABC) transporters. Furthermore, atypical MDR is mediated by several different, some unknown, mechanisms. To overcome chemoresistance problems, antineoplastic drugs are often combined with other modes of therapy, e.g. hyperthermia, where good response has been reported in several experimental tumour models and in advanced cancer patients. The success of this combined anticancer treatment may be limited by an increase in chemoresistance and thermoresistance. A model system to study resistance phenomena is the use of chemoresistant and thermoresistant cancer cell lines. We have established chemoresistant cancer cell lines (gastric and pancreatic carcinoma, fibrosarcoma, melanoma) and now thermoresistant cell lines derived from gastric and pancreatic carcinoma cells and their counterparts that were resistant towards daunorubicin (classical MDR) and mitoxantrone (atypical MDR). Using proteomics, in this paper we evaluate the drug resistance of chemoresistant melanoma cells (parental cell line MeWo and sublines exhibiting drug resistance towards etoposide, cisplatin, fotemustine and vindesine) as a paradigm for analysis of drug resistance phenomena. Additionally, we investigate heat resistance and the interaction of chemoresistance and thermoresistance to identify common pathways using the parental and drug resistant stomach cancer cell lines EPG85-257, EPG85-257RNOV, EPG85-257RDB and their thermoresistant counterparts. Possible implications of differential protein expression will be discussed.

TITLE:Proteomic analysis of lipopolysaccharide-induced apoptosis in PC12 cells 

[2-s2.0-0036744390] Refers [2-s2.0-0033978227] LEVEL: 2
We employed rat pheochromocytoma PC12 cells as our model system to identify cellular proteins that accompany Escherichia coli lipopolysaccharide (LPS)-induced apoptosis, based on a proteomic approach. Cell viability tests revealed that nai?ve PC12 cells underwent cell death in a dose-dependent manner after treatment with LPS. Flow cytometric analysis confirmed that apoptosis was primarily responsible for the observed cell death. Two-dimensional electrophoresis in conjunction with N-terminal sequencing, immunoblot, matrix-assisted laser desorption/ionization-time of flight analysis or computer matching with protein databases further revealed that the LPS-induced apoptosis is accompanied by an augmented level of calreticulin, calcium binding protein 50, endoplasmic reticulum protein 60 (ERP60), heat shock protein 60 (HSP60) or HSP90, and a reduced level of amphoterin, cytochrome c oxidase polypeptide Vla-liver or ERP29. These proteins are associated with endoplasmic reticulum, mitochondria or cell membrane, and are with known or potential roles in apoptosis. Their identification therefore provides an impetus for further delineation of the cellular and molecular basis of apoptotic cell death and sepsis based on proteomic profiling of PC12 cells.

TITLE:Multiple functional categories of proteins identified in an in vitro cellular ubiquitin affinity extract using shotgun peptide sequencing 

[2-s2.0-0042734593] Refers [2-s2.0-0033978227] LEVEL: 2
To construct a high information content assay for examination of the function of the cellular ubiquitin system, we added his-tagged ubiquitin, ATP, and an ATP-regenerating system to endogenous human cellular ubiquitin system enzymes, and labeled cellular proteins with hexa-histidine tagged ubiquitin in vitro. Labeling depended on ATP, the ATP recycling system, the proteasome inhibitor MG132, and the ubiquitin protease inhibitor ubiquitin aldehyde, and was inhibited by iodoacetamide. Quadruplicate affinity extracted proteins were digested with trypsin, and the peptides were analyzed by 2D capillary LC-MS/MS, SEQUEST, MEDUSA, and support vector machine calculations. Identified proteins included 22 proteasome subunits or associated proteins, 18 E1, E2, or E3 ubiquitin system enzymes or related proteins, 4 ubiquitin domain proteins and 36 proteins in functional clusters associated with redox processes, endocytosis/vesicle trafficking, the cytoskeleton, DNA damage/repair, calcium binding, and mRNA splicing. This suggests a link between the ubiquitin system and these cellular processes. This map of cellular ubiquitin-associated proteins may be useful for further studies of ubiquitin system function.

TITLE:Warfarin and the vitamin K-dependent Ī³-carboxylation system 

[2-s2.0-3042705857] Refers [2-s2.0-0033978227] LEVEL: 2
Insight into the molecular basis for genetic warfarin resistance has recently been accomplished by the identification of an 18-kDa protein of the endoplasmic reticulum that is targeted by the drug. When expressed in eukaryotic and insect cells, the protein reduces vitamin K1 2,3-epoxide in a warfarin-sensitive reaction. This finding strongly suggests that the protein is part of the vitamin K cycle, which is essential for the production of vitamin K-dependent proteins. Identification of the 18-kDa protein has aided the understanding of the vitamin K-dependent ?-carboxylation system at the molecular level.

TITLE:ERp29 is a ubiquitous resident of the endoplasmic reticulum with a distinct role in secretory protein production 

[2-s2.0-0036200181] Refers [2-s2.0-0033978227] LEVEL: 2
ERp29 was recently characterized biochemically as a novel protein that resides in mammalian endoplasmic reticulum (ER). Here we applied immunochemical procedures at the cellular level to investigate the hypothesized role of ERp29 in secretory protein production. ERp29 was localized exclusively to the ER/nuclear envelope of MDCK cells using confocal immunocytochemistry and comparative markers of the ER lumen, ER/Golgi membrane, nuclei, and mitochondria. A predominant association with rough ER was revealed by sucrose-gradient analysis of rat liver microsomes. Immunohistochemistry showed ERp29 expression in 35 functionally distinct cell types of rat, establishing ERp29 as a general ER marker. The ERp29 expression profile largely paralleled that of protein disulfide isomerase (PDI), the closest relative of ERp29, consistent with a role in secretory protein production. However strikingly different ERp29/PDI ratios were observed in various cell types, suggesting independent regulation and functional roles. Together, these findings associate ERp29 primarily with the early stages of secretory protein production and implicate ERp29 in a distinct functional role that is utilized in most cells. Our identification of several ERp29-enriched cell types suggests a potential selectivity of ERp29 for non-collagenous substrates and provides a physiological foundation for future investigations.

TITLE:Numerous proteins in mammalian cells are prone to iron-dependent oxidation and proteasomal degradation 

[2-s2.0-0036970951] Refers [2-s2.0-0033978227] LEVEL: 2
The mechanisms that underlie iron toxicity in cells and organisms are poorly understood. Previous studies of regulation of the cytosolic iron sensor, iron-regulatory protein 2 (IRP2), indicate that iron-dependent oxidation triggers ubiquitination and proteasomal degradation of IRP2. To determine if oxidization by iron is involved in degradation of other proteins, we have used a carbonyl assay to identify oxidized proteins in lysates from RD4 cells treated with either an iron source or iron chelator. Protein lysates from iron-loaded or iron-depleted cells were resolved on two-dimensional gels and these iron manipulations were also repeated in the presence of proteasomal inhibitors. Eleven abundant proteins were identified as prone to iron-dependent oxidation and subsequent proteasomal degradation. These proteins included two putative iron-binding proteins, hNFU1 and calreticulin; two proteins involved in metabolism of hydrogen peroxide, peroxiredoxin 2 and superoxide dis-mutase 1; and several proteins identified in inclusions in neurodegenerative diseases, including HSP27, UCHL1, actin and tropomyosin. Our results indicate that cells can recognize and selectively eliminate iron-dependently oxidized proteins, but unlike IRP2, levels of these proteins do not significantly decrease in iron-treated cells. As iron overload is a feature of many human neurological diseases, further characterization of the process of degradation of iron-dependently oxidized proteins may yield insights into mechanisms of human disease. Copyright © 2002 S. Karger AG, Basel.

TITLE:Proteomics of the injured rat sciatic nerve reveals protein expression dynamics during regeneration 

[2-s2.0-14944359702] Refers [2-s2.0-0033978227] LEVEL: 2
Using proteomics, We investigated the temporal expression profiles of proteins in rat sciatic nerve after experimental crush. Extracts of sciatic nerves collected at 5, 10, and 35 days after injury were analyzed by two-dimensional gel electrophoresis and quantitative image analysis. Of the ?1,500 protein spots resolved on each gel, 121 showed significant regulation during at least one time point. Using cluster analysis, these proteins were grouped into two expression profiles of down-regulation and four of up-regulation. These profiles mainly reflected differences in cellular origins in addition to different functional roles. Mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute-phase proteins, antioxidant proteins, and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization, and in lipid metabolism. Several proteins not previously implicated in nerve regeneration were identified, e.g. translationally controlled tumor protein, annexin A9/31, vitamin D-binding protein, ?-crystallin B, ?-synuclein, dimethylargininases, and reticulocalbin. Real-time PCR analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. In conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:Inhibition of serum- and calcium-induced terminal differentiation of human keratinocytes by HPV 16 E6: Study of the association with p53 degradation, inhibition of p53 transactivation, and binding to E6BP 

[2-s2.0-0036060364] Refers [2-s2.0-0033978227] LEVEL: 2
Transfection of the E6 gene of human papillovirus (HPV) 16 into primary human keratinocytes (PHKs) generates proliferating cell colonies which are resistant to serum- and calcium-induced terminal differentiation. The extreme C-terminus of E6 was shown to be dispensable for this activity. To map further the amino acid sequences required for inducing resistance to serum and calcium, and to address the functional significance of E6 interactions with p53 and E6BP (ERC-55) in this function, we evaluated the activities of a series of E6 mutants. Small deletions within the central portion of the second putative zinc-finger abolished, or markedly reduced, E6 biological activity, while mutations affecting the cysteine residues in the base of the finger were less effective in this respect. When these mutants were assayed for their ability to degrade p53 in vitro and in vivo and to inhibit p53 transcriptional activation (TA), we found that there was a dissociation of these activities in some mutants. We mapped one mutant which was highly efficient in p53 degradation and inhibition of p53 TA, yet displayed severely reduced activity in the biological assay, and conversely, a subset of mutants that showed moderate activities in the colony assay while being severely impaired in p53 degradation and inhibition of p53 TA. These data argue that p53 inactivation or even elimination are not sufficient, and may not be essential, for altering the response of PHKs to serum and calcium. When these E6 mutants were evaluated for E6BP binding in vitro, there was a similar dissociation between the biological and biochemical activities of several mutants. We mapped mutants with moderate activity in the biological assay that lacked the ability to bind to E6BP and a mutant that showed high biological activity with only marginal binding to E6BP. Thus, there is no absolute correlation between the ability of E6 mutant proteins to induce alterations in keratinocyte differentiation responses to calcium and serum and to induce p53 degradation, inhibit p53 mediated transactivation, or bind E6BP. Evidently there are additional cellular targets for E6 which mediate this alteration in cellular differentiation. © 2002 Elsevier Science.

TITLE:Monocrotaline pyrrole targets proteins with and without cysteine residues in the cytosol and membranes of human pulmonary artery endothelial cells 

[2-s2.0-28444450886] Refers [2-s2.0-0033978227] LEVEL: 2
A single injection of monocrotaline produces a pulmonary insult in rats with a phenotype similar to human primary pulmonary hypertension. Although extensively used as a model, the mechanism(s) by which this chemical insult mimics a condition with genetic and environmental links remains an enigma, although formation of protein adducts has been implicated. Monocrotaline (MCT) is non-toxic and must undergo hepatic dehydrogenation to the soft electrophile monocrotaline pyrrole as prerequisite to damaging endothelial cells lining arterioles at remote pulmonary sites. In this report we extend our earlier investigation (J. Biol. Chem. 2000, 275, 29091-29099) by examining protein adducts to lower abundance adducts, a pI range not covered before, and subcellular localization of adduct-forming proteins associated with plasma membranes. Human pulmonary artery endothelial cells were exposed to [ 14C]MCT pyrrole (MCTP) and protein targets were identified using 2-DE with IPG 4-11. Adducted proteins were identified by pI, apparent molecular weight, and PMF using MALDI-TOF MS. Results of this study show that the majority of adducts form on proteins that contain reactive thiols in a CXXC motif, such as protein disulfide isomerase A3 (ERp57), protein disulfide isomerase (PDI), and endothelial PDI. These same proteins were the major adduct-forming proteins associated with the plasma membrane. Other proteins found to be targets were thioredoxin, galectin-1, reticulocalbin 1 and 3, cytoskeletal tropomyosin, mitochondrial ATP synthase ?-chain, annexin A2 and cofilin-1. For the first time, MCTP adducts were observed on proteins not known to contain cysteine residues. However, known reactive proteins including nudeophosmin did not form detectable adducts, potentially indicating that MCTP did not reach the interior of nucleus to the same extent as other cellular sites. These findings suggest that molecular events underlying MCTP toxicity are initiated at the plasma membrane or readily accessible subcellular regions including the cytosol and membranes of the endoplasmic reticulum and mitochondria. © 2005 WILEY-VCH Verlag GmbH & Co. KGaA.

TITLE:HPV16 E6 natural variants exhibit different activities in functional assays relevant to the carcinogenic potential of E6 

[2-s2.0-33744935290] Refers [2-s2.0-0033978227] LEVEL: 2
Genetic studies have revealed natural amino acid variations within the human papillomavirus (HPV) type 16 E6 oncoprotein. To address the functional significance of E6 polymorphisms, 10 HPV16 E6 variants isolated from cervical lesions of Swedish women were evaluated for their activities in different in vitro and in vivo assays relevant to the carcinogenic potential of E6. Small differences between E6 prototype and variants, and among variants, were observed in transient expression assays that assessed p53 degradation, Bax degradation, and inhibition of p53 transactivation. More variable levels of activities were exhibited by the E6 proteins in assays that evaluated binding to the E6-binding protein (E6BP) or the human discs large protein (hDlg). The E6 prototype expressed moderate to high activity in the above assays. The L83V polymorphism, previously associated with risk for cancer progression in some populations, expressed similar levels of activity as that of the E6 prototype in most functional assays. On the other hand, L83V displayed more efficient degradation of Bax and binding to E6BP, but lower binding to hDlg. Results of this study indicate that naturally occurring amino acid variations in HPV16 E6 can alter activities of the protein important for its carcinogenic potential. © 2006 Elsevier Inc. All rights reserved.

TITLE:Identification and comparative analysis of the peptidyl-prolyl cis/trans isomerase repertoires of H. sapiens, D. melanogaster, C. elegans, S. cerevisiae and Sz. pombe 

[2-s2.0-24944532876] Refers [2-s2.0-0033978227] LEVEL: 2
The peptidyl-prolyl cis/trans isomerase (PPIase) class of proteins comprises three member families that are found throughout nature and are present in all the major compartments of the cell. Their numbers appear to be linked to the number of genes in their respective genomes, although we have found the human repertoire to be smaller than expected due to a reduced cyclophilin repertoire. We show here that whilst the members of the cyclophilin family (which are predominantly found in the nucleus and cytoplasm) and the parvulin family (which are predominantly nuclear) are largely conserved between different repertoires, the FKBPs (which are predominantly found in the cytoplasm and endoplasmic reticulum) are not. It therefore appears that the cyclophilins and parvulins have evolved to perform conserved functions, while the FKBPs have evolved to fill ever-changing niches within the constantly evolving organisms. Many orthologous subgroups within the different PPIase families appear to have evolved from a distinct common ancestor, whereas others, such as the mitochondrial cyclophilins, appear to have evolved independently of one another. We have also identified a novel parvulin within Drosophila melanogaster that is unique to the fruit fly, indicating a recent evolutionary emergence. Interestingly, the fission yeast repertoire, which contains no unique cyclophilins and parvulins, shares no PPIases solely with the budding yeast but it does share a majority with the higher eukaryotes in this study, unlike the budding yeast. It therefore appears that, in comparison with Schizosaccharomyces pombe, Saccharomyces cerevisiae is a poor representation of the higher eukaryotes for the study of PPIases. Copyright © 2005 John Wiley & Sons, Ltd.

TITLE:Gene expression profile of human chondrocyte HCS-2/8 cell line by EST sequencing analysis 

[2-s2.0-11144354761] Refers [2-s2.0-0033978227] LEVEL: 2
Large-scale single-pass sequencing of randomly selected cDNA clones from cell type specific libraries has proven to be a powerful approach for the discovery of novel gene functions, identification of novel gene family members, and definition of gene expression profiles. HCS-2/8 chondrocyte has been used as a cell culture model to study chondrocyte differentiation. Here we performed 3350 single-pass sequencing reactions obtained from the 5? ends of cDNAs from HCS-2/8 cells. To define the expression profiles of HCS-2/8 chondrocytes, we analyzed the identity of these representative cDNA sequences using database searches (BLAST). The sequences represent 1927 unique genes with known function (i.e., unigene clusters), 38 transcripts that are similar to genes with known function, 739 expressed genes with unknown function (i.e., expressed sequence tags), and 18 cDNAs which have not previously been sequenced. Interestingly, many transcripts were expressed from chromosome 12 compared with total genes, while the fewer numbers of cDNAs were derived from genes on chromosomes 14, 18 and Y. The chondrocytic phenotype of HCS-2/8 cells is reflected by abundant expression of genes related to cell structure and motility and the 20 most frequently expressed unigenes reflect a chondrocyte-related gene expression signature. Thus, our data establish a representative set of more than 2000 genes expressed in a chondrocytic cell line. This finding provides a framework for understanding cell growth and differentiation of chondrocytes and their metabolic function in the formation and remodeling of cartilage. © 2004 Elsevier B.V. All rights reserved.

TITLE:Characterization of the tissue-specific expression of the S100P gene which encodes an EF-hand Ca2+-binding protein 

[2-s2.0-0344875984] Refers [2-s2.0-0033978227] LEVEL: 2
S100 proteins are a calcium-binding protein family containing two EF-hand domains exclusively expressed in vertebrates and play roles in many cellular activities. Human S100P gene was first cloned as a 439 bp cDNA in placenta and it was found to be associated with human prostate cancer. Here we describe the cloning of the 1297 bp full-length cDNA, and the characterization of the tissue-specific expression of the human S100P gene. It is abundantly expressed in many tissues including placenta by Northern blot and RT-PCR analysis, unlike the expression pattern of other S100 family genes.

TITLE:Characterization of isoforms and genomic organization of mouse calumenin 

[2-s2.0-1242338800] Refers [2-s2.0-0033978227] LEVEL: 2
Calumenin is a multiple EF-hand protein located in endo/sarcoplasmic reticulum of mammalian heart and other tissues [J. Biol. Chem. 272 (1997) 18232; Genomics 49 (1998) 331; Biochim. Biophys. Acta 1386 (1998) 121]. In the present study, a new isoform of mouse calumenin (mouse calumenin 2) was cloned by RT-PCR and genomic DNA PCR. The deduced amino acid sequence of mouse calumenin 2 is 315 aa long with the calculated MW of 37,064 and pI of 4.26. It has 92% aa sequence identity to previously identified mouse calumenin [J. Biol. Chem. 272 (1997) 18232] (mouse calumenin 1). The difference in the aa sequence was restricted to the first two EF-hand regions (residues 74-138). Northern blot analysis shows that mouse calumenin 2 is highly expressed in heart, lung, testis and unpregnant uterus. The expression of mouse calumenin 2 appears to decrease when fetal development is progressed. Genomic DNA PCR, sequencing and data mining of mouse genome database were utilized to examine the exon-intron boundaries of mouse calumenin genes. Both mouse calumenin 1 and 2 genes encompass six exons, and five of them (Exon1, 3, 4, 5 and 6) are identical. However, mouse calumenin 1 contains Exon2-1, whereas mouse calumenin 2 contains a neighboring Exon2-2. The calumenin genes are localized on mouse chromosome 6 having conserved synteny with human chromosome 7q32. For comparison, the genomic organization of human calumenin was also examined using the published human genome database (UCSC Genome Bioinformatics at Like mouse calumenin genes, two human calumenin genes also consist of five identical exons (Exon1, 3, 4, 5 and 6) and a different Exon2. The present study suggests that the genomic organization of calumenin genes is well conserved between human and mouse. © 2003 Elsevier B.V. All rights reserved.

TITLE:Golgi retention of human protein NEFA is mediated by its N-terminal Leu/Ile-rich region 

[2-s2.0-0035861857] Refers [2-s2.0-0033978227] LEVEL: 2
The subcellular localization of the human Ca2+-binding EF-hand/leucine zipper protein NEFA was studied in HeLa cells by immunofluorescence microscopy. Double immunostaining using mouse anti-NEFA monoclonal antibody 1H8D12 and rabbit anti-ERD2 polyclonal antibody proved that NEFA is localized in the Golgi apparatus. The result was confirmed by the expression of NEFA-green fluorescent protein (GFP) fusion protein in the Golgi in the same cell line. Cycloheximide treatment proved NEFA to be a Golgi-resident protein. Seven NEFA deletion mutants were constructed to ascertain the peptide region relevant for Golgi retention. The expression of each NEFA-GFP variant was detected by fluorescence microscopy and immunoblotting. Only the ?N mutant, lacking the N-terminal Leu/Ile-rich region, failed to be retained in the Golgi after cycloheximide treatment. The other six deletion mutants in which either the basic region, the complete EF-hand pair domain, the two EF-hand motifs separately, the leucine zipper and the leucine zipper plus the C-terminal region is deleted, were localized to the Golgi. The peptide sequence within the Leu/Ile-rich region is discussed as a novel Golgi retention motif. © 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

TITLE:Calumin, a novel Ca2+-binding transmembrane protein on the endoplasmic reticulum 

[2-s2.0-34248593731] Refers [2-s2.0-0033978227] LEVEL: 2
We have identified a novel endoplasmic reticulum (ER)-resident protein, named "calumin", which is expressed in various tissues. This protein has a molecular mass of ?60 kDa and is composed of an ER-luminal domain rich in acidic residues, a single transmembrane segment, and a large cytoplasmic domain. Biochemical experiments demonstrated that the amino-terminal luminal domain is capable of binding Ca2+ with a high capacity and moderate affinity. In embryonic fibroblasts derived from calumin-knockout mice exhibiting embryonic and neonatal lethality, fluorometric Ca2+ imaging detected insufficient Ca2+ contents in intracellular stores and attenuated store-operated Ca2+ entry. Moreover, the mutant fibroblasts were highly sensitive to cell death induced by ER stress. These observations suggest that calumin plays an essential role in ER Ca2+ handling and is also implicated in signaling from the ER, which is closely associated with cell-fate decision. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Differential liver protein expression during schistosomiasis 

[2-s2.0-33846808872] Refers [2-s2.0-0033978227] LEVEL: 2
The arrival of eggs in the liver during Schistosoma mansoni infection initiates a protective granulomatous response; however, as the infection progresses, this response results in chronic liver fibrosis. To better understand the impact of schistosomiasis on liver function, we used a proteomic approach to identify proteins whose expression was significantly altered in schistosome-infected mice 8 weeks postinfection. Identification of differentially expressed proteins by mass fingerprinting revealed that schistosome infection markedly reduced the abundance of proteins associated with several normal liver functions (i.e., citric acid cycle, fatty acid cycle, and urea cycle), while proteins associated with stress responses, acute phase reactants, and structural components were all significantly more abundant. The expression patterns of several immunity-related proteins (peroxiredoxin 1, arginase 1, and galectin 1) suggested that different protein forms are associated with schistosome infection. These findings indicate that acute schistosomiasis lias a significant impact on specific liver functions and, moreover, that the alterations in specific protein isoforms and upregulation of unique proteins may be valuable as new markers of disease. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

TITLE:Proteomic profiling of fibroblasts reveals a modulating effect of extracellular calumenin on the organization of the actin cytoskeleton 

[2-s2.0-33745697841] Refers [2-s2.0-0033978227] LEVEL: 2
CREC proteins constitute a family of EF-hand calcium binding proteins localized to the secretory pathway. Calumenin is the only member known to be secreted. Recently, it was shown that thrombin-activated thrombocytes liberate calumenin, which also is found in atherosclerotic lesions but not in normal vasculature. To study the possible effects of calumenin extracellularly, we used proteomic profiling of fibroblasts cultured in absence and in presence of calumenin. Using 2-DE and MS/MS, we show that normal fibroblasts contain several 28-29-kDa N-terminal and a 16-kDa C-terminal fragment of ?- or ?-actin. Extracellularly added calumenin decreases the levels of both the N-terminal and C-terminal actin fragments, and, in addition, decreases the expression level of septin 2, which interacts with the actin cytoskeleton and is involved in cytokinesis. Labeling of S-phase fibroblasts with bromo-2?deoxy-uridine indicates that calumenin added to the medium also modulates the cell cycle. Our study thus indicates that calumenin may have an automne or a paracrine effect on the cells in its vicinity, and, therefore, may be involved in the pathophysiology of thrombosis or in wound healing. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

TITLE:A proteomic approach reveals transient association of reticulocalbin-3, a novel member of the CREC family, with the precursor of subtilisin-like proprotein convertase, PACE4 

[2-s2.0-33646772541] Refers [2-s2.0-0033978227] LEVEL: 2
SPCs (subtilisin-like proprotein convertases) are a family of seven structurally related serine endoproteases that are involved in the proteolytic activation of proproteins. In an effort to examine the substrate protein for PACE4 (paired basic amino-acid-cleaving enzyme-4), an SPC, a potent protein inhibitor of PACE4, an ?1-antitrypsin RVRR (Arg-Val-Arg-Arg) variant, was expressed in GH4C1 cells. Ectopic expression of the RVRR variant caused accumulation of the 48 kDa protein in cells. Sequence analysis indicates that the 48 kDa protein is a putative Ca2+-binding protein, RCN-3 (reticulocalbin-3), which had previously been predicted by bioinformatic analysis of cDNA from the human hypothalamus. RCN-3 is a member of the CREC (Cab45/reticulocalbin/ERC45/calumenin) family of multiple EF-hand Ca 2+-binding proteins localized to the secretory pathway. The most interesting feature of the RCN-3 sequence is the presence of five Arg-Xaa-Xaa-Arg motifs, which represents the target sequence of SPCs. Biosynthetic studies showed that RCN-3 is transiently associated with proPACE4, but not with mature PACE4. Inhibition of PACE4 maturation by a Ca2+ ionophore resulted in accumulation of the proPACE4-RCN-3 complex in cells. Furthermore, autoactivation and secretion of PACE4 was increased upon co-expression with RCN-3. Our findings suggest that selective and transient association of RCN-3 with the precursor of PACE4 plays an important role in the biosynthesis of PACE4. © 2006 Biochemical Society.

TITLE:Lumenal protein sorting to the constitutive secretory pathway of a regulated secretory cell 

[2-s2.0-33744542154] Refers [2-s2.0-0033978227] LEVEL: 2
Newly synthesized secretory granule content proteins are delivered via the Golgi complex for storage within mature granules, whereas constitutive secretory proteins are not stored. Most soluble proteins traveling anterograde through the trans-Golgi network are not excluded from entering immature secretory granules, whether or not they have granule-targeting signals. However, the 'sorting-for-entry' hypothesis suggests that soluble lumenal proteins lacking signals enter transport intermediates for the constitutive secretory pathway. We aimed to investigate how these constitutive secretory proteins are sorted. In a pancreatic ?-cell line, we stably expressed two lumenal proteins whose normal sorting information has been deleted: alkaline phosphatase, truncated to eliminate its glycosylphosphatidylinositol membrane anchor (SEAP); and Cab45361, a Golgi lumenal resident, truncated to eliminate its intracellular retention (Cab308Myc). Both truncated proteins are efficiently secreted, but whereas SEAP enters secretory granules, Cab308Myc behaves as a true constitutive marker excluded from granules. Interestingly, upon permeabilization of organelle membranes with saponin, SEAP is extracted as a soluble protein whereas Cab308Myc remains associated with the membrane. These are among the first data to support a model in which association with the lumenal aspect of Golgi and/or post-Golgi membranes can serve as a means for selective sorting of constitutive secretory proteins.

TITLE:Gene expression alteration during redox-dependent enhancement of arsenic cytotoxicity by emodin in HeLa cells 

[2-s2.0-27244437283] Refers [2-s2.0-0033978227] LEVEL: 2
Emodin (1,3,8-trihydroxy-6-methylanthraquinone) could enhance the sensitivity of tumor cells to arsenic trioxide (As2O 3)-induced apoptosis via generation of ROS, but the molecular mechanism has not been elucidated. Here, we carried out cDNA microarray-based global transcription profiling of HeLa cells in response to As2O 3/emodin cotreatment, comparing with As2O3-only treatment. The results showed that the expression of a number of genes was substantially altered at two time points. These genes are involved in different aspects of cell function. In addition to redox regulation and apoptosis, ROS affect genes encoding proteins associated with cell signaling, organelle functions, cell cycle, cytoskeleton, etc. These data suggest that based on the cytotoxicity of As2O3, emodin mobilize every genomic resource through which the As2O3-induced apoptosis is facilitated.

TITLE:Differential analysis of CD4+ Th memory clones with identical T-cell receptor (TCR)-Ī±Ī² rearrangement (non-transgenic), but distinct lymphokine phenotype, reveals diverse and novel gene expression 

[2-s2.0-4744365527] Refers [2-s2.0-0033978227] LEVEL: 2
This study describes a subtractive hybridization analysis to identify differences in gene expression between sibling Th memory clones, elicited by virus infection and expressing identical T-cell receptor (TCR)-?? rearrangements but distinct lymphokine phenotype: clone Bpp9 secretes interleukin (IL)-4, IL-5 and IL-10; clone Bpp19 secretes interferon (IFN)-?, low levels of IL-4, and IL-5 on TCR ligation. cDNA sequencing of difference products (DP) identified both novel and known regulatory (DNA: RNA-binding) or signalling proteins (kinases: phosphatases). Of the 10 novel genes identified, three were putative membrane proteins, one a predicted nuclear protein containing a PEST sequence motif, one a predicted transporter fragment and one contained a zinc-finger motif. One of the membrane proteins was found only in RNA from the activated IFN-?-producing clone, i.e. not in other tissues. In addition, 2 high frequency of granzyme A, B, C and G transcripts (for clone Bpp9) or transcripts for CD94 and NKG2A (for clone Bpp19) were expressed differentially, together with transcripts that mapped to, so far, unassigned regions of the mouse genome that may be further novel genes. The transcriptional profiles presented here may therefore include candidate regulators of Th diversity and effector function.

TITLE:Cloning, characterization, and expression of calcyphosine 2, a novel human gene encoding an EF-hand Ca2+-binding protein 

[2-s2.0-0036291303] Refers [2-s2.0-0033978227] LEVEL: 2
Calcyphosine is a calcium-binding protein containing four EF-hand domains, initially identified as thyroid protein p24. It was first cloned and its counterparts in rabbit, human, and mouse, crayfish and lobster of invertebrate were also cloned. Here we describe the cloning and characterization of a novel human calcyphosine gene. The 3829-bp cDNA encodes a EF-hand Ca2+-binding protein homologous to the dog calcyphosine. It also contains two EF-hand Ca2+-binding motif. It is abundantly expressed in many tissues including by RT-PCR analysis and believed to play important role in calcium signaling. It was mapped to human genome 12q15. © 2002 Elsevier Science (USA).

TITLE:Proteomics-based strategy to delineate the molecular mechanisms of the metastasis suppressor gene BRMS1 

[2-s2.0-35648989121] Refers [2-s2.0-0033978227] LEVEL: 2
The breast cancer metastasis suppressor 1 (BRMS1) gene has been shown to suppress metastasis without affecting the growth of the primary tumor in mouse models. It has also been shown to suppress the metastasis of tumors derived from breast, melanoma, and, more recently, ovarian carcinoma (see ref 1). However, how BRMS1 exerts its metastasis suppressor function remains unknown. To shed light into its metastatic mechanism of action, the sensitive 2D-DIGE analysis coupled with MS has been used to identify proteins differentially expressed by either overexpressing (Mel-BRMS1) or silencing BRMS1 (sh635) in a melanoma cell line. After comparison of the protein profiles from WT, Mel-BRMS1, and sh635 cells, 79 spots were found to be differentially expressed. Mass spectrometry analysis allowed the unambiguous identification of 55 polypeptides, corresponding to 43 different proteins. Interestingly, more than 75% of the identified proteins were down-regulated in Mel-BRMS1 cells compared to WT. In contrast, all the identified proteins in sh635 cells extracts were up-regulated compared to WT. Most of the deregulated proteins are involved in cell growth/maintenance and signal transduction among other cell processes. Six differentially expressed proteins (Hsp27, Alpha1 protease inhibitor, Cofilin1, Cathepsin D, Bone morphogenetic protein receptor2, and Annexin2) were confirmed by immunoblot and functional assays. Excellent correlation was found between DIGE analysis and immunoblot results, indicating the reliability of the analysis. Available evidence on the reported functions of the identified proteins supports the emerging role of BRMS1 as negative regulator of the metastasis development. This work opens an avenue for the molecular mechanisms' characterization of metastasis suppressor genes with the aim to understand their roles. © 2007 American Chemical Society.

TITLE:siRNA silencing of calumenin enhances functional factor IX production 

[2-s2.0-33845249178] Refers [2-s2.0-0033978227] LEVEL: 2
To improve production of functional fully ?-carboxylated recombinant human clotting factor IX (r-hFIX), cell lines stably overexpressing r-hFIX have been engineered to also overexpress proteins of the ?-carboxylation system. Here we demonstrate that siRNA silencing of calumenin, an inhibitor of the ?-carboxylation system, enhances production of functional r-hFIX produced by engineered BHK21 cells. The production yield of functional r-hFIX was 80% in engineered cells where calumenin had been silenced 78%. We propose that this high-yield expression system can easily be adapted to overproduce functional forms of all members of the vitamin K-dependent protein family. © 2006 by The American Society of Hematology.

TITLE:Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm 

[2-s2.0-33645108870] Refers [2-s2.0-0033978227] LEVEL: 2
Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions. © 2006 Elsevier Inc. All rights reserved.

TITLE:Differentiation of neuroblastoma cell line N1E-115 involves several signaling cascades 

[2-s2.0-21144436160] Refers [2-s2.0-0033978227] LEVEL: 2
No systematic searches for differential expression of signaling proteins (SP) in undifferentiated vs. differentiated cell lineages were published and herein we used protein profiling for this purpose. The N1E-115 cell line was cultivated and an aliquot was differentiated with dimethylsulfoxide (DMSO), that is known to lead to a neuronal phenotype. Cell lysates were prepared, run on two-dimensional gel electrophoresis followed by MALDI-TOF-TOF identification of proteins and maps of identified SPs were generated. Seven SPs were comparable, 27 SPs: GTP-binding/Ras-related proteins, kinases, growth factors, calcium binding proteins, phosphatase-related proteins were observed in differentiated N1E-115 cells and eight SPs of the groups mentioned above were observed in undifferentiated cells only. Switching-on/off of several individual SPs from different signaling cascades during the differentiation process is a key to understand mechanisms involved. The findings reported herein are challenging in vitro and in vivo studies to confirm a functional role for deranged SPs. © 2005 Springer Science+Business Media, Inc.

TITLE:Pathobiology of dynorphins in trauma and disease 

[2-s2.0-20244386405] Refers [2-s2.0-0037399166] LEVEL: 2
Dynorphins, endogenous opioid neuropeptides derived from the prodynorphin gene, are involved in a variety of normative physiologic functions including antinociception and neuroendocrine signaling, and may be protective to neurons and oligodendroglia via their opioid receptor-mediated effects. However, under experimental or pathophysiological conditions in which dynorphin levels are substantially elevated, these peptides are excitotoxic largely through actions at glutamate receptors. Because the excitotoxic actions of dynorphins require supraphysiological concentrations or prolonged tissue exposure, there has likely been little evolutionary pressure to ameliorate the maladaptive, non-opioid receptor mediated consequences of dynorphins. Thus, dynorphins can have protective and/or proapoptotic actions in neurons and glia, and the net effect may depend upon the distribution of receptors in a particular region and the amount of dynorphin released. Increased prodynorphin gene expression is observed in several disease states and disruptions in dynorphin processing can accompany pathophysiological situations. Aberrant processing may contribute to the net negative effects of dysregulated dynorphin production by tilting the balance towards dynorphin derivatives that are toxic to neurons and/or oligodendroglia. Evidence outlined in this review suggests that a variety of CNS pathologies alter dynorphin biogenesis. Such alterations are likely maladaptive and contribute to secondary injury and the pathogenesis of disease.

TITLE:Solution structure and calcium-binding properties of EF-hands 3 and 4 of calsenilin 

[2-s2.0-35648937651] Refers [2-s2.0-0037399166] LEVEL: 2
Calsenilin is a member of the recoverin branch of the EF-hand superfamily that is reported to interact with presenilins, regulate prodynorphin gene expression, modulate voltage-gated Kv4 potassium channel function, and bind to neurotoxins. Calsenilin is a Ca+2-binding protein and plays an important role in calcium signaling. Despite its importance in numerous neurological functions, the structure of this protein has not been reported. In the absence of Ca+2, the protein has limited spectral resolution that increases upon the addition of Ca+2. Here, we describe the three-dimensional solution structure of EF-hands 3 and 4 of calsenilin in the Ca+2-bound form. The Ca+2-bound structure consists of five ?-helices and one two-stranded antiparallel ?-sheet. The long loop that connects EF hands 3 and 4 is highly disordered in solution. In addition to its structural effects, Ca+2 binding also increases the protein's propensity to dimerize. These changes in structure and oligomerization state induced upon Ca+2 binding may play important roles in molecular recognition during calcium signaling. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society.

TITLE:The DREAM of pain relief 

[2-s2.0-33745895597] Refers [2-s2.0-0037399166] LEVEL: 2
Chronic pain has manifested itself as an independent disease. Different molecules acting in nociceptive pathways in the periphery and the nervous system are currently under investigation. Recently the multifunctional protein DREAM (Downstream Regulatory Element Antagonist Modulator)/calsenilin/KChIP3 has been implicated to play a role in the mechanisms of pain modulation and the hypothesis "No DREAM - No pain" was raised. In addition to the binding to DRE (Downstream Regulatory Element) sequences, DREAM/calsenilin/KChIP3 was shown to interact with presenilin, a protein thought to be a key molecule in Alzheimer's disease, and Kv4alpha-subunits assembling potassium channels. DREAM/calsenilin/KChIP3 has been described as a Ca2+-dependent transcriptional repressor, which is targeted to the regulatory DNA sequence of the prodynorphin gene. By actively suppressing gene expression of the endogenous opioid receptor ligand dynorphin, DREAM/calsenilin/KChIP3 is modulating the kappa opioid receptor system, which mediates analgesia. DREAM knock-out mice showed elevated levels of dynorphin and consequently displayed attenuated chemical induced and inflammatory pain. The data derived from the DREAM knock-out model led to the identification of a novel target in chronic pain management. In this review we focus on DREAM/calsenilin/KChIP3 and its role in pain relief. © 2006 Bentham Science Publishers Ltd.

TITLE:Enforced cytokinesis without complete nuclear division in embryonic cells depleting the activity of DNA topoisomerase IIĪ± 

[2-s2.0-12444269092] Refers [2-s2.0-0036201868] LEVEL: 2
Background: There are two distinct DNA topoisomerase II (topo II) isoforms, designated topo II? and topo II?, in mammalian cells. The function of topo II? in the development of mammalian cells has not been elucidated because of a lack of topo II? mutants. Results: We generated mice with a targeted disruption of the topo II? gene. The development of topo II?-/- embryos was terminated at the 4- or 8-cell stage. When wild-type embryos at the 2- or 4-cell stage were treated with ICRF-193, a catalytic inhibitor of topo II, nuclear division occurred followed by cytokinesis to form 4 or 8 cells, respectively, then development was terminated. Microscope analysis of 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei of both topo II?-/- and ICFR-193-treated embryonic cells revealed a droplet-like structure connecting the terminals of two adjacent nuclei forming a bridge-like structure. Phosphorylated histone H3, a marker for the M phases, disappeared from the nuclei of the topo II?-depleted embryonic cells. Laser scanning cytometry of the topo II?-depleted cells revealed the presence of 2N DNA cells. Conclusions: Our results indicate that topo II? has an essential role in the early stages of mouse development and that depletion of topo II? from the embryonic cells causes incomplete nuclear division followed by enforced cytokinesis.

TITLE:DNA topoisomerase II is required for the G0-to-S phase transition in Drosophila Schneider cells, but not in yeast 

[2-s2.0-5644241026] Refers [2-s2.0-0036201868] LEVEL: 2
We previously reported that DNA topoisomerase II (topo II) is required for the G0-to-S phase transition in mammalian cells [Hossain et al. (2002) ICRF-193, a catalytic inhibitor of DNA topoisomerase II, inhibits re-entry into the cell division cycle from quiescent state in mammalian cells. Genes Cells 7, 285-294]. In this study, we examined whether the requirement for topo II is evolutionarily conserved in Drosophila and yeast. ICRF-193, a catalytic inhibitor of topo II, inhibited DNA synthesis in Drosophila Schneider cells released from the G0 (stationary) phase, whereas the drug did not inhibit DNA synthesis in Schneider cells released from the M phase. Depletion of topo II mRNA by RNA-interference (RNAi) in G0-phase Schneider cells resulted in significant inhibition of DNA synthesis after release from G0-arrest. In the yeast topo II temperature-sensitive (ts) mutant, the initial cycle of DNA synthesis occurred at a restrictive temperature after release from starvation-induced G0 phase and doubling of the DNA content in the cells was confirmed by both flow cytometry and fluorescence spectrophotometry. DNA synthesis in yeast cells after release from the G0 phase was also observed in the presence of ICRF-193. Doubling of the DNA content was observed during spore germination of topo II ts mutant yeast at a restrictive temperature as determined by fluorescence spectrophotometry. These results indicate that topo II is required for the G0-to-S phase transition in Drosophila Schneider cells, but not in yeast. © Blackwell Publishing Limited.

TITLE:Reactive oxygen species: Metabolism, oxidative stress, and signal transduction 

[2-s2.0-3242715114] Refers [2-s2.0-0034954408] LEVEL: 3
Several reactive oxygen species (ROS) are continuously produced in plants as byproducts of aerobic metabolism. Depending on the nature of the ROS species, some are highly toxic and rapidly detoxified by various cellular enzymatic and nonenzymatic mechanisms. Whereas plants are surfeited with mechanisms to combat increased ROS levels during abiotic stress conditions, in other circumstances plants appear to purposefully generate ROS as signaling molecules to control various processes including pathogen defense, programmed cell death, and stomatal behavior. This review describes the mechanisms of ROS generation and removal in plants during development and under biotic and abiotic stress conditions. New insights into the complexity and roles that ROS play in plants have come from genetic analyses of ROS detoxifying and signaling mutants. Considering recent ROS-induced genome-wide expression analyses, the possible functions and mechanisms for ROS sensing and signaling in plants are compared with those in animals and yeast.

TITLE:Hydrogen peroxide signalling 

[2-s2.0-0036779326] Refers [2-s2.0-0034954408] LEVEL: 3
Recent biochemical and genetic studies confirm that hydrogen peroxide is a signalling molecule in plants that mediates responses to abiotic and biotic stresses. Signalling roles for hydrogen peroxide during abscisic-acid-mediated stomatal closure, auxin-regulated root gravitropism and tolerance of oxygen deprivation are now evident. The synthesis and action of hydrogen peroxide appear to be linked to those of nitric oxide. Downstream signalling events that are modulated by hydrogen peroxide include calcium mobilisation, protein phosphorylation and gene expression. Calcium and Rop signalling contribute to the maintenance of hydrogen peroxide homeostasis.

TITLE:Arabidopsis gp91phox homologues Atrbohd and Atrbohf are required for accumulation of reactive oxygen intermediates in the plant defense response 

[2-s2.0-0037039157] Refers [2-s2.0-0034954408] LEVEL: 3
Reactive oxygen intermediates (ROI) are strongly associated with plant defense responses. The origin of these ROI has been controversial. Arabidopsis respiratory burst oxidase homologues (rboh genes) have been proposed to play a role in ROI generation. We analyzed lines carrying dSpm insertions in the highly expressed AtrbohD and AtrbohF genes. Both are required for full ROI production observed during incompatible interactions with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000(avrRpm1) and the oomycete parasite Peronospora parasitica. We also observed reduced cell death, visualized by trypan blue stain and reduced electrolyte leakage, in the Atrboh mutants after DC3000(avrRpm1) inoculation. However, enhanced cell death is observed after infection of mutant lines with P. parasitica. Paradoxically, although atrbohD mutation eliminated the majority of total ROI production, atrbohF mutation exhibited the strongest effect on cell death.

TITLE:NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in arabidopsis 

[2-s2.0-0037507299] Refers [2-s2.0-0034954408] LEVEL: 3
Reactive oxygen species (ROS) have been proposed to function as second messengers in abscisic acid (ABA) signaling in guard cells. However, the question whether ROS production is indeed required for ABA signal transduction in vivo has not yet been addressed, and the molecular mechanisms mediating ROS production during ABA signaling remain unknown. Here, we report identification of two partially redundant Arabidopsis guard cell-expressed NADPH oxidase catalytic subunit genes, AtrbohD and AtrbohF, in which gene disruption impairs ABA signaling. atrbohD/F double mutations impair ABA-induced stomatal closing, ABA promotion of ROS production, ABA-induced cytosolic Ca2+ increases and ABA-activation of plasma membrane Ca2+-permeable channels in guard cells. Exogenous H2O2 rescues both Ca2+ channel activation and stomatal closing in atrbohD/F. ABA inhibition of seed germination and root elongation are impaired in atrbohD/F, suggesting more general roles for ROS and NADPH oxidases in ABA signaling. These data provide direct molecular genetic and cell biological evidence that ROS are rate-limiting second messengers in ABA signaling, and that the AtrbohD and AtrbohF NADPH oxidases function in guard cell ABA signal transduction.

TITLE:Small GTPases: Versatile signaling switches in plants 

[2-s2.0-0036270260] Refers [2-s2.0-0034954408] LEVEL: 3
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TITLE:Nicotiana benthamiana gp91phox homologs Nbrboha and Nbrbohb participate in H2O2 accumulation and resistance to Phytophthora infestans 

[2-s2.0-0037343106] Refers [2-s2.0-0034954408] LEVEL: 3
Active oxygen species (AOS) are responsible for triggering defense responses in plants. Respiratory burst oxidase homologs (rboh genes) have been implicated in AOS generation. We have isolated two rboh cDNAs, NbrbohA and NbrbohB, from Nicotiana benthamiana leaves. NbrbohA was expressed constitutively at a low level and the transcripts were increased after mechanical stress of control leaf infiltration, whereas NbrbohB was induced specifically by the protein elicitor INF1 from the potato pathogen Phytophthora infestans. We examined the function of the Nbrboh genes in AOS generation and in the hypersensitive response (HR) using virus-induced gene silencing (VIGS). VIGS indicated that both genes are required for H2O2 accumulation and for resistance to Phytophthora. VIGS of Nbrboh genes also led to a reduction and delay of HR cell death caused by INF1. We further demonstrate that the induction of HR-like cell death by overexpression of a constitutively active mutant of a mitogen-activated protein kinase kinase, MEKDD, is compromised by VIGS of NbrbohB. We found that MEKDD induced NbrbohB but not NbrbohA. This work provides genetic evidence for the involvement of a mitogen-activated protein kinase cascade in the regulation of rboh genes.

TITLE:RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygen deprivation tolerance 

[2-s2.0-0037076971] Refers [2-s2.0-0034954408] LEVEL: 3
Transient soil flooding limits cellular oxygen to roots and reduces crop yield. Plant response to oxygen deprivation involves increased expression of the alcohol dehydrogenase gene (ADH) and ethanolic fermentation. Disruption of the Arabidopsis gene that encodes Rop (RHO-like small G protein of plants) guanosine triphosphatase (GTPase) activating protein 4 (ROPGAP4), a Rop deactivator, elevates ADH expression in response to oxygen deprivation but decreases tolerance to stress. Rop-dependent production of hydrogen peroxide via a diphenylene iodonium chloride-sensitive calcium-dependent reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is necessary for induction of both ADH and RopGAP4 expression. Tolerance to oxygen deprivation requires Rop activation and RopGAP4-dependent negative feedback regulation. This Rop signal transduction rheostat balances the ability to increase ethanolic fermentation with survival.

TITLE:Changes in hydrogen peroxide homeostasis trigger an active cell death process in tobacco 

[2-s2.0-0345103736] Refers [2-s2.0-0034954408] LEVEL: 3
In transgenic tobacco plants with reduced catalase activity, high levels of hydrogen peroxide (H2O2) can accumulate under photorespiratory conditions. Such a perturbation in H2O2 homeostasis induced cell death in clusters of palisade parenchyma cells, primarily along the veins. Ultrastructural alterations, such as chromatin condensation and disruption of mitochondrial integrity, took place before cell death. Furthermore, enhanced transcript levels of mitochondrial defense genes accompanied these mitochondrial changes. Pharmacological data indicated that the initiation and execution of cell death require de novo protein synthesis and that the signal transduction pathway leading to cell death involved changes in ion homeostasis, (de)phosphorylation events and an oxidative burst, as observed during hypersensitive responses. This oxidase-dependent oxidative burst is essential for cell death, but it is not required for the accumulation of defense proteins, suggesting a more prominent role for the oxidative burst in abiotic stress-induced cell death.

TITLE:Functions of the respiratory burst oxidase in biotic interactions, abiotic stress and development 

[2-s2.0-20444458715] Refers [2-s2.0-0034954408] LEVEL: 3
The production of reactive oxygen intermediates (ROI) is among the earliest temporal events following pathogen recognition in plants. Initially, ROI were thought to be cell-death executioners. Emerging evidence, however, suggests a broader role for ROI as signals that mediate responses to infection, the abiotic environment, developmental cues, and programmed cell death in different cell types. The Respiratory burst oxidase homolog (Rboh) gene family encodes the key enzymatic subunit of the plant NADPH oxidase. Rboh proteins are the source of ROI produced following pathogen recognition and in a variety of other processes. © 2005 Elsevier Ltd. All rights reserved.

TITLE:The plasma membrane oxidase NtrbohD is responsible for AOS production in elicited tobacco cells 

[2-s2.0-0036323236] Refers [2-s2.0-0034954408] LEVEL: 3
A cDNA encoding a protein, NtrbohD, located on the plasma membrane and homologue to the flavocytochrome of the neutrophil NADPH oxidase, was cloned in tobacco. The corresponding mRNA was accumulated when tobacco leaves and cells were treated with the fungal elicitor cryptogein. After elicitation with cryptogein, tobacco cells transformed with antisense constructs of NtrbohD showed the same extracellular alkalinization as the control, but no longer produced active oxygen species (AOS). This work represents the first demonstration of the function of a homologue of gp91-phox in AOS production in elicited tobacco cells.

TITLE:The heterotrimeric G protein Ī± subunit acts upstream of the small GTPase Rac in disease resistance of rice 

[2-s2.0-0036791063] Refers [2-s2.0-0034954408] LEVEL: 3
We used rice dwarf1 (d1) mutants lacking a single-copy G? gene and addressed G?'s role in disease resistance. d1 mutants exhibited a highly reduced hypersensitive response to infection by an avirulent race of rice blast. Activation of PR gene expression in the leaves of the mutants infected with rice blast was delayed for 24 h relative to the wild type. H2O2 production and PR gene expression induced by sphingolipid elicitors (SE) were strongly suppressed in d1 cell cultures. Expression of the constitutively active OsRac1, a small GTPase Rac of rice, in d1 mutants restored SE-dependent defense signaling and resistance to rice blast. G? mRNA was induced by an avirulent race of rice blast and SE application on the leaf. These results indicated the role of G? in R gene-mediated disease resistance of rice. We have proposed a model for the defense signaling of rice in which the heterotrimeric G protein functions upstream of the small GTPase OsRac1 in the early steps of signaling.

TITLE:Reactive oxygen intermediates in plant-microbe interactions: Who is who in powdery mildew resistance? 

[2-s2.0-0038271896] Refers [2-s2.0-0034954408] LEVEL: 3
Reactive oxygen intermediates (ROIs) such as hydrogen peroxide (H2O2) and the superoxide anion radical (O2·-) accumulate in many plants during attack by microbial pathogens. Despite a huge number of studies, the complete picture of the role of ROIs in the host-pathogen interaction is not yet fully understood. This situation is reflected by the controversially discussed question as to whether ROIs are key factors in the establishment and maintenance of either host cell inaccessibility or accessibility for fungal pathogens. On the one hand, ROIs have been implicated in signal transduction as well as in the execution of defence reactions such as cell wall strengthening and a rapid host cell death (hypersensitive reaction). On the other hand, ROIs accumulate in compatible interactions, and there are reports suggesting a function of ROIs in restricting the spread of leaf lesions and thus in suppressing cell death. Moreover, in situ analyses have demonstrated that different ROIs may trigger opposite effects in plants depending on their spatiotemporal distribution and subcellular concentrations. This demonstrates the need to determine the particular role of individual ROIs in distinct stages of pathogen development. The well-studied interaction of cereals with fungi from the genus Blumeria is an excellent model system in which signal transduction and defence reactions can be further elucidated in planta. This review article gives a synopsis of the role of ROI accumulation, with particular emphasis on the pathosystem Hordeum vulgare L.-Blumeria graminis.

TITLE:Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans 

[2-s2.0-0345689448] Refers [2-s2.0-0034954408] LEVEL: 3
NADPH oxidases (Nox) have been characterized as higher eukaryotic enzymes used deliberately to produce reactive oxygen species (ROS). The recent discovery of new functional members of the Nox family in plants and animals has led to the recognition of the increasing importance of ROS as signals involved in regulation of diverse cellular processes such as defence, growth and signalling. Here, we address the role of NADPH oxidase-generated ROS in the biology of the filamentous fungus Aspergillus nidulans. We characterize the noxA gene and show that it encodes a member of a novel NADPH oxidase subfamily ubiquitous in lower eukaryotes. Deletion of noxA specifically blocks differentiation of sexual fruit bodies (cleistothecia), without affecting hyphal growth or asexual development. Accordingly, the noxA gene is induced during sexual development, peaking at the time of cleistothecia differentiation and in parallel with the hu?lle cell-associated catalase peroxidase gene cpeA. This expression pattern is not dependent on transcription factors SteA and StuA, which are essential for cleistothecia formation. In contrast, nox4-dependent premature sexual development correlates with noxA derepression in ?sakA null mutants, connecting stress MAPK signalling to the regulated production of ROS. Using a nitroblue tetrazolium (NBT) assay to detect superoxide, we found that hu?lle cells and cleistothecia initials produce superoxide in a process inhibited by NADPH oxidase inhibitor DPI and markedly reduced in ?noxA mutants. Furthermore, using H2DCFDA, we detected that H2O2 and possibly other ROS are generated in a NoxA-dependent fashion, mainly in the external walls from cleistothecia initials. The essential role of NoxA-generated ROS in A. nidulans sexual differentiation and the presence of one or two noxA homologues in all analysed filamentous fungi suggest that NADPH oxidase-generated ROS play important roles in fungal physiology and differentiation.

TITLE:Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings 

[2-s2.0-0036943993] Refers [2-s2.0-0034954408] LEVEL: 3
The roles of the plasma-membrane (PM) NADPH oxidase in abscisic acid (ABA)- and water stress-induced antioxidant defense were investigated in leaves of maize (Zea mays L.) seedlings. Treatment by exogenous ABA (100 ?M ABA) or osmotic stress (-0.7 MPa induced by polyethylene glycol) significantly increased the activity of the PM NADPH oxidase, the production of leaf O2-, the activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), and the contents of antioxidant metabolites (ascorbate and reduced glutathione). Pretreatment with three different inhibitors of NADPH oxidase (diphenylene iodonium, imidazole and pyridine) or an inhibitor of ABA biosynthesis (tungstate) reduced the increase in the activity of the PM NADPH oxidase and the production of leaf O2-, and the capacity of antioxidant defense systems mediated by ABA. The inhibitory effects above caused by tungstate were reversed by exogenous ABA. These data indicate that NADPH oxidase is involved m the ABA-induced production of active oxygen species (AOS), and our results depict a minimal chain of events initiated by water stress-reduced ABA accumulation, which then triggers the production of AOS by membrane-bound NADPH oxidase, resulting in the induction of antioxidant defense systems against oxidative damage in plants.

TITLE:Reactive oxygen species activation of plant Ca2+ channels. A signaling mechanism in polar growth, hormone transduction, stress signaling, and hypothetically mechanotransduction 

[2-s2.0-3042571946] Refers [2-s2.0-0034954408] LEVEL: 3
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TITLE:Stress response, cell death and signalling: The many faces of reactive oxygen species 

[2-s2.0-0141857861] Refers [2-s2.0-0034954408] LEVEL: 3
Plants respond to pathogens and abiotic stresses by transient increases in the production of reactive oxygen species (ROS) and ion fluxes, which activate both local programmed cell death and systemic increases in stress- and pathogen-resistance. The present essay explores the emerging complexity of the multiple roles that ROS play in intra- and intercellular communication in both stressed and unstressed organisms.

TITLE:Oxidative burst and cell death in ozone-exposed plants 

[2-s2.0-0036619353] Refers [2-s2.0-0034954408] LEVEL: 3
The phytotoxic air pollutant ozone spontaneously generates reactive oxygen species (ROS) in the leaf apoplast, provokes hypersensitive response-like lesions and induces defence reactions that significantly overlap with pathogen and other oxidative stress responses. Consequently, ozone has been used as a tool to unravel in planta ROS-induced plant defence and cell death mechanisms. Ozone exposure stimulates an oxidative burst in leaves of sensitive plants, resulting in the generation and accumulation of hydrogen peroxide or superoxide anions in distinct species. Accumulation of these ROS precedes the induction of cell death, and both responses co-occur spatially in the periveinal regions of the leaves. The review summarizes some of the recent results that have been obtained concerning the molecular basis of apoplastic ROS production in monocot and dicot species. Signal molecules, in particular ethylene and salicylic acid, control and potentiate the oxidative burst and subsequent cell death in its initiation and propagation phases while jasmonate leads to lesion containment. Amplification mechanisms that result in the production of excess ROS and hypersensitive cell death are discussed as major factors in ozone sensitivity of plant species and cultivars. © 2002 E?ditions scientifiques et me?dicales Elsevier SAS. All rights reserved.

TITLE:Reactive oxygen species signaling in response to pathogens 

[2-s2.0-33745662410] Refers [2-s2.0-0034954408] LEVEL: 3
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TITLE:An integrative genetic linkage map of winter wheat (Triticum aestivum L.) 

[2-s2.0-0242405201] Refers [2-s2.0-0034954408] LEVEL: 3
We constructed a genetic linkage map based on a cross between two Swiss winter wheat (Triticum aestivum L.) varieties, Anna and Forno. Two-hundred and forty F5 single-seed descent (SSD)-derived lines were analysed with 112 restriction fragment length polymorphism (RFLP) anonymous probes, 18 wheat cDNA clones coding for putative stress or defence-related proteins and 179 simple-sequence repeat (SSR) primer-pairs. The 309 markers revealed 396 segregating loci. Linkage analysis defined 27 linkage groups that could all be assigned to chromosomes or chromosome arms. The resulting genetic map comprises 380 loci and spans 3,086 cM with 1,131 cM for the A genome, 920 cM for the B genome and 1,036 cM for the D genome. Seventeen percent of the loci showed a significant (P < 0.05) deviation from a 1:1 ratio, most of them in favour of the Arina alleles. This map enabled the mapping of QTLs for resistance against several fungal diseases such as Stagonospora glume blotch, leaf rust and Fusarium head blight. It will also be very useful for wheat genetic mapping, as it combines RFLP and SSR markers that were previously located on separate maps.

TITLE:ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation 

[2-s2.0-23644456321] Refers [2-s2.0-0034954408] LEVEL: 3
Pollen exposure induces allergic airway inflammation in sensitized subjects. The role of antigenic pollen proteins in the induction of allergic airway inflammation is well characterized, but the contribution of other constituents in pollen grains to this process is unknown. Here we show that pollen grains and their extracts contain intrinsic NADPH oxidases. The pollen NADPH oxidases rapidly increased the levels of ROS in lung epithelium as well as the amount of oxidized glutathione (GSSG) and 4-hydroxynonenal (4-HNE) in airway-lining fluid. These oxidases, as well as products of oxidative stress (such as GSSG and 4-HNE) generated by these enzymes, induced neutrophil recruitment to the airways independent of the adaptive immune response. Removal of pollen NADPH oxidase activity from the challenge material reduced antigen-induced allergic airway inflammation, the number of mucin-containing cells in airway epithelium, and antigen-specific IgE levels in sensitized mice. Furthermore, challenge with Amb a 1, the major antigen in ragweed pollen extract that does not possess NADPH oxidase activity, induced low-grade allergic airway inflammation. Addition of GSSG or 4-HNE to Amb a 1 challenge material boosted allergic airway inflammation. We propose that oxidative stress generated by pollen NADPH oxidases (signal 1) augments allergic airway inflammation induced by pollen antigen (signal 2).

TITLE:Nod factor inhibition of reactive oxygen efflux in a host legume 

[2-s2.0-0042510530] Refers [2-s2.0-0034954408] LEVEL: 3
Hydrogen peroxide (H2O2) efflux was measured from Medicago truncatula root segments exposed to purified Nod factor and to poly-GalUA (PGA) heptamers. Nod factor, at concentrations > 100 pM, reduced H2O2 efflux rates to 60% of baseline levels beginning 20 to 30 min after exposure, whereas the PGA elicitor, at > 75 nM, caused a rapid increase in H2O2 efflux to >200% of baseline rates. Pretreatment of plants with Nod factor alters the effect of PGA by limiting the maximum H2O2 efflux rate to 125% of that observed for untreated plants. Two Nod factor-related compounds showed no ability to modulate peroxide efflux, and tomato (Lycopersicon esculentum), a nonlegume, showed no response to 1 nM Nod factor. Seven M. truncatula mutants, lacking the ability to make nodules, were tested for Nod factor effects on H2O2 efflux. The nfp mutant was blocked for suppression of peroxide efflux, whereas the dmi1 and dmi2 mutants, previously shown to be blocked for early Nod factor responses, showed a wild-type peroxide efflux modulation. These data demonstrate that exposure to Nod factor suppresses the activity of the reactive oxygen-generating system used for plant defense responses.

TITLE:Cross-talk between calcium and reactive oxygen species originated from NADPH oxidase in abscisic acid-induced antioxidant defence in leaves of maize seedlings 

[2-s2.0-0038046132] Refers [2-s2.0-0034954408] LEVEL: 3
The signal interactions between calcium (Ca2+) and reactive oxygen species (ROS) originated from plasma membrane NADPH oxidase in abscisic acid (ABA)-induced antioxidant defence were investigated in leaves of maize (Zea mays L.) seedlings. Treatment with ABA led to significant increases in the activity of plasma membrane NADPH oxidase, the production of leaf O2-, and the activities of several antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). However, such increases were blocked by the pretreatment with Ca2+ chelator EGTA or Ca2+ channel blockers La3+ and verapamil, and NADPH oxidase inhibitors such as diphenylene iodonium (DPI), imidazole and pyridine. Treatment with Ca2+ also significantly induced the increases in NADPH oxidase activity, O2- production and the activities of antioxidant enzymes, and the increases were arrested by pretreatment with the NADPH oxidase inhibitors. Treatment with oxidative stress induced by paraquat, which generates O2-, led to the induction of antioxidant defence enzymes, and the upregulation was suppressed by the pretreatment of Ca2+ chelator and Ca2+ channel blockers. Our data suggest that a cross-talk between Ca2+ and ROS originated from plasma membrane-bound NADPH oxidase is involved in the ABA signal transduction pathway leading to the induction of antioxidant enzyme activity, and Ca2+ functions upstream as well as downstream of ROS production in the signal transduction event in plants.

TITLE:Plant respiratory burst oxidase homologs impinge on wound responsiveness and development in Lycopersicon esculentum 

[2-s2.0-1542437689] Refers [2-s2.0-0034954408] LEVEL: 3
Plant respiratory burst oxidase homologs (Rboh) are homologs of the human neutrophil pathogen-related gp91phox. Antisense technology was employed to ascertain the biological function of Lycopersicon esculentum (tomato) Rboh. Lines with diminished Rboh activity showed a reduced level of reactive oxygen species (ROS) in the leaf, implying a role for Rboh in establishing the cellular redox milieu. Surprisingly, the antisense plants acquired a highly branched phenotype, switched from indeterminate to determinate growth habit, and had fasciated reproductive organs. Wound-induced systemic expression of proteinase inhibitor II was compromised in the antisense lines, indicating that ROS intermediates supplied by Rboh are required for this wound response. Extending these observations by transcriptome analysis revealed ectopic leaf expression of homeotic MADS box genes that are normally expressed only in reproductive organs. In addition, both Rboh-dependent and -independent wound-induced gene induction was detected as well as transcript changes related to redox maintenance. The results provide novel insights into how the steady state cellular level of ROS is controlled and portrays the role of Rboh as a signal transducer of stress and developmental responses.

TITLE:Functional analysis of barley RAC/ROP G-protein family members in susceptibility to the powdery mildew fungus 

[2-s2.0-0345531096] Refers [2-s2.0-0034954408] LEVEL: 3
Small monomeric G-proteins of the plant ras (rat sarcome oncogene product) related C3 botulinum toxin substrate (RAC)/Rho of giants (ROP) family are molecular switches in signal transduction of many cellular processes. RAC/ROPs regulate hormone effects, subcellular gradients of Ca2+, the organisation of the actin cytoskeleton and the production of reactive oxygen intermediates. Therefore, we followed a genetic bottom-up strategy to study the role of these proteins during the interaction of barley (Hordeum vulgare L.) with the fungal biotrophic pathogen Blumeria graminis f.sp. hordei (Bgh). We identified six barley RAC/ ROP proteins and studied their gene expression. Five out of six Rac/Rop genes were expressed constitutively in the leaf epidermis, which is the site of interaction with Bgh. None of the genes showed enhancement of mRNA abundance after inoculation with Bgh. After microprojectile mediated transformation of single barley epidermal cells with constitutively activated mutant RAC/ROP proteins, we found an RAC/ ROP-specrfic enhancement of pathogen accessibility, tagging HvRACB, HvRAC3 and HvROP6 as host proteins potentially involved in the establishment of susceptibility to Bgh. Confocal laser scanning microscopy (CLSM) of green fluorescent protein (GFP):HvRAC/ROP-transformed cells revealed varying strengths of plasma membrane association of barley RAC/ROPs. The C-terminal CAAX motif for presumable prenylation or the C-terminal hypervariable region (HVR), respectively, were required for membrane association of the RAC/ROPs. Proper intracellular localisation was essential for HvRACB and HvRAC3 function. Together, our data support the view that different paths of host signal transduction via RAC/ROP G-proteins are involved in processes supporting parasitic entry into epidermal host cells.

TITLE:Cell signalling following plant/pathogen interactions involves the generation of reactive oxygen and reactive nitrogen species 

[2-s2.0-0036619357] Refers [2-s2.0-0034954408] LEVEL: 3
It is now clear that reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), and reactive nitrogen species, such as nitric oxide (NO), are produced by plant cells in response to a variety of stresses, including pathogen challenge. Such molecules may be involved in direct defence mechanisms, such as cross-linking of plant cell walls, or as antimicrobial agents. However, it is also apparent that cells generate such reactive species as signalling molecules, produced at controlled levels, and leading to defined responses. Signalling responses to ROS and NO include the activation of mitogen-activated protein kinases, and the up- and down-regulation of gene expression, often leading to localised programmed cell death, characteristic of the hypersensitive response. Therefore, ROS and NO are key molecules which may help to orchestrate events following pathogen challenge. Here we review the generation and role of both reactive oxygen and reactive nitrogen species in plant cells. © 2002 E?ditions scientifiques et me?dicales Elsevier SAS. All rights reserved.

TITLE:Are leaf hydrogen peroxide concentrations commonly overestimated? The potential influence of artefactual interference by tissue phenolics and ascorbate 

[2-s2.0-0036619733] Refers [2-s2.0-0034954408] LEVEL: 3
We have examined the authenticity of two methods for determination of H2O2 in leaf tissue. We show that the high concentrations of ascorbic acid present in leaf extracts interfere with both techniques. In the chromogenic peroxidase-coupled assay, H2O2 is determined by oxidation of 3-methyl-2-benzothiazoline hydrazone (MBTH) and 3-(dimethylamino) benzoic acid (DMAB). The method yields two phases of absorbance increase as these substrates are oxidized. We show (a) that only the first phase is dependent on extracted H2O2; (b) that the slow phase is due to phenolic-dependent generation of H2O2 during the assay; and (c) that ascorbate inhibits both phases. These effects could explain both the high values and the variable results found in the literature. The chemiluminescence method involves H2O2 enhancement of ferricyanide-induced chemiluminescence of luminol (3-amino-phthal-hydrazide). Chemiluminescence of luminol is strongly inhibited by added ascorbate, suggesting that failure to remove ascorbate from extracts will cause this method to underestimate H2O2. Using the fast phase of the peroxidase-coupled assay to estimate H2O2 in extracts from which ascorbate and phenolic compounds had been removed, we obtained leaf contents of H2O2 within the range of 40-120 nmol g-1 FW. © 2002 E?ditions scientifiques et me?dicales Elsevier SAS. All rights reserved.

TITLE:Biotic interactions of marine algae 

[2-s2.0-0036017857] Refers [2-s2.0-0034954408] LEVEL: 3
Marine algae encompass lineages that diverged about one billion years ago. Recent results suggest that they feature natural immunity traits that are conserved, as well as others that appear to be phylum- or environment-specific. In particular, marine plants resemble terrestrial plants and animals in their basic mechanisms for pathogen recognition and signaling, suggesting that these essential cell functions arose in the sea. Specific traits are based on the synthesis of unique secondary defense metabolites, often making use of the variety of halides found in the sea.

TITLE:Ultraviolet-B radiation co-opts defense signaling pathways 

[2-s2.0-0345358537] Refers [2-s2.0-0034954408] LEVEL: 3
Plants in the field exposed to ambient solar ultraviolet-B (UV-B) radiation (280-320 nm) often show an increased resistance to herbivorous insects compared with control plants grown under filters that exclude the UV-B component of solar radiation. This corresponds with a significant overlap in gene expression between the UV-B and the wounding/herbivory response. Furthermore, wound-responsive signaling components such as mitogen-activated protein kinases are activated by UV-B. A mechanistic explanation for these overlaps might be that UV-B co-opts cell surface receptors for defense signals such as systemin and oligosaccharide elicitors in a ligand-independent manner.

TITLE:Small GTPase 'Rop': Molecular switch for plant defense responses 

[2-s2.0-0038198644] Refers [2-s2.0-0034954408] LEVEL: 3
The conserved Rho family of GTPases (Rho, Rac, and Cdc42) in fungi and mammals has emerged as a key regulator of diverse cellular activities, such as cytoskeletal rearrangements, programmed cell death, stress-induced signaling, and cell growth and differentiation. In plants, a unique class of Rho-like proteins, most closely related to mammalian Rac, has only been found and termed 'Rop' (Rho-related GTPase from plant [Li et al. (1998) Plant Physiol. 118, 407-417; Yang (2002) Plant Cell 14, S375-S388]). ROPs have been implicated in regulating various plant cellular responses including defense against pathogens. It has been shown that ROPs, like mammalian Rac, trigger hydrogen peroxide production and hence the 'oxidative burst', a crucial component associated with the cell death, most likely via activation of nicotinamide adenine dinucleotide phosphate oxidase in both monocotyledonous and dicotyledonous species. Recent studies have established that ROPs also function as a molecular switch for defense signaling pathway(s) linked with disease resistance. As discerning the defense pathway remains one of the priority research areas in the field of plant biology, this review is therefore particularly focused on recent progresses that have been made towards understanding the plant defense responses mediated by ROPs. © 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

TITLE:Two NADPH oxidase isoforms are required for sexual reproduction and ascospore germination in the filamentous fungus Podospora anserina 

[2-s2.0-4744362719] Refers [2-s2.0-0034954408] LEVEL: 3
NADPH oxidases are enzymes that produce reactive oxygen species (ROS) using electrons derived from intracellular NADPH. In plants and mammals, ROS have been proposed to be second messengers that signal defence responses or cell proliferation. By inactivating PaNox1 and PaNox2, two genes encoding NADPH oxidases, we demonstrate the crucial role of these enzymes in the control of two key steps of the filamentous fungus Podospora anserina life cycle. PaNox1 mutants are impaired in the differentiation of fruiting bodies from their progenitor cells, and the deletion of the PaNox2 gene specifically blocks ascospore germination. Furthermore, we show that PaNox1 likely acts upstream of PaASK1, a MAPKKK previously implicated in stationary phase differentiation and cell degeneration. Using nitro blue tetrazolium (NBT) and diaminobenzidine (DAB) assays, we detect a regulated secretion of both superoxide and peroxide during P. anserina vegetative growth. In addition, two oxidative bursts are shown to occur during fruiting body development and ascospore germination. Analysis of mutants establishes that PaNox1, PaNox2, and PaASK1, as well as a still unknown additional source of ROS, modulate these secretions. Altogether, our data point toward a role for NADPH oxidases in signalling fungal developmental transitions with respect to nutrient availability. These enzymes are conserved in other multicellular eukaryotes, suggesting that early eukaryotes were endowed with a redox network used for signalling purposes. © 2004 Elsevier Inc. All rights reserved.

TITLE:The germinlike protein GLP4 exhibits superoxide dismutase activity and is an important component of quantitative resistance in wheat and barley 

[2-s2.0-1642424407] Refers [2-s2.0-0034954408] LEVEL: 3
Germinlike proteins (GLP) are encoded in plants by a gene family with proposed functions in plant development and defense. Genes of GLP subfamily 4 of barley (HvGLP4, formerly referred to as HvOxOLP) and the wheat orthologue TaGLP4 (formerly referred to as TaGLP2a) were previously found to be expressed in pathogen-attacked epidermal tissue of barley and wheat leaves, and the corresponding proteins are proposed to accumulate in the apoplast. Here, the role of HvGLP4 and TaGLP4 in the defense of barley and wheat against Blumeria graminis (DC.) E. O. Speer, the cereal powdery mildew fungus, was examined in an epidermal transient expression system and in transgenic Arabidopsis thaliana plants overexpressing His-tagged HvGLP4. Leaf extracts of transgenic Arabidopsis overexpressing HvGLP4 contained a novel His-tagged protein with superoxide dismutase activity and HvGLP4 epitopes. Transient overexpression of TaGLP4 and HvGLP4 enhanced resistance against B. graminis in wheat and barley, whereas transient silencing by RNA interference reduced basal resistance in both cereals. The effect of GLP4 overexpression or silencing was strongly influenced by the genotype of the plant. The data suggest that members of GLP subfamily 4 are components of quantitative resistance in both barley and wheat, acting together with other, as yet unknown, plant components.

TITLE:Early signaling events induced by elicitors of plant defenses 

[2-s2.0-33745748478] Refers [2-s2.0-0034954408] LEVEL: 3
Plant pathogen attacks are perceived through pathogen-issued compounds or plant-derived molecules that elicit defense reactions. Despite the large variety of elicitors, general schemes for cellular elicitor signaling leading to plant resistance can be drawn. In this article, we review early signaling events that happen after elicitor perception, including reversible protein phosphorylations, changes in the activities of plasma membrane proteins, variations in free calcium concentrations in cytosol and nucleus, and production of nitric oxide and active oxygen species. These events occur within the first minutes to a few hours after elicitor perception. One specific elicitor transduction pathway can use a combination or a partial combination of such events which can differ in kinetics and intensity depending on the stimulus. The links between the signaling events allow amplification of the signal transduction and ensure specificity to get appropriate plant defense reactions. This review first describes the early events induced by cryptogein, an elicitor of tobacco defense reactions, in order to give a general scheme For signal transduction that will be use as a thread to review signaling events monitored in different elicitor or plant models. © 2006 The American Phytopathological Society.

TITLE:Production of reactive oxygen species by plant NADPH oxidases 

[2-s2.0-33745646568] Refers [2-s2.0-0034954408] LEVEL: 3
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TITLE:Calcium in plant defence-signalling pathways: Tansley review 

[2-s2.0-33745198422] Refers [2-s2.0-0034954408] LEVEL: 3
In plant cells, the calcium ion is a ubiquitous intracellular second messenger involved in numerous signalling pathways. Variations in the cytosolic concentration of Ca2+ ([Ca2+]cyt) couple a large array of signals and responses. Here we concentrate on calcium signalling in plant defence responses, particularly on the generation of the calcium signal and downstream calcium-dependent events participating in the establishment of defence responses with special reference to calcium-binding proteins. © New Phytologist (2006).

TITLE:Cryptogein-Induced Initial Events in Tobacco BY-2 Cells: Pharmacological Characterization of Molecular Relationship among Cytosolic Ca2+ Transients, Anion Efflux and Production of Reactive Oxygen Species 

[2-s2.0-1542290919] Refers [2-s2.0-0034954408] LEVEL: 3
Ion fluxes and the production of reactive oxygen species (ROS) are early events that follow elicitor treatment or microbial infection. However, molecular mechanisms for these responses as well as their relationship have been controversial and still largely unknown. We here simultaneously monitored the temporal sequence of initial events at the plasma membrane in suspension-cultured tobacco cells (cell line BY-2) in response to a purified proteinaceous elicitor, cryptogein, which induced hypersensitive cell death. The elicitor induced transient rise in cytosolic Ca2+ concentration ([Ca2+]cyt) showing two distinct peaks, followed by biphasic (rapid/transient and slow/prolonged) Cl- efflux and H + influx. Pharmacological analyses suggested that the two phases of the [Ca2+]cyt response correspond to Ca2+ influx through the plasma membrane and an inositol 1,4,5-trisphophate-mediated release of Ca2+ from intracellular Ca2+ stores, respectively, and the [Ca2+]cyt transients and the Cl - efflux were mutually dependent events regulated by protein phosphorylation. The elicitor also induced production of ROS including ?O2- and H2O2, which initiated after the [Ca2+]cyt rise and required Ca2+ influx, Cl- efflux and protein phosphorylation. An inhibitor of NADPH oxidase, diphenylene iodonium, completely inhibited the elicitor-induced production of ?O2- and H2O2, but did not affect the [Ca2+] cyt transients. These results suggest that cryptogein-induced plasma membrane Ca2+ influx is independent of ROS, and NADPH oxidase dependent ROS production is regulated by these series of ion fluxes.

TITLE:Cauliflower mosaic virus, a compatible pathogen of Arabidopsis, engages three distinct defense-signaling pathways and activates rapid systemic generation of reactive oxygen species 

[2-s2.0-33644655397] Refers [2-s2.0-0034954408] LEVEL: 3
We analyzed expression of marker genes for three defense pathways during infection by Cauliflower mosaic virus (CaMV), a compatible pathogen of Arabidopsis (Arabidopsis thaliana), using luciferase reporter transgenes and directly by measuring transcript abundance. Expression of PR-1, a marker for salicylic acid signaling, was very low until 8 d postinoculation and then rose sharply coinciding with the rise in virus levels. In contrast, as early as 2 h postinoculation, transcriptional up-regulation of GST1-a marker for reactive oxygen species-and PDF1.2-a marker for jasmonic acid/ethylene defense signaling-was detectable in the virus-inoculated leaf and systemically. In parallel with the activation of GST1, H2O2 accumulated locally and systemically in virus- but not mock-inoculated plants. However, in plants inoculated with infectious CaMV DNA rather than virus particles, the onset of systemic luciferase activity was delayed by 24 to 48 h, suggesting that virion structural proteins act as the elicitor. This phenomenon, which we term the rapid systemic response, preceded virus movement from the inoculated leaf; therefore, the systemic signal is not viral. Systemic, but not local, H 2O2 accumulation was abolished in rbohDF double mutants and in etr1-1 and ein2-1 mutants, implicating NADPH oxidase and ethylene signaling in the generation and transduction of the response. Ethylene, but not rbohDF mutants, also showed reduced susceptibility to CaMV, whereas in NahG transgenics, virus levels were similar to wild type. These findings implicate reactive oxygen species and ethylene in signaling in response to CaMV infection, but suggest that salicylic acid does not play an effective role. © 2005 American Society of Plant Biologists.

TITLE:Polyamines inhibit NADPH oxidase-mediated superoxide generation and putrescine prevents programmed cell death induced by polyamine oxidase-generated hydrogen peroxide 

[2-s2.0-18044390928] Refers [2-s2.0-0034954408] LEVEL: 3
Our previous results indicate that during protoplast isolation an oxidative burst occurs [A.K. Papadakis and KA Roubelakis-Angelakis (1999) Plant Physiol 127:197-205] and that suppression of totipotency is correlated with reduced antioxidant activity and low redox state [A.K. Papadakis et al. (2001b) Plant Physiol 126:434-444]. Polyamines are known to affect cell development and to act as antioxidants. Polyamines applied during isolation of tobacco (Nicotiana tabacum L.) protoplasts reduced the accumulation of O2?- but not that of H2O2. This antioxidant effect is probably due to the inhibition of microsomal membrane NADPH oxidase, which occurred in a concentration-dependent manner, with spermine exerting the highest inhibitory effect. However, during protoplast culture, polyamine oxidase activity increased severalfold in spermidine- and spermine-treated protoplasts, concomitant with H2O2 titers. A cell death program was executed in untreated protoplasts, as documented by membrane malfunction, induced DNase activity, DNA fragmentation and a positive TUNEL reaction. Protoplast cell death was prevented in protoplasts treated with putrescine, but not by treatment with spermidine or spermine, which rather had the opposite effect. The data presented suggest that PAs may be implicated in the expression of plant protoplast totipotency. © Springer-Verlag 2004.

TITLE:Sensing and signalling in response to oxygen deprivation in plants and other organisms 

[2-s2.0-24744457970] Refers [2-s2.0-0034954408] LEVEL: 3
? Aims and Scope: All aerobic organisms require molecular di-oxygen (O2) for efficient production of ATP though oxidative phosphorylation. Cellular depletion of oxygen results in rapid molecular and physiological acclimation. The purpose of this review is to consider the processes of low oxygen sensing and response in diverse organisms, with special consideration of plant cells. ? Conclusions: The sensing of oxygen deprivation in bacteria, fungi, metazoa and plants involves multiple sensors and signal transduction pathways. Cellular responses result in a reprogramming of gene expression and metabolic processes that enhance transient survival and can enable long-term tolerance to sub-optimal oxygen levels. The mechanism of sensing can involve molecules that directly bind or react with oxygen (direct sensing), or recognition of altered cellular homeostasis (indirect sensing). The growing knowledge of the activation of genes in response to oxygen deprivation has provided additional information on the response and acclimation processes. Conservation of calcium fluxes and reactive oxygen species as second messengers in signal transduction pathways in metazoa and plants may reflect the elemental importance of rapid sensing of cellular restriction in oxygen by aerobic organisms. © The Author 2005. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.

TITLE:Abscisic acid and antioxidant defense in plant cells 

[2-s2.0-4444298287] Refers [2-s2.0-0034954408] LEVEL: 3
Water stress is one of the most important environmental factors that affect plant growth and development, and limit plant production. Plants can respond and adapt to water stress by perceiving the stimulus, generating and transmitting the signals, and initiating various defense mechanisms. The plant hormone abscisic acid (ABA), as a stress signal, plays important roles in the regulation of plant responses to water stress. ABA not only regulates water balance by inducing stomatal closure, but also enhances water stress tolerance by inducing the expression of genes that encode dehydration tolerance proteins. Increasing evidence indicates that ABA-enhanced water stress tolerance is related to the induction of antioxidant defense systems by ABA. In this review, recent advances on the roles of ABA in the induction of the generation of reactive oxygen species (ROS), the expression of antioxidant enzyme genes, and the capacity of antioxidant defense systems are presented. Special attention is given to the cross-talk mechanisms between Ca2+ and ROS that originates from NADPH oxidase in the ABA-induced antioxidant defense in plants.

TITLE:Cross-Talk between Reactive Oxygen Species and Calcium in Living Cells 

[2-s2.0-0346271905] Refers [2-s2.0-0034954408] LEVEL: 3
The results of many investigations have shown that calcium is essential for production of reactive oxygen species (ROS). Elevation of intracellular calcium level is responsible for activation of ROS-generating enzymes and formation of free radicals by the mitochondria respiratory chain. On the other hand, an increase in intracellular calcium concentration may be stimulated by ROS. H2O2 has been recently shown to accelerate the overall channel opening process in voltage-dependent calcium channels in plant and animal cells. The 1,4,5-inositol-triphosphate-receptors as well as the ryanodine receptors of sarcoplasmic reticulum have also been demonstrated to be redox-regulated. Activity of Ca2+-ATPases and Na+/Ca 2+ exchangers of animal cells are modulated by the intracellular redox state. Simultaneously, Ca2+ may activate antioxidant enzymes, such as plant catalase and glutathione reductase, and increase the level of Superoxide dismutase in animal cells. Reviewed data support the speculation that Ca2+ and ROS are two cross-talking messengers in various cellular processes.

TITLE:Identification of a putative voltage-gated Ca2+ channel as a key regulator of elicitor-induced hypersensitive cell death and mitogen-activated protein kinase activation in rice 

[2-s2.0-21244456133] Refers [2-s2.0-0034954408] LEVEL: 3
Elicitor-triggered transient membrane potential changes and Ca2+ influx through the plasma membrane are thought to be important during defense signaling in plants. However, the molecular bases for the Ca2+ influx and its regulation remain largely unknown. Here we tested effects of overexpression as well as retrotransposon (Tos17)-insertional mutagenesis of the rice two-pore channel 1 (OsTPC1), a putative voltage-gated Ca 2+-permeable channel, on a proteinaceous fungal elicitor-induced defense responses in rice cells. The overexpressor showed enhanced sensitivity to the elicitor to induce oxidative burst, activation of a mitogen-activated protein kinase (MAPK), OsMPK2, as well as hypersensitive cell death. On the contrary, a series of defense responses including the cell death and activation of the MAPK were severely suppressed in the insertional mutant, which was complemented by overexpression of the wild-type gene. These results suggest that the putative Ca2+-permeable channel determines sensitivity to the elicitor and plays a role as a key regulator of elicitor-induced defense responses, activation of MAPK cascade and hypersensitive cell death. © 2005 Blackwell Publishing Ltd.

TITLE:Multiple mediators of plant programmed cell death: Interplay of conserved cell death mechanisms and plant-specific regulators 

[2-s2.0-0037235145] Refers [2-s2.0-0034954408] LEVEL: 3
Programmed cell death (PCD) is a process aimed at the removal of redundant, misplaced, or damaged cells and it is essential to the development and maintenance of multicellular organisms. In contrast to the relatively well-described cell death pathway in animals, often referred to as apoptosis, mechanisms and regulation of plant PCD are still ill-defined. Several morphological and biochemical similarities between apoptosis and plant PCD have been described, including DNA laddering, caspase-like proteolytic activity, and cytochrome c release from mitochondria. Reactive oxygen species (ROS) have emerged as important signals in the activation of plant PCD. In addition, several plant hormones may exert their respective effects on plant PCD through the regulation of ROS accumulation, The possible plant PCD regulators discussed in this review are integrated in a model that combines plant-specific regulators with mechanisms functionally conserved between animals and plants. © 2002 Wiley Periodicals, Inc.

TITLE:NADPH oxidase homologs are required for normal cell differentiation and morphogenesis in Dictyostelium discoideum 

[2-s2.0-20444381691] Refers [2-s2.0-0034954408] LEVEL: 3
Membrane-associated NADPH oxidase complexes catalyse the production of the superoxide anion radical from oxygen and NADPH. In mammalian systems, NADPH oxidases form a family of at least seven isoforms that participate in host defence and signalling pathways. We report here the cloning and the characterisation of slime mould Dictyostelium discoideum homologs of the mammalian heme-containing subunit of flavocytochrome b (gp91phox) (NoxA, NoxB and NoxC), of the small subunit of flavocytochrome b (p22 phox) and of the cytosolic factor p67phox. Null-mutants of either noxA, noxB, noxC or p22phox show aberrant starvation-induced development and are unable to produce spores. The overexpression of NoxA myc2 in noxA null strain restores spore formation. Remarkably, the gene alg-2B, coding for one of the two penta EF-hand proteins in Dictyostelium, acts as a suppressor in noxA, noxB, and p22phox null-mutant strains. Knockout of alg-2B allows noxA, noxB or p22phox null-mutants to return to normal development. However, the knockout of gene encoding NoxC, which contains two penta EF-hands, is not rescued by the invalidation of alg-2B. These data are consistent with a hypothesis connecting superoxide and calcium signalling during Dictyostelium development. © 2005 Elsevier B.V. All rights reserved.

TITLE:Involvement of the small GTPase rac in the defense responses of tobacco to pathogens 

[2-s2.0-12444288639] Refers [2-s2.0-0034954408] LEVEL: 3
During the hypersensitive response (HR), plants accumulate reactive oxygen species (ROS) that are likely generated at least in part by an NADPH oxidase similar to that found in mammalian neutrophils. An essential regulator of mammalian NADPH oxidase is the small GTP-binding protein Rac. To investigate whether Rac also regulates the pathogen-induced oxidative burst in plants, a dominant negative form of the rice OsRac1 gene was overexpressed in tobacco carrying the N resistance gene. Following infection with Tobacco mosaic virus (TMV), DN-OsRac1 plants developed smaller lesions than wild-type plants, accumulated lower levels of lipid peroxidation products, and failed to activate expression of antioxidant genes. These results, combined with the demonstration that superoxide and hydrogen peroxide levels were reduced in DN-OsRac1 tobacco developing a synchronous HR triggered by transient expression of the TMV p50 helicase domain or the Pto and AvrPto proteins, suggest that ROS production is impaired. The dominant negative effect of DN-OsRac1 could be rescued by transiently overexpressing the wild-type OsRac1 protein. TMV-induced salicylic acid accumulation also was compromised in DN-OsRac1 tobacco. Interestingly, while systemic acquired resistance to TMV was not impaired, nonhost resistance to Pseudomonas syringae pv. maculicola ES4326 was suppressed. Thus, the effect DN-OsRac1 expression exerts on the resistance signaling pathway appears to vary depending on the identity of the inoculated pathogen.

TITLE:Role of chloroplast trienoic fatty acids in plaint disease defense responses 

[2-s2.0-11144226808] Refers [2-s2.0-0034954408] LEVEL: 3
Trienoic fatty acids (TAs) are the major polyunsaturated fatty acid species in the membrane lipids in plant cells. TAs are crucial for the adaptation to abiotic stresses, especially low- or high-temperature stress. We show that TAs in chloroplast membrane lipids are involved in defense responses against avirulent bacterial pathogens. Avirulent pathogen invasion of plants induces a transient production of reactive oxygen intermediates (ROI), programmed cell death and subsequent disease resistance. The Arabidopsis fad7fad8 mutation, which prevents the synthesis of TAs in chloroplast lipids, caused the reduction in ROI accumulation in leaves inoculated with Pseudomonas syringae pv. tomato DC3000 (avrRpm1). Linolenic acid, the most abundant TA, activated the NADPH oxidase that is responsible for ROI generation. TAs were transferred from chloroplast lipids to extrachloroplast lipids coincident with ROI accumulation after inoculation with Pst DC3000 (avrRpm1). Furthermore, the fad7fad8 mutant exhibited reduced cell death and was compromised in its resistance to several avirulent P. syringae strains. These results suggest that TAs derived from chloroplast lipids play an important role in the regulation of plant defense responses.

TITLE:Is the nectar redox cycle a floral defense against microbial attack? 

[2-s2.0-3042825308] Refers [2-s2.0-0034954408] LEVEL: 3
Many angiosperms use a remarkable reproductive strategy that relies on attracting animals (insect, avian or mammalian pollinators) to transfer pollen between plants. Relying on other organisms for sexual reproduction seems evolutionarily untenable, but the great diversity of angiosperms illustrates how highly successful this strategy is. To attract pollinators, plants offer a variety of rewards. Perhaps the primary floral reward is floral nectar. Plant nectar has long been considered a simple sugar solution but recent work has demonstrated that nectar is a complex biological fluid containing significant and important biochemistry with the potential function of inhibiting microbial growth. These results lead the way to novel insights into the mechanisms of floral defense and the co-evolution of angiosperms and their pollinators.

TITLE:Superoxide and hydrogen peroxide play different roles in the nonhost interaction of barley and wheat with inappropriate formae speciales of Blumeria graminis 

[2-s2.0-1642546567] Refers [2-s2.0-0034954408] LEVEL: 3
Nonhost resistance of cereals to inappropriate formae speciales of Blumeria graminis is little understood. However, on the microscopic level, nonhost defense to B. graminis is reminiscent of host defense preventing fungal development by penetration resistance and the hypersensitive cell death response (HR). We analyzed histochemically the accumulation of superoxide anion radicals (O2.-) and hydrogen peroxide (H2O 2) at sites of B. graminis attack in nonhost barley and wheat. Superoxide visualized by subcellular reduction of nitroblue tetrazolium accumulated in association with successful fungal penetration in attacked cells and in cells neighboring HR. In contrast, H2O2 accumulated in cell wall appositions beneath fungal penetration attempts or in the entire epidermal cell during HR. The data provide evidence for different roles and sources of superoxide and H2O2 in the nonhost interaction of cereals with inappropriate formae speciales of B. graminis.

TITLE:Effects of feeding Spodoptera littoralis on lima bean leaves. III. Membrane depolarization and involvement of hydrogen peroxide 

[2-s2.0-33646926166] Refers [2-s2.0-0034954408] LEVEL: 3
In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H2O 2) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H2O2 was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H 2O2 was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H2O2 in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H2O2 prompted a transient transmembrane potential (Vm) depolarization, with a V m depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca 2+-sensing aequorin system, increasing amounts of added H 2O, correlated with a higher cytosolic calcium ([Ca2+]cyt) concentration. In MD and HW leaves, H2O2 also triggered the increase of [Ca2+]cyt but MD-elicited [Ca 2+]cyt increase was more pronounced when compared to HW leaves after addition of exogenous H2O2. The results clearly indicate mat Vm depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules. © 2006 American Society of Plant Biologists.

TITLE:Hydrogen peroxide is a common signal for darkness- and ABA-induced stomatal closure in Pisum sativum 

[2-s2.0-7744220678] Refers [2-s2.0-0034954408] LEVEL: 3
The requirement for hydrogen peroxide (H2O2) generation and action during stomatal closure induced by darkness and abscisic acid (ABA) was investigated in pea (Pisum sativum L.). Stomatal closure induced by darkness or ABA was inhibited by the H2O2-scavenging enzyme catalase or the antioxidant N-acetyl cysteine (NAC), or by diphenylene iodonium (DPI), an inhibitor of the H2O2-generating enzyme NADPH oxidase. Exogenous H2O2 induced stomatal closure in a dose- and time-dependent manner, and H2O2 was also required for ABA-inhibition of stomatal opening in the light. H 2O2 accumulation in guard cells was increased by darkness or ABA, as assessed with the fluorescent dye dichlorodihydrofluorescein diacetate (H2-DCFDA) and confocal microscopy. Such increases were inhibited by catalase, NAC or DPI, consistent with the effects of these compounds on stomatal apertures. Employing polymerase chain reaction (PCR) with degenerate oligonucleotide primers, several NADPH oxidase homologues were identified from pea genomic DNA that had substantial identity to the Arabidopsis thaliana (L.) Heynh. rboh (respiratory burst oxidase homologue) genes. Furthermore, an antibody raised against the tomato rboh identified immunoreactive proteins in epidermal, mesophyll and guard cells.

TITLE:The plant Mo-hydroxylases aldehyde oxidase and xanthine dehydrogenase have distinct reactive oxygen species signatures and are induced by drought and abscisic acid 

[2-s2.0-21244461224] Refers [2-s2.0-0034954408] LEVEL: 3
The plant molybdenum-cofactor (Moco) and flavin-containing enzymes, xanthine dehydrogenase (XDH; EC and aldehyde oxidase (AO; EC are thought to play important metabolic roles in purine metabolism and hormone biosynthesis, respectively. Their animal counterparts contribute to reactive oxygen species (ROS) production in numerous pathologies and here we examined these enzymes as potential sources of ROS in plants. Novel in-gel assay techniques and Moco sulfurase mutants, lacking a sulfur ligand in their Moco active center, were employed to demonstrate that the native tomato and Arabidopsis XDHs are capable of producing O2-, but not H2O2, while the animal counterpart was shown to produce both, O2- and H2O2. Superoxide production was dependent on Moco sulfuration when using hypoxanthine/xanthine but not NADH as substrates. The activity was inhibited by diphenylene iodonium (DPI), a suicide inhibitor of FAD containing enzymes. Analysis of XDH in an Arabidopsis Atxdh1 T-DNA insertion mutant and RNA interference lines revealed loss of O2- activity, providing direct molecular evidence that plant XDH generates superoxides. Contrary to XDH, AO activity produced only H2O2 dissimilar to native animal AO, that can produce O2- as well. Surprisingly, H2O2 accumulation was not sensitive to DPI. Plant ROS production and transcript levels of AO and XDH were rapidly upregulated by application of abscisic acid and in water-stressed leaves and roots. These results, supported by in vivo measurement of ROS accumulation, indicate that plant AO and XDH are possible novel sources for ROS increase during water stress. © 2005 Blackwell Publishing Ltd.

TITLE:Properties of guaiacol peroxidase activities isolated from corn root plasma membranes 

[2-s2.0-0038715037] Refers [2-s2.0-0034954408] LEVEL: 3
Although several investigations have demonstrated a plasma membrane (PM)-bound peroxidase activity in plants, this study is the first, to our knowledge, to purify and characterize the enzymes responsible. Proteins were extracted from highly enriched and thoroughly washed PMs. Washing and solubilization procedures indicated that the enzymes were tightly bound to the membrane. At least two distinct peroxidase activities could be separated by cation exchange chromatography (pmPOX1 and pmPOX2). Prosthetic groups were identified in fractions with peroxidase activity by absorption spectra, and the corresponding protein bands were identified by heme staining. The activities of the peroxidase enzymes responded different to various substrates and effectors and had different thermal stabilities and pH and temperature optima. Because the enzymes were localized at the PM and were not effected by p-chloromercuribenzoate, they were probably class III peroxidases. Additional size exclusion chromatography of pmPOX1 revealed a single activity peak with a molecular mass of 70 kD for the native enzyme, whereas pmPOX2 had two activity peaks (155 and 40 kD). Further analysis of these fractions by a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with heme staining confirmed the estimated molecular masses of the size exclusion chromatography.

TITLE:NADPH oxidases in Eukaryotes: Red algae provide new hints! 

[2-s2.0-33644537394] Refers [2-s2.0-0034954408] LEVEL: 3
The red macro-alga Chondrus crispus is known to produce superoxide radicals in response to cell-free extracts of its green algal pathogenic endophyte Acrochaete operculata. So far, no enzymes involved in this metabolism have been isolated from red algae. We report here the isolation of a gene encoding a homologue of the respiratory burst oxidase gp91phox in C. crispus, named Ccrboh. This single copy gene encodes a polypeptide of 825 amino acids. Search performed in available genome and EST algal databases identified sequences showing common features of NADPH oxidases in other algae such as the red unicellular Cyanidioschyzon merolae, the economically valuable red macro-alga Porphyra yezoensis and the two diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Domain organization and phylogenetic relationships with plant, animal, fungal and algal NADPH oxidase homologues were analyzed. Transcription analysis of the C. crispus gene revealed that it was over-transcribed during infection of C. crispus gametophyte by the endophyte A. operculata, and after incubation in presence of atrazine, methyl jasmonate and hydroxyperoxides derived from C20 polyunsaturated fatty acids (PUFAs). These results also illustrate the interest of exploring the red algal lineage for gaining insight into the deep evolution of NADPH oxidases in Eukaryotes. © Springer-Verlag 2005.

TITLE:Role of ABA, salicylic acid, calcium and hydrogen peroxide on antioxidant enzymes induction in wheat seedlings 

[2-s2.0-22444433279] Refers [2-s2.0-0034954408] LEVEL: 3
Experiments were conducted to study the regulatory role of salicylic acid (SA), abscisic acid (ABA), calcium (Ca2+) and hydrogen peroxide (H2O2) on antioxidant enzymes induction in wheat genotypes C 306 and Hira. Results revealed that SA (1 mM), ABA (0.5 mM), Ca2+ (5 mM) and H2O2 (0.05 mM) were most effective in increasing the activities of superoxide dismutase (SOD), ascorbate peroxidase (APOX), catalase (CAT) and NADPH oxidase. Hydrogen peroxide contents estimated immediately after termination of treatments also increased by all the treatments. The response was more with Ca2+ + ABA, followed by Ca2+ alone, both in terms of antioxidant enzymes and H 2O2 contents. EGTA, a calcium chelator, decreased the activity of all the antioxidant enzymes below control level as well as decreased H2O2 contents. Similarly, DPI, a specific inhibitor of membrane linked NADPH oxidase, not only inhibited NADPH oxidase activity, but also inhibited the increase of SOD and APOX activity. Results revealed that SA induced H2O2 accumulation in germinating seedlings was not associated with inhibition of CAT or APOX. It is suggested that abiotic stress signal is transduced via ABA, Ca2+ and H2O2, which might be responsible for the activation of some common transcription factor associated with SOD, APOX and CAT. © 2005 Elsevier Ireland Ltd. All rights reserved.

TITLE:The role of reactive oxygen species in cell growth: Lessons from root hairs 

[2-s2.0-33745590665] Refers [2-s2.0-0034954408] LEVEL: 3
Reactive oxygen species (ROS) play a diversity of roles in plants. In recent years, a role for NADPH oxidase-derived ROS during cell growth and development has been discovered in a number of plant model systems. These studies indicate that ROS are required for cell expansion during the morphogenesis of organs such as roots and leaves. Furthermore, there is evidence that ROS are required for root hair growth where they control the activity of calcium channels required for polar growth. The role of ROS in the control of root hair growth is reviewed here and results are highlighted that may provide insight into the mechanism of plant cell growth in general. © The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.

TITLE:The small GTPase AtRAC2/ROP7 is specifically expressed during late stages of xylem differentiation in Arabidopsis 

[2-s2.0-23944522096] Refers [2-s2.0-0034954408] LEVEL: 3
The RAC/ROP family of small GTPases are central regulators of important cellular processes in plants. AtRAC2/ROP7 is an ancient member of the RAC/ROP gene family in Arabidopsis thaliana whose functions are generally unknown. In order to study the spatial expression pattern of the AtRAC2/ROP7 gene, transgenic plants expressing GUS or GFP under the control of the AtRAC2/ROP7 promoter were analysed. Functional analysis of AtRAC2/ROP7 was done using transgenic plants overexpressing wild-type and constitutively activated AtRAC2/ROP7 (Val15Gly), and an AtRAC2/ROP7T-DNA insertion mutant. The AtRAC2/ROP7 promoter directs a highly specific xylem-specific expression in the root, hypocotyl, stem, and leaves. The expression is developmentally limited to the late stages of xylem differentiation, and coincides with the formation of secondary cell walls. Leaf epidermal cells of transgenic plants overexpressing constitutively active AtRAC2/ ROP7 exhibited highly impaired lobe formation, suggesting that AIRAC2/ROP7 is able to regulate polar cell expansion. Finally, GFP-AtRAC2/ROP7 fusion proteins were localized to the plasma membrane. The results indicate a role for AtRAC2/ROP7 in the development of secondary cell walls of xylem vessels. © The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.

TITLE:N-acetylchitooligosaccharides elicit rice defence responses including hypersensitive response-like cell death, oxidative burst and defence gene expression 

[2-s2.0-11844287641] Refers [2-s2.0-0034954408] LEVEL: 3
Rice, a monocotyledonous model plant, can be used to dissect the molecular mechanism of plant defence activation in cereal crops. In the present work, we investigated acetylchitooligosaccharide (COS)-mediated defence activation in rice. COS strongly induced cell death and defence gene expression in rice suspension cells in a dose- and degree of polymerization-dependent manner. COS treatment could also induce visible cell death on rice leaves. Rapid production of H2O2 was observed preceding cell death in the COS-stimulated rice cells. The H2O2 generation and consequential cell death could be blocked by diphenylene iodonium, indicating that the COS-mediated oxidative burst depends on an NADPH oxidase. The rice defence-related genes, RCH10 and PAL were strongly induced by COS, and OsCatB was first repressed and then induced by COS. Consequentially, rice disease resistance was enhanced by COS against the blast fungus. Taken together, these results strongly suggested that COS activates the rice defence responses via a mechanism similar to the hypersensitive response involved in the plant-microbe interactions. Our study shows the feasibility of this system to dissect elicitor-mediated activation of non-specific defence in rice. © 2004 Elsevier Ltd. All rights reserved.

TITLE:Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase 

[2-s2.0-34250728188] Refers [2-s2.0-0034954408] LEVEL: 3
Reactive oxygen species (ROS) are implicated in plant innate immunity. NADPH oxidase (RBOH; for Respiratory Burst Oxidase Homolog) plays a central role in the oxidative burst, and EF-hand motifs in the N terminus of this protein suggest possible regulation by Ca2+. However, regulatory mechanisms are largely unknown. We identified Ser-82 and Ser-97 in the N terminus of potato (Solanum tuberosum) St RBOHB as potential phosphorylation sites. An anti-phosphopeptide antibody (pSer82) indicated that Ser-82 was phosphorylated by pathogen signals in planta. We cloned two potato calcium-dependent protein kinases, St CDPK4 and St CDPK5, and mass spectrometry analyses showed that these CDPKs phosphorylated only Ser-82 and Ser-97 in the N terminus of St RBOHB in a calcium-dependent manner. Ectopic expression of the constitutively active mutant of St CDPK5, St CDPK5VK, provoked ROS production in Nicotiana benthamiana leaves. The CDPK-mediated ROS production was disrupted by knockdown of Nb RBOHB in N. benthamiana. The loss of function was complemented by heterologous expression of wild-type potato St RBOHB but not by a mutant (S82A/S97A). Furthermore, the heterologous expression of St CDPK5VK phosphorylated Ser-82 of St RBOHB in N. benthamiana. These results suggest that St CDPK5 induces the phosphorylation of St RBOHB and regulates the oxidative burst. © 2007 American Society of Plant Biologists.

TITLE:Calcium-calmodulin is required for abscisic acid-induced antioxidant defense and functions both upstream and downstream of H2O2 production in leaves of maize (Zea mays) plants 

[2-s2.0-33845547595] Refers [2-s2.0-0034954408] LEVEL: 3
? Using pharmacological and biochemical approaches, the role of calmodulin (CaM) and the relationship between CaM and hydrogen peroxide (H 2O2) in abscisic acid (ABA)-induced antioxidant defense in leaves of maize (Zea mays) plants were investigated. ? Treatment with ABA or H2O2 led to significant increases in the concentration of cytosolic Ca2+ in the protoplasts of mesophyll cells and in the expression of the calmodulin 1 (CaM1) gene and the content of CaM in leaves of maize plants, and enhanced the expression of the antioxidant genes superoxide dismutase 4 (SOD4), cytosolic ascorbate peroxidase (cAPX), and glutathione reductase 1 (GR1) and the activities of the chloroplastic and cytosolic antioxidant enzymes. The up-regulation of the antioxidant enzymes was almost completely blocked by pretreatments with two CaM antagonists. ? Pretreatments with CaM antagonists almost completely inhibited ABA-induced H2O2 production throughout ABA treatment, but pretreatment with an inhibitor or scavenger of reactive oxygen species (ROS) did not affect the initial increase in the contents of CaM induced by ABA. ? Our results suggest that Ca2+-CaM is involved in ABA-induced antioxidant defense, and that cross-talk between Ca2+-CaM and H2O2 plays a pivotal role in ABA signaling. © The Authors (2006).

TITLE:Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant 

[2-s2.0-15944385366] Refers [2-s2.0-0034954408] LEVEL: 3
Podospora anserina and Coprinopsis cinerea (syn. Coprinus cinereus) are endowed with a defence system able to differentiate self vs. non-self and involving the generation of peroxide. Indeed, they produce peroxide when confronted with a filamentous fungus, only in non-self confrontations. Both species are not able to recognize yeasts and show a differential response to bacteria. The accumulation of peroxides in the ascomycete Podospora anserina requires an NADPH oxidase and a MAP kinase cascade, previously shown to be involved in fruit body formation, cell differentiation and cell degeneration. Confrontation is accompanied by the death of the contestant hyphae only in specific combinations of species. As in animals and plants, data suggest that peroxide is likely involved in signalling rather than playing a direct toxic role. Fungi display more complex behaviours than generally acknowledged, i.e. they are able to recognize potential contestants and built up defence reactions involving evolutionary conserved enzymes. © The British Mycological Society.

TITLE:Three types of tobacco calmodulins characteristically activate plant NAD kinase at different Ca2+ concentrations and pHs 

[2-s2.0-9244254318] Refers [2-s2.0-0034954408] LEVEL: 3
We previously reported that three types of tobacco calmodulin (CaM) isoforms originated from 13 genes are differently regulated at the transcript and protein levels in response to wounding and tobacco mosaic virus-induced hypersensitive reaction (HR); wound-inducible type I and HR-inducible type III levels increased after wounding and HR, respectively, while type II, whose expression is constitutive and wound responsible, remained unchanged. Here, we show that these CaMs differentially activate target enzymes; rat NO synthase was activated most effectively by type III, moderately by type I and weakly by type II, and plant NAD kinase (NADK) was activated in the inverse order. Furthermore, we found that a suitable Ca2+ concentration differs by type; type II activated NADK at lower Ca2+ of around 0.1 ?M, which is the cytosolic concentration in unstimulated cells, type I did so at 1-5 ?M, which is the increased Ca2+ concentration in stimulated cells, while type III did not at any Ca2+ level. NADK activation was highest over a pH range of 7.1-6.8 for which the cytosolic pH reportedly changed from 7.5 after being stimulated. Thus, tobacco CaMs, especially type I, effectively activate NADK in stimuli-induced conditions.

TITLE:Possible functions of extracellular peroxidases in stress-induced generation and detoxification of active oxygen species 

[2-s2.0-23944488439] Refers [2-s2.0-0034954408] LEVEL: 3
Extracellular peroxidases are classified as free, or ionically or covalently bound to the cell wall. In addition, peroxidase-like activities have often been demonstrated at the outer surface of protoplasts and plasma membrane preparations. Under certain conditions apoplastic peroxidases have been shown to contribute to the formation of superoxide and hydrogen peroxide during the 'oxidative burst' through the oxidation of a reductant. However, the identity of this reductant remains unclear. It has been suggested that the production of these active oxygen species may play important roles in plant responses to biotic and abiotic stress. Extracellular release of pre-existing and de novo synthesis of apoplastic peroxidases is regulated by changing environmental conditions. While the oxidative burst could potentially be harmful to a plant's own cells, tissues can rapidly metabolize even high concentrations of hydrogen peroxide. Recent work has shown that when extracellular hydrogen peroxide exceeds the supplies of reductants, class II and class III peroxidases can display catalase-like activity. Under these conditions, hydrogen peroxide is able to act as both oxidizing and reducing substrate. It seems likely therefore, that a further role of extracellular peroxidases is to protect plants from the consequences of the oxidative burst that they themselves are responsible for producing. © 2004 Kluwer Academic Publishers.

TITLE:Cell wall-associated mechanisms of disease resistance and susceptibility 

[2-s2.0-35148869094] Refers [2-s2.0-0034954408] LEVEL: 3
The plant cuticle and cell wall separate microbial pathogens from the products of plant metabolism. While microbial pathogens try to breach these barriers for colonization, plants respond to attempted penetration by a battery of wall-associated defense reactions. Successful pathogens circumvent or suppress plant nonself recognition and basal defense during penetration and during microbial reproduction. Additionally, accommodation of fungal infection structures within intact cells requires host reprogramming. Recent data high-light that both early plant defense to fungal penetration and host reprogramming for susceptibility can function at the host cell periphery. Genetic evidence has also widened our understanding of how fungal pathogens are restricted during penetration at the plant cell wall. This review summarizes the current view of how plants monitor and model their cell periphery during interaction with microbial invaders. Copyright © 2007 by Annual Reviews. All rights reserved.

TITLE:NADPH oxidase-dependent reactive oxygen species formation required for root hair growth depends on ROP GTPase 

[2-s2.0-34548421877] Refers [2-s2.0-0034954408] LEVEL: 3
Reactive oxygen species (ROS) production by an NADPH oxidase (NOX) encoded by AtrbohC/RHD2 is required for root hair growth in Arabidopsis thaliana. ROP (RHO of plants) GTPases are also required for normal root hair growth and have been proposed to regulate ROS production in plants. Therefore, the role of ROP GTPase in NOX-dependent ROS formation by root hairs was investigated. Plants overexpressing wild-type ROP2 (ROP2 OX), constitutively active (CA-rop2), or dominant negative (DN-rop2) rop2 mutant proteins were used. Superoxide formation by root hairs was detected by superoxide dismutase-sensitive nitroblue tetrazolium reduction, and ROS production in the root hair differentiation zone was detected by dihydrofluorescein diacetate oxidation. Both probes showed that ROS production was increased in ROP2 OX and CA-rop2 plants, and decreased in DN-rop2 plants, relative to wild-type plants. When CA-rop2 was expressed in the NOX loss-of-function rhd2-1 mutant, ROS formation and root hair growth were impaired, suggesting that RHD2 is required for this ROP2-dependent ROS formation. © 2007 The Author(s).

TITLE:Allelochemical stress causes inhibition of growth and oxidative damage in Lycopersicon esculentum Mill 

[2-s2.0-33748955965] Refers [2-s2.0-0034954408] LEVEL: 3
The aim of this study was to analyse the effect of allelochemical stress on Lycopersicon esculentum growth. Our results showed that allelochemical stress caused by Sicyos deppei aqueous leachate inhibited root growth but not germination, and produced an imbalance in the oxidative status of cells in both ungerminated seeds and in primary roots. We observed changes in activity of catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione reductase (GR) and the plasma membrane NADPH oxidase, as well as in the levels of H2O2 and O2?- in seeds at 12 and 24 h, and in primary roots at 48 and 72 h of treatment, which could account for the oxidative imbalance. There were changes in levels of expression of the mentioned enzymes, but without a correlation with their respective activities. Higher levels of membrane lipid peroxidation were observed in primary roots at 48 and 72 h of treatment. No effect on the expression of metacaspase and the PR1 was observed as indicators of cell death or induction of plant defence. This paper contributes to the understanding of plant-plant interactions through the phytotoxic allelochemicals released in an aqueous leachate of the weed S. deppei, which cause a negative effect on other plants. © 2006 The Authors.

TITLE:Polyamine oxidase is one of the key elements for oxidative burst to induce programmed cell death in tobacco cultured cells 

[2-s2.0-33748804928] Refers [2-s2.0-0034954408] LEVEL: 3
Programmed cell death plays a critical role during the hypersensitive response in the plant defense system. One of components that triggers it is hydrogen peroxide, which is generated through multiple pathways. One example is proposed to be polyamine oxidation, but direct evidence for this has been limited. In this article, we investigated relationships among polyamine oxidase, hydrogen peroxide, and programmed cell death using a model system constituted of tobacco (Nicotiana tabacum) cultured cell and its elicitor, cryptogein. When cultured cells were treated with cryptogein, programmed cell death occurred with a distinct pattern of DNA degradation. The level of hydrogen peroxide was simultaneously increased, along with polyamine oxidase activity in apoplast. With the same treatment in the presence of ?-difluoromethyl-Orn, an inhibitor of polyamine biosynthesis, production of hydrogen peroxide was suppressed and programmed cell death did not occur. A gene encoding a tobacco polyamine oxidase that resides in the apoplast was isolated and used to construct RNAi transgenic cell lines. When these lines were treated with cryptogein, polyamines were not degraded but secreted into culture medium and hydrogen peroxide was scarcely produced, with a concomitant suppression of cell death. Activities of mitogen-activated protein kinases (wound- and salicylic acid-induced protein kinases) were also suppressed, indicating that phosphorylation cascade is involved in polyamine oxidation-derived cell death. These results suggest that polyamine oxidase is a key element for the oxidative burst, which is essential for induction of programmed cell death, and that mitogen-activated protein kinase is one of the factors that mediate this pathway. © 2006 American Society of Plant Biologists.

TITLE:Subcellular localization of Strboh proteins and NADPH-dependent O 2 --generating activity in potato tuber tissues 

[2-s2.0-33645736979] Refers [2-s2.0-0034954408] LEVEL: 3
Rapid generation of reactive oxygen species (ROS) at the cell surface has been implicated in plant defence responses. Genetic evidence indicates that a plant NADPH oxidase (Rboh; respiratory burst oxidase homologue) is associated with oxidative burst. However, there is not enough physiological evidence of Rboh localization available yet. Isozyme-specific antibodies against potato StrbohA and StrbohB (St; Solanum tuberosum) were prepared to investigate the localization of these proteins. Immunoblot analyses using potato microsomal proteins revealed that StrbohA was expressed constitutively at a low level, whereas the accumulation of StrbohB protein was induced by the cell wall elicitor of the potato pathogen Phytophthora infestans. It is demonstrated here that StrbohA and StrbohB are distributed in plasma membrane fractions which have been separated by sucrose density-gradient centrifugation using their specific antibodies. Green fluorescent protein-tagged Strboh proteins were also located on the plasma membrane by transient expression assay in onion epidermal cells. Additionally, NADPH-dependent O2--generating activities in plasma membrane fractions were diphenylene iodonium-sensitive and NaN 3-insensitive. These data suggest that StrbohA and StrbohB are predominantly localized on the plasma membrane and regulate ROS production in defence signalling. © The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.

TITLE:Controlled salicylic acid levels and corresponding signaling mechanisms in plants 

[2-s2.0-13944267076] Refers [2-s2.0-0034954408] LEVEL: 3
Salicylic acid (SA) is a natural signaling molecule involved in plant defense response against pathogen infection. This article covers the recent key works contributing to our understanding of SA signaling and biosynthesis leading to a controlled SA level in plants.

TITLE:Fusicoccin stimulates the production of H2O2 in sycamore cell cultures and induces alternative respiration and cytochrome c leakage from mitochondria 

[2-s2.0-0344668730] Refers [2-s2.0-0034954408] LEVEL: 3
Fusicoccin (FC) is a well known toxin acting as a 14-3-3 protein-mediated activator of the plasma membrane H+-ATPase and it has been widely used to study the regulatory mechanism and the physiological role of this enzyme's activity. Recently, FC has been shown to induce other responses similar to those occurring under a stress condition, perhaps not strictly dependent on the activation of proton extrusion. In this paper we report that in cultured sycamore (Acer pseudoplatanus L.) cells FC induces H 2O2 overproduction as well as other novel, presumably related responses, such as the activation of the alternative oxidase and the leakage of cytochrome c from the mitochondria, accompanied by a decrease of the cytochrome pathway capacity. The relationship between H2O 2 production and other phenomena has also been studied by means of exogenously added H2O2.

TITLE:Superoxide synthase and dismutase activity of plasma membranes from maize roots 

[2-s2.0-0038348656] Refers [2-s2.0-0034954408] LEVEL: 3
Superoxide synthase and superoxide dismutase activity have been monitored in isolated maize (Zea mays) root plasma membranes spectrophotometrically by determination of nitro-blue tetrazolium and cytochrome c reduction, respectively. Superoxide production was induced by NADH and NADPH, with similar kinetics and approaching saturation at 0.06 mM in the case of NADPH and 0.1 mM in the case of NADH, with rates of 18.6 ± 5.0 and 21.8 ± 7.2 nmol/min · mg of protein, respectively. These activities exhibited a broad pH optimum between pH 6.5 and 7.5. Diphenylene iodonium inhibited about 25% (10 ?M DPI) and 40% (100 ?M DPI) of this activity, imidazole inhibited about 20%, while KCN, a peroxidase inhibitor, did not show any significant inhibition. Superoxide-dismutating activity was shown to occur in the same isolates and depended on the quantity of plasma membrane protein present. Growth of plants on salicylic acid prior to membrane isolation induced a rise in the activity of both of the enzymes by 20-35%, suggesting their coordinated action.

TITLE:Physiological roles of nonselective cation channels in plants: From salt stress to signalling and development 

[2-s2.0-34447515608] Refers [2-s2.0-0034954408] LEVEL: 3
Nonselective cation channels (NSCCs) catalyse passive fluxes of cations through plant membranes. NSCCs do not, or only to a small extent, select between monovalent cations, and several are also permeable to divalent cations. Although a number of NSCC genes has been identified in plant genomes, a direct correlation between gene products and in vivo observed currents is still largely absent for most NSCCs. In this review, physiological functions and molecular properties of NSCCs are critically discussed. Recent studies have demonstrated that NSCCs are directly involved in a multitude of stress responses, growth and development, uptake of nutrients and calcium signalling. NSCCs can also function in the perception of external stimuli and as signal transducers for reactive oxygen species, pathogen elicitors, cyclic nucleotides, membrane stretch, amino acids and purines. © The Authors (2007).

TITLE:Involvement of plasma-membrane NADPH oxidase in nickel-induced oxidative stress in roots of wheat seedlings 

[2-s2.0-27144547997] Refers [2-s2.0-0034954408] LEVEL: 3
The roles of the plasma-membrane (PM) NADPH oxidase in nickel-induced oxidative stress were investigated in roots of wheat (Triticum durum D.) seedlings. Treatment with nickel resulted in significant increase in level of membrane lipid peroxidation, content of H2O2, the production rate of O2?- and the activity of the PM NADPH oxidase in wheat roots. The effects caused by nickel were inhibited pronouncedly by pretreatments with three widely used NADPH oxidase inhibitors (diphenylene idonium, imidazole and pyridine). Moreover, the change patterns of the Ni-induced increase and inhibitor-caused decrease in these parameters were quite similar. These data suggest that the Ni-induced enhancements in levels of H2O2 and O2?-, which appear to cause membrane lipid peroxidation, originate mainly from PM NADPH oxidase. A native in-gel assay also confirmed that PM NADPH oxidase participates in the Ni-induced production of reactive oxygen species in wheat. In addition, pharmacological experiments suggest that Ca2+ may be involved in the oxidative stress induced by nickel in wheat roots. © 2005 Elsevier Ireland Ltd. All rights reserved.

TITLE:Function of the rice gp91phox homologs OsrbohA and OsrbohE genes in ROS-dependent plant immune responses 

[2-s2.0-28444458822] Refers [2-s2.0-0034954408] LEVEL: 3
Reactive oxygen species (ROS) are proposed to function as diffusible signaling molecules in plant immune response. Rice respiratory burst oxidase homologs (Osrboh genes) are proposed to play a role in ROS generation. We examined a role in rice immune responses of four Osrboh homologs, OsrbohA, OsrbohB, OsrbohD, and OsrbohE. OsrbohA and OsrbohD transcripts were induced after inoculation with an incompatible N1141 strain of Acidovorax avenae, whereas OsrbohaB and OsrbohE mRNA levels did not obviously change even after inoculation with the incompatible strain. We examined the function of the Osrboh genes in ROS generation and in the plant immune response using RNAi-based knockdown in rice cells. OsrbohA and OsrbohE knockdown lines showed that rapid H2O2 generation is caused by OsrbohA, whereas OsrbohE is involved in late H2O2 production during the immune response. Hypersensitive cell death was decreased only in the OsrbohA knockdown line. We further demonstrated that among immune related genes, the induction of EL2 and LOX genes is controlled by ROS generated by OsrbohE, whereas expression of Cht-1 gene is regulated by both OsrbohA and OsrbohE. These results indicate that the ROS molecules generated by OsrbohA and OsrbohE regulate different signaling pathways in the plant immune response. Copyright © 2005 The Japanese Society for Plant Cell and Molecular Biology.

TITLE:The suppression of salinity-associated oxygen radicals production, in pepper (Capsicum annuum) fruit, by manganese, zinc and calcium in relation to its sensitivity to blossom-end rot 

[2-s2.0-13244295772] Refers [2-s2.0-0034954408] LEVEL: 3
We investigated the possibility that oxidative stress contributes to blossom-end rot (BER) initiation in bell pepper (Capsicum annuum L.) grown under high salinity. Pepper plants (cv. Mazurka, Rijk Zwaan, the Netherlands) were grown in a greenhouse and irrigated with nutrient solution made up with either desalinated water (control - rising from E.C. 1.9 to 2.4 dS m-1) or saline water (salinity - rising from E.C. 3.2 to 7.0 dS m-1). Irrigation was by a circulation system. BER symptoms were observed throughout the experiment but were highly enhanced in the salinity-grown plants during the spring and summer. The fruit calcium concentration was not affected by salinity, but manganese concentrations in both leaves and fruits were significantly reduced under these conditions. Under salinity there was an enhancement of apoplast reactive oxygen species (ROS) production, which was partly a result of increase in NAD(P)H oxidase activity in the pericarp of pepper fruit at the stage that it was most sensitive to BER. Apoplast ROS production and extracted NAD(P)H oxidase activity were inhibited by manganese, zinc and to a lesser extent by calcium. These cations also negated the enhancement of ROS production caused by incubation of fruit pericarp discs in NaCl solutions. Manganese, zinc and calcium also inhibited NAD(P)H oxidase activity, extracted following their infiltration into fruit pericarp discs. The results suggest that generation and scavenging of oxygen free radicals in the apoplast may contribute to the appearance of BER symptoms in pepper fruits under saline conditions. It is suggested that manganese may serve as antioxidant in pepper fruit and that manganese addition to peppers grown under salinity may alleviate BER symptoms in the fruits.

TITLE:The influence of Cocksfoot mottle virus on antioxidant metabolism in the leaves of Dactylis glomerata L. 

[2-s2.0-0141993490] Refers [2-s2.0-0034954408] LEVEL: 3
Antioxidant metabolism in the leaves of Dactylis glomerata L. plants which had never been infected with Cocksfoot mottle virus (CfMV) and were susceptible to disease was compared to that of plants that had fully recovered from a previous inoculation, had no detectable CfMV present and showed immunity to disease. In susceptible plants, H2O2 levels declined immediately following inoculation with CfMV and then gradually increased as lipid peroxidation and the symptoms of infection were observed. Recovered plants showed only a brief increase in H2O2 levels, immediately following inoculation, with no significant increase in lipid peroxidation. Inoculation of susceptible plants with CfMV caused a decline in the oxidised forms of ascorbate and glutathione and a decline in the total glutathione pool. No changes in the ascorbate or glutathione pools were observed in recovered plants. With the exception of catalase (CAT), which increased in activity for the first 5 days following inoculation and then declined, the activities of antioxidant enzyme showed a biphasic response to CfMV in susceptible plants. A decline in antioxidant enzyme activity was observed during the early stages of virus establishment, followed by increased activity in response to cellular damage. Only transient increases in ascorbate peroxidase (APOX), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) activities occurred in recovered plants. © 2003 Elsevier Ltd. All rights reserved.

TITLE:Different responses of two tobacco cultivars and their cell suspension cultures to quercinin, a novel elicitin from Phytophthora quercina 

[2-s2.0-0038138066] Refers [2-s2.0-0034954408] LEVEL: 3
In this study we describe the response of two tobacco cultivars (Nicotiana tabacum L. cv. Bel B and Bel W3) and their cell suspension cultures to quercinin, a novel elicitin produced by the oak pathogen Phytophthora quercina. N-terminal sequencing of the purified protein proved that it belongs to the basic ?-elicitins with threonine on position 13. Both tobacco leaves and cells of the cultivar Bel W3 showed hypersensitive cell death after quercinin treatment. Leaves of Bel B also developed quercinin-induced necrosis but higher concentrations of quercinin were necessary as compared to Bel W3. Also Bel B cells showed cell death induction only at the highest quercinin concentration (20 nM). In cell suspension experiments we also measured the quercinin-induced oxidative burst, which occurred in both cultivars. H2O2 production in Bel B increased with increasing quercinin concentration and was inhibited only at the highest elicitin concentration (20 nM) whereas the oxidative burst in Bel W3 was completely abolished by 5 nM quercinin. Furthermore we demonstrated that neither H2O2 nor superoxide were responsible for cell death induction since neither the inhibitor diphenyleneiodonium (DPI) nor the enzymes catalase (CAT) and superoxide dismutase (SOD) influenced the hypersensitive reaction (HR) in Bel W3 cells. Due to the different response of Bel W3 and Bel B towards the P. quercina elicitin, our system represents an interesting tool to elucidate signaling pathways in tobacco leading to hypersensitive cell death. © 2003 E?ditions scientifiques et me?dicales Elsevier SAS. All rights reserved.

TITLE:A RHOse by any other name: A comparative analysis of animal and plant Rho GTPases 

[2-s2.0-33745584806] Refers [2-s2.0-0034954408] LEVEL: 3
Rho GTPases are molecular switches that act as key regulators of a many cellular processes, including cell movement, morphogenesis, host defense, cell division and gene expression. Rho GTPases are found in all eukaryotic kingdoms. Plants lack clear homologs to conventional Rho GTPases found in yeast and animals; instead, they have over time developed a unique subfamily, ROPs, also known as RAC. The origin of ROP-like proteins appears to precede the appearance of land plants. This review aims to discuss the evolution of ROP/RAC and to compare plant ROP and animal Rho GTPases, focusing on similarities and differences in regulation of the GTPases and their downstream effectors. © 2006 IBCB, SIBS, CAS All rights reserved.

TITLE:Identification and characterization of differentially expressed ESTs of Gossypium barbadense infected by Verticillium dahliae with suppression subtractive hybridization 

[2-s2.0-20844451571] Refers [2-s2.0-0034954408] LEVEL: 3
Cotton wilt defense reaction is a complicated continuous process and involves a battery of genes. In this study, we adopted suppression subtractive hybridization (SSH) technique to isolate differentially expressed ESTs from Gossypium barbadense variety 7124 during Verticillium wilt defense process. An array of 1165 clones from the subtractive library has been screened with reverse northern blotting, of which 131 ESTs were considered as over-expressed and 16 ESTs were down-regulated. Sequence analysis and blast search showed that 83 ESTs were homologous to 45 unique sequences in the databases. Among all these differentially expressed ESTs, at least three kinds of genes were characterized. The majority of ESTs with deduced identity to aerobic metabolism enzymes strongly expressed in the infection process. Likewise, ESTs similar to those reported for pathogen- related protein genes were also picked out in this study. These ESTs in combination with other kinase-like genes and a defensin-like EST constituted an assembly of genes responded during pathogens' infection. These results imply that sea-island cotton undergoes strong oxidative stress and results in a series of defense responses when attacked by V. dahliae. To our knowledge, this is the first report on the isolation of global ESTs during sea-island cotton defense reaction.

TITLE:Nutrient sensing and signaling: NPKS 

[2-s2.0-34347262497] Refers [2-s2.0-0034954408] LEVEL: 3
Plants often grow in soils that contain very low concentrations of the macronutrients nitrogen, phosphorus, potassium, and sulfur. To adapt and grow in nutrient-deprived environments plants must sense changes in external and internal mineral nutrient concentrations and adjust growth to match resource availability. The sensing and signal transduction networks that control plant responses to nutrient deprivation are not well characterized for nitrogen, potassium, and sulfur deprivation. One branch of the signal transduction cascade related to phosphorus-deprivation response has been defined through the identification of a transcription factor that is regulated by sumoylation. Two different microRNAs play roles in regulating gene expression under phosphorus and sulfur deprivation. Reactive oxygen species increase rapidly after mineral nutrient deprivation and may be one upstream mediator of nutrient signaling. A number of molecular analyses suggest that both short-term and longer-term responses will be important in understanding the progression of signaling events when the external, then internal, supplies of nutrients become depleted. Copyright © 2007 by Annual Reviews. All rights reserved.

TITLE:Regulation of reactive oxygen species production by a 14-3-3 protein in elicited tobacco cells 

[2-s2.0-34247647461] Refers [2-s2.0-0034954408] LEVEL: 3
The regulation of the system responsible for the production of reactive oxygen species (ROS) during plant-micro-organism interaction is still largely unknown. The protein NtrbohD has been recently demonstrated as the plasma membrane oxidase responsible for ROS production in elicited tobacco cells. Here, its C-terminus part was used as a bait in a two-hybrid screen in order to identify putative regulators of this system. This led to the isolation of a cDNA coding for a member of the 14-3-3 protein family. The corresponding transcript was induced after infiltration of tobacco leaves with the fungal elicitor cryptogein. Tobacco cells transformed with an antisense construct of this 14-3-3 no longer accumulated ROS, which constitutes a functional validation of the two-hybrid screen. This work provides new insights to the understanding of the regulation of ROS production in a signalling context and gives a new light to the possible role of 14-3-3 proteins in plant-micro-organisms interactions. Journal compilation © 2007 Blackwell Publishing Ltd.

TITLE:Distinct signalling pathways for induction of MAP kinase activities by cadmium and copper in rice roots 

[2-s2.0-33847617937] Refers [2-s2.0-0034954408] LEVEL: 3
Plant growth is severely affected by toxic concentrations of heavy metals. On characterizing the heavy metal-induced signalling pathways, the effects of cadmium (CdCl2) and copper (CuCl2) on MBP (myelin basic protein) kinase activities in Oryza sativa L. cv. TNG67 were analysed and it was found that Cd2+-induced 42 kDa MBP kinase has the characteristics of a mitogen-activated protein (MAP) kinase. This study confirmed that the 42 kDa kinase-active band contains, at least, the activities of OsMPK3 and OsMPK6. Then, the heavy metal signal transduction pathways leading to MAP kinase activation in rice roots were examined. Pretreatment with sodium benzoate, a hydroxyl radical scavenger, attenuated Cd2+- or Cu 2+-induced MAP kinase activation. The Cd2+-, but not Cu2+-, induced MAP kinase activities were suppressed by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and Cd2+ induced NADPH oxidase-like activities, suggesting that NADPH oxidases may be involved in Cd2+-induced MAP kinase activation. Using a Ca2+ indicator, it was demonstrated that Cd2+ and Cu2+ induce Ca2+ accumulation in rice roots. The Cd2+- and Cu 2+-induced MAP kinase activation required the involvement of Ca 2+-dependent protein kinase (CDPK) and phosphatidylinositol 3-kinase (PI3 kinase) as shown by the inhibitory effect of a CDPK antagonist, W7, and a PI3 kinase inhibitor, wortmannin, respectively. Furthermore, bongkrekic acid (BK), a mitochondrial permeability transition pore opening blocker, suppressed Cd2+-, but not Cu2+-, induced MAP kinase activation, indicating that Cd2+-induced MAP kinase activities are dependent on the functional state of mitochondria. Collectively, these findings imply that Cd2+ and Cu2+ may induce MAP kinase activation through distinct signalling pathways. Moreover, it was found that the 42 kDa MAP kinase activities are higher in Cd-tolerant cultivars than in Cd-sensitive cultivars. Therefore, the Cd-induced 42 kDa MAP kinase activation may confer Cd tolerance in rice plants. © The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.

TITLE:Respiratory burst oxidase homologue A of barley contributes to penetration by the powdery mildew fungus Blumeria graminis f. sp. hordei 

[2-s2.0-33845235998] Refers [2-s2.0-0034954408] LEVEL: 3
Reactive oxygen intermediates (ROI) are closely related to defence reactions of plants against pathogens. A prominent role in the production of ROI has been attributed to the plant respiratory burst oxidase homologues (RBOH) of the human phagocyte GP91(phox). A barley RBOH, which encodes a putative superoxide (O2.-) producing NADPH oxidase, is described here. Histochemical analysis of the barley-Blumeria graminis f. sp. hordei (Bgh) interaction showed that O2.- is produced locally at the site of penetration. In contrast, hydrogen peroxide (H2O2) is produced in non-penetrated cell wall appositions. A barley RBOHA cDNA was isolated and a minor induction of expression of RBOHA was observed during the interactions of barley with Bgh. Transient RNA interference-mediated gene silencing of HvRBOHA during the penetration process of Bgh led to an increase of basal penetration resistance. The results support a potential role of HvRBOHA in cellular accessibility to Blumeria graminis. © The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.

TITLE:The coexistence of the oxidative and reductive systems in roots: The role of plasma membranes 

[2-s2.0-25144474926] Refers [2-s2.0-0034954408] LEVEL: 3
Different components of the plasma membrane bound and associated redox system, which participate in the energy transfer from the predominantly reducing intercellular environment to the extracellular oxidizing environment, are reviewed. Special attention is given to plant root cells. An analysis of the plasma membrane-associated redox components, such as the cytochromes, quinones, and different types of oxidoreductases (dehydrogenases, oxidases, peroxidases, and superoxide dismutases), is made, as well as their coupling with naturally occurring extracellular substrates, such as oxygen and its reactive forms, phenols, ascorbate, nitrate, ferric ion, and organic acids. The participation of different free radical species in most of the plasma membrane-bound redox reactions is documented. © 2005 New York Academy of Sciences.

TITLE:The plant peroxidases of class III | [Les peroxydases veĢgeĢtales de classe III] 

[2-s2.0-15444379901] Refers [2-s2.0-0034954408] LEVEL: 3
Peroxidases are heme proteins involved in the oxidization of a large variety of substrates, through the reaction with hydrogen peroxide. Plant peroxidases are class III peroxidases, according to nomenclature. Primary and tertiary structure, as well as principles of reactions catalyzed by such peroxidases are discussed. Class III peroxidases are involved in different physiological functions, such as cell wall metabolism, hormonal metabolism, responses to abiotic stresses, and plant-microorganisms interactions. Biotechnological applications are presented.

TITLE:Pathogen-induced defense and innate immunity in macroalgae 

[2-s2.0-37549036710] Refers [2-s2.0-0034954408] LEVEL: 3
Animals and vascular plants are known to defend themselves facultatively against pathogens, with innate receptors mediating their resistance. Macroalgal defense against microorganisms, in contrast, has until recently been regarded mainly as constitutive. Indeed, many macroalgae appear to be chemically defended at constantly high levels, and this is possibly one of the reasons why the first evidence of pathogen-aroused resistance in a macroalga was detected only a decade ago. Here, I summarize the results of studies that indicate the existence of pathogen-activated or pathogen-induced macroalgal defense. Most indications so far come from molecular investigations, which revealed major functional similarities among the defense systems of distant macroalgal clades and the innate immune systems of vascular plants and metazoans. Homologies exist in the primary and secondary defense-activating signals, as well as in the enzymes that are involved and the cellular responses that are activated. This strongly suggests that innate immunity also exists in relatively distinct macroalgal clades. However, a macroalgal receptor still needs to be isolated and characterized, and the molecular concept of macroalgal receptor-mediated immunity needs to be complemented with an ecological perspective on pathogen-induced defense, to develop a joint neuroecological perspective on seaweed-microbe interactions. © 2007 Marine Biological Laboratory.

TITLE:Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth 

[2-s2.0-34248374154] Refers [2-s2.0-0034954408] LEVEL: 3
? Tip-localized reactive oxygen species (ROS) were detected in growing pollen tubes by chloromethyl dichlorodihydrofluorescein diacetate oxidation, while tip-localized extracellular superoxide production was detected by nitroblue tetrazolium (NBT) reduction. ? To investigate the origin of the ROS we cloned a fragment of pollen specific tobacco NADPH oxidase (NOX) closely related to a pollen specific NOX from Arabidopsis. Transfection of tobacco pollen tubes with NOX-specific antisense oligodeoxynucleotides (ODNs) resulted in decreased amount of NtNOX mRNA, lower NOX activity and pollen tube growth inhibition. ? The ROS scavengers and the NOX inhibitor diphenylene iodonium chloride (DPI) inhibited growth and ROS formation in tobacco pollen tube cultures. Exogenous hydrogen peroxide (H2O2) rescued the growth inhibition caused by NOX antisense ODNs. Exogenous CaCl2 increased NBT reduction at the pollen tube tip, suggesting that Ca2+ increases the activity of pollen NOX in vivo. ? The results show that tip-localized ROS produced by a NOX enzyme is needed to sustain the normal rate of pollen tube growth and that this is likely to be a general mechanism in the control of tip growth of polarized plant cells. © The Authors (2007).

TITLE:Salinity-induced decrease in NADPH oxidase activity in the maize leaf blade elongation zone 

[2-s2.0-33846572916] Refers [2-s2.0-0034954408] LEVEL: 3
We reported previously that salinity-induced elongation constraints in the expansion zone of maize leaves are associated with reduced reactive oxygen species (ROS) production and could be alleviated by the addition of ROS. The NaCl effect was salt-specific and not osmotic. This paper explores the causes for such reduction. The decrease in ROS levels under salinity was not accompanied by increases in soluble apoplastic antioxidant activities such as superoxide dismutase, peroxidases and ascorbate. In experimental systems devoid of cell walls (protoplasts and membrane fractions) superoxide anion (O2-) production was inhibited by 50 and 100 mM NaCl, 50 ?M DPI, 10 mM EGTA, and 5 mM verapamil, a Ca2+ channel inhibitor. Inhibitory effects of NaCl and reduced Ca2+ supply were also observed in in gel assessment of O2--generating activity. The main activity band excised from the ND-PAGE was recognized by an antibody against the C-terminal portion of the tomato gp91phox homolog. These results indicate the {radical dot}O2--generating activity negatively affected by NaCl was compatible with that of plasma membrane NADPH oxidase. © 2006 Elsevier GmbH. All rights reserved.

TITLE:Tobacco nectaries express a novel NADPH oxidase implicated in the defense of floral reproductive tissues against microorganisms 

[2-s2.0-33846391335] Refers [2-s2.0-0034954408] LEVEL: 3
Hydrogen peroxide produced from the nectar redox cycle was shown to be a major factor contributing to inhibition of most microbial growth in floral nectar; however, this obstacle can be overcome by the floral pathogen Erwinia amylovora. To identify the source of superoxide that leads to hydrogen peroxide accumulation in nectary tissues, nectaries were stained with nitroblue tetrazolium. Superoxide production was localized near nectary pores and inhibited by diphenylene iodonium but not by cyanide or azide, suggesting that NAD(P)H oxidase is the source of superoxide. Native PAGE assays demonstrated that NADPH (not NADH) was capable of driving the production of superoxide, diphenyleneiodonium chloride was an efficient inhibitor of this activity, but cyanide and azide did not inhibit. These results confirm that the production of superoxide was due to an NADPH oxidase. The nectary enzyme complex was distinct by migration on gels from the leaf enzyme complex. Temporal expression patterns demonstrated that the superoxide production (NADPH oxidase activity) was coordinated with nectar secretion, the expression of Nectarin I (a superoxide dismutase in nectar), and the expression of NOX1, a putative gene for a nectary NADPH oxidase that was cloned from nectaries and identified as an rbohD-like NADPH oxidase. Further, in situ hybridization studies indicated that the NADPH oxidase was expressed in the early stages of flower development although superoxide was generated at later stages (after Stage 10), implicating posttranslational regulation of the NADPH oxidase in the nectary. © 2006 American Society of Plant Biologists.

TITLE:Phosphatidylinositol 3-phosphate is required for abscisic acid-induced hydrogen peroxide production in rice leaves 

[2-s2.0-19944367615] Refers [2-s2.0-0034954408] LEVEL: 3
Rice leaves produce H2O2 in response to abscisic acid (ABA), which results in induction of senescence and accumulation of NH 4+. The upstream steps of the ABA-induced H 2O2 production pathway in rice leaves remain largely unclear. In animal cells, H2O2 production in neutrophils is activated by phosphatidylinositol 3-phosphate (PI3P), a product of phosphatidylinositol 3-knase (PI3K). In the present study, we examined whether PI3P plays a role in H2O2 production in rice leaves exposed to ABA. We found that PI3K inhibitors LY 294002 (LY) or wortmannin (WM) inhibited ABA-induced H2O2 production, senescence and NH4+ accumulation. Hydrogen peroxide almost completely rescued the inhibitory effect of LY or WM. It appears that PI3P plays a role in ABA-induced H2O2 production, senescence, and NH 4+ accumulation in rice leaves. © Springer 2005.

TITLE:The use of model systems to study biological functions of Nox/Duox enzymes 

[2-s2.0-16244373376] Refers [2-s2.0-0034954408] LEVEL: 3
ROS (reactive oxygen species; including superoxide and H2O 2) are conventionally thought of as being broadly reactive and cytotoxic. Phagocytes utilize an NADPH oxidase to generate large amounts of ROS, and exploit their toxic properties as a host-defence mechanism to kill invading microbes. However, the recent discovery of the Nox and Duox enzymes that are expressed in many non-phagocytic cells implies that the 'deliberate' generation of ROS has additional cellular roles, which are currently incompletely understood. Functions of ROS in mammals have been inferred primarily from cell-culture experiments, and include signalling for mitogenic growth, apoptosis and angiogenesis. Nox/Duox enzymes may also provide H2O2 as a substrate for peroxidase enzymes (or, in the case of Duox, for its own peroxidase domain), thereby supporting peroxidative reactions. A broad comparison of biological functions of ROS and Nox enzymes across species and kingdoms provides insights into possible functions in mammals. To further understand novel biological roles for Nox/Duox enzymes, we are manipulating the expression of Nox/Duox enzymes in model organisms including Caenorhabditis elegans, Drosophila melanogaster and mouse. This chapter focuses on new insights into the roles of Nox enzymes gained from these approaches. © 2004 The Biochemical Society.

TITLE:Activation of plasma membrane NADPH oxidase and generation of H2O2 mediate the induction of PAL activity and saponin synthesis by endogenous elicitor in suspension-cultured cells of Panax ginseng 

[2-s2.0-2342460979] Refers [2-s2.0-0034954408] LEVEL: 3
Endogenous elicitor, termed cellulase-degraded cell wall (CDW), was prepared from the cell wall of suspension-cultured ginseng (Panax ginseng C.A. Meyer) cells via cellulase degradation. CDW activated the NADPH oxidase activity of isolated plasma membranes and stimulated in vivo H2O2 generation in ginseng cell suspensions. CDW also increased the activity of phenylalanine ammonia lyase (PAL), expression of a P. ginseng squalene epoxidase (sqe) gene and saponin synthesis. NADPH oxidase inhibitors inhibited both in vitro NADPH oxidase activity and in vivo H2O2 generation. Induction of PAL activity, saponin synthesis and sqe gene expression were all inhibited by such inhibitor treatments and reduced by incubation with catalase and H2O2 scavengers. These data indicate that activation of NADPH oxidase and generation of H2O2 are essential signalling events mediating defence responses induced by the endogenous elicitor(s) present in CDW.

TITLE:Activation by fatty acids of the production of active oxygen species by tobacco cells 

[2-s2.0-1542787250] Refers [2-s2.0-0034954408] LEVEL: 3
Among the different transduction steps leading from hypoosmotic stress to oxidative burst in suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells, phospholipase activation was evidenced. Using thin layer chromatography and phospholipase inhibitors the involved lipase was strongly suggested to be a phospholipase A2 (EC Fatty acids like arachidonate and linolenate stimulated the oxidative response and prevented its inhibition by a phospholipase inhibitor, confirming the physiological relevance of the phospholipase action. A production of active oxygen species by plasma membrane vesicles was demonstrated, using two different probes. The producing system characterized in vitro was NADPH-dependent, strongly depressed by iodonium diphenyl and activated by fatty acids like the oxidative response assayed in vivo. Several other anionic amphiphiles like SDS were able to mimic the activation of the oxidative response by fatty acids, both in vivo and in vitro, suggesting that negative charges may be involved in the action mode of fatty acids. Inversely, a cationic detergent was an efficient inhibitor of the hypoosmotically induced oxidative burst and the inhibition was fully reversed by SDS. The possible identification of the active oxygen synthase involved in hypoosmotic signalling with a plasma membrane-located NADPH oxidase is discussed. © 2002 E?ditions scientifiques et me?dicales Elsevier SAS. All rights reserved.

TITLE:Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension 

[2-s2.0-39149121438] Refers [2-s2.0-0034954408] LEVEL: 3
Reactive oxygen species (ROS) produced by NADPH oxidase play critical roles in various cellular activities, including plant innate immunity response. In contrast with the large multiprotein NADPH oxidase complex of phagocytes, in plants, only the homologs of the catalytic subunit gp91phox and the cytosolic regulator small GTPase Rac are found. Plant homologs of the gp91 phox subunit are known as Rboh (for respiratory burst oxidase homolog). Although numerous Rboh have been isolated in plants, the regulation of enzymatic activity remains unknown. All rboh genes identified to date possess a conserved N-terminal extension that contains two Ca2+ binding EF-hand motifs. Previously, we ascertained that a small GTPase Rac (Os Rac1) enhanced pathogen-associated molecular pattern-induced ROS production and resistance to pathogens in rice (Oryza sativa). In this study, using yeast two-hybrid assay, we found that interaction between Rac GTPases and the N-terminal extension is ubiquitous and that a substantial part of the N-terminal region of Rboh, including the two EF-hand motifs, is required for the interaction. The direct Rac-Rboh interaction was supported by further studies using in vitro pull-down assay, a nuclear magnetic resonance titration experiment, and in vivo fluorescence resonance energy transfer (FRET) microscopy. The FRET analysis also suggests that cytosolic Ca2+ concentration may regulate Rac-Rboh interaction in a dynamic manner. Furthermore, transient coexpression of Os Rac1 and rbohB enhanced ROS production in Nicotiana benthamiana, suggesting that direct Rac-Rboh interaction may activate NADPH oxidase activity in plants. Taken together, the results suggest that cytosolic Ca2+ concentration may modulate NADPH oxidase activity by regulating the interaction between Rac GTPase and Rboh. © 2007 American Society of Plant Biologists.

TITLE:NADPH oxidase-dependent hydrogen peroxide production, induced by salinity stress, may be involved in the regulation of total calcium in roots of wheat 

[2-s2.0-35348814922] Refers [2-s2.0-0034954408] LEVEL: 3
Hydrogen peroxide (H2O2) is often generated by cells and tissues under environmental stress. In this work, we provide evidence that plasma membrane (PM) NADPH oxidase-dependent H2O2 production might act as an intermediate step in the NaCl-induced elevation of calcium (Ca) in roots of wheat. Remarkable increases in the content of total Ca were observed not only in roots exposed to NaCl but also in roots of seedlings exposed to exogenous H2O2. In roots, H2O2 production increased upon exposure to salt stress. PM vesicles were isolated from roots, and NADPH oxidase activity was determined by measuring superoxide anion (O2-) production. NADPH oxidase-dependent O2- production was 11.6 nmol mg-1 protein min-1 in control vesicles, but 19.6 nmol after NaCl treatment (24 h), indicating that salt stress resulted in the activation of the PM NADPH oxidase. Furthermore, the NaCl-induced increase in total Ca was partially abolished by the addition of 150 U/mL catalase (CAT), a H2O2 scavenger, and also by 10 ?M diphenylane iodonium (DPI), a NADPH oxidase inhibitor. This data suggest that NADPH oxidase-dependent H2O2 production might be involved in the modulation of the Ca content in wheat roots. In conclusion, our results show that salinity stress increases the total Ca content of wheat roots, which is partly due to PM NADPH oxidase-dependent H2O2 generation. © 2006 Elsevier GmbH. All rights reserved.

TITLE:Involvement of hydrogen peroxide and nitric oxide in salt resistance in the calluses from Populus euphratica 

[2-s2.0-34249828656] Refers [2-s2.0-0034954408] LEVEL: 3
Nitric oxide (NO) and hydrogen peroxide (H2O2) function as signalling molecules in plants under abiotic and biotic stresses. Calluses from Populus euphratica, which show salt tolerance, were used to study the interaction of NO and H2O2 in plant adaptation to salt resistance. The nitric oxide synthase (NOS) activity was identified in the calluses, and this activity was induced under 150 mM NaCl treatment. Under 150 mM NaCl treatment, the sodium (Na) percentage decreased, but the potassium (K) percentage and the K/Na ratio increased in P. euphratica calluses. Application of glucose/glucose oxidase (G/GO, a H2O2 donor) and sodium nitroprusside (SNP, a NO donor) revealed that both H2O2 and NO resulted in increased K/Na ratio in a concentration-dependent manner. Diphenylene iodonium (DPI, an NADPH oxidase inhibitor) counteracted H 2O2 and NO effect by increasing the Na percentage, decreasing the K percentage and K/Na ratio. NG-monomethyl-L-Arg monoacetate (NMMA, an NO synthase inhibitor) and 2-phenyl-4,4,5,5-tetramethyl- imidazoline-1-oxyl-3-oxyde (PTIO, a specific NO scavenger) only reversed NO effect, but did not block H2O2 effect. The increased activity of plasma membrane (PM) H+-ATPase caused by salt stress was reversed by treatment with DPI and NMMA. Exogenous H2O2 increased the activity of PM H+-ATPase, but the effect could not be diminished by NMMA and PTIO. The NO-induced increase of PM H+-ATPase can be reversed by NMMA and PTIO, but not by DPI. Western blot analysis demonstrated that NO and H2O2 stimulated the expression of PM H+-ATPase in P. euphratica calluses. These results indicate that NO and H2O2 served as intermediate molecules in inducing salt resistance in the calluses from P. euphratica under slat stress by increasing the K/Na ratio, which was dependent on the increased PM H +-ATPase activity. © 2007 The Authors.

TITLE:Pollen NAD(P)H Oxidases and Their Contribution to Allergic Inflammation 

[2-s2.0-33846786788] Refers [2-s2.0-0034954408] LEVEL: 3
This article provides an overview of NADPH oxidase and its role in allergic inflammation. A background and historical perspectives of NADPH oxidase are first provided, followed by a detailed overview of mammalian NADPH oxidase subunits and their functional organization. Plant NADPH oxidase, the authors' discovery of NADPH oxidase in pollens, and their contribution to allergic inflammation are then discussed, concluding with a discussion of future directions and outstanding questions that require attention. © 2007 Elsevier Inc. All rights reserved.

TITLE:Ca2+-responsive cis-elements in plants 

[2-s2.0-34547713430] Refers [2-s2.0-0034954408] LEVEL: 3
External physical and chemical stimuli are transduced via second messengers, following primary interaction with specific membrane or soluble receptors. Ca2+ is an important second messenger in plants as in other eukaryotes, mediating responses to numerous environmental stimuli and affecting a multitude of cellular processes including gene expression. However, there is yet very little information concerning the cis-elements that mediate Ca2+-responsive gene expression. In this article we discuss a recent investigation combining bioinformatics with experimental data, revealing DNA regulatory elements that convey specific cytosolic Ca2+ transients to the transcription machinery. ©2007 Landes Bioscience.

TITLE:Characterization of active oxygen-producing proteins in response to hypo-osmolarity in tobacco and Arabidopsis cell suspensions: Identification of a cell wall peroxidase 

[2-s2.0-33645745877] Refers [2-s2.0-0034954408] LEVEL: 3
The oxidative response induced by hypo-osmolarity is characterized in tobacco and Arabidopsis cells in order to identify the corresponding active oxygen-producing proteins. The pharmacological profiles of the oxidative responses were clearly different in the two plant materials, leading to the identification of distinct active oxygen producers in tobacco and Arabidopsis cells. In tobacco cells, a 100 kDa protein, localized in the plasma membrane, was demonstrated to produce active oxygen in the presence of NADPH. This production can be activated by fatty acids and is strongly depressed by diphenylene iodonium, as measured by an in vivo response. In Arabidopsis, 30 kDa and 34 kDa proteins localized in the cell wall were shown to be able to produce active oxygen in the presence of cofactors and the production is prevented by peroxidase inhibitors, as is the in vivo response. The two purified proteins were identified by mass spectrometry and both correspond to the peroxidase gene At5g64120.

TITLE:Identification and characterization of differentially expressed ESTs of Gossypium barbadense infected by Verticillium dahliae with suppression subtractive hybridization 

[2-s2.0-18844379893] Refers [2-s2.0-0034954408] LEVEL: 3
The wilt defense reaction of cotton is a complicated continuous process and involves a battery of genes. In this study, we adopted the suppression subtractive hybridization (SSH) technique to isolate differentially expressed ESTs from Gossypium barbadense variety 7124 during the Verticillium wilt defense process. An array of 1165 clones from the subtractive library has been screened with reverse northern blotting, of which 131 ESTs were considered as overexpressed and 16 ESTs were downregulated. Sequence analysis and blast search showed that 83 ESTs were homologous to 45 unique sequences in the databases. Among all these differentially expressed ESTs, at least three kinds of genes were characterized. The majority of ESTs with a deduced identity as aerobic metabolism enzymes were strongly expressed in the infection process. Likewise, ESTs similar to those reported for pathogen-related protein genes were also picked out in this study. These ESTs, in combination with other kinase-like genes and a defensin-like EST, constituted an assembly of genes which responded during pathogenic infection. These results imply that sea-island cotton undergoes strong oxidative stress and results in a series of defense responses when attacked by V. dahliae. To our knowledge, this is the first report on the isolation of global ESTs during the sea-island cotton defense reaction. © 2005 Pleiades Publishing, Inc.

TITLE:Microdomains of intracellular Ca2+: Molecular determinants and functional consequences 

[2-s2.0-33644847375] Refers [2-s2.0-12944314765] LEVEL: 3
Calcium ions are ubiquitous and versatile signaling molecules, capable of decoding a variety of extracellular stimuli (hormones, neurotransmitters, growth factors, etc.) into markedly different intracellular actions, ranging from contraction to secretion, from proliferation to cell death. The key to this pleiotropic role is the complex spatiotemporal organization of the [Ca2+] rise evoked by extracellular agonists, which allows selected effectors to be recruited and specific actions to be initiated. In this review, we discuss the structural and functional bases that generate the subcellular heterogeneity in cellular Ca2+ levels at rest and under stimulation. This complex choreography requires the concerted action of many different players; the central role is, of course, that of the calcium ion, with the main supporting characters being all the entities responsible for moving Ca2+ between different compartments, while the cellular architecture provides a determining framework within which all the players have their exits and their entrances. In particular, we concentrate on the molecular mechanisms that lead to the generation of cytoplasmic Ca2+ microdomains, focusing on their different subcellular location, mechanism of generation, and functional role. Copyright © 2006 the American Physiological Society.

TITLE:ER stress and neurodegenerative diseases 

[2-s2.0-33645141853] Refers [2-s2.0-12944314765] LEVEL: 3
Endoplasmic reticulum (ER) stress is caused by disturbances in the structure and function of the ER with the accumulation of misfolded proteins and alterations in the calcium homeostasis. The ER response is characterized by changes in specific proteins, causing translational attenuation, induction of ER chaperones and degradation of misfolded proteins. In case of prolonged or aggravated ER stress, cellular signals leading to cell death are activated. ER stress has been suggested to be involved in some human neuronal diseases, such as Parkinson's disease, Alzheimer's and prion disease, as well as other disorders. The exact contributions to and casual effects of ER stress in the various disease processes, however, are not known. Here we will discuss the possible role of ER stress in neurodegenerative diseases, and highlight current knowledge in this field that may reveal novel insight into disease mechanisms and help to design better therapies for these disorders. © 2006 Nature Publishing Group. All rights reserved.

TITLE:Endoplasmic reticulum stress response and neurodegeneration 

[2-s2.0-24644487812] Refers [2-s2.0-12944314765] LEVEL: 3
The endoplasmic reticulum (ER) is a subcellular compartment playing a central role in calcium storage and signaling. Disturbances of ER calcium homeostasis constitute a severe form of stress interfering with central functions of this structure including the folding and processing of newly synthesized membrane and secretory proteins. Blocking the folding and processing reactions results in the accumulation of unfolded proteins forming potentially toxic aggregates. To restore ER functioning, specific stress responses are activated one of which is the unfolded protein response (UPR). UPR is characterized by a shutdown of global protein synthesis and activation of expression of genes coding for ER-resident proteins that are involved in the folding and processing reactions. ER calcium homeostasis is therefore inevitably associated with major cellular functions, including gene transcription and translation. ER calcium homeostasis und ER functions are believed to be impaired in various degenerative diseases of the brain including Alzheimer's, Parkinson's and Huntington's disease, and amyotrophic lateral sclerosis. ER functioning has also been shown to be disturbed in acute pathological states of the brain such as ischemia and trauma, which have been identified as risk factors for the development of degenerative diseases. This implies that there are common underlying pathomechanisms. This review will summarize new observations suggesting that impairment of ER functioning may be a common denominator of pathological processes resulting in neuronal cell injury in acute disorders and degenerative diseases of the brain. © 2005 Elsevier Ltd. All rights reserved.

TITLE:Presynaptic calcium stores and synaptic transmission 

[2-s2.0-20444410101] Refers [2-s2.0-12944314765] LEVEL: 3
Following the gradual recognition of the importance of intracellular calcium stores for somatodendritic signaling in the mammalian brain, recent reports have also indicated a significant role of presynaptic calcium stores. Ryanodine-sensitive stores generate local, random calcium signals that shape spontaneous transmitter release. They amplify spike-driven calcium signals in presynaptic terminals, and consequently enhance the efficacy of transmitter release. They appear to be recruited by an association with certain types of calcium-permeant ion channels, and they induce specific forms of synaptic plasticity. Recent research also indicates a role of inositoltrisphosphate- sensitive presynaptic calcium stores in synaptic plasticity. © 2005 Elsevier Ltd. All rights reserved.

TITLE:The endoplasmic reticulum: Folding, calcium homeostasis, signaling, and redox control 

[2-s2.0-33750902737] Refers [2-s2.0-12944314765] LEVEL: 3
The endoplasmic reticulum (ER) plays a major role in regulating synthesis, folding, and orderly transport of proteins. It is also essentially involved in various cellular signaling processes, primarily by its function as a dynamic Ca2+ store. Compared to the cytosol, oxidizing conditions are found in the ER that allow oxidation of cysteine residues in nascent polypeptide chains to form intramolecular disulfide bonds. However, compounds and enzymes such as PDI that catalyze disulfide bonds become reduced and have to be reoxidized for further catalytic cycles. A number of enzymes, among them products of the ERO1 gene, appear to provide oxidizing equivalents, and oxygen appears to be the final oxidant in aerobic living organisms. Thus, protein oxidation in the ER is connected with generation of reactive oxygen species (ROS). Changes in the redox state and the presence of ROS also affect the Ca2+ homeostasis by modulating the functionality of ER-based channels and buffering chaperones. In addition, a close relationship exists between oxidative stress and ER stress, which both may activate signaling events leading to a rebalance of folding capacity and folding demand or to cell death. Thus, redox homeostasis appears to be a prerequisite for proper functioning of the ER. © Mary Ann Liebert, Inc.

TITLE:Calcium signalling: Past, present and future 

[2-s2.0-24644481046] Refers [2-s2.0-12944314765] LEVEL: 3
Ca2+ is a universal second messenger controlling a wide variety of cellular reactions and adaptive responses. The initial appreciation of Ca2+ as a universal signalling molecule was based on the work of Sydney Ringer and Lewis Heilbrunn. More recent developments in this field were critically influenced by the invention of the patch clamp technique and the generation of fluorescent Ca2+ indicators. Currently the molecular Ca2+ signalling mechanisms are being worked out and we are beginning to assemble a reasonably complete picture of overall Ca2+ homeostasis. Furthermore, investigations of organellar Ca2+ homeostasis have added complexity to our understanding of Ca2+ signalling. The future of the Ca2+ signalling field lies with detailed investigations of the integrative function in vivo and clarification of the pathology associated with malfunctions of Ca2+ signalling cascades. © 2005 Elsevier Ltd. All rights reserved.

TITLE:Ca2+ signalling and Ca2+-activated ion channels in exocrine acinar cells 

[2-s2.0-24644481459] Refers [2-s2.0-12944314765] LEVEL: 3
The development of the calcium signalling field, from its early beginnings some 40 years ago to the present, is described. Calcium signalling in exocrine gland acinar cells and the effects of neurotransmitter- or hormone-elicited rises in the cytosolic calcium ion concentration on ion channel gating are reviewed. The highly polarized arrangement of the organelle systems in living acinar cells is described as well as its importance for the physiologically relevant local and polarized calcium signalling events. © 2005 Elsevier B.V. All rights reserved.

TITLE:Intraluminal calcium as a primary regulator of endoplasmic reticulum function 

[2-s2.0-24644438715] Refers [2-s2.0-12944314765] LEVEL: 3
The concentration of Ca2+ inside the lumen of endoplasmic reticulum (ER) regulates a vast array of spatiotemporally distinct cellular processes, from intracellular Ca2+ signals to intra-ER protein processing and cell death. This review summarises recent data on the mechanisms of luminal Ca2+-dependent regulation of Ca2+ release and uptake as well as ER regulation of cellular adaptive processes. In addition we discuss general biophysical properties of the ER membrane, as trans-endomembrane Ca2+ fluxes are subject to basic electrical forces, determined by factors such as the membrane potential of the ER and the ease with which Ca2+ fluxes are able to change this potential (i.e. the resistance of the ER membrane). Although these electrical forces undoubtedly play a fundamental role in shaping [Ca2+]ER dynamics, at present there is very little direct experimental information about the biophysical properties of the ER membrane. Further studies of how intraluminal [Ca2+] is regulated, best carried out with direct measurements, are vital for understanding how Ca2+ orchestrates cell function. Direct monitoring of [Ca2+]ER under conditions where the cytosolic [Ca2+] is known may also help to capture elusive biophysical information about the ER, such as the potential difference across the ER membrane. © 2005 Elsevier Ltd. All rights reserved.

TITLE:D1-D2 dopamine receptor heterooligomers with unique pharmacology are coupled to rapid activation of Gq/11 in the striatum 

[2-s2.0-33846328669] Refers [2-s2.0-12944314765] LEVEL: 3
We demonstrate a heteromeric D1-D2 dopamine receptor signaling complex in brain that is coupled to Gq/11 and requires agonist binding to both receptors for G protein activation and intracellular calcium release. The D1 agonist SKF83959 was identified as a specific agonist for the heteromer that activated Gq/11 by functioning as a full agonist for the D1 receptor and a high-affinity partial agonist for a pertussis toxin-resistant D2 receptor within the complex. We provide evidence that the D1-D2 signaling complex can be more readily detected in mice that are 8 months in age compared with animals that are 3 months old, suggesting that calcium signaling through the D1-D2 dopamine receptor complex is relevant for function in the postadolescent brain. Activation of Gq/11 through the heteromer increases levels of calcium/calmodulin-dependent protein kinase II? in the nucleus accumbens, unlike activation of Gs/olf-coupled D1 receptors, indicating a mechanism by which D1-D2 dopamine receptor complexes may contribute to synaptic plasticity. © 2006 by The National Academy of Sciences of the USA.

TITLE:Enhanced caffeine-induced Ca2+ release in the 3xTg-AD mouse model of Alzheimer's disease 

[2-s2.0-24644460177] Refers [2-s2.0-12944314765] LEVEL: 3
Alzheimer's disease (AD) is the most prevalent form of dementia among the elderly and is a complex disorder that involves altered proteolysis, oxidative stress and disruption of ion homeostasis. Animal models have proven useful in studying the impact of mutant AD-related genes on other cellular signaling pathways, such as Ca2+ signaling. Along these lines, disturbances of intracellular Ca2+ ([Ca2+]i) homeostasis are an early event in the pathogenesis of AD. Here, we have employed microfluorimetric measurements of [Ca2+]i to investigate disturbances in Ca2+ homeostasis in primary cortical neurons from a triple transgenic mouse model of Alzheimer's disease (3xTg-AD). Application of caffeine to mutant presenilin-1 knock-in neurons (PS1KI) and 3xTg-AD neurons evoked a peak rise of [Ca2+]i that was significantly greater than those observed in non-transgenic neurons, although all groups had similar decay rates of their Ca2+ transient. This finding suggests that Ca 2+ stores are greater in both PS1KI and 3xTg-AD neurons as calculated by the integral of the caffeine-induced Ca2+ transient signal. Western blot analysis failed to identify changes in the levels of several Ca2+ binding proteins (SERCA-2B, calbindin, calsenilin and calreticulin) implicated in the pathogenesis of AD. However, ryanodine receptor expression in both PS1KI and 3xTg-AD cortex was significantly increased. Our results suggest that the enhanced Ca2+ response to caffeine observed in both PS1KI and 3xTg-AD neurons may not be attributable to an alteration of endoplasmic reticulum store size, but to the increased steady-state levels of the ryanodine receptor. © 2005 International Society for Neurochemistry.

TITLE:'Rejuvenation' protects neurons in mouse models of Parkinson's disease 

[2-s2.0-34347359673] Refers [2-s2.0-12944314765] LEVEL: 3
Why dopamine-containing neurons of the brain's substantia nigra pars compacta die in Parkinson's disease has been an enduring mystery. Our studies suggest that the unusual reliance of these neurons on L-type Cav1.3 Ca2+ channels to drive their maintained, rhythmic pacemaking renders them vulnerable to stressors thought to contribute to disease progression. The reliance on these channels increases with age, as juvenile dopamine-containing neurons in the substantia nigra pars compacta use pacemaking mechanisms common to neurons not affected in Parkinson's disease. These mechanisms remain latent in adulthood, and blocking Cav1.3 Ca2+ channels in adult neurons induces a reversion to the juvenile form of pacemaking. Such blocking ('rejuvenation') protects these neurons in both in vitro and in vivo models of Parkinson's disease, pointing to a new strategy that could slow or stop the progression of the disease. ©2007 Nature Publishing Group.

TITLE:Early and simultaneous emergence of multiple hippocampal biomarkers of aging is mediated by Ca2+-induced Ca2+ release 

[2-s2.0-33645450459] Refers [2-s2.0-12944314765] LEVEL: 3
Age-dependent changes in multiple Ca2+-related electrophysiological processes in the hippocampus appear to be consistent biomarkers of aging, and several also correlate with cognitive decline. These findings have led to the hypothesis that a common mechanism of Ca2+ dyshomeostasis underlies aspects of aging-dependent brain impairment. However, some key predictions of this view remain untested, including that multiple Ca2+-related biomarkers should emerge concurrently during aging and their onset should also precede/coincide with initial signs of cognitive decline. Moreover, blocking a putative common source of dysregulated Ca 2+ should eliminate aging differences. Here, we tested these predictions using combined electrophysiological, imaging, and pharmacological approaches in CA1 neurons to determine the ages of onset (across 4-, 10-, 12-, 14-, and 23-month-old F344 rats) of several established biomarkers, including the increases in the slow afterhyperpolarization, spike accommodation, and [Ca2+]i rise during repetitive synaptic stimulation. In addition, we tested the hypothesis that altered Ca2+-induced Ca 2+ release (CICR) from ryanodine receptors, which can be triggered by L-type Ca2+ channels, provides a common source of dysregulated Ca2+ in aging. Results showed that multiple aging biomarkers were first detectable at about the same age (12 months of age; approximately midlife), sufficiently early to influence initial cognitive decline. Furthermore, selectively blocking CICR with ryanodine slowed the Ca2+ rise during synaptic stimulation more in aged rat neurons and, notably, reduced or eliminated aging differences in the biomarkers. Thus, this study provides the first evidence that altered CICR plays a role in driving the early and simultaneous emergence in hippocampus of multiple Ca2+-related biomarkers of aging. Copyright © 2006 Society for Neuroscience.

TITLE:Expansion of the calcium hypothesis of brain aging and Alzheimer's disease: Minding the store 

[2-s2.0-33947493213] Refers [2-s2.0-12944314765] LEVEL: 3
Evidence accumulated over more than two decades has implicated Ca2+ dysregulation in brain aging and Alzheimer's disease (AD), giving rise to the Ca2+ hypothesis of brain aging and dementia. Electrophysiological, imaging, and behavioral studies in hippocampal or cortical neurons of rodents and rabbits have revealed aging-related increases in the slow afterhyperpolarization, Ca2+ spikes and currents, Ca2+ transients, and L-type voltage-gated Ca2+ channel (L-VGCC) activity. Several of these changes have been associated with age-related deficits in learning or memory. Consequently, one version of the Ca2+ hypothesis has been that increased L-VGCC activity drives many of the other Ca2+-related biomarkers of hippocampal aging. In addition, other studies have reported aging- or AD model-related alterations in Ca2+ release from ryanodine receptors (RyR) on intracellular stores. The Ca2+-sensitive RyR channels amplify plasmalemmal Ca2+ influx by the mechanism of Ca2+- induced Ca2+ release (CICR). Considerable evidence indicates that a preferred functional link is present between L-VGCCs and RyRs which operate in series in heart and some brain cells. Here, we review studies implicating RyRs in altered Ca2+ regulation in cell toxicity, aging, and AD. A recent study from our laboratory showed that increased CICR plays a necessary role in the emergence of Ca2+-related biomarkers of aging. Consequently, we propose an expanded L-VGCC/Ca2+ hypothesis, in which aging/pathological changes occur in both L-type Ca2+ channels and RyRs, and interact to abnormally amplify Ca2+ transients. In turn, the increased transients result in dysregulation of multiple Ca2+-dependent processes and, through somewhat different pathways, in accelerated functional decline during aging and AD. © 2007 The Authors Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007.

TITLE:NMDA receptors in glia 

[2-s2.0-33846025401] Refers [2-s2.0-12944314765] LEVEL: 3
The amino acid L-Glutamate acts as the most ubiquitous mediator of excitatory synaptic transmission in the central nervous system. Glutamatergic transmission is central for diverse brain functions, being particularly important for learning, memory, and cognition. In brain pathology, excessive release of glutamate triggers excitotoxic neural cell death through necrotic or apoptotic pathways. Glutamate effects are mediated by several classes of glutamate receptors, expressed in virtually all cells of neural origin. Specifically important for both physiological information processing and cell damage are glutamate receptors of NMDA (N-methyl-D-aspartate) type, which, for a long time, were considered to be expressed exclusively in neurons. Recent studies have found functional NMDA receptors in brain macroglia, in astrocytes, and oligodendrocytes. Glial and neuronal NMDA receptors are functionally and structurally different; the glial receptors are weakly (if at all) sensitive to the extracellular magnesium block, which may indicate a predominant expression of the NR3 receptor subunit. In the cortex, astroglial NMDA receptors are activated upon physiological synaptic transmission. The physiological relevance of NMDA receptors in the white matter remains unknown; their activation upon ischemia triggers Ca2+-dependent damage of oligodendrocytes and myelin. The discovery of glial NMDA receptors further indicates the complex nature of intercellular signaling mechanisms in the brain, which involve all types of neural cells, connected through diverse types of chemical and electrical synapses. Copyright © 2007 Sage Publications.

TITLE:Endoplasmic reticulum stress inhibition protects against excitotoxic neuronal injury in the rat brain 

[2-s2.0-33846577831] Refers [2-s2.0-12944314765] LEVEL: 3
Elevated brain glutamate with activation of neuronal glutamate receptors accompanies neurological disorders, such as epilepsy and brain trauma. However, the mechanisms by which excitotoxicity triggers neuronal injury are not fully understood. We have studied the glutamate receptor agonist kainic acid (KA) inducing seizures and excitotoxic cell death. KA caused the disintegration of the endoplasmic reticulum (ER) membrane in hippocampal neurons and ER stress with the activation of the ER proteins Bip, Chop, and caspase-12. Salubrinal, inhibiting eIF2? (eukaryotic translation initiation factor 2 subunit ?) dephosphorylation, significantly reduced KA-induced ERstress and neuronal death in vivo and in vitro. KA-induced rise in intracellular calcium was not affected by Salubrinal. The results show that ER responses are essential parts of excitotoxicity mediated by glutamate receptor activation and that Salubrinal decreases neuronal death in vivo. Inhibition of ER stress by small molecular compounds may be beneficial for treatment of various neuronal injuries and brain disorders. Copyright © 2007 Society for Neuroscience.

TITLE:Intracellular zinc elevation measured with a "calcium-specific" indicator during ischemia and reperfusion in rat hippocampus: A question on calcium overload 

[2-s2.0-33749831752] Refers [2-s2.0-12944314765] LEVEL: 3
Much of our current evidence concerning of the role of calcium (Ca 2+) as a second messenger comes from its interaction with fluorescent probes; however, many Ca2+ probes also have a higher affinity for another divalent cation: zinc (Zn2+). In this study, using a selective Zn2+ probe (Newport Green), we investigated the accumulation of intracellular Zn2+ transients in acute rat hippocampal slices during ischemia, simulated by oxygen and glucose deprivation (OGD). Subsequent reperfusion with glucose-containing oxygenated medium resulted in an additional increase in intracellular Zn2+. Such observations compelled us to investigate the contribution of Zn2+ to the alleged intracellular Ca2+ overload occurring in ischemia and reperfusion. Using confocal fluorescent microscopy of Calcium Green-1, a widely used Ca 2+ indicator, we detected increases in fluorescence intensity during OGD and reperfusion. However, application of a Zn2+ chelator, at the peak of the fluorescence elevation (interpreted as Ca2+ overload), resulted in a significant drop in intensity, suggesting that rising Zn 2+ is the primary source of the increasing Calcium Green-1 fluorescence. Finally, staining with the cell viability indicator propidium iodide revealed that Zn2+ is responsible for the ischemic neuronal cell death, because Zn2+ chelation prevented cells from sustaining ischemic damage. Current cellular models of ischemic injury center on Ca 2+-mediated excitotoxicity. Our results indicate that Zn2+ elevation contributes to conventionally recognized Ca2+ overload and also suggest that the role of Ca2+ in neurotoxicity described previously using Ca2+ probes may need to be re-examined to determine whether effect previously attributed to Ca2+ could, in part, be attributable to Zn2+. Copyright © 2006 Society for Neuroscience.

TITLE:Junctophilin-mediated channel crosstalk essential for cerebellar synaptic plasticity 

[2-s2.0-34247210841] Refers [2-s2.0-12944314765] LEVEL: 3
Functional crosstalk between cell-surface and intracellular ion channels plays important roles in excitable cells and is structurally supported by junctophilins (JPs) in muscle cells. Here, we report a novel form of channel crosstalk in cerebellar Purkinje cells (PCs). The generation of slow afterhyperpolarization (sAHP) following complex spikes in PCs required ryanodine receptor (RyR)-mediated Ca2+-induced Ca2+ release and the subsequent opening of small-conductance Ca2+-activated K+ (SK) channels in somatodendritic regions. Despite the normal expression levels of these channels, sAHP was abolished in PCs from mutant mice lacking neural JP subtypes (JP-DKO), and this defect was restored by exogenously expressing JPs or enhancing SK channel activation. The stimulation paradigm for inducing long-term depression (LTD) at parallel fiber-PC synapses adversely established long-term potentiation in the JP-DKO cerebellum, primarily due to the sAHP deficiency. Furthermore, JP-DKO mice exhibited impairments of motor coordination and learning, although normal cerebellar histology was retained. Therefore, JPs support the Ca2+-mediated communication between voltage-gated Ca2+ channels, RyRs and SK channels, which modulates the excitability of PCs and is fundamental to cerebellar LTD and motor functions. © 2007 European Molecular Biology Organization | All Rights Reserved.

TITLE:Regional interaction of endoplasmic reticulum Ca2+ signals between soma and dendrites through rapid luminal Ca2+ diffusion 

[2-s2.0-33751335638] Refers [2-s2.0-12944314765] LEVEL: 3
The endoplasmic reticulum (ER) Ca2+ store plays a key role in integration and conveyance of Ca2+ signals in highly polarized neurons. The interconnected ER network in neurons generates Ca2+ signals in local domains, but the regional interaction is unclear. Here, we show that continuous or repetitive applications of caffeine produced robust Ca 2+ release from the ER Ca2+ store in dendritic areas without severe store depletion, but that similar stimuli applied to soma caused rapid store depletion in acutely isolated midbrain dopamine neurons. Partial emptying of the ER Ca2+ store within a dendrite caused a similar level of store depletion in unstimulated dendrites, as well as in soma. Photobleaching and local stimulation experiments revealed that Ca2+ and the dye trapped within the ER diffused rapidly from the soma to dendrites up to 90 ?m, which we could resolve, suggesting that the ER network acts as a functional tunnel for rapid Ca2+ transport. These data imply that the ER in soma acts as a Ca2+ reservoir supplying Ca2+ to the dendritic store, and that the dendritic store, hence, is able to respond to Ca2+-mobilizing input signals endurably. Copyright © 2006 Society for Neuroscience.

TITLE:Regulation of store-operated calcium entry by calcium-independent phospholipase A2 in rat cerebellar astrocytes 

[2-s2.0-33748689020] Refers [2-s2.0-12944314765] LEVEL: 3
We have studied store-operated Ca2+ entry (SOCE) in Bergmann glia and granule cell layer astrocytes in acute brain slices of the rat cerebellum, using the Ca2+-sensitive fluorescent dye Fluo-4 and confocal laser scanning microscopy. Astrocytes were identified by their morphology, location, and their Ca2+ response in K+-free solution. Depletion of Ca2+ stores by cyclopiazonic acid (CPA) (20 ?M) induced SOCE in both types of astrocyte. A similar Ca2+ influx was elicited by the calmodulin antagonist calmidazolium (CMZ) (1 ?M). The SOCE channel blocker 2-aminoethoxy-diphenylborate (2-APB) (100 ?M) and the Ca2+ release-activated channel blocker 3,5-bistrifluoromethyl pyrazole derivative (BTP2) (20 ?M) suppressed the CPA- and the CMZ-induced Ca2+ influx. Pretreatment of acute slices with the specific Ca 2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL) (25 ?M) blocked the CPA- and the CMZ-induced Ca2+ influx. The lysophospholipid products of iPLA 2, lysophosphatidylcholine (250 nM) and lysophosphatidylinositol (250 nM), but not lysophosphatidic acid (250 nM), induced a BTP2- and 2-APB-sensitive, but BEL-insensitive, Ca2+ influx. CPA or CMZ enhanced the BEL-sensitive enzymatic activity of iPLA2 in cerebellar astrocyte culture. Inhibition of iPLA2 expression by specific antisense oligodeoxynucleotide of iPLA2 reduced the SOCE and the Ca2+ store refilling in cultured astrocytes. Spontaneous Ca 2+ oscillations in astrocytes in situ were reduced after inhibiting SOCE channels or iPLA2 activity. The results suggest that the depletion of Ca2+ stores activates iPLA2 to open Ca 2+ channels in the plasma membrane by the formation of lysophospholipids in astrocytes, presumably to refill the stores and allow normal Ca2+ signaling. Copyright © 2006 Society for Neuroscience.

TITLE:Ca2+-dependent mechanisms of presynaptic control at central synapses 

[2-s2.0-33745902534] Refers [2-s2.0-12944314765] LEVEL: 3
Classically, a high-power association relates the neurotransmitter release probability to the concentration of presynaptic Ca2+. Activated by the action potential waveform, voltage-gated Ca2+ channels mediate Ca2+ entry into presynaptic terminals. Inside the terminal, Ca 2+ ions rapidly bind to endogenous intracellular buffers and could trigger Ca2+ release from internal Ca2+ stores. The resulting space-time profile of free Ca2+ determines the time course and probability of neurotransmitter release through the interaction with molecular release triggers strategically located in the vicinity of release sites. Following a rapid concentration transient, excess Ca2+ has to be removed from the cytosol through the process involving Ca2+ uptake by the endoplasmatic reticulum stores, sequestration by mitochondria, and/or extrusion into the extracellular medium. The ongoing synaptic activity could affect any of the multiple factors that shape presynaptic Ca2+ dynamics, thus arbitrating use-dependent modification of the neurotransmitter release probability. Here we present an overview of major players involved in Ca2+-dependent presynaptic regulation of neurotransmitter release and discuss the relationships arising between their actions. Copyright © 2006 Sage Publications.

TITLE:Calcium ions and integration in neural circuits 

[2-s2.0-33745048549] Refers [2-s2.0-12944314765] LEVEL: 3
Integration in the nervous system is achieved by signal processing within dynamic functional ensembles formed by highly complex neuronal-glial cellular circuits. The interactions between electrically excitable neuronal networks and electrically non-excitable glial syncytium occur through either chemical transmission, which involves the release of transmitters from presynaptic terminals or from astroglial cells, or via direct intercellular contacts, gap junctions. Calcium ions act as a universal intracellular signalling system, which controls many aspects of neuronal-glial communications. In neurones, calcium signalling events regulate the exocytosis of neurotransmitters and establish the link between excitation of postsynaptic cells and integrative intracellular events, which control synaptic strength, expression of genes and memory function. In glial cells metabotropic receptor mediated release of calcium ions from the intracellular endoplasmic reticulum calcium store provide specific form of glial excitability. Glial calcium signals ultimately result in vesicular secretion of 'glio' transmitters, which affect neuronal networks thus closing the glial-neuronal circuits. Cellular signalling through calcium ions therefore can be regarded as a molecular mechanism of integration in the nervous system. © 2006 Scandinavian Physiological Society.

TITLE:Depolarization of mitochondria in isolated CA1 neurons during hypoxia, glucose deprivation and glutamate excitotoxicity 

[2-s2.0-33644958401] Refers [2-s2.0-12944314765] LEVEL: 3
During cerebral ischemia neuronal injury is induced by a combination of hypoxia, hypoglycemia and glutamate excitotoxicity. To evaluate the relative importance of these factors on the mitochondrial function, acutely isolated rat hippocampal CA1 neurons were loaded with Rhodamine 123 to monitor the mitochondrial membrane potential (??m). During 15 min of hypoxia, a rapid and complete mitochondrial depolarization was observed in all neurons also when complex V of the respiratory chain was blocked by oligomycin. Glucose deprivation caused 77% of the neurons to loose the ?? m completely, whereas most oligomycin-treated neurons retained their ??m. During oxygen and glucose deprivation, a similar mitochondrial response was seen as in hypoxia. Although 15 min of high glutamate concentration (1 mM) provoked a rapid and irreversible increase in [Ca 2+]i, only 25% of the neurons lost the ??m. All oligomycin-treated neurons, however, lost the ??m during glutamate exposure. In conclusion, the mitochondrial function of acutely isolated CA1 neurons is more sensitive to hypoxia than to glucose deprivation and glutamate excitotoxicity. © 2005 Elsevier B.V. All rights reserved.

TITLE:Intracellular Ca2+ regulation in rat motoneurons during development 

[2-s2.0-31544461787] Refers [2-s2.0-12944314765] LEVEL: 3
Changes in intracellular Ca2+ concentration ([Ca2+]i) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca2+ stores, via both ryanodine-sensitive and IP3-sensitive intracellular Ca2+ channels control [Ca2+]i homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca2+]i increased progressively from days 1-7 in culture. These responses were blocked by ryanodine and nicardipine but not by ?-conotoxin-GVIA or ?-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Cav1 Ca2+ channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca2+ oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca2+ stores from the endoplasmic reticulum, induced an increase in [Ca2+]i in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5-6 days in vitro. Xestospongin-C, a membrane-permeable IP3 receptor inhibitor blocked the CPA-induced [Ca2+]i response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP3 Ca2+ channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca2+]i homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca2+ channel responsible for a Ca2+-induced Ca2+ release in embryonic motoneurons following voltage-dependent Ca2+ entry via L-type Ca2+ channels. © 2005 Elsevier Ltd. All rights reserved.

TITLE:Store calcium mediates cholinergic effects on mIPSCs in the rat main olfactory bulb 

[2-s2.0-33645976858] Refers [2-s2.0-12944314765] LEVEL: 3
The significance of endoplasmic reticulum (ER) store calcium in modulating transmitter release is slowly gaining recognition. One transmitter system that might play an important role in store calcium modulation of transmitter release in the CNS is acetylcholine (ACh). The main olfactory bulb (OB) receives rich cholinergic innervation from the horizontal limb of the diagonal band of Broca and blocking cholinergic signaling in the bulb inhibits the ability of animals to discriminate between closely related odors. Here we show that exposing OB slices to carbamylcholine (CCh), a hydrolysis-resistant analog of Ach, increases ?-aminobutyric acid (GABA) release at dendrodendritic synapses onto the mitral cells. This increase in transmitter release is mediated by the activation of the M1 class of muscarinic receptors and requires the mobilization of calcium from the ER. The site of action of CCh for this effect is developmentally regulated. In animals younger than postnatal day 10, the major action of CCh appears to be on mitral cells, enhancing GABA release by reciprocal signaling resulting from increased glutamate release from mitral cells. In animals older than postnatal day 10, CCh appears to modulate transmitter release from dendrites of the interneurons themselves. Our results point to modulation of inhibition as an important role for cholinergic signaling in the OB. Our data also strengthen the emerging idea of a role for store calcium in modulating transmitter release at CNS synapses. Copyright © 2006 The American Physiological Society.

TITLE:Calmodulin regulation and identification of calmodulin binding region of type-3 ryanodine receptor calcium release channel 

[2-s2.0-27744486301] Refers [2-s2.0-12944314765] LEVEL: 3
Ryanodine receptors (RyRs) are a family of intracellular Ca2+ channels that are regulated by calmodulin (CaM). At low Ca2+ concentrations (< 1 ?M), CaM activates RyR1 and RyR3 and inhibits RyR2. At elevated Ca2+ concentrations (> 1 ?M), CaM inhibits all three RyR isoforms. Here we report that the regulation of recombinant RyR3 by CaM is sensitive to redox regulation. RyR3 in the presence of reduced glutathione binds CaM with 10-15-fold higher affinity, at low and high Ca2+ concentrations, compared to in the presence of oxidized glutathione. However, compared to RyR1 assayed at low Ca2+ concentrations under both reducing and oxidizing conditions, CaM binds RyR3 with reduced affinity but activates RyR3 to a greater extent. Under reducing conditions, RyR1 and RyR3 activities are inhibited with a similar affinity at [Ca2+] > 1 ?M. Mutagenesis studies demonstrate that RyR3 contains a single conserved CaM binding site. Corresponding amino acid substitutions in the CaM binding site differentially affect CaM binding and CaM regulation of RyR3 and those of the two other isoforms. The results support the suggestion that other isoform dependent regions have a major role in the regulation of RyRs by CaM [Yamaguchi et al. (2004) J. Biol. Chem. 279, 36433-36439]. © 2005 American Chemical Society.

TITLE:Failure of calcium microdomain generation and pathological consequences 

[2-s2.0-33750496237] Refers [2-s2.0-12944314765] LEVEL: 3
Normal physiological regulation depends on Ca2+ microdomains, because there is a need to spatially separate Ca2+ regulation of different cellular processes. It is only possible to generate local Ca2+ signals transiently; so, there is an important functional link between Ca2+ spiking and microdomains. The pancreatic acinar cell provides a useful cell biological model, because of its clear structural and functional polarization. Although local Ca2+ spiking in the apical (granular) microdomain regulates fluid and enzyme secretion, prolonged global elevations of the cytosolic Ca2+ concentration are associated with the human disease acute pancreatitis, in which proteases in the granular region become inappropriately activated and digest the pancreas and its surroundings. A major cause of pancreatitis is alcohol abuse and it has now been established that fatty acid ethyl esters and fatty acids, non-oxidative alcohol metabolites, are principally responsible for causing the acinar cell damage. The fatty acid ethyl esters release Ca2+ from the endoplasmic reticulum and the fatty acids inhibit markedly mitochondrial ATP generation, which prevents the acinar cell from disposing of the excess Ca2+ in the cytosol. Because of the abolition of ATP-dependent Ca2+ pump activity, all intracellular Ca2+ concentration gradients disappear and the most important part of the normal regulatory machinery is thereby destroyed. The end stage is necrosis. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Mechanism of nitric oxide action on inhibitory GABAergic signaling within the nucleus tractus solitarii 

[2-s2.0-33845662659] Refers [2-s2.0-12944314765] LEVEL: 3
The cellular mechanisms mediating nitric oxide (NO) modulation of the inhibitory transmission in the nucleus tractus solitarii (NTS) remain unclear, even though this could be extremely important for various physiological and pathological processes. Specifically, in the NTS NO-evoked glutamate and ?-aminobutyric acid (GABA) release might contribute to pathological hypertension. In cultured rat brainstem slices, NTS GABAergic neurons were targeted using an adenoviral vector to express enhanced green fluorescent protein and studied with a combination of patch clamp and confocal microscopy. Low nanomolar concentrations of NO increased intracellular Ca2+ concentration ([Ca2+]i) in somata, dendrites, and putative axons of GABAergic neurons, with axons being the most sensitive compartment. This effect was cGMP mediated and not related to depolarization or indirect presynaptic effects on glutamatergic transmission. Blockade of the cyclic adenosine diphosphate ribose (cADPR)/ryanodine-sensitive stores but not the inositol triphosphate-sensitive stores, inhibited NO effect. Since cADPR/ryanodine-sensitive stores are implicated in the Ca2+-induced Ca2+ release, NO can be expected to potentiate GABA release. In support of this notion, a cADPR antagonist abolished the NO-induced potentiation of GABAergic inhibitory postsynaptic potentials in the NTS. Thus, the NO-cGMP-cADPR-Ca2+ pathway, previously described in sea urchin eggs, also operates in mammalian GABAergic neurons. Potentiation of GABA release by NO may have implications for numerous brain functions. © FASEB.

TITLE:GABAB heterodimeric receptors promote Ca2+ influx via store-operated channels in rat cortical neurons and transfected chinese hamster ovary cells 

[2-s2.0-31144441055] Refers [2-s2.0-12944314765] LEVEL: 3
The GABAB receptors are generally considered to be classical Gi-coupled receptors that lack the ability to mobilize intracellular Ca2+ without the aid of promiscuous G proteins. Here, we report the ability of GABAB receptors to promote calcium influx into primary cultures of rat cortical neurons and transfected Chinese hamster ovary cells. Chinese hamster ovary cells were transfected with GABAB1(a) or GABAB1(b) subunits along with GABAB2 subunits. In experiments using the fluorometric imaging plate reader platform, GABA and selective agonists promoted increases in intracellular Ca2+ levels in transfected Chinese hamster ovary cells and cortical neurons with the expected order of potency. These effects were fully antagonized by selective GABA B receptor antagonists. To investigate the intracellular pathways responsible for mediating these effects we employed several pharmacological inhibitors. Pertussis toxin abolished GABAB mediated Ca2+ increases, as did the phospholipase C? inhibitor U73122. Inhibitor 2-aminethoxydiphenyl borane acts as an antagonist at inositol 1,4,5-trisphosphate receptors and at store-operated channels. In all cell types, 2-aminethoxydiphenyl borane prevented Ca2+ mobilization. The selective store-operated channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4- methoxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride prevented increases in intracellular Ca2+ levels as did performing the assays in Ca 2+ free buffers. In conclusion, GABAB receptors expressed in Chinese hamster ovary cells and endogenously expressed in rat cortical neurons promote Ca2+ entry into the cell via the activation of store-operated channels, using a mechanism that is dependent on Gi/o heterotrimeric proteins and phospholipase C?. These findings suggest that the neuronal effects mediated by GABAB receptors may, in part, rely on the receptor's ability to promote Ca2+ influx. © 2005 Published by Elsevier Ltd on behalf of IBRO.

TITLE:Type-3 ryanodine receptor involved in Ca2+-induced Ca2+ release and transmitter exocytosis at frog motor nerve terminals 

[2-s2.0-27744540670] Refers [2-s2.0-12944314765] LEVEL: 3
Ca2+-induced Ca2+ release (CICR) occurs in frog motor nerve terminals after ryanodine receptors (RyRs) are primed for activation by conditioning large Ca2+ entry. We studied which type of RyR exists, whether CICR occurs without conditioning Ca2+ entry and how RyRs are primed. Immunohistochemistry revealed the existence of RyR3 in motor nerve terminals and axons and both RyR1 and RyR3 in muscle fibers. A blocker of RyR, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) slightly decreased rises in intracellular Ca2+ ([Ca2+]i) induced by a short tetanus (50 Hz, 1-2 s), but not after treatment with ryanodine. Repetitive tetani (50 Hz for 15 s every 20 s) produced repetitive rises in [Ca2+] i, whose amplitude overall waxed and waned. TMB-8 blocked the waxing and waning components. Ryanodine suppressed a slow increase in end-plate potentials (EPPs) induced by stimuli (33.3 Hz, 15 s) in a low Ca2+, high Mg2+ solution. KN-62, a blocker of Ca2+/calmoduline-activated protein kinase II (CaMKII), slightly reduced short tetanus-induced rises in [Ca2+] i, but markedly the slow waxing and waning rises produced by repetitive tetani in both normal and low Ca2+, high Mg2+ solutions. Likewise, KN-62, but not KN-04, an inactive analog, suppressed slow increases in EPP amplitude and miniature EPP frequency during long tetanus. Thus, CICR normally occurs weakly via RyR3 activation by single impulse-induced Ca2+ entry in frog motor nerve terminals and greatly after the priming of RyR via CaMKII activation by conditioning Ca2+ entry, thus, facilitating transmitter exocytosis and its plasticity. © 2005 Elsevier Ltd. All rights reserved.

TITLE:Cellular abnormalities linked to endoplasmic reticulum dysfunction in cerebrovascular disease-therapeutic potential 

[2-s2.0-28144448251] Refers [2-s2.0-12944314765] LEVEL: 3
Unfolded proteins accumulate in the lumen of the endoplasmic reticulum (ER) as part of the cellular response to cerebral hypoxia/ischemia and also to the overexpression of the mutant genes responsible for familial forms of degenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyothrophic lateral sclerosis, and Huntington's disease, as well as other disorders that are caused by an expanded CAG repeat. This accumulation arises from an imbalance between the load of proteins that need to be folded and processed in the ER lumen and the ER folding/processing capacity. To withstand such potentially lethal conditions, stress responses are activated that includes the shutdown of translation to reduce the ER work load and the activation of the expression of genes coding for proteins involved in the folding and processing reactions, to increase folding/processing capacity. In transient cerebral ischemia, ER stress-induced suppression of protein synthesis is believed to be too severe to permit sufficient activation of the genetic arm of the ER stress response. Mutations associated with Alzheimer's disease down-regulate the ER stress response and make cells more vulnerable to conditions associated with ER stress. When the functioning of the ER is severely impaired and affected cells can no longer withstand these stressful conditions, programmed cell death is induced, including a mitochondria-driven apoptotic pathway. Raising the resistance of cells to conditions that interfere with ER functions and activating the degradation and refolding of unfolded proteins accumulated in the ER lumen are possible strategies for blocking the pathological process leading to cell death at an early stage. © 2005 Elsevier Inc. All rights reserved.

TITLE:Excitotoxic motoneuron disease in chick embryo evolves with autophagic neurodegeneration and deregulation of neuromuscular innervation 

[2-s2.0-34548088683] Refers [2-s2.0-12944314765] LEVEL: 3
In the chick embryo, in ovo application of NMDA from embryonic day (E) 5 to E9 results in selective damage to spinal cord motoneurons (MNs) that undergo a long-lasting degenerative process without immediate cell death. This contrasts with a single application of NMDA on E8, or later, which induces massive necrosis of the whole spinal cord. Chronic MN degeneration after NMDA implies transient incompetence to develop programmed cell death, altered protein processing within secretory pathways, and late activation of autophagy. Chronic NMDA treatment also results in an enlargement of thapsigargin-sensitive Ca 2+ stores. In particular MN pools, such as sartorius-innervating MNs, the neuropeptide CGRP is accumulated in somas, peripheral axons and neuromuscular junctions after chronic NMDA treatment, but not in embryos paralyzed by chronic administration of curare. Intramuscular axonal branching is also altered severely after NMDA: it usually increases, but in some cases a marked reduction can also be observed. Moreover, innervated muscle postsynaptic sites increase by NMDA, but to a lesser extent than by curare. Because some of these results show interesting homologies with MN pathology in human sporadic ALS, the model presented here provides a valuable tool for advancing in the understanding of some cellular and molecular processes particularly involved in this disease. © 2007 Wiley-Liss, Inc.

TITLE:The importance of being subtle: Small changes in calcium homeostasis control cognitive decline in normal aging 

[2-s2.0-34248997850] Refers [2-s2.0-12944314765] LEVEL: 3
Aging is a complex, multifactorial process. One of the features of normal aging of the brain is a decline in cognitive functions and much experimental attention has been devoted to understanding this process. Evidence accumulated in the last decade indicates that such functional changes are not due to gross morphological alterations, but to subtle functional modification of synaptic connectivity and intracellular signalling and metabolism. Such synaptic modifications are compatible with a normal level of activity and allow the maintenance of a certain degree of functional reserve. This is in contrast to the changes in various neurodegenerative diseases, characterized by significant neuronal loss and dramatic and irreversible functional deficit. This whole special issue has been initiated with the intention of focusing on the processes of normal brain aging. In this review, we present data that shows how subtle changes in Ca2+ homeostasis or in the state of various Ca 2+-dependent processes or molecules, which occur in aging can have significant functional consequences. © 2007 The Authors Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007.

TITLE:Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites 

[2-s2.0-34247882821] Refers [2-s2.0-12944314765] LEVEL: 3
The free [Ca2+] in endoplasmic/sarcoplasmic reticulum Ca 2+ stores regulates excitability of Ca2+ release by stimulating the Ca2+ release channels. Just how the stored Ca 2+ regulates activation of these channels is still disputed. One proposal attributes luminal Ca2+-activation to luminal facing regulatory sites, whereas another envisages Ca2+ permeation to cytoplasmic sites. This study develops a unified model for luminal Ca 2+ activation for single cardiac ryanodine receptors (RyR 2) and RyRs in coupled clusters in artificial lipid bilayers. It is shown that luminal regulation of RyR2 involves three modes of action associated with Ca2+ sensors in different parts of the molecule; a luminal activation site (L-site, 60 ?M affinity), a cytoplasmic activation site (A-site, 0.9 ?M affinity), and a novel cytoplasmic inactivation site (I2-site, 1.2 ?M affinity). RyR activation by luminal Ca 2+ is demonstrated to occur by a multistep process dubbed luminal-triggered Ca2+ feedthrough. Ca2+ binding to the L-site initiates brief openings (1 ms duration at 1-10 s-1) allowing luminal Ca2+ to access the A-site, producing up to 30-fold prolongation of openings. The model explains a broad data set, reconciles previous conflicting observations and provides a foundation for understanding the action of pharmacological agents, RyR-associated proteins, and RyR 2 mutations on a range of Ca2+-mediated physiological and pathological processes. © 2007 by the Biophysical Society.

TITLE:Regulation of synaptic input to hypothalamic presympathetic neurons by GABAB receptors 

[2-s2.0-33748801278] Refers [2-s2.0-12944314765] LEVEL: 3
The hypothalamic paraventricular (PVN) neurons projecting to the spinal cord and brainstem play an important role in the control of homeostasis and the sympathetic nervous system. Although GABAB receptors are present in the PVN, their function in the control of synaptic inputs to PVN presympathetic neurons is not clear. Using retrograde tracing and whole-cell patch-clamp recordings in rat brain slices, we determined the role of presynaptic GABAB receptors in regulation of glutamatergic and GABAergic inputs to spinally projecting PVN neurons. The GABAB receptor agonist baclofen (1-50 ?M) dose-dependently decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs). The effect of baclofen on sEPSCs and sIPSCs was completely blocked by 10 ?M CGP52432, a selective GABAB receptor antagonist. Baclofen also significantly reduced the frequency of both miniature excitatory and miniature inhibitory postsynaptic currents (mEPSCs and mIPSCs). Furthermore, uncoupling pertussis toxin-sensitive Gi/o proteins with N-ethylmaleimide abolished baclofen-induced inhibition of mEPSCs and mIPSCs. However, the inhibitory effect of baclofen on the frequency of mIPSCs and mEPSCs persisted in the presence of either Cd2+, a voltage-gated Ca2+ channel blocker, or 4-aminopyridine, a blocker of voltage-gated K+ channels. Our results suggest that activation of presynaptic GABAB receptors inhibits synaptic GABA and glutamate release to PVN presympathetic neurons. This presynaptic action of GABAB receptors is mediated by the N-ethylmaleimide-sensitive Gi/o proteins, but independent of voltage-gated Ca2+ and K+ channels. © 2006 IBRO.

TITLE:Main determinants of presynaptic Ca2+ dynamics at individual mossy fiber-CA3 pyramidal cell synapses 

[2-s2.0-33745932961] Refers [2-s2.0-12944314765] LEVEL: 3
Synaptic transmission between hippocampal mossy fibers (MFs) and CA3 pyramidal cells exhibits remarkable use-dependent plasticity. The underlying presynaptic mechanisms, however, remain poorly understood. Here, we have used fluorescent Ca2+ indicators Fluo-4, Fluo-5F, and Oregon Green BAPTA-1 to investigate Ca2+ dynamics in individual giant MF boutons (MFBs) in area CA3 traced from the somata of granule cells held in whole-cell mode. In an individual MFB, a single action potential induces a brief peak of free Ca2+ (estimated in the range of 8-9 ?M) followed by an elevation to ?320 nM, which slowly decays to its resting level of ?110 nM. Changes in the somatic membrane potential influence presynaptic Ca2+ entry at proximal MFBs in the hilus. This influence decays with distance along the axon, with a length constant of ?200 ?m. In giant MFBs in CA3, progressive saturation of endogenous Ca2+ buffers during repetitive spiking amplifies rapid Ca2+ peaks and the residual Ca2+ severalfold, suggesting a causal link to synaptic facilitation. We find that internal Ca2+ stores contribute to maintaining the low resting Ca2+ providing ?22% of the buffering/extrusion capacity of giant MFBs. Rapid Ca2+ release from stores represents up to 20% of the presynaptic Ca2+ transient evoked by a brief train of action potentials. The results identify the main components of presynaptic Ca 2+ dynamics at this important cortical synapse. Copyright © 2006 Society for Neuroscience.

TITLE:Glial calcium signaling in physiology and pathophysiology 

[2-s2.0-33748544273] Refers [2-s2.0-12944314765] LEVEL: 3
Neuronal-glial circuits underlie integrative processes in the nervous system. Function of glial syncytium is, to a very large extent, regulated by the intracellular calcium signaling system. Glial calcium signals are triggered by activation of multiple receptors, expressed in glial membrane, which regulate both Ca2+ entry and Ca2+ release from the endoplasmic reticulum. The endoplasmic reticulum also endows glial cells with intracellular excitable media, which is able to produce and maintain long-ranging signaling in a form of propagating Ca2+ waves. In pathological conditions, calcium signals regulate glial response to injury, which might have both protective and detrimental effects on the nervous tissue. ©2006 CPS and SIMM.

TITLE:Ryanodine receptor interaction with the SNARE-associated protein snapin 

[2-s2.0-33745480157] Refers [2-s2.0-12944314765] LEVEL: 3
The ryanodine receptor (RyR) is a widely expressed intracellular calcium (Ca2+)-release channel regulating processes such as muscle contraction and neurotransmission. Snapin, a ubiquitously expressed SNARE-associated protein, has been implicated in neurotransmission. Here, we report the identification of snapin as a novel RyR2-interacting protein. Snapin binds to a 170-residue predicted ryanodine receptor cytosolic loop (RyR2 residues 4596-4765), containing a hydrophobic segment required for snapin interaction. Ryanodine receptor binding of snapin is not isoform specific and is conserved in homologous RyR1 and RyR3 fragments. Consistent with peptide fragment studies, snapin interacts with the native ryanodine receptor from skeletal muscle, heart and brain. The snapin-RyR1 association appears to sensitise the channel to Ca2+ activation in [3H]ryanodine-binding studies. Deletion analysis indicates that the ryanodine receptor interacts with the snapin C-terminus, the same region as the SNAP25-binding site. Competition experiments with native ryanodine receptor and SNAP25 suggest that these two proteins share an overlapping binding site on snapin. Thus, regulation of the association between ryanodine receptor and snapin might constitute part of the elusive molecular mechanism by which ryanodine-sensitive Ca2+ stores modulate neurosecretion.

TITLE:Biophysical re-equilibration of Ca2+ fluxes as a simple biologically plausible explanation for complex intracellular Ca2+ release patterns 

[2-s2.0-30644464804] Refers [2-s2.0-12944314765] LEVEL: 3
Physiological regulation of Ca2+ release from the endoplasmic reticulum (ER) is critical for cell function. Recent direct measurements of free [Ca2+] inside the ER ([Ca2+]ER) revealed that [Ca2+]ER itself is a key regulator of ER Ca2+ handling. However, the role of this new regulatory process in generating various patterns of Ca2+ release remains to be elucidated in detail. Here, we incorporate the recently quantified experimental correlations between [Ca2+]ER and Ca2+ movements across the ER membrane into a mathematical model ER Ca2+ handling. The model reproduces basic experimental dynamics of [Ca2+]ER. Although this was not goal in model design, the model also exhibits mechanistically unclear experimental phenomena such as "quantal" Ca2+ release, and "store charging" by increasing resting cytosolic [Ca2+]. While more complex explanations cannot be ruled out, on the basis of our data we propose that "quantal release" and "store charging" could be simple re-equilibration phenomena, predicted by the recently quantified biophysical dynamics of Ca2+ movements across the ER membrane. © 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

TITLE:Serca isoform expression in the mammalian retina 

[2-s2.0-29044450013] Refers [2-s2.0-12944314765] LEVEL: 3
The sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) is a key intracellular calcium transporter, which regulates cellular calcium concentration [Ca2+] by transporting Ca2+ ions from the cytosol into the endoplasmic reticulum. SERCA-mediated Ca2+ sequestration controls proper folding of newly synthesized proteins within the ER as well as the timing and spatial patterning of depolarization-evoked Ca 2+ responses in the cytoplasm. To understand the spatial and temporal properties of Ca2+ homeostasis in retinal neurons better, I studied expression and distribution of all three SERCA isoforms in the mouse retina using isoform-specific antibodies. No immunostaining was observed with the SERCA1 antibody. SERCA2 was expressed in photoreceptor inner segments, amacrine and ganglion cells of the mouse retina. Similar SERCA2 localization was observed in adult rat, macaque and ground squirrel retinas. Analysis of distribution of SERCA2 immunofluorescence in the developing mouse retina revealed prominent SERCA2 signals throughout postnatal development. The N89 antibodys used to identify the SERCA3 isoforms labelled cone outer segments, inner segments of photoreceptors and cell processes in the inner nuclear layer of the mouse retina. These results imply that the SERCA2 isoform controls Ca2+ sequestration into the endoplasmic reticulum in most classes of retinal neuron. A potential role for SERCA3 in cone function is suggested. © 2005 Elsevier Ltd. All rights reserved.

TITLE:Calcium in Alzheimer's disease pathogenesis: Too much, too little or in the wrong place? 

[2-s2.0-32444438356] Refers [2-s2.0-12944314765] LEVEL: 3
Our understanding of the molecular genetics and biochemical pathology of Alzheimer's disease has progressed tremendously in the past decade. The metabolism of amyloid ?-peptide is being unraveled, and specific anti-amyloid therapies are now in clinical trials worldwide. The precise biophysical structure of the amyloid ?-peptide that causes neuronal dysfunction remains under investigation, as does the interaction between amyloid peptides and tau hyperphosphorylation, but these two molecules likely play key roles in neuronal dysfunction in Alzheimer's disease. Despite these advances, the cell biology of neuronal dysfunction and cell death in the Alzheimer's disease brain remains poorly understood. This brief review will explore the role of calcium (Ca2+) in neuronal death occurring during Alzheimer's disease. The evidence for glutamate receptor-mediated Ca2+ overload, or excitotoxicity, and other derangements of Ca2+ homeostasis in cell culture and animal models of Alzheimer's disease is reviewed. Finally, we raise the possibility that some of the neuronal death observed in Alzheimer's disease might be associated with a reduction in rather than an increase in cytosolic Ca2+ levels, an idea with potentially important therapeutic implications. © 2005 - IOS Press and the authors. All rights reserved.

TITLE:Release and sequestration of Ca2+ by a caffeine- and ryanodine-sensitive store in a sub-population of human SH-SY5Y neuroblastoma cells 

[2-s2.0-23844522066] Refers [2-s2.0-12944314765] LEVEL: 3
We have used single cell fluorescence imaging techniques to examine the role that ryanodine receptors play in the stimulus-induced Ca2+ responses of SH-SY5Y cells. The muscarinic agonist methacholine (1 mM) resulted in a Ca2+ signal in 95% of all cells. Caffeine (30 mM) however stimulated a Ca2+ signal in only 1-7% of N-type (neuroblastic) cells within any given field. The caffeine response was independent of extracellular Ca2+, regenerative in nature, and abolished in a use-dependent fashion by ryanodine. In caffeine-responsive cells, the magnitude of the methacholine-induced Ca2+ signal was inhibited by 75.07 ± 5.51% by pretreatment with caffeine and ryanodine, suggesting that the caffeine-sensitive store may act as a Ca2+ source after muscarinic stimulation. When these data were combined with equivalent data from non-caffeine-responsive cells, the degree of apparent inhibition was significantly reduced. In contrast, after store depletion by caffeine, the Ca2+ signal induced by 55 mM K+ was potentiated 2.5-fold in the presence of ryanodine, suggesting that the store may act a Ca2+ sink after depolarisation. We conclude that a caffeine- and ryanodine-sensitive store can act as a Ca2+ source and sink in SH-SY5Y cells, and that effects of the store can become obscured if data from caffeine-insensitive cells are not excluded. © 2005 2005 Elsevier Ltd. All rights reserved.

TITLE:Aryl-indolyl maleimides as inhibitors of CaMKIIĪ“. Part 1: SAR of the aryl region 

[2-s2.0-41149166383] Refers [2-s2.0-12944314765] LEVEL: 3
A family of aryl-substituted maleimides was prepared and studied for their activity against calmodulin dependant kinase. Inhibitory activities against the enzyme ranged from 34 nM to >20 ?M and were dependant upon both the nature of the aryl group and the hydrogen bond donating potential of the maleimide ring. Key interactions with the kinase ATP site and hinge region were found to be consistent with homology modeling predictions. © 2008 Elsevier Ltd. All rights reserved.

TITLE:Ca2+ signal summation and NFATc1 nuclear translocation in sympathetic ganglion neurons during repetitive action potentials 

[2-s2.0-34247396628] Refers [2-s2.0-12944314765] LEVEL: 3
NFATc-mediated gene expression constitutes a critical step during neuronal development and synaptic plasticity. Although considerable information is available regarding the activation and functionality of specific NFATc isoforms, in neurons little is known about how sensitive NFAT nuclear translocation is to specific patterns of electrical activity. Here we used high-speed fluo-4 confocal imaging to monitor action potential (AP)-induced cytosolic Ca2+ transients in rat sympathetic neurons. We have recorded phasic and repetitive AP patterns, and corresponding Ca2+ transients initiated by either long (100-800 ms) current-clamp pulses, or single brief (2 ms) electrical field stimulation. We address the functional consequences of these AP and Ca2+ transient patterns, by using an adenoviral construct to express NFATc1-CFP and evaluate NFATc1-CFP nuclear translocation in response to specific patterns of electrical activity. Ten Hertz trains stimulation induced nuclear translocation of NFATc1, whereas 1 Hz trains did not. However, 1 Hz train stimulation did result in NFATc1 translocation in the presence of 2 mM Ba2+, which inhibits M-currents and promotes repetitive firing and the accompanying small (?0.6 ?F/F0) repetitive and summating Ca2+ transients. Our results demonstrate that M-current inhibition-mediated spike frequency facilitation enhances cytosolic Ca2+ signals and NFATc1 nuclear translocation during trains of low frequency electrical stimulation. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Mpp4 is required for proper localization of plasma membrane calcium ATPases and maintenance of calcium homeostasis at the rod photoreceptor synaptic terminals 

[2-s2.0-34447535286] Refers [2-s2.0-12944314765] LEVEL: 3
Membrane palmitoylated protein 4 (Mpp4) is a member of the membrane-associated guanylate kinase family. We show that Mpp4 localizes specifically to the plasma membrane of photoreceptor synaptic terminals. Plasma membrane Ca2+ ATPases (PMCAs), the Ca2+ extrusion pumps, interact with an Mpp4-dependent presynaptic membrane protein complex that includes Veli3 and PSD95. In mice lacking Mpp4, PMCAs were lost from rod photoreceptor presynaptic membranes. Synaptic ribbons were enlarged, a phenomenon known to correlate with higher Ca2+. SERCA2 (sarcoplasmic-endoplasmic reticulum Ca2+ ATPase, type 2), which pumps cytosolic Ca2+ into intracellular Ca2+ stores and localizes next to the ribbons, was increased. The distribution of IP3RII (InsP3 receptor, type 2), which releases Ca2+ from the stores, was shifted away from the synaptic terminals. Synaptic transmission to second-order neurons was maintained but was reduced in amplitude. These data suggest that loss of Mpp4 disrupts a Ca2+ extrusion mechanism at the presynaptic membranes, with ensuing adaptive responses by the photoreceptor to restore Ca2+ homeostasis. We propose that Mpp4 organizes a presynaptic protein complex that includes PMCAs and has a role in modulating Ca2+ homeostasis and synaptic transmission in rod photoreceptors. © The Author 2007. Published by Oxford University Press. All rights reserved.

TITLE:The amyloid precursor protein potentiates CHOP induction and cell death in response to ER Ca2+ depletion 

[2-s2.0-33846279033] Refers [2-s2.0-12944314765] LEVEL: 3
Here we investigated the role of the amyloid precursor protein (APP) in regulation of Ca2+ store depletion-induced neural cell death. Ca2+ store depletion from the endoplasmic reticulum (ER) was induced by the SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase) inhibitor thapsigargin which led to a rapid induction of the unfolded protein response (UPR) and a delayed activation of executioner caspases in the cultures. Overexpression of APP potently enhanced cytosolic Ca2+ levels and cell death after ER Ca2+ store depletion in comparison to vector-transfected controls. GeneChipR and RT-PCR analysis revealed that the expression of classical UPR chaperone genes was not altered by overexpression of APP. Interestingly, the induction of the ER stress-responsive pro-apoptotic transcription factor CHOP was significantly upregulated in APP-overexpressing cells in comparison to vector-transfected controls. Chelation of intracellular Ca2+ with BAPTA-AM revealed that enhanced CHOP expression after store depletion occurred in a Ca2+-dependent manner in APP-overexpressing cells. Prevention of CHOP induction by BAPTA-AM and by RNA interference was also able to abrogate the potentiating effect of APP on thapsigargin-induced apoptosis. Application of the store-operated channel (SOC)-inhibitors SK & F96365 and 2-APB downmodulated APP-triggered potentiation of cytosolic Ca2+ levels and apoptosis after treatment with thapsigargin. Our data demonstrate that APP significantly modulates Ca2+ store depletion-induced cell death in a SOC- and CHOP-dependent manner, but independent of the UPR. © 2006 Elsevier B.V. All rights reserved.

TITLE:Cell type-specific expression of a genetically encoded calcium indicator reveals intrinsic calcium oscillations in adult gonadotropin-releasing hormone neurons 

[2-s2.0-33846637395] Refers [2-s2.0-12944314765] LEVEL: 3
The gonadotropin-releasing hormone (GnRH) neurons exhibit a unique pattern of episodic activity to control fertility in all mammals. To enable the measurement of intracellular calcium concentration ([Ca2+] i) in adult GnRH neurons in situ, we generated transgenic mice in which the genetically encodable calcium indicator ratiometric Pericam was expressed by ?95% of GnRH neurons. Real-time monitoring of [Ca 2+]i within adult male GnRH neurons in the acute brain slice revealed that ?70% of GnRH neurons exhibited spontaneous, 10-15 s duration [Ca2+]i transients with a mean frequency of 7 per hour. The remaining 30% of GnRH neurons did not exhibit calcium transients nor did a population of non-GnRH cells located within the lateral septum that express Pericam. Pharmacological studies using antagonists to the inositol-1,4,5-trisphosphate receptor (InsP3R) and several calcium channels, demonstrated that [Ca2+]i transients in GnRH neurons were generated by an InsP3R-dependent store-release mechanism and were independent of plasma membrane ligand- or voltage-gated calcium channels. Interestingly, the abolition of action potential-mediated transmission with tetrodotoxin reduced the number of [Ca2+]i transients in GnRH neurons by 50% (p < 0.05), suggesting a modulatory role for synaptic inputs on [Ca2+]i transient frequency. Using a novel transgenic strategy that enables [Ca2+]i to be examined in a specific neuronal phenotype in situ, we provide evidence for spontaneous [Ca2+]i fluctuations in adult GnRH neurons. This represents the initial description of spontaneous [Ca2+]i transients in mature neurons and shows that they arise from an InsP 3R-generating mechanism that is further modulated by synaptic inputs. Copyright © 2007 Society for Neuroscience.

TITLE:Spatially confined diffusion of calcium in dendrites of hippocampal neurons revealed by flash photolysis of caged calcium 

[2-s2.0-33750519045] Refers [2-s2.0-12944314765] LEVEL: 3
The extent of diffusion of a locally evoked calcium surge in dendrites of cultured hippocampal neurons was studied by flash photolysis of caged EGTA. Cells were transfected with pDsRed for visualization, preincubated with caged NP-EGTA (AM) and Fluo-4 (AM) at room temperature and imaged in a PASCAL confocal microscope. Pulses of UV laser light within an active sphere of about 1 ?m2 produced a rise of Fluo-4 fluorescence transients in dendrites which peaked at 1 ms and decayed exponentially with a fast (8-10 ms) time constant. A slower decay component was uncovered following incubation with thapsigargin. Lateral diffusion of [Ca2+]i did not vary significantly among different size dendrites being symmetric and reaching about 3-3.5 ?m at a diffusion rate of 0.8 ?m/ms on both sides of the photolysis center. Fluo-4 was also replaced by the membrane-bound Fluo-NOMO (AM) or by the 'heavy' Calcium Green dextran (CGd) loaded through a patch pipette. Similar rates of diffusion were found in these cases, indicating that the diffusion is not of the dye complexed to calcium but of genuine free calcium ions. Interestingly, presence of a dendritic spine at the focus of photolysis significantly reduced [Ca2+]i spread while the focal transient remained unaffected. Finally, [Ca2+]i diffused about twice as far from the photolysis sphere in glass tubes of a similar diameter to that of a dendrite, indicating that intrinsic calcium uptake mechanisms in the dendrite determine the diffusion of calcium away from its original site of rise. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Sarco/endoplasmic reticulum Ca2+ATPase type 3 isoforms (SERCA3b and SERCA3f): Distinct roles in cell adhesion and ER stress 

[2-s2.0-33744551667] Refers [2-s2.0-12944314765] LEVEL: 3
Sarco/endoplasmic reticulum Ca2+ATPases (SERCAs) pump free Ca2+ from the cytosol into the endoplasmic reticulum. The human SERCA3 family counts six members named SERCA3a to 3f. However, the exact role of these different isoforms in cellular physiology remains undetermined. In this study, we compared some physiological consequences of SERCA3b and SERCA3f overexpression in HEK-293 cells. We observed that overexpression of SERCA3b affected cell adhesion capacity associated with a major disorganization of F-actin and a decrease in focal adhesion. Furthermore, we found that SERCA3f overexpression resulted in an increase in endoplasmic reticulum stress markers (including processing of X-box-binding protein-1 (XBP-1) mRNA and expression of chaperone glucose-regulated protein 78 (GRP78)). This was associated with the activation of caspase cascade and a higher spontaneous cell death. In conclusion, these data point for the first time to distinct physiological roles of SERCA3 isoforms in cell functions. © 2006 Elsevier Inc. All rights reserved.

TITLE:Aryl-indolyl maleimides as inhibitors of CaMKIIĪ“. Part 2: SAR of the amine tether 

[2-s2.0-41149142050] Refers [2-s2.0-12944314765] LEVEL: 3
A family of aryl-substituted maleimides was prepared and studied for their activity against calmodulin-dependant kinase. Inhibitory activities against the enzyme ranged from 34 nM to >20 ?M and were dependant upon both the nature of the aryl group and the tether joining the basic amine to the indolyl maleimide core. Key interactions with the kinase ATP site and hinge region, predicted by homology modeling, were confirmed. © 2008 Elsevier Ltd. All rights reserved.

TITLE:Evolution of calcium homeostasis: From birth of the first cell to an omnipresent signalling system 

[2-s2.0-34547941644] Refers [2-s2.0-12944314765] LEVEL: 3
Specific Ca2+ homeostatic system appeared very early in the history of the cell, as a survival system preventing Ca2+-mediated cell damage. This homeostatic system produced a steep (?20,000 times) concentration gradient between extracellular and intracellular compartments, which has both survival importance (even relatively short increases in cytosolic Ca2+ concentrations higher then 100 nM are incompatible with life) and signalling function. Evolution utilised this gradient together with an ability of Ca2+ to interact with many biological molecules to create the most widespread and versatile signalling system, controlling the majority of cellular processes and executing complex routines of intercellular communications. © 2007 Elsevier Ltd. All rights reserved.

TITLE:Fast regulation of axonal growth cone motility by electrical activity 

[2-s2.0-34447624504] Refers [2-s2.0-12944314765] LEVEL: 3
Axonal growth cones are responsible for the correct guidance of developing axons and the establishment of functional neural networks. They are highly motile because of fast and continuous rearrangements of their actin-rich cytoskeleton. Here we have used live imaging of axonal growth cones of hippocampal neurons in culture and quantified their motility with a temporal resolution of 2 s. Using novel methods of analysis of growth cone dynamics, we show that transient activation of kainate receptors by bath-applied kainate induced a fast and reversible growth cone stalling. This effect depends on electrical activity and can be mimicked by the transient discharge of action potentials elicited in the neuron by intracellular current injections at the somatic level through a patch pipette. Growth cone stalling induced by electrical stimulation is mediated by calcium entry from the extracellular medium as well as by calcium release from intracellular stores that define spatially restricted microdomains directly affecting cytoskeletal dynamics. We propose that growth cone motility is dynamically controlled by transient bursts of spontaneous electrical activity, which constitutes a prominent feature of developing neural networks in vivo. Copyright © 2007 Society for Neuroscience.

TITLE:Glutamate-mediated neuronal-glial transmission 

[2-s2.0-34249776663] Refers [2-s2.0-12944314765] LEVEL: 3
The brain is the most complex organ of the human body. It is composed of several highly specialized and heterogeneous populations of cells, represented by neurones (e.g. motoneurons, projection neurons or interneurons), and glia represented by astrocytes, oligodendrocytes and microglia. In recent years there have been numerous studies demonstrating close bidirectional communication of neurons and glia at structural and functional levels. In particular, the excitatory transmitter glutamate has been shown to evoke a variety of responses in astrocytes and oligodendrocytes in the healthy as well as the diseased brain. Here we overview the multitude of glutamate sensing molecules expressed in glia and describe some general experiments which have been performed to identify the glutamate-responsive molecules, i.e. the ionotropic and metabotropic glutamate receptors as well as the glutamate transporters. We also discuss a transgenic mouse model that permits detailed and specific investigations of the role of glial glutamate receptors. © 2007 The Authors Journal compilation © 2007 Anatomical Society of Great Britain and Ireland.

TITLE:Caenorhabditis elegans as a model system for Parkinson's disease 

[2-s2.0-34347221219] Refers [2-s2.0-12944314765] LEVEL: 3
Parkinson's disease (PD) is one of the most common age-related neurodegenerative diseases that is characterized by selective loss of dopaminergic neurons. Despite recent findings from mammalian model systems, molecular mechanisms of the pathophysiology are poorly understood. Given the high conservation of molecular pathways from invertebrates to mammalians, combined with technical advantages, such as high-throughput approaches, Caenorhabditis elegans represents a powerful system for the identification of factors involved in neurodegeneration. In this review we describe that C. elegans can be used to advance our understanding of the genetic mechanisms implicated in these disorders. Copyright © 2007 S. Karger AG.

TITLE:Integrin-binding RGD peptides induce rapid intracellular calcium increases and MAPK signaling in cortical neurons 

[2-s2.0-33846370905] Refers [2-s2.0-12944314765] LEVEL: 3
Integrins mediate cell adhesion to the extracellular matrix and initiate intracellular signaling. They play key roles in the central nervous system (CNS), participating in synaptogenesis, synaptic transmission and memory formation, but their precise mechanism of action remains unknown. Here we show that the integrin ligand-mimetic peptide GRGDSP induced NMDA receptor-dependent increases in intracellular calcium levels within seconds of presentation to primary cortical neurons. These were followed by transient activation and nuclear translocation of the ERK1/2 mitogen-activated protein kinase. RGD-induced effects were reduced by the NMDA receptor antagonist MK801, and ERK1/2 signaling was specifically inhibited by ifenprodil and PP2, indicating a functional connection between integrins, Src and NR2B-containing NMDA receptors. GRGDSP peptides were not significantly neuroprotective against excitotoxic insults. These results demonstrate a previously undescribed, extremely rapid effect of RGD peptide binding to integrins on cortical neurons that implies a close, functionally relevant connection between adhesion receptors and synaptic transmission. © 2006 Elsevier Inc. All rights reserved.

TITLE:A store-operated Ca2+ influx pathway in the bag cell neurons of Aplysia 

[2-s2.0-33751218000] Refers [2-s2.0-12944314765] LEVEL: 3
Although store-operated Ca2+ influx has been well-studied in nonneuronal cells, an understanding of its nature in neurons remains poor. In the bag cell neurons of Aplysia californica, prior work has suggested that a Ca2+ entry pathway can be activated by Ca2+ store depletion. Using fura-based imaging of intracellular Ca2+ in cultured bag cell neurons, we now characterize this pathway as store-operated Ca 2+ influx. In the absence of extracellular Ca2+, the endoplasmic reticulum Ca2+-ATPase inhibitors, cyclopiazonic acid (CPA) or thapsigargin, depleted intracellular stores and elevated intracellular free Ca2+. With the subsequent addition of extracellular Ca 2+, a prominent Ca2+ influx was observed. The ryanodine receptor agonist, chloroethylphenol (CEP), also increased intracellular Ca 2+ but did not initiate store-operated Ca2+ influx, despite overlap between CEP- and CPA-sensitive stores. Bafilomycin A, a vesicular H+-ATPase inhibitor, liberated intracellular Ca 2+ from acidic stores and attenuated subsequent Ca2+ influx, presumably by replenishing CPA-depleted stores. Store-operated Ca 2+ influx was partially blocked by low concentrations of La 3+ or BTP2, and strongly inhibited by either 1-[b-[3-(4- methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365) or a high concentration of Ni2+. Regarding IP3 receptor blockers, 2-aminoethyldiphenyl borate, but not xestospongin C, prevented store-operated Ca2+ influx. However, jasplakinolide, an actin stabilizer reported to inhibit this pathway in smooth muscle cell lines, was ineffective. The bag cell neurons initiate reproductive behavior through a prolonged afterdischarge associated with intracellular Ca2+ release and neuropeptide secretion. Store-operated Ca2+ influx may serve to replenish stores depleted during the afterdischarge or participate in the release of peptide that triggers behavior. Copyright © 2006 The American Physiological Society.

TITLE:Modulation of Gq-protein-coupled inositol trisphosphate and Ca2+ signaling by the membrane potential 

[2-s2.0-33749175607] Refers [2-s2.0-12944314765] LEVEL: 3
Gq-protein-coupled receptors (GqPCRs) are widely distributed in the CNS and play fundamental roles in a variety of neuronal processes. Their activation results in phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and Ca2+ release from intracellular stores via the phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3) signaling pathway. Because early GqPCR signaling events occur at the plasma membrane of neurons, they might be influenced by changes in membrane potential. In this study, we use combined patch-clamp and imaging methods to investigate whether membrane potential changes can modulate GqPCR signaling in neurons. Our results demonstrate that GqPCR signaling in the human neuronal cell line SH-SY5Y and in rat cerebellar granule neurons is directly sensitive to changes in membrane potential, even in the absence of extracellular Ca2+. Depolarization has a bidirectional effect on GqPCR signaling, potentiating thapsigargin-sensitive Ca2+ responses to muscarinic receptor activation but attenuating those mediated by bradykinin receptors. The depolarization-evoked potentiation of the muscarinic signaling is graded, bipolar, non-inactivating, and with no apparent upper limit, ruling out traditional voltage-gated ion channels as the primary voltage sensors. Flash photolysis of caged IP3/GPIP2 (glycerophosphoryl-myo- inositol 4,5-bisphosphate) places the voltage sensor before the level of the Ca2+ store, and measurements using the fluorescent bioprobe eGFP-PHPLC? (enhanced green fluorescent protein-pleckstrin homology domain-PLC?) directly demonstrate that voltage affects muscarinic signaling at the level of the IP3 production pathway. The sensitivity of GqPCR IP3 signaling in neurons to voltage itself may represent a fundamental mechanism by which ionotropic signals can shape metabotropic receptor activity in neurons and influence processes such as synaptic plasticity in which the detection of coincident signals is crucial. Copyright © 2006 Society for Neuroscience.

TITLE:The sigma-2 receptor agonist PB28 inhibits calcium release from the endoplasmic reticulum of SK-N-SH neuroblastoma cells 

[2-s2.0-33646815696] Refers [2-s2.0-12944314765] LEVEL: 3
In this paper we demonstrate that PB28 abolishes the Ca2+ release through the inositol 1,4,5-trisphosphate (InsP3) receptors and ryanodine receptors in SK-N-SH cells. Sigma receptors are divided into the subtypes sigma-1 and sigma-2, which are expressed in tumor cell lines and characterized by distinct pharmacological profiles. The sigma-1 receptor has been recently cloned, whereas the sigma-2 receptor is less well characterized. The endogenous ligand(s) of both subtypes remain unclear. In isolated guinea pig ileum, PB28 inhibits the contraction induced by carbachol dose dependently and in a non-competitive manner. In SK-N-SH cells PB28 challenge does not affect the intracellular Ca2+ concentration but incubation with PB28 for 45 min abolishes the cytosolic Ca2+ increases evoked by carbachol or histamine. This effect, not sensitive to cycloheximide, is caused by direct inhibition of the InsP3 receptors, since PB28 abolishes the response elicited by InsP3 administration in permeabilized SK-N-SH cells. Finally, incubation for 45 min with PB28 also abolishes the cytosolic Ca2+ increase evoked by caffeine. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Calcium signaling in physiology and pathophysiology 

[2-s2.0-33748546948] Refers [2-s2.0-12944314765] LEVEL: 3
Calcium ions are the most ubiquitous and pluripotent cellular signaling molecules that control a wide variety of cellular processes. The calcium signaling system is represented by a relatively limited number of highly conserved transporters and channels, which execute Ca2+ movements across biological membranes and by many thousands of Ca2+-sensitive effectors. Molecular cascades, responsible for the generation of calcium signals, are tightly controlled by Ca2+ ions themselves and by genetic factors, which tune the expression of different Ca2+-handling molecules according to adaptational requirements. Ca2+ ions determine normal physiological reactions and the development of many pathological processes. ©2006 CPS and SIMM.

TITLE:Sub-second calcium coupling between outside medium and subplasmalemmal stores during overstimulation/depolarisation-induced ciliary beat reversal in Paramecium cells 

[2-s2.0-33646349985] Refers [2-s2.0-12944314765] LEVEL: 3
As amply documented by electrophysiology, depolarisation in Paramecium induces a Ca2+ influx selectively via ciliary voltage-dependent Ca2+-channels, thus inducing ciliary beat reversal. Subsequent downregulation of ciliary Ca2+ has remained enigmatic. We now analysed this aspect, eventually under overstimulation conditions, by quenched-flow/cryofixation, combined with electron microscope X-ray microanalysis which registers total calcium concentrations, [Ca]. This allows to follow Ca-signals within a time period (?30 ms) smaller than one ciliary beat (?50 ms) and beyond. Particularly under overstimulation conditions (?10-5 M Ca2+ before, 0.5 mM Ca2+ during stimulation) we find in cilia a [Ca] peak at ?80 ms and its decay to near-basal levels within 110 ms (90%) to 170 ms (100% decay). This [Ca] wave is followed, with little delay, by a [Ca] wave into subplasmalemmal Ca-stores (alveolar sacs), culminating at ?100 ms, with a decay to original levels within 170 ms. Also with little delay [Ca] slightly increases in the cytoplasm below. This implies rapid dissipation of Ca2+ through the ciliary basis, paralleled by a rapid, transient uptake by, and release from cortical stores, suggesting fast exchange mechanisms to be analysed as yet. This novel type of coupling may be relevant for some phenomena described for other cells. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Arachidonic acid is a physiological activator of the ryanodine receptor in pancreatic Ī²-cells 

[2-s2.0-33646339378] Refers [2-s2.0-12944314765] LEVEL: 3
Pancreatic ?-cells have ryanodine receptors but little is known about their physiological regulation. Previous studies have shown that arachidonic acid releases Ca2+ from intracellular stores in ?-cells but the identity of the channels involved in the Ca2+ release has not been elucidated. We studied the mechanism by which arachidonic acid induces Ca2+ concentration changes in pancreatic ?-cells. Cytosolic free Ca2+ concentration was measured in fura-2-loaded INS-1E cells and in primary ?-cells from Wistar rats. The increase of cytosolic Ca2+ concentration induced by arachidonic acid (150 ?M) was due to both Ca2+ release from intracellular stores and influx of Ca2+ from extracellular medium. 5,8,11,14-Eicosatetraynoic acid, a non-metabolizable analogue of arachidonic acid, mimicked the effect of arachidonic acid, indicating that arachidonic acid itself mediated Ca2+ increase. The Ca2+ release induced by arachidonic acid was from the endoplasmic reticulum since it was blocked by thapsigargin. 2-Aminoethyl diphenylborinate (50 ?M), which is known to inhibit 1,4,5-inositol-triphosphate-receptors, did not block Ca2+ release by arachidonic acid. However, ryanodine (100 ?M), a blocker of ryanodine receptors, abolished the effect of arachidonic acid on Ca2+ release in both types of cells. These observations indicate that arachidonic acid is a physiological activator of ryanodine receptors in ?-cells. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Cytochrome c binds to inositol (1,4,5) trisphosphate and ryanodine receptors in vivo after transient brain ischemia in gerbils 

[2-s2.0-33645881942] Refers [2-s2.0-12944314765] LEVEL: 3
Previously we have shown that the biphasic efflux of mitochondrial protein cytochrome c to cytoplasm is one of the important events of the delayed postichemic neuronal death. We concluded that early and transient appearance of cytochrome c in cytoplasm of cells recovering after ischemia was decisive for initiation of the pathological signaling cascade leading to neuronal death, but the precise mechanism remained unknown. In vitro cytochrome c was identified as a messenger that coordinates mitochondrial-endoplasmatic reticulum interactions that drive apoptosis. Here we show that in vivo cytochrome c interacts with inositol (1,4,5) trisphosphate receptor type 1 in gerbil hippocampus subjected to transient brain ischemia and short reperfusion. Moreover, cytochrome c binds also to ryanodine receptor type 2, the role of which in postischemic neuronal death is suggested. The complexes could be coimmunoprecipitated by antibodies against any of the two proteins. Our data verified that the mechanism observed in vitro applies to the pathological in vivo situation. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Activation of protein kinase C in sensory neurons accelerates Ca 2+ uptake into the endoplasmic reticulum 

[2-s2.0-30644465044] Refers [2-s2.0-12944314765] LEVEL: 3
The rate of Ca2+ clearance from the neuronal cytoplasm affects the amplitude, duration, and localization of Ca2+ signals and influences a variety of Ca2+-dependent functions. We reported previously that activation of protein kinase C (PKC) accelerates Ca+ efflux in rat sensory neurons mediated by the plasma membrane Ca +-ATPase isoform 4 (PMCA4). Here we show that sarco-endoplasmic reticulum Ca+-ATPase (SERCA)-mediated Ca+ uptake into intracellular stores is also accelerated by PKC activation. The rate of intracellular Ca+ concentration ([Ca+]i) clearance was studied after small (<350 nM) action potential-induced Ca + loads in rat dorsal root ganglion neurons. Under these conditions, mitochondrial Ca+ uptake and Na+/Ca+ exchange do not significantly influence [Ca+]i recovery. Phorbol dibutyrate (PDBu) increased the rate of [Ca+]i clearance by 71% in a manner sensitive to the selective PKC inhibitors GF109203x (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide) and calphostin. PKC-dependent acceleration was still observed (?39%) when the PKC-sensitive PMCA isoform was knocked down by expression of an antisense PMCA4 cDNA (AS4). Direct measurement of Ca+ in the endoplasmic reticulum (ER) lumen revealed that PKC increased the rate of store refilling more than twofold after depletion by treatment with cyclopiazonic acid. ER refilling was less complete in PDBu-treated cells, although, in AS4-expressing cells, PDBu accelerated the rate without reducing the ER capacity, suggesting that PMCA and SERCA compete for Ca+. Thus, activation of PKC accelerates the clearance of Ca+ from the cytoplasm by the concerted stimulation of Ca+ sequestration and Ca+ efflux. Copyright © 2006 Society for Neuroscience.

TITLE:Aryl-indolyl maleimides as inhibitors of CaMKIIĪ“. Part 3: Importance of the indole orientation 

[2-s2.0-41149139078] Refers [2-s2.0-12944314765] LEVEL: 3
A family of aryl-substituted maleimides was prepared and studied for their activity against calmodulin dependant kinase. Inhibitory activities against the enzyme ranged from 10 nM to >20 ?M and were dependant upon both the nature of the aryl group and the tether joining the basic amine to the indolyl maleimide core of the inhibitors. Key interactions with the kinase ATP site and hinge region, predicted by homology modeling, were confirmed. © 2008 Elsevier Ltd. All rights reserved.

TITLE:Light-evoked calcium responses of isolated melanopsin-expressing retinal ganglion cells 

[2-s2.0-37149056035] Refers [2-s2.0-12944314765] LEVEL: 3
A small number (<2%) of mammalian retinal ganglion cells express the photopigment melanopsin and are intrinsically photosensitive (ipRGCs). Light depolarizes ipRGCs and increases intracellular calcium levels ([Ca 2+]i) but the signaling cascades underlying these responses have yet to be elucidated. To facilitate physiological studies on these rare photoreceptors, highly enriched ipRGC cultures from neonatal rats were generated using anti-melanopsin-mediated plate adhesion (immunopanning). This novel approach enabled experiments on isolated ipRGCs, eliminating the potential confounding influence of rod/cone-driven input. Light induced a rise in [Ca2+]i (monitored using fura-2 imaging) in the immunopanned ipRGCs and the source of this Ca2+ signal was investigated. The Ca2+ responses were inhibited by 2-aminoethoxydiphenyl borate, SKF-96365 (1-2-(4-methoxyphenyl)-2-[3-(4- methoxyphenyl)propoxy]ethyl-1H-imidazole), flufenamic acid, lanthanum, and gadolinium, consistent with the involvement of canonical transient receptor potential (TRP) channels in ipRGC phototransduction. However, the contribution of direct Ca2+ flux through a putative TRP channel to ipRGC [Ca 2+]i was relatively small, as most (?90%) of the light-induced Ca2+ responses could be blocked by preventing action potential firing with tetrodotoxin. The L-type voltage-gated Ca2+ channel (VGCC) blockers verapamil and (+)-cis-diltiazem significantly reduced the light-evoked Ca2+ responses, while the internal Ca2+ stores depleting agent thapsigargin had negligible effect. These results indicate that Ca2+ influx through VGCCs, activated after action potential firing, was the primary source for light-evoked elevations in ipRGC [Ca2+]i. Furthermore, concurrent Ca2+ imaging and cell-attached electrophysiological recordings demonstrated that the Ca 2+ responses were highly correlated to spike frequency, thereby establishing a direct link between action potential firing and somatic [Ca 2+]i in light-stimulated ipRGCs. Copyright © 2007 Society for Neuroscience.

TITLE:The pathogenesis of Alzheimers disease - Is it a lifelong "calciumopathy"? 

[2-s2.0-34548823253] Refers [2-s2.0-12944314765] LEVEL: 3
Alzheimer's disease (AD) is a fatal neurodegenerative disorder that has no known cure, nor is there a clear mechanistic understanding of the disease process itself. Although amyloid plaques, neurofibrillary tangles, and cognitive decline are late-stage markers of the disease, it is unclear how they are initially generated, and if they represent a cause, effect, or end phase in the pathology process. Recent studies in AD models have identified marked dysregulations in calcium signaling and related downstream pathways, which occur long before the diagnostic histopathological or cognitive changes. Under normal conditions, intracellular calcium signals are coupled to effectors that maintain a healthy physiological state. Consequently, sustained up-regulation of calcium may have pathophysiological consequences. Indeed, upon reviewing the current body of literature, increased calcium levels are functionally linked to the major features and risk factors of AD: ApoE4 expression, presenilin and APP mutations, beta amyloid plaques, hyperphosphorylation of tau, apoptosis, and synaptic dysfunction. In turn, the histopathological features of AD, once formed, are capable of further increasing calcium levels, leading to a rapid feed-forward acceleration once the disease process has taken hold. The views proposed here consider that AD pathogenesis reflects long-term calcium dysregulations that ultimately serve an enabling role in the disease process. Therefore, "Calcinists" do not necessarily reject ?Aptist or Tauist doctrine, but rather believe that their genesis is associated with earlier calcium signaling dysregulations. Copyright © 2007 Sage Publications.

TITLE:Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites 

[2-s2.0-34447515461] Refers [2-s2.0-12944314765] LEVEL: 3
1. In muscle, intracellular calcium concentration, hence skeletal muscle force and cardiac output, is regulated by uptake and release of calcium from the sarcoplasmic reticulum. The ryanodine receptor (RyR) forms the calcium release channel in the sarcoplasmic reticulum. 2. The free [Ca2+] in the sarcoplasmic reticulum regulates the excitability of this store by stimulating the Ca2+ release channels in its membrane. This process involves Ca2+-sensing mechanisms on both the luminal and cytoplasmic sides of the RyR. In the cardiac RyR, these have been shown to be a luminal Ca 2+ activation site (L-site; 60 ?mol/L affinity), a cytoplasmic activation site (A-site; 0.9 ?mol/L affinity) and a cytoplasmic Ca 2+ inactivation site (I2-site; 1.2 ?mol/L affinity). 3. Cardiac RyR activation by luminal Ca2+ occurs by a multistep process dubbed 'luminal-triggered Ca2+ feed-through'. Binding of Ca 2+ to the L-site initiates brief (1 msec) openings at a rate of up to 10 /s. Once the pore is open, luminal Ca2+ has access to the A-site (producing up to 30-fold prolongation of openings) and to the I2-site (causing inactivation at high levels of Ca2+ feed-through). 4. The present paper reviews the evidence for the principal aspects of the 'luminal-triggered Ca2+ feed-through' model, the properties of the various Ca2+-dependent gating mechanisms and their likely role in controlling sarcoplasmic reticulum (SR) Ca2+ release in cardiac muscle. 5. The model makes the following important predictions: (i) there will be a close link between luminal and cytoplasmic regulation of RyRs and any cofactor that prolongs channel openings triggered by cytoplasmic Ca2+ will also promote RyR activation by luminal Ca2+; (ii) luminal Mg2+ (1 mmol/L) is essential for the control of SR excitability in cardiac muscle by luminal Ca2+; and (iii) the different RyR isoforms in skeletal and cardiac muscle will be controlled quite differently by the luminal milieu. For example, Mg2+ in the SR lumen (approximately 1 mmol/L) can strongly inhibit RyR2 by competing with Ca2+ for the L-site, whereas RyR1 is not affected by luminal Mg2+. © 2007 The Author.

TITLE:Nitric oxide regulates growth cone filopodial dynamics via ryanodine receptor-mediated calcium release 

[2-s2.0-34548700121] Refers [2-s2.0-12944314765] LEVEL: 3
Nitric oxide (NO) is a gaseous intercellular messenger involved in numerous processes during development, including wiring of the nervous system. Neuronal growth cones are responsible for establishing the correct connectivity in the nervous system, but how NO might affect neuronal pathfinding is not fully understood. We have demonstrated in a previous study that local application of a NO donor, NOC-7, via micropipette onto individual growth cones from Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number and an increase in the intracellular calcium concentration ([Ca2+]i). Moreover, these NO-induced effects were demonstrated to be mediated via an intracellular cascade involving soluble guanylyl cyclase, protein kinase G (PKG) and cyclic adenosine diphosphate ribose (cADPR). We now demonstrate that the increase in the [Ca2+] i that results from local NO application is mediated via release from ryanodine receptor (RyR)-sensitive intracellular stores. We also show that PKG and RyRs are localized within growth cones and microinjection of cADPR mimics the effects of NO, providing further support that the NO-induced effects are mediated via cADPR. Lastly, we provide evidence that calcium influx across the plasma membrane is a necessary component of the NO-induced calcium increase; however, this calcium influx is secondary to the RyR-induced calcium release from intracellular stores. This study details a signalling pathway by which NO can cause changes in growth cone morphology and thus provides a mechanism by which NO could affect neuronal wiring by acting locally on individual growth cones during the pathfinding process. © The Authors (2007).

TITLE:Ryanodine receptor Ca2+-release channels are an output pathway for the circadian clock in the rat suprachiasmatic nuclei 

[2-s2.0-34547636661] Refers [2-s2.0-12944314765] LEVEL: 3
Ryanodine-sensitive intracellular Ca2+ channels (RyRs) are present in suprachiasmatic nuclei (SCN) neurons, but the functions served by these channels are not known. Here we addressed whether mobilization of intracellular Ca2+ stores through the RyRs may be a link between the molecular clock and the firing rate in SCN neurons. Activation of the RyRs by administration of either 1 mm caffeine or 100 nm ryanodine increased the firing frequency, whereas inhibition of RyRs by 10 ?m dantrolene or 80 ?m ryanodine decreased firing rate. Similar results were obtained in experiments conducted at either midday or midnight. Furthermore, these effects were not mediated by synaptic transmission as blockade of GABA A, AMPA and NMDA receptors did not prevent the excitatory or inhibitory effects induced by either dose of ryanodine on SCN firing. We conclude that gating of RyRs is a key element of the intricate output pathway from the circadian clock within SCN neurons in rats. © The Authors (2007).

TITLE:Janus a god with two faces: Death and survival utilise same mechanisms conserved by evolution 

[2-s2.0-34250731741] Refers [2-s2.0-12944314765] LEVEL: 3
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TITLE:Differential contribution of extracellular and intracellular calcium sources to basal transmission and long-term potentiation in the sympathetic ganglion of the rat 

[2-s2.0-34249849353] Refers [2-s2.0-12944314765] LEVEL: 3
Calcium involved in basal ganglionic transmission and long-term potentiation (LTP) can arise either by influx from the extracellular medium or release from intracellular stores. No attempts have yet been made to concurrently explore the contributions of extracellular and intracellular Ca2+ to basal ganglionic transmission or LTP. Here, we investigate this subject using the superior cervical ganglion of the rat. To explore the extracellular Ca2+ contribution, we evaluated basal transmission and LTP at different extracellular Ca2+ concentrations. To assess intracellular Ca2+ release, we explored the contribution of the calcium-induced calcium release process by overactivation or blockade of ryanodine-sensitive Ca2+ receptor channel with caffeine, and also by blocking either IP3R with Xestospongin C or the sarco (endo)plasmic reticulum Ca2+-ATPase pump with thapsigargin. Extracellular Ca2+ affected ganglionic basal transmission and LTP to different extents. While 25% of the physiological Ca2+ concentration supported 80% of basal transmission, 50% of normal Ca2+ was required to achieve 80% of LTP. Notably, disruption of intracellular Ca2+ release by all the drugs tested apparently did not affect basal ganglionic transmission but impaired LTP. We conclude that basal transmission requires only a small level of Ca 2+ entry, while LTP expression not only requires more Ca2+ entry but is also dependent on Ca2+ release from intracellular stores. © 2007 Wiley Periodicals, Inc.

TITLE:Changes in functional properties of the caffeine-sensitive Ca2+ store during differentiation of human SH-SY5Y neuroblastoma cells 

[2-s2.0-33846629894] Refers [2-s2.0-12944314765] LEVEL: 3
We have used single cell fluorescence imaging techniques to examine how functional properties of the caffeine-sensitive Ca2+ store change during differentiation of a sub-population of caffeine-sensitive SH-SY5Y cells. Application of caffeine (30 mM) 1-10.5 min after a 'priming' depolarisation pulse of 55 mM K+ revealed that the caffeine-sensitive store in undifferentiated cells remained replete, whereas that in 9-cis retinoic acid (9cRA)-differentiated cells spontaneously dissipated with a t1/2 of 2.8 min, and was essentially completely depleted ?10 min after priming. In 9cRA-differentiated cells that were stimulated with methacholine (10 ?M) 1 min after priming, the amplitude, rate of rise and propagation velocity of the Ca2+ wave in the neurites were all constant, whereas these kinetic parameters all progressively decreased as the wave travelled along the neurites in cells that were stimulated 10 min after priming. Use-dependent block with ryanodine inhibited the global Ca2+ signal in 9cRA-differentiated cells stimulated with methacholine 1 min after priming (71 ± 8%) but not 10 min after priming. Depolarisation was more effective at priming the caffeine-sensitive Ca2+ store in 9cRA-differentiated cells, which lack a functional store-operated Ca2+ entry pathway. We conclude that differentiation of caffeine-sensitive SH-SY5Y cells is accompanied by an increase in lability of the caffeine-sensitive Ca2+ store, and that spontaneous dissipation of Ca2+ from the store limits the time course of its molecular 'memory' during which it can amplify the hormone-induced Ca2+ signal by Ca2+-induced Ca2+ release. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Morphological analysis of endoplasmic reticulum dynamics in growing amphibian oocytes 

[2-s2.0-34047231580] Refers [2-s2.0-12944314765] LEVEL: 3
Morphological analysis of endoplasmic reticulum (ER) dynamics was carried out in early amphybian oocytes at different stages of their development. Comparative morphometric analysis of ER membrane relative surface area and relative volume has shown that at the 1st-2nd stages of oogenesis the accumulation of ER membranes occurred mainly in the peripheral region of the cell cytoplasm. At the 3rd-4th stages of oocyte growth characterized by active transcription, the increase in the relative volume of ER and relative surface area of ER membranes occurs in the middle and near-nuclear region of cytoplasm, and reduction of these parameters takes place in the peripheral part of the cell. Simultaneously observed fusion of numerous ER vesicles with the outer nuclear membrane may serve as an evidence of active formation of new fragments of nuclear envelope. The 5 th-6th stages of oocyte development are characterized by an increase in the relative volume of ER in the whole oocyte. It has been supposed that the revealed reorganization of endoplasmic reticulum in growing amphibians oocytes might be due to yolk accumulation (vitellogenesis), redistribution of calcium ions, and synthesis and storage of lipids and proteins. These processes are necessary for the future embryo development, and also for assembly of new fragments of growing nuclear envelope.

TITLE:From Galvani to patch clamp: The development of electrophysiology 

[2-s2.0-33751073392] Refers [2-s2.0-12944314765] LEVEL: 3
The development of electrophysiology is traced from the early beginnings represented by the work of the Dutch microscopist, Jan Swammerdam, in the 17th century through the first notion of an aqueous transmembrane pore as a substrate of excitability made by Luigi Galvani in late 18th century to the invention late in the 20th century of the patch-clamp technique by Erwin Neher and Bert Sakmann. © 2006 Springer-Verlag.

TITLE:Differential distribution of voltage-gated calcium channels in dopaminergic neurons of the rat retina 

[2-s2.0-33745005072] Refers [2-s2.0-12944314765] LEVEL: 3
We studied by immunocytochemistry and Western blots the identity and cellular distribution of voltage-gated calcium channels within dopaminergic neurons of the rat retina. The aim was to associate particular calcium channel subtypes with known activities of the neuron (e.g., transmitter release from axon terminals). Five voltage-gated calcium channels were identified: ?1A, ?1B, ?1E, ?1F, and ?1H. All of these, except the ?1B subtype, were found within dopaminergic perikarya. The ?1B channels were concentrated at axon terminal rings, together with ?1A calcium channels. In contrast, ?1H calcium channels were most abundant in the dendrites, and alF calcium channels were restricted to the perikaryon. The ?1E calcium channel was present at such a low density that its cellular distribution beyond the perikaryon could not be determined. Our findings are consistent with the available pharmacological data indicating that ?1A and ?1B calcium channels control the major fraction of dopamine release in the rat retina. © 2006 Wiley-Liss, Inc.

TITLE:Ca2+ sparks and Ca2+ glows in superior cervical ganglion neurons 

[2-s2.0-33748570160] Refers [2-s2.0-12944314765] LEVEL: 3
Aim: Ca2+ release from the endoplasmic reticulum (ER) is an integral component of neuronal Ca2+ signaling. The present study is to investigate properties of local Ca2+ release events in superior cervical ganglion (SCG) neurons. Methods: Primary cultured SCG neurons were prepared from neonatal rats (P3-P7). Low concentration of caffeine was used to induce Ca2+ release from the ER Ca2+ store, and intracellular Ca2+ was recorded by high-resolution line scan confocal imaging and the Ca2+ indicator Fluo-4. Results: Two populations of local Ca2+ release events with distinct temporal characteristics were evoked by 1.5 mmol/L caffeine near the surface membrane in the soma and the neurites of SCG neurons. Brief events similar to classic Ca2+ sparks lasted a few hundreds of milliseconds, whereas long-lasting events displayed duration up to tens of seconds. Typical somatic and neurite sparks were of 0.3- and 0.52-fold increase in local Fluo-4 fluorescence, respectively. Typical Ca2+ glows were brighter (?F/F0 approximately 0.6), but were highly confined in space. The half maximum of full duration of neurite sparks was much longer than those in the soma (685 vs 381 ms). Conclusion: Co-existence of Ca2+ sparks and Ca2+ glows in SCG neurons indicates distinctive local regulation of Ca2+ release kinetics. The local Ca2+ signals of variable, site-specific temporal length may bear important implications in encoding a "memory" of the trigger signal. ©2006 CPS and SIMM.

TITLE:Can 4-chloro-m-cresol be substituted for caffeine as an activator of calcium oscillation in bullfrog sympathetic ganglion cells? 

[2-s2.0-33645232213] Refers [2-s2.0-12944314765] LEVEL: 3
4-Chloro-m-cresol (cresol) and caffeine have been shown to be potent activators of the Ca2+ release mediated by the ryanodine-sensitive Ca2+ release channel and therefore increase the cytosolic free calcium concentration in skeletal muscles. To distinguish the effects of cresol and caffeine in neurones, the responses of the intracellular ([Ca2+]i) and intraluminal free calcium concentrations to cresol were investigated using bullfrog sympathetic ganglion cells and then compared with those to caffeine. Cresol generated a gradual rise (slow response) with and without a fast transient rise (fast response) in [Ca2+ ]i. A low extracellular Ca2+ concentration abolished the slow response but not the fast response, thus indicating that the slow response was caused by a Ca2+ influx across the cell membrane. The fast response was inhibited by ryanodine, thus confirming that the source may therefore be the Ca2+ release through the ryanodine-sensitive calcium store. Unlike caffeine, the long-term application of cresol did not cause any calcium oscillation; neither did it cause a decrease in the basal calcium levels. © 2006 Elsevier Ltd. All rights reserved.

TITLE:The Ca2+-releasing messenger NAADP, a new player in the nervous system 

[2-s2.0-33645035130] Refers [2-s2.0-12944314765] LEVEL: 3
Many physiological processes are controlled by a great diversity of Ca 2+ signals. Within cell, Ca2+ signals depend upon Ca 2+ entry and/or Ca2+ release from internal Ca2+ stores. The control of Ca2+-store mobilization is ensured by a family of messengers comprising inositol 1,4,5 trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). From recent works, new concepts have emerged where activation of the cells by outside stimuli, acting at the plasma membrane, results in the synthesis of multiple Ca 2+-releasing messengers which may interact and shape complex Ca 2+ signals in the cytosol as well as in the nucleus. This contribution will cover the most recent advances on NAADP signalling with some emphasis on neurons. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Inhibition of rat brain inositol 1,4,5-trisphosphate 3-kinase A expression by kainic acid 

[2-s2.0-28444444206] Refers [2-s2.0-12944314765] LEVEL: 3
Defects in intracellular calcium homeostasis may cause aberrant neuronal activation and subsequent neuronal death. Because inositol trisphosphate (IP3) regulates the release of calcium from the endoplasmic reticulum and the IP3 kinase A isoform (IP3K-A) reduces intracellular IP3, regulation of IP3K could be involved in neuronal activation and/or neuronal death. In this study, we found that kainic acid (KA) treatment in vitro and in vivo reduced the level of IP3K-A mRNA. Since KA treatment induces aberrant neuronal activation and neuronal death, we tested whether the reduction of IP3K-A mRNA was required for KA-induced neuronal death. Overexpression of adenovirus-derived IP 3K-A failed to rescue neurons from KA-induced death. Because neuronal activation by KCl in vitro is sufficient to reduce IP3K-A expression, we conclude that the KA-derived reduction of IP3K-A expression is due to the aberrant neuronal activation, and the reduction in the IP3K-A mRNA level is not required for the toxic effect of KA. © 2005 Elsevier Ireland Ltd. All rights reserved.

TITLE:Non-stimulated Ca2+ leak pathway in cerebellar granule neurones 

[2-s2.0-23044434658] Refers [2-s2.0-12944314765] LEVEL: 3
The aim of this study was to investigate the pathways of calcium influx routes in non-stimulated cerebellar granule neurones by use of standard microspectrofluorimetric techniques. Repetitive application of Ca 2+-free solutions for various time intervals induced decreases of resting cytosolic free Ca2+ concentration ([Ca2+] i) which were followed, on Ca2+ readmission, by a full recovery, always to the initial resting [Ca2+]i levels. Use of drugs to deplete calcium stores (thapsigargin, alone or combined with low levels of ionomycin) did not cause release of Ca2+ from the intracellular stores nor enhanced the activity of the Ca2+ entry pathway. This influx was mainly independent of voltage operated calcium channels, since both L-type channel blockers (nitrendipine) and the hyperpolarizing agent pinacidil (a K+-channel opener) were without effect. Contribution from glutamate receptors to this influx was eliminated since a combination of blockers of NMDA and AMPA glutamate receptors (NBQX and D-AP5) did not affect the properties of the Ca2+ response. The Ca2+ leak pathway was sensitive to micromolar levels of lanthanum and gadolinium, and to the compound 2-APB, features shared by several channels of the TRP superfamily. In summary, our results show the presence of a Ca 2+ permeable pathway, active and patent in resting conditions in cerebellar granule neurones, and which is different from the voltage-operated calcium channels and not operated by depletion of the stores. © 2005 Elsevier Inc. All rights reserved.

TITLE:Metastasis suppressor pathways - An evolving paradigm 

[2-s2.0-0037980379] Refers [2-s2.0-0036280779] LEVEL: 3
A greater understanding of the processes of tumor invasion and metastasis, the principal cause of death in cancer patients, is essential to determine newer therapeutic targets. Metastasis suppressor genes, by definition, suppress metastasis without affecting tumorigenicity and, hence, present attractive targets as prognostic or therapeutic markers. This short review focuses on those twelve metastasis suppressor genes for which functional data exist. We also outline newly identified genes that bear promising traits of having metastasis suppressor activity, but for which functional data have not been completed. We also summarize the biochemical mechanism(s) of action (where known), and present a working model assembling potential metastasis suppression pathways. © 2003 Elsevier Ireland Ltd. All rights reserved.

TITLE:Identification of Serum Amyloid A Protein As a Potentially Useful Biomarker to Monitor Relapse of Nasopharyngeal Cancer by Serum Proteomic Profiling 

[2-s2.0-1642453766] Refers [2-s2.0-0036280779] LEVEL: 3
Purpose: Nasopharyngeal cancer (NPC) is a common cancer in Hong Kong, and relapse can occur frequently. Using protein chip profiling analysis, we aimed to identify serum biomarkers that were useful in the diagnosis of relapse in NPC. Experimental Design: Profiling analysis was performed on 704 sera collected from 42 NPC patients, 39 lung cancer patients, 30 patients with the benign metabolic disorder thyrotoxicosis (TX), and 35 normal individuals (NM). Protein profile in each NPC patient during clinical follow up was correlated with the relapse status. Results: Profiling analysis identified two biomarkers with molecular masses of 11.6 and 11.8 kDa, which were significantly elevated in 22 of 31 (71%) and 21 of 31 (68%) NPC patients, respectively, at the time of relapse (RP) as compared with 11 patients in complete remission (CR; RP versus CR, P = 0.009), 30 TX (RP versus TX, P < 0.001), or 35 NM (RP versus NM, P < 0.001). The markers were also elevated in 16 of 39 (41%) lung cancer patients at initial diagnosis. By tryptic digestion, followed by tandem mass spectrometry fragmentation, the markers were identified as two isoforms of serum amyloid A (SAA) protein. Monitoring the patients longitudinally for SAA level both by protein chip and immunoassay showed a dramatic SAA increase, which correlated with relapse and a drastic fall correlated with response to salvage chemotherapy. Serum SAA findings were compared with those of serum Epstein-Barr virus DNA in three relapsed patients showing a similar correlation with relapse and chemo-response. Conclusions: SAA could be a useful biomarker to monitor relapse of NPC.

TITLE:Serum Protein Profiles to Identify Head and Neck Cancer 

[2-s2.0-1842481344] Refers [2-s2.0-0036280779] LEVEL: 3
Purpose: New and more consistent biomarkers of head and neck squamous cell carcinoma (HNSCC) are needed to improve early detection of disease and to monitor successful patient management. The purpose of this study was to determine whether a new proteomic technology could correctly identify protein expression profiles for cancer in patient serum samples. Experimental Design: Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry Protein-Chip system was used to screen for differentially expressed proteins in serum from 99 patients with HNSCC and 102 normal controls. Protein peak clustering and classification analyses of the surface-enhanced laser desorption/ionization spectral data were performed using the Biomarker Wizard and Biomarker Patterns software (version 3.0), respectively (Ciphergen Biosystems, Fremont, CA). Results: Several proteins, with masses ranging from 2,778 to 20,800 Da, were differentially expressed between HNSCC and the healthy controls. The serum protein expression profiles were used to develop and train a classification and regression tree algorithm, which reliably achieved a sensitivity of 83.3% and a specificity of 100% in discriminating HNSCC from normal controls. Conclusions: We propose that this technique has potential for the development of a screening test for the detection of HNSCC.

TITLE:Identification of Patients with Head and Neck Cancer Using Serum Protein Profiles 

[2-s2.0-0345743495] Refers [2-s2.0-0036280779] LEVEL: 3
Background: New and more consistent biomarkers of head and neck squamous cell carcinoma (HNSCC) are needed to improve early detection of disease and to monitor successful patient management. Objective: To determine if a new proteomic technology can correctly identify protein expression profiles for cancer in patient serum samples as well as detect the presence of a known tumor marker. Design: Direct proteomic analysis and comparison. Methods: The surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF) ProteinChip system was used to screen for differentially expressed proteins in serum samples from 99 patients with HNSCC, 25 "healthy" smokers, and 102 healthy (normal) controls. Protein peak clustering and classification analyses of the SELDI spectral data were performed. Results: Several proteins, with masses ranging from 2778 to 20800 Da, were differentially expressed between patients with HNSCC and the normal controls. The serum protein expression profiles were used to develop a classification tree algorithm, which achieved a sensitivity of 83.3% and a specificity of 90% in discriminating HNSCC from normal and healthy smoker controls. The positive and negative predictive values were 80% and 92%, respectively. A peak with an average mass of 10068 Da was detected in sera from HNSCC patients and identified as the known biomarker metallopanstimulin-1 (MPS-1), based on mass. Peak relative intensity of the 10068-Da protein correlated consistently with MPS-1 levels detected by radioimmunoassay in serum samples of HNSCC patients and controls. The 10068-Da peak was provisionally identified as MPS-1 by SELDI immunoassay. Conclusion: We propose that this technique may allow for the development of a reliable screening test for the early detection and diagnosis of HNSCC, as well as the potential identification of tumor biomarkers.

TITLE:Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H and MHCC97-L, with different metastasis potentials 

[2-s2.0-1842528203] Refers [2-s2.0-0036280779] LEVEL: 3
To better understand the mechanism underlying hepatocellular carcinoma (HCC) metastasis and to search for potential markers for HCC prognosis, differential proteome analysis on two HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional (2-D) gel electrophoresis followed by matrix-assisted laser desorption/time of flight mass spectrometry and liquid chromatography ion trap mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 56 protein spots showed significant differential expression in MHCC97-H and MHCC97-L cells (Student's t-test, P < 0.05) and 4 protein spots were only detected in MHCC97-H cells. Fourteen protein spots were further identified using in-gel tryptic digestion, peptide mass fingerprinting and tandem mass spectrometry. The expressions of pyruvate kinase M2, ubiquitin carboxy-terminal hydrolase L1, laminin receptor 67 kDa, S100 calcium-binding protein A4, thioredoxin and cytokeratin 19 were elevated in MHCC97-H cells. However, manganese superoxide dismutase, calreticulin precursor, cathepsin D, lactate dehydrogenase B, non-metastatic cell protein 1, cofilin 1 and calumenin precursor were down-regulated in MHCC97-H cells. Intriguingly, most of these identified proteins have been reported to be associated with tumor metastasis. The functional implications of alterations in the levels of these proteins are discussed.

TITLE:Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis 

[2-s2.0-0037879089] Refers [2-s2.0-0036280779] LEVEL: 3
Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one-dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP-dissociation inhibitor 1, tropomyosin, fibroblast type, IL-18 and annexin I were significantly up-regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down-regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense/IL-18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, while IL-18 antisense remarkably decreased cell invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play a role in metastasis by inhibiting E-cadherin expression.

TITLE:Annexin A1 Down-Regulation in Head and Neck Cancer Is Associated with Epithelial Differentiation Status 

[2-s2.0-1542784505] Refers [2-s2.0-0036280779] LEVEL: 3
Annexin A1 (ANXA1) protein expression was evaluated by Western blot in a series of 32 head and neck squamous cell carcinomas (HNSCCs) in a search for molecular alterations that could serve as useful diagnostic/prognostic markers. ANXA1 down-regulation was observed in 24 cases (75%) compared with patient-matched normal epithelium. In relation to clinicopathological variables, ANXA1 down-regulation was significantly associated with advanced T stages (P = 0.029), locoregional lymph node metastases (P = 0.038), advanced disease stage (P = 0.006), hypopharyngeal localization (P = 0.038), and poor histological differentiation (P = 0.005). ANXA1 expression was also analyzed by immunohistochemistry in paraffin-embedded sections from 22 of 32 HNSCCs and 8 premalignant lesions. All dysplastic tissues showed significantly reduced ANXA1 expression compared to a strong positive signal observed in adjacent normal epithelia (except basal and suprabasal cells). A close association was observed between ANXA1 expression and the histological grade in HNSCC. Well-differentiated tumors presented a positive ANXA1 signal in highly keratinized areas whereas moderately and poorly differentiated tumors exhibited very weak or negative staining. Our findings clearly identify ANXA1 as an effective differentiation marker for the histopathological grading of HNSCCs and for the detection of epithelial dysplasia.

TITLE:Laser capture microdissection-based in vivo genomic profiling of wound keratinocytes identifies similarities and differences to squamous cell carcinoma 

[2-s2.0-0037663263] Refers [2-s2.0-0036280779] LEVEL: 3
Keratinocytes undergo a dramatic phenotypic conversion during reepithelialization of skin wounds to become hyperproliferative, migratory, and invasive. This transient healing response phenotypically resembles malignant transformation of keratinocytes during squamous cell carcinoma progression. Here we present the first analysis of global changes in keratinocyte gene expression during skin wound healing in vivo, and compare these changes to changes in gene expression during malignant conversion of keratinized epithelium. Laser capture microdissection was used to isolate RNA from wound keratinocytes from incisional mouse skin wounds and adjacent normal skin keratinocytes. Changes in gene expression were determined by comparative cDNA array analyses, and the approach was validated by in situ hybridization. The analyses identified 48 candidate genes not previously associated with wound reepithelialization. Furthermore, the analyses revealed that the phenotypic resemblance of wound keratinocytes to squamous cell carcinoma is mimicked at the level of gene expression, but notable differences between the two tissue-remodeling processes were also observed. The combination of laser capture microdissection and cDNA array analysis provides a powerful new tool to unravel the complex changes in gene expression that underlie physiological and pathological remodeling of keratinized epithelium.

TITLE:Differential polypeptide display: The search for the elusive target 

[2-s2.0-1542509482] Refers [2-s2.0-0036280779] LEVEL: 3
Proteomics, as a tool to identify proteins in biological samples, is gaining rapidly importance in the postgenomic era. Here we discuss the current and potential role of different techniques in the field of proteomics such as two-dimensional gel electrophoresis off-line coupled to MALDI-MS (2D-PAGE-MALDI-MS), high performance liquid chromatography mass spectrometry (HPLC-MS), surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) and a newly developed technique, capillary electrophoresis mass spectrometry (CE-MS). The developments of the last years are presented discussed. © 2003 Elsevier B.V. All rights reserved.

TITLE:A proteomic approach to cisplatin resistance in the cervix squamous cell carcinoma cell line A431 

[2-s2.0-5644294264] Refers [2-s2.0-0036280779] LEVEL: 3
Since drug resistance is a complex and multifactorial event involving activation/repression of multiple biochemical pathways, we used a proteomic approach to study cisplatin resistance and drug response in human tumor cell lines. The cervix squamous cell carcinoma cell line A431 and its cisplatin-resistant subline, A431/Pt, were used as a model system. The experimental set-up involved not just a two-way comparison of the control vs. the drug-resistant cell line, but also an acute cisplatin treatment of both cell lines, leading to a four-way comparison, as follows: 1) A431 vs. A431/Pt cells; 2) A431 vs. A431 cisplatin exposed cells; 3) A431/Pt vs. A431/Pt cisplatin exposed cells; 4) A431 cisplatin exposed cells vs. A431/Pt cisplatin exposed cells. We found modulation of proteins, which could be classified under various categories, such as molecular chaperones (e.g. heat-shock proteins HSP60, HSP90, HSC71, heat-shock cognate 71 kDa protein), Ca2+-binding proteins (e.g. calmodulin, calumenin), proteins involved in drug detoxification (such as peroxiredoxins PRX 2 and PRX 6, and glutathione-S-transferase, GST), anti-apoptotic proteins (such as 14-3-3 switched on in cisplatin-exposed cells) and ion channels (such as VDAC-1, voltage-dependent anion-selective channel). In particular, the basal levels of HSC71 and HSP60 were increased in A431/Pt cells as compared to A431 cells, and cisplatin exposure resulted in up-regulation of HSP60 and HSP90 only in A431 cells. Moreover, cisplatin exposure up-regulated the anti-apoptotic 14-3-3 protein in both cell lines, GST in sensitive cells and PRX6 in A431/Pt cells. These findings are consistent with a constitutive expression of defence factors by resistant cells and with activation by cisplatin of mechanisms acting to protect cells from drug-induced damage. This pattern of response, also observed in parental cells, could reflect an intrinsic resistance of this tumor type.

TITLE:Proteomics of buccal squamous cell carcinoma: The involvement of multiple pathways in tumorigenesis 

[2-s2.0-3543055313] Refers [2-s2.0-0036280779] LEVEL: 3
Squamous cell carcinoma (SCC) of the buccal mucosa is an aggressive oral cancer. It mainly occurs in Central and Southeast Asia, and is closely related to the practice of tobacco smoking and betel squid chewing. The high recurrence and low survival rates of buccal SCC require our continued efforts to understand the pathogenesis of the disease for designing better therapeutic strategies. We used proteomic technology to analyze buccal SCC tissues aiming at identifying tumor-associated proteins for the utilization as biomarkers or molecular targets. With the exception of alpha B-crystallin being substantially reduced, a number of proteins were found to be significantly over-expressed in cancer tissues. These increased proteins included glycolytic enzymes, heat-shock proteins, tumor antigens, cytoskeleton proteins, enzymes involved in detoxification and anti-oxidation systems, and proteins involved in mitochondrial and intracellular signaling pathways. These extensive protein variations indicate that multiple cellular pathways were involved in the process of tumorigenesis, and suggest that multiple protein molecules should be simultaneously targeted as an effective strategy to counter the disease. At least, SCC antigen, G protein, glutathione S-transferase, manganese superoxide dismutase, annexins, voltage-dependent anion channel, cyclophilin A, stratifin and galectin 7 are candidates for targeted proteins. The present findings also demonstrated that rich protein information can be produced by means of proteomic analysis for a better understanding of the oncogenesis and pathogenesis in a global way, which in turn is a basis for the rational designs of diagnostic and therapeutic methods.

TITLE:Proteomic identification of heat shock protein 70 as a candidate target for enhancing apoptosis induced by farnesyl transferase inhibitor 

[2-s2.0-0142213549] Refers [2-s2.0-0036280779] LEVEL: 3
Farnesyl transferase inhibitors (FTIs) are novel antitumor drugs with clinical activity. FTIs inhibit cell growth not only by preventing direct Ras farnesylation but also through a Ras-independent pathway. Proteomics has been shown to be a powerful tool to monitor and analyze molecular networks and fluxes within the living cells and to identify the proteins that participate in these networks upon perturbation of the cellular environment. To observe early and dynamic protein changes in the cellular response to FTI in ovarian cancer cells, total proteins were extracted from 2774 cells treated or not with 10 ?M manumycin, an FTI, for 3, 6 and 16 h. The proteins in the cells that were differentially expressed following treatment with manumycin for 3, 6 and 16 h were noted by two-dimensional electrophoresis and further identified by peptide mass fingerprinting as stress proteins. Both heat shock protein 70 (HSP70) and altered HSP70 were significantly up-regulated as early as 16 h in 2774 cells after exposure to manumycin. Since HSP70 plays an important role in protecting cells under stress, we treated the 2774 cells with the HSP inhibitor quercetin in combination with FTI. Quercetin dramatically enhanced the manumycin-mediated apoptosis in 2774 cells. Inducible HSP70 by manumycin in surviving ovarian cancer cells was also inhibited by quercetin as demonstrated by enzyme-linked immunosorbent assay. The inhibition of HSP70 by quercetin was correlated with enhancement of manumycin-induced mediated apoptosis in 2774 cells. The inhibition of HSP70 by 50 ?M quercetin was also correlated with a decreased expression of procaspase-3 and enhancement of specific cleavage of poly (ADP-ribose) polymerase into apoptotic fragment in 2774 cells treated with manumycin. The interaction between the HSP70 inhibitor and FTI confirms the functional significance of the up-regulation of HSP70 as a protective mechanism against FTI-induced apoptosis and provides the framework for combination treatment of ovarian cancer.

TITLE:Proteome analysis of gastric cancer metastasis by two-dimensional gel electrophoresis and matrix assisted laser desorption/lonization-mass spectrometry for identification of metastasis-related proteins 

[2-s2.0-7044235835] Refers [2-s2.0-0036280779] LEVEL: 3
A well-described animal model was used to understand the molecular mechanisms of carcinogenesis and metastasis of gastric cancer at the protein level. Gastric cancer was induced in 12 Wistar rats by oral administration of N-methyl-N?-Nitro-N-Nitrosoguanidine (MNNG). The protein expression patterns of normal gastric tissue, gastric cancer, and corresponding metastases were analyzed by proteomics in matched tissues of 3 rats. Proteins in the region of molecular masses of 15-75 kDa and an isoelectric point of 3-7 were separated by two-dimensional electrophoresis (2-DE) and identified by peptide fingerprinting with matrix-assisted laser desorption/ionization-time-of-flight- mass spectrometry (MALDI-TOF-MS). Twenty-seven spots corresponding to 25 different proteins served as landmarks for comparison between tissues. The identified proteins included cytoskeletal proteins, stress associated proteins, proteins involved in signal transduction, cell proliferation and differentiation, and metabolism. Eleven proteins were up-regulated and 2 proteins were down-regulated in tumor tissue when compared with normal tissue. Twelve proteins were up-regulated and 8 proteins were down-regulated in the metastases when compared with the primary tumor. The overexpression of HSP27 in gastric cancer was confirmed by immunohistochemical analysis of human gastric cancer specimens. Combining well-defined animal models with proteome analysis will improve our understanding of the fundamental changes that contribute to the process of carcinogenesis and the formation of metastases in gastric cancer.

TITLE:Molecular predictors of clinical outcome in patients with head and neck squamous cell carcinoma 

[2-s2.0-28544438498] Refers [2-s2.0-0036280779] LEVEL: 3
Head and neck squamous cell carcinoma (HNSCC) involves the upper aerodigestive tract and can destroy the structure and function of organs involved in voice, speech, taste, smell and hearing, as well as vital structures necessary for survival. HNSCC has long been a treatment challenge because of the high rate of recurrences and of advanced disease at the time of diagnosis. Molecular identification of tissue biomarkers in diagnostic biopsy specimens may not only identify patients at risk for developing HNSCC but may also select patients that may benefit from more aggressive treatment modalities. Several biomarkers studied to date such as the proteins p53, cyclin D1, p16, Cox-2 enzyme, epidermal growth factor and vascular endothelial growth factor receptors, matrix metalloproteinases and the Fhit marker for genomic instability could be manipulated for the therapeutic benefit of these patients. This review presents the most updated information on molecular biomarkers with the greatest prognostic potential in HNSCC and discusses some factors that contribute to the controversy concerning their prognostic importance. © 2005 Blackwell Publishing Ltd.

TITLE:Identification of novel candidate oncogenes and tumor suppressors in malignant pleural mesothelioma using large-scale transcriptional profiling 

[2-s2.0-21144456382] Refers [2-s2.0-0036280779] LEVEL: 3
Malignant pleural mesothelioma (MPM) is a highly lethal, poorly understood neoplasm that is typically associated with asbestos exposure. We performed transcriptional profiling using high-density oligonucleotide microarrays containing ?22,000 genes to elucidate potential molecular and pathobiological pathways in MPM using discarded human MPM tumor specimens (n = 40), normal lung specimens (n = 4), normal pleura specimens (n = 5), and MPM and SV40-immortalized mesothelial cell lines (n = 5). In global expression analysis using unsupervised clustering techniques, we found two potential subclasses of mesothelioma that correlated loosely with tumor histology. We also identified sets of genes with expression levels that distinguish between multiple tumor subclasses, normal and tumor tissues, and tumors with different morphologies. Microarray gene expression data were confirmed using quantitative reverse transcriptase-polymerase chain reaction and protein analysis for three novel candidate oncogenes (NME2, CRI1, and PDGFC) and one candidate tumor suppressor (GSN). Finally, we used bioinformatics tools (ie, software) to create and explore complex physiological pathways. Combined, all of these data may advance our understanding of mesothelioma tumorigenesis, pathobiology, or both. Copyright © American Society for Investigative Pathology.

TITLE:Overexpression of alpha enolase in hepatitis C virus-related hepatocellular carcinoma: Association with tumor progression as determined by proteomic analysis 

[2-s2.0-17844380283] Refers [2-s2.0-0036280779] LEVEL: 3
To identify proteins that could be molecular targets for diagnosis and treatment of hepatitis C virus-related hepatocellular carcinoma (HCV-related HCC), we used a proteomic approach to analyze protein expression in samples of human liver. Twenty-six pairs of tumorous and corresponding nontumorous liver samples from patients with HCV-related HCC and six normal liver samples were analyzed by two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry. One of the numerous spots that showed stronger intensity in tumorous than in nontumorous samples was identified as alpha enolase, a key enzyme in the glycolytic pathway. Expression of this protein increased with tumor dedifferentiation and was significantly higher in poorly differentiated HCC than in well-differentiated HCC. This pattern was reproduced by immunoblot analysis and immunohistochemistry. Expression of alpha enolase also correlated positively with tumor size and venous invasion. These results suggest that alpha enolase is one of the candidates for biomarkers for tumor progression that deserves further investigation in HCV-related HCC. © 2005 WILEY-VCH Verlag GmbH & Co. KGaA.

TITLE:Differential proteomic analysis of human hepatocellular carcinoma cell line metastasis-associated proteins 

[2-s2.0-5644251923] Refers [2-s2.0-0036280779] LEVEL: 3
Purpose: The comparative study of differentially expression of protein profiles of hepatocellular carcinoma cell lines with various metastasic potential and screening key molecules related to hepatocellular carcinoma metastasis and recurrence. Methods: Using two-dimensional electrophoresis and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), we analyzed differentially displayed proteomics of human hepatocellular carcinoma cell lines Hep3B, MHCC97L, MHCC97H with different metastasic potential. Results: Approximate 1,000 protein spots were detected on silver-stained gel by ImageMaster (977 ± 113 spots in Hep3B, 1092 ± 40 in MHCC97L, and 889 ± 14 in MHCC97H). Fifty distinct different protein spots were analyzed with online LC-ESI-MS/MS. Only 26 protein spots had a positive result, including annexin1, S100A4, and so on. In comparison with nonmetastasis Hep3B cell lines, there were 16 proteins overexpressed in MHCC97H and MHCC97L, 10 proteins underexpressed in MHCC97H and MHCC97L. Applying cell immunohistochemistry and RT-PCR, we further validated two interesting and different proteins, annexin1 and S100A4. Conclusion: The protein profile of metastatic hepatocellular carcinoma cell lines displayed obvious differences compared with non-metastatic liver cancer cell lines. The results imply that various different proteins may lead to HCC metastasis together.

TITLE:Comparative proteome analysis of native differentiated and cultured dedifferentiated human RPE cells 

[2-s2.0-0041342074] Refers [2-s2.0-0036280779] LEVEL: 3
PURPOSE. Dedifferentiation of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). This study was designed to improve the understanding of RPE cell dedifferentiation in vitro. The protein expression pattern of native differentiated RPE cells was compared with that of cultured, thereby dedifferentiated, RPE cells. METHODS. Differentiated native human RPE cells and monolayers of dedifferentiated cultured primary human RPE cells were processed for two-dimensional (2-D) electrophoresis. Total cellular proteins were separated by isoelectric focusing using immobilized pH gradients (IPG 3-10) and electrophoresis on 9% to 15% gradient polyacrylamide gels. Proteins were visualized by silver staining. Silver-stained gel spots were excised, digested in situ, and analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy (MS). The resultant peptide mass fingerprints were searched against the public domain NCBInr, MSDB, and EnsemblC databases to identify the respective proteins. RESULTS. One hundred seventy nine protein spots were analyzed and classified into functional categories. Proteins associated with highly specialized functions of the RPE, which are required for interaction with photoreceptor cells, including RPE65, cellular retinaldehyde-binding protein (CRALBP), and cellular retinol-binding protein (CRBP), were absent in dedifferentiated cultured RPE cells, whereas proteins involved in phagocytosis and exocytosis, including cathepsin D and clathrin were still present. Dedifferentiated RPE cells displayed a strong shift toward increased expression of proteins associated with cell shape, cell adhesion, and stress fiber formation, including cytokeratin 19, gelsolin, and tropomyosins, and also acquired increased expression of factors involved in translation and tumorigenic signal transduction such as annexin I and translation initiation factor (eIF)-5A. CONCLUSIONS. Dedifferentiation of human RPE cells in vitro results in downregulation of proteins associated with highly specialized functions of the RPE and induces the differential expression of proteins related to cytoskeleton organization, cell shape, cell migration, and mediation of proliferative signal transduction. These in vitro data suggest that the dedifferentiated status of RPE cells per se may initiate PVR. Further investigation of candidate proteins may identify additional targets for treatment or prevention of diseases associated with RPE dedifferentiation.

TITLE:Proteomics in cancer research 

[2-s2.0-0036741723] Refers [2-s2.0-0036280779] LEVEL: 3
With the human genome sequence now determined, the field of molecular medicine is moving beyond genomics to proteomics. In the field of cancer research, the key question is: how can oncologists best use techniques of proteomics in basic research and clinical application? In the postgenomic era, proteomics promises the discovery of biomarkers and tumor markers for early detection and diagnosis, novel protein-based drug targets for anticancer therapy, and new endpoints for the assessment of therapeutic efficacy and toxicity. This review paper will explore key themes in proteomics and their application in clinical cancer research.

TITLE:Serum, salivary and tissue proteomics for discovery of biomarkers for head and neck cancers 

[2-s2.0-12344295469] Refers [2-s2.0-0036280779] LEVEL: 3
Initial clinically oriented applications of emerging proteomic technologies that aim to identify biomarkers for head and neck squamous cell carcinoma diagnostics have yielded promising results. The development of new proteomic diagnostics remains critical for the early detection of head and neck squamous cell carcinoma at more treatable stages. Prognostic markers for disease recurrence and treatment sensitivities are also required. In this overview of current biomarker identification strategies for head and neck squamous cell carcinoma, different combinations of mass spectrometry platforms, laser capture microscopy and 2D gel electrophoresis procedures are summarized as applied to readily available clinical specimens (tissue, blood and saliva). Issues related to assay reproducibility, management of large data sets and future improvements in clinical proteomics are also addressed.

TITLE:Biomarkers of human cutaneous squamous cell carcinoma from tissues and cell lines identified by DNA microarrays and qRT-PCR 

[2-s2.0-0037757606] Refers [2-s2.0-0036280779] LEVEL: 3
Squamous cell carcinoma (SCC) is the second most common form of skin cancer in Caucasians. Here we report on the identification of biomarkers of human cutaneous SCC cell lines in vitro and tissue samples in vivo using DermArray and PharmArray DNA microarrays, consisting of ca. 7400 unique human cDNAs. Differentially expressed genes were identified in two facial skin SCC cell lines (SCC 12 and SCC 13) compared to normal keratinocytes, and three cutaneous SCC tissue samples compared to normal skin. Quantitative validations of up- and down-regulated biomarkers were performed by qRT-PCR on 23 biomarker genes for the cell lines and 20 biomarker genes for the tumor tissues. In addition, three oral SCC cell lines were also included in the qRT-PCR validations for comparison, and the biomarker profiles were highly similar between the cutaneous and the oral SCC cell lines for all 23 biomarkers examined. The expression profiles for a variety of non-cutaneous SCC types, such as head-and-neck, oral, and lung, have been previously published. This report is the first to describe biomarkers for cutaneous SCC in two contexts, in vitro and in vivo. Although there was minimal overlap between the two different contexts using DNA microarrays, five genes were found common to both the cell lines and tissues, namely fibronectin 1, annexin A5, glyceraldehyde 3-phosphate dehydrogenase, zinc-finger protein 254, and huntingtin-associated protein interacting protein. Some of our previously published biomarkers of normal keratinocytes were down-regulated in SCC, suggestive of the dedifferentiated status of the transformed cells. While recent reports have identified some of the same genes as SCC biomarkers, for instance in head-and-neck cancer, thereby validating our approach, we have identified some novel biomarkers for cutaneous disease. These biomarker lists may be useful in molecular diagnostics of non-melanoma skin cancer, and a subset of the biomarkers might serve as suitable targets for drug discovery efforts of therapies for SCC. © 2003 Elsevier Science (USA). All rights reserved.

TITLE:Proteomic analysis of pancreatic ductal carcinoma cells treated with 5-aza-2ā€²-deoxycytidine 

[2-s2.0-0346458828] Refers [2-s2.0-0036280779] LEVEL: 3
A pancreatic adenocarcinorna cell line (PaCa44), which contains, among other alterations, a methylated p16 promoter, was treated with a chemoterapeutic agent, 5-aza-2?-deoxycytidine (DAC), in order to evaluate the effect of this drug on cell growth and protein expression. Cell proliferation was strongly inhibited by a 24 h DAC treatment and this inhibition lasted for at least 10 days. Master maps of control and treated PaCa44 cells were generated by analysis with the PDQuest software. The comparison between such maps showed up- and downregulation of 45 polypeptide chains, of which 32 were downregulated and 13 upregulated, out of a total of 700 spots detected by a medium-sensitivity stain, micellar Coomassie Brilliant Blue. Fingerprinting by mass spectrometry analysis enabled the identification of 36 of these spots. Among the major changes in DAC-treated cells: cofilin and profilin 1 are silenced; coactosin, peptidyl-propyl cis-trans isomerase A and cystatin B are decreased by 22, 16- and 15-fold, respectively; stress-70 protein, superoxide dismutase and protein disulfide isomerase A3 are increased by 13-, 11-, and 5-fold, respectively. The significance of some of these major changes is discussed.

TITLE:Proteomic profiling for cancer progression: Differential display analysis for the expression of intracellular proteins between regressive and progressive cancer cell lines 

[2-s2.0-16344381930] Refers [2-s2.0-0036280779] LEVEL: 3
Tumor development and progression consist of a series of complex processes involving multiple changes in gene expression (Paolo et al. Physiol. Rev., 1993, 73, 161-195; Lance et al. Cell., 1991, 64, 327-336). Tumor cells acquire an invasive and metastatic phenotype that is the main cause of death for cancer patients. Therefore, for early diagnosis and effective therapeutic intervention, we need to detect the alterations associated with transition from benign to malignant tumor cells on a molecular basis. To unravel alterations concerned with tumor progression, the proteomic approach has attracted great attention because it can identify qualitative and quantitative changes in protein composition, including post-translational modifications. In this study, we performed proteomic differential display analysis for the expression of intracellular proteins in the regressive cancer cell line QR-32 and the inflammatory cell-promoting progressive cancer cell line QRsP-11 of murine fibrosarcoma by two-dimensional gel electrophoresis and mass spectrometry using an Agilent 1100 LC/MSD Trap XCT. We found 11 protein spots whose expression was different between QR-32 and QRsP-11 cells and identified nine proteins, seven of which, calreticulin precursor, tropomyosin 1 ? chain, annexin A5, heat shock protein (HSP)90-?, HSP90-?, PEBP, and Prx II, were over-expressed, and two, Anp32e and HDGF, which were down-regulated. The results suggest an important complementary role for proteomics in identification of molecular abnormalities in tumor progression. © 2005 WILEY-VGH Verlag GmbH & Co. KGaA.

TITLE:Proteomic characterization of harvested pseudopodia with differential gel electrophoresis and specific antibodies 

[2-s2.0-14744296225] Refers [2-s2.0-0036280779] LEVEL: 3
Malignant gliomas (astrocytomas) are lethal tumors that invade the brain. Invasive cell migration is initiated by extension of pseudopodia into interstitial spaces. In this study, U87 glioma cells formed pseudopodia in vitro as cells pushed through 3 ?m pores of polycarbonate membranes. Harvesting pseudopodia in a novel two-step method provided material for proteomic analysis. Differences in the protein profiles of pseudopodia and whole cells were found using differential gel electrophoresis (DIGE) and immunoblotting. Proteins from two-dimensional (2D) gels with MR's of 20-100 kDa and p/'s of 3.0-10.0 were identified by peptide mass fingerprinting analysis using mass spectrometry. For DIGE, lysates of pseudopodia and whole cells were each labeled with electrophilic forms of fluorescent dyes, Cy3 or Cy5, and analyzed as mixtures. Analysis was repeated with reciprocal labeling. Differences in protein distributions were detected by manual inspection and computer analysis. Topographical digital maps of the scanned gels were used for algorithmic spot matching, normalization of background, quantifying spot differences, and elimination of artifacts. Pseudopodial proteins in Coomassie-stained 2D gels included isoforms of glycolytic enzymes as the largest group, seven of 24 proteins. Peptide mass fingerprint analysis of DIGE gels demonstrated increased isoforms of annexin (Anx) I, AnxII, enolase, pyruvate kinase, and aldolase, and decreased mitochondrial manganese superoxide dismutase and transketolase in pseudopodia. Specific antibodies showed restricted immunoreactivity of the hepatocyte growth factor (HGF) ? chain to pseudopodia, indicating localization of its active form. Met (the HGF receptor), actin, and total AnxI were increased in pseudopodial lysates on immunoblots. Increased constituents of the pseudopodial proteome in glioma cells, identified in this study as actin, HGF, Met, and isoforms of AnxI, AnxII, and several glycolytic enzymes, represent therapeutic targets to consider for suppression of tumor invasion. © 2005 USCAP, Inc All rights reserved.

TITLE:Gene doping 

[2-s2.0-33645689379] Refers [2-s2.0-0036280779] LEVEL: 3
Together with the rapidly increasing knowledge on genetic therapies as a promising new branch of regular medicine, the issue has arisen whether these techniques might be abused in the field of sports. Previous experiences have shown that drugs that are still in the experimental phases of research may find their way into the athletic world. Both the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) have expressed concerns about this possibility. As a result, the method of gene doping has been included in the list of prohibited classes of substances and prohibited methods. This review addresses the possible ways in which knowledge gained in the field of genetic therapies may be misused in elite sports. Many genes are readily available which may potentially have an effect on athletic performance. The sporting world will eventually be faced with the phenomena of gene doping to improve athletic performance. A combination of developing detection methods based on gene arrays or proteomics and a clear education program on the associated risks seems to be the most promising preventive method to counteract the possible application of gene doping. © Georg Thieme Verlag KG.

TITLE:Differential proteome analysis of conditioned media to detect Smad4 regulated secreted biomarkers in colon cancer 

[2-s2.0-22044442559] Refers [2-s2.0-0036280779] LEVEL: 3
Smad4 is a tumor suppressor gene primarily involved in carcinogenesis of the pancreas and colon. The functional inactivation of Smad4 is a late step genetically. In pancreatic carcinogenesis, loss of Smad4 marks the transition to invasive growth. In colorectal cancers, the frequency of Smad4 inactivation is markedly increased in metastatic cancers. We have established cell biological models, re-expressing Smad4 in deficient human cancer cells, in which we could show that Smad4 is adequate to suppress tumor growth through suppression of angiogenic and invasive properties. Thus, pairs of Smad4-re-expressing and Smad4-deficient cells are prone to model the progression from premalignant stages to carcinomas in the carcinogenic process and may provide access to Smad4 targets of high clinical relevance. We present here a "differential secretome analysis", comparing all the proteins released in vitro from the Smad4-deficient and Smad4-re-expressing SW480 human colon carcinoma cells. The differential secretome catalog comprises more than 25 proteins including proteases and protease inhibitors, as well as established tumor biomarkers. In conclusion, this approach proved to be a sensitive tool to specifically detect Smad4 targets relevant for tumor-stroma interactions. It is also able to reflect complex alterations of cellular physiology. Moreover, the results support our hypothesis that human tumor markers detectable in serum may be identified through differential secretome analyses. © 2005 WILEY-VCH Verlag GmbH & Co. KGaA.

TITLE:Identification of metastasis-associated proteins through protein analysis of metastatic MDA-MB-435 and metastasis-suppressed BRMS1 transfected-MDA-MB-435 cells 

[2-s2.0-2942750477] Refers [2-s2.0-0036280779] LEVEL: 3
BRMS1 (breast cancer metastasis suppressor 1) was recently identified as a novel breast cancer metastasis suppressor gene. To further characterize BRMS1-mediated metastasis suppression, we applied two-dimensional proteomic and mass spectrometry (LC-tandem MS and MALDI-TOF) analysis to identify proteins differentially expressed between highly metastatic MDA-MB-435 cells and metastasis-suppressed BRMS1-transfected MDA-MB-435 cells. Quadruplicate independent 2D gels were run and analyzed under identical conditions. Following in-gel trypsin digestion of seven differentially expressed proteins, amino acid sequence and mass profiles of the peptides were generated. Proteins were identified from the NCBI non-redundant database using the search program TurboSequest. Differential expression was confirmed for five proteins, including annexin I and alpha B-crystallin, by Northern blot analysis and immunostaining. Furthermore, we showed that both proteins were expressed in vivo in lungs containing metastasized MDA-MB-435 cells but not expressed in normal lung tissue of athymic mice. Our results suggest that annexin I and alpha B-crystallin are important cellular proteins that are down regulated through BRMS1 mediated metastasis suppression.

TITLE:Expression of biomarkers modulating prostate cancer angiogenesis: Differential expression of annexin II in prostate carcinomas from India and USA 

[2-s2.0-2942624260] Refers [2-s2.0-0036280779] LEVEL: 3
Background: Prostate cancer (PCa) incidences vary with genetic, geographical and ethnic dietary background of patients while angiogenesis is modulated through exquisite interplay of tumor-stromal interactions of biological macromolecules. We hypothesized that comprehensive analysis of four biomarkers modulating angiogenesis in PCa progression in two diverse populations might explain the variance in the incidence rates. Results: Immunohistochemical analysis of 42 PCa biopsies reveals that though Anx-II expression is lost in both the Indian and American population with Gleason scores (GS) ranging between 6 and 10, up to 25 % of cells in the entire high grade (GS > 8) PD PCa samples from US show intense focal membrane staining for Anx-II unlike similarly graded specimens from India. Consistent with this observation, the prostate cancer cell lines PC-3, DU-145 and MDA PCa 2A, but not LNCaP-R, LNCAP-UR or MDA PCa 2B cell lines, express Anx-II. Transcriptional reactivation of Anx-II gene with Aza-dC could not entirely account for loss of Anx-II protein in primary PCa. Cyclooxygenase-2 (COX-2) was moderately expressed in most of high grade PIN and some MD PCa and surrounding stroma. COX-2 was not expressed in PD PCa (GS ?7-10), while adjacent smooth muscles cells stained weakly positive. Decorin expression was observed only in high grade PIN but not in any of the prostate cancers, atrophy or BPH while stromal areas of BPH stained intensively for DCN and decreased with advancing stages of PCa. Versican expression was weak in most of the MD PCa, moderate in all of BPH, moderately focal in PD PC, weak and focal in PIN, atrophy and adjacent stroma. Conclusions: Expression of pro- and anti-angiogenic modulators changes with stage of PCa but correlates with angiogenic status. Focal membrane staining of Anx-II reappears in high grade PCa specimens only from US indicating differential expression of Anx-II. COX-2 stained stronger in American specimens compared to Indian specimens. The sequential expression of DCN and VCN in progressive stages was similar in specimens from India and USA indicating no population-based differences. The mechanistic and regulatory role of Anx-II in PCa progression warrants further investigation. © 2003 Banerjee et al; licensee BioMed Central Ltd.

TITLE:Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry 

[2-s2.0-33745090846] Refers [2-s2.0-0036280779] LEVEL: 3
To better understand the molecular mechanisms underlying breast cancer metastasis and search for potential markers for metastatic progression, we have developed a highly metastatic variant of human MDA-MB-435 breast cancer cell line through in vivo stepwise selection of pulmonary metastatic cells caused by parental MDA-MB-435 cells in the athymic mice. Comparative proteomic analysis using 2-DE and LC-IT-MS revealed that 102 protein spots were reproducibly altered more than three-fold between the selected variant and its parental counterpart. Eleven differentially expressed protein spots were identified with high confidence using SEQUEST with uninterpreted tandem mass raw data. Cathepsin D precursor, peroxiredoxin 6 (PDX6), heat shock protein 27 (HSP27), HSP60, tropomyosin 1 (TPM1), TPM2, TPM3, TPM4, 14-3-3 protein epsilon, and tumor protein D54 were up-regulated in the highly metastatic variant, whereas alpha B-crystalline (CRAB) was only detected in its parental counterpart. Differential expression was confirmed for four proteins including PDX6, CRAB, TPM4, and HSP60 by real-time quantitative PCR and Western blotting analysis in our model. Immunohistochemical analysis in 80 breast cancer donors demonstrated a significant association of TPM4 (p = 0.002), HSP60 (p = 0.001), PDX6 (p = 0.002) but not CRAB (p = 0.113) staining with the presence of lymph node metastasis. In addition, TPM4 staining was also associated with clinical stage (p = 0.000), but no significant association was found between TPM4, PDX6, CRAB, and HSP60 expression and tumor size, hormone receptor, and HER-2 status (p > 0.05). The functional implication of these identified proteins was also discussed. These proteomic data are valuable and informative for understanding breast cancer metastasis and searching for potential markers for metastatic progression. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

TITLE:Identification of over-expressed proteins in oral squamous cell carcinoma (OSCC) patients by clinical proteomic analysis 

[2-s2.0-33845284524] Refers [2-s2.0-0036280779] LEVEL: 3
Background: Oral cancer is a worldwide problem. It is a universal aggressive disease in the population of smoking and drinking. The oral cancer mortality has been ranked 5th place in Taiwan in male cancer patients. A number of protein markers for oral cancer are still not applicable in large populations. Proteomic technologies provide excellent tools for rapid screening of a large number of potential biomarkers in malignant cells. Method: Proteomics and real-time quantitative RT-PCR were used to analyze over-expressed proteins in 10 OSCC patients. Result: Forty-one proteins were identified as commonly over-expressed in OSCC tissues. In OSCC tissues, ?B-crystallin, tropomyosin 2, myosin light chain 1, heat shock protein 27 (HSP27), stratifin, thioredoxin-dependent peroxide reductase, flavin reductase, vimentin, rho GDP-dissociation inhibitor 2 (rho GDI-2), glutathione S-transferase Pi (GST-pi) and superoxide dismutase [Mn] (MnSOD) were significantly over-expressed (an average of 7.2, 6.0, 5.7, 4.3, 3.6, 3.4, 3.0, 3.0, 2.6, 2.5, 2.1-fold, respectively). In real-time quantitative RT-PCR analysis, the gene expressions of ?B-crystallin, HSP27 and MnSOD were also increased in the cancer tissues, consistent with proteomic results. Conclusion: The identified proteins in this experiment may be used in future studies of carcinogenesis or as diagnostic markers and therapeutic targets for OSCC. © 2006 Elsevier B.V. All rights reserved.

TITLE:Proteome analysis of a human uveal melanoma primary cell culture by 2-DE and MS 

[2-s2.0-29544435751] Refers [2-s2.0-0036280779] LEVEL: 3
We present here the first proteomics analysis of uveal melanoma (UM) cells. These cells represent a good model for the identification of polypeptide markers, which could be developed as diagnostic tools. UM is the most common primary intraocular tumour in adults. In contrast to other cancers, the survival rate of patients with this malignancy has changed little over the past few decades; a better understanding of the molecular biology of UM oncogenesis and metastasis is needed to build the basis for the identification of novel drug targets. In the study presented here, proteins from a UM primary cell culture were separated by 2-DE using a pI 3-10 gradient; 270 spots were analysed by LC-MS/MS, identifying 683 proteins derived from 393 different genes. Of those, 69 (18%) are related to cancer processes involving cell division, proliferation, invasion, metastasis, oncogenesis, drug resistance and others. To our knowledge, 96% of the proteins identified, including 16 hypothetical proteins, have never been reported in UM before. This study represents the first step towards the establishment of a UM protein database as a valuable resource for the study of this malignancy. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA.

TITLE:Discovery and Immunologic Validation of New Antigens for Therapeutic Cancer Vaccines 

[2-s2.0-1542286638] Refers [2-s2.0-0036280779] LEVEL: 3
Immunotherapy using both active and passive approaches is increasingly being used as a modality to treat human cancer. The last decade has seen a tremendous burst of activity in antigen discovery in cancer, and many new targets have now been identified for both monoclonal antibody therapy and active immunization. In addition, advances have been made in our understanding of the immune response against cancer and how new vaccine vectors, such as poxviruses, alphaviruses and bacterial vectors, can be used to overcome some of the traditional hurdles (e.g. self-tolerance and immune suppression in cancer patients) that have hindered the generation of effective antitumor immune responses. Improvements in genomics technology in the area of DNA microarrays and differential display and subtractive hybridization together with a new wave of mass spectrometry-based proteomics tools, as well as more sensitive assays to validate the immunoreactivity of new antigens, have all accelerated the rate of new antigen discovery in cancer. This rapid progress should initiate a major paradigm shift in how we treat cancer within the next 10 years, where, instead of being a novelty, the combination of targeted T cell-based vaccines and antiangiogenesis therapies will be routinely combined with traditional chemotherapy. The successful combination of these approaches will change the face of cancer from a relatively acute, life-threatening disease to a manageable chronic disorder with long survival times. Copyright © 2004 S. Karger AG, Basel.

TITLE:Annexin I regulates SKCO-15 cell invasion by signaling through formyl peptide receptors 

[2-s2.0-33745822755] Refers [2-s2.0-0036280779] LEVEL: 3
Annexin 1 (AnxA1) is a multifunctional phospholipid-binding protein associated with the development of metastasis in some invasive epithelial malignancies. However, the role of AnxA1 in the migration/invasion of epithelial cells is not known. In this study, experiments were performed to investigate the role of AnxA1 in the invasion of a model epithelial cell line, SKCO-15, derived from colorectal adenocarcinoma. Small interfering RNA-mediated knockdown of AnxA1 expression resulted in a significant reduction in invasion through Matrigel-coated filters. Localization studies revealed a translocation of AnxA1 to the cell surface upon the induction of cell migration, and functional inhibition of cell surface AnxA1 using antiserum (LCO1) significantly reduced cell invasion. Conversely, SKCO-15 cell invasion was increased by ?2-fold in the presence of recombinant full-length AnxA1 and the AnxA1 N-terminal-derived peptide mimetic, Ac2-26. Because extracellular AnxA1 has been shown to regulate leukocyte migratory events through interactions with n-formyl peptide receptors (nFPRs), we examined the expression of FPR-1, FPRL-1, and FPRL-2 in SKCO-15 cells by reverse transcriptase-PCR and identified expression of all three receptors in this cell line. Treatment of SKCO-15 cells with AnxA1, Ac2-26, and the classical nFPR agonist, formylmethionylleucylphenylalanine, induced intracellular calcium release consistent with nFPR activation. Furthermore, the nFPR antagonist, Boc2, abrogated the AnxA1 and Ac2-26-induced intracellular calcium release and increase in SKCO-15 cell invasion. Together, these results support an autocrine/paracrine role for membrane AnxA1 in stimulating SKCO-15 cell migration through nFPR activation. The findings in this study suggest that activation of nFPRs stimulates epithelial cell motility important in the development of metastasis as well as wound healing. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:Mass spectrometry in diagnostic oncoproteomics 

[2-s2.0-24944529073] Refers [2-s2.0-0036280779] LEVEL: 3
Diagnostic oncoproteomics is the application of proteomic techniques for the diagnosis of malignancies. A new mass spectrometric technology involves surface enhanced laser desorption ionization combined with time-of flight mass analysis (SELDI-TOF-MS), using special protein chips. After the description of the relevant principles of the technique, including approaches to proteomic pattern diagnostics, applications are reviewed for the diagnosis of ovarian, breast, prostate, bladder, pancreatic, and head and neck cancers, and also several other malignancies. Finally, problems and prospects of the approach are discussed. Copyright © Taylor & Francis Inc.

TITLE:The RacGEF Tiam1 inhibits migration and invasion of metastatic melanoma via a novel adhesive mechanism 

[2-s2.0-7244251723] Refers [2-s2.0-0036280779] LEVEL: 3
Rho-like GTPases such as RhoA, Rac1 and Cdc42 are key regulators of actin-dependent cell functions including cell morphology, adhesion and migration. Tiam1 (T lymphoma invasion and metastasis 1), a guanine nucleotide exchange factor that activates Rac, is an important regulator of cell shape and invasiveness in epithelial cells and fibroblasts. Overexpression of Tiam1 in metastatic melanoma cells converted the constitutive mesenchymal phenotype into an epithelial-like phenotype. This included the induction of stringent cell-cell contacts mediated by the Ig-like receptor ALCAM (activated leukocyte cell adhesion molecule) and actin redistribution to cell-cell junctions. This phenotypic switch was dependent on increased Rac but not Rho activity, and on the redistribution and adhesive function of ALCAM, whereas cadherins were not involved. Although cell proliferation was significantly enhanced, the gain of cell-cell junctions strongly counteracted cell motility and invasion as shown for two- and three-dimensional collagen assays as well as invasion into human skin reconstructs. The reverse transition from mesenchymal invasive to a resident epithelial-like phenotype implicates a role for Tiam1/Rac signaling in the control of cell-cell contacts through a novel ALCAM-mediated mechannism.

TITLE:Identification and analysis of altered Ī±1,6-fucosylated glycoproteins associated with hepatocellular carcinoma metastasis 

[2-s2.0-33751108967] Refers [2-s2.0-0036280779] LEVEL: 3
Tumor metastasis might be associated with the expression levels of cellular glycoproteins and the alteration of their glycan parts. In order to screen the aberrantly ?1,6-fucosylated glycoproteins related to hepatocellular carcinoma (HCC) metastasis, a high-throughput glycomic approach which consisted of 2-DE, electronic transfer of proteins, lectin affinity blot and precipitation, and MALDI-TOF-MS/MS, was established. Lens culinaris agglutinin (LCA) affinity glycoprotein profiles of higher and lower metastatic HCC cell lines were compared and analyzed. Seven out of 34 identified glycoproteins were differentially displayed; they were cytokeratin 8 (CK8), annexin I, annexin II, heterogeneous nuclear ribonucleoprotein A/B, PDZ and LIM domain 1, RNA-binding motif protein 4, and poly(rC)-binding protein 1. On comparison with Hep3B, CK8 showed a higher affinity to Ricinus communis agglutinin 1 (RCA-I) and LCA, and annexin I presented a higher affinity to LCA and Con A by the lectin-binding assay. Furthermore, the up-regulation of CK8, annexin I, and annexin II were found by Western blot and immunofluorescence analysis in higher metastatic HCC cell lines. This implied that the alteration of CK8, annexin I, and annexin II both in their expression levels and their glycan parts might be related to metastatic ability, and play a critical role in the process of HCC metastasis. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

TITLE:Angiogenesis-associated protein annexin II in breast cancer: Selective expression in invasive breast cancer and contribution to tumor invasion and progression 

[2-s2.0-33750812269] Refers [2-s2.0-0036280779] LEVEL: 3
Many advanced human tumors including breast cancer overproduce plasmin that is known to promote angiogenesis and metastasis. The mechanism of this effect is poorly understood. Here we report that annexin II, an endothelial co-receptor for tPA (tissue-type plasminogen activator) and plasminogen, was undetectable in normal and hyperplastic ductal epithelial cells and ductal complexes. By contrast, it was consistently expressed in invasive breast cancer and ductal carcinoma in situ (DCIS) indicating its involvement in breast cancer. Using the well established invasive/metastatic MDA-MB231 cell line and the noninvasive/nonmetastatic MCF-7 human breast cancer cell line, we investigated the mechanism by which annexin II regulates breast cancer progression and metastasis. Western and Northern blot analyses demonstrate selective expression of annexin II in MDA-MB231 cells but not in poorly invasive MCF-7 cells suggesting its participation in invasive breast cancer. Since annexin II is a receptor for plasminogen, we tested whether MDA-MB231 cells are capable of producing plasmin in vitro. MDA-MB231 cell membranes induced plasmin generation in a time-dependent manner while those from MCF-7 cells failed to convert plasminogen to plasmin. The generated plasmin is capable of degrading ECM consequently facilitating cell invasion and migration, biological functions required for angiogenesis and metastasis. Plasmin generation and its dependent invasion and migration can be blocked by a monoclonal antibody to annexin II or angiostatin, potent inhibitors of angiogenesis, breast cancer, and metastasis. Our findings indicate that annexin II-dependent localized plasmin generation by human breast cancer cells could contribute to angiogenesis and metastasis. These results suggest that annexin II may be an attractive target for new anti-angiogenic and anti-breast cancer therapies. © 2006 Elsevier Inc. All rights reserved.

TITLE:The characterization of the invasion phenotype of uveal melanoma tumour cells shows the presence of MUC18 and HMG-1 metastasis markers and leads to the identification of DJ-1 as a potential serum biomarker 

[2-s2.0-33746377043] Refers [2-s2.0-0036280779] LEVEL: 3
Uveal malignant melanoma (UM) is the most frequent primary intraocular tumour in adult humans. Because the survival rate of patients with UM has changed little in the past few decades, a better understanding of the molecular events governing UM development and the identification of markers indicating the potential for metastasis at the time of diagnosis are necessary to design improved and more specific treatments. In this study, we investigated UM tumour development by comparing two recently established UM cultures with different invasion potential by two-dimensional gel electrophoresis. Protein features expressed differentially were identified by mass spectrometric analysis. Potential markers were assayed in both cultures and in long-term established UM cell lines (UW-1, OCM-1, SP6.5 and 92.1) by Western blotting and their role in invasion analysed using Matrigel membranes. Comparative analysis revealed that UM cultures with low-and high-grade invasion potential differ in their cellular metabolism and, more interestingly, in several cancer-associated proteins, including those implicated in cell adhesion and migration, proliferation and various oncogenes. Our data indicate a correlation between MUC18 and HMG-1 expression and the invasiveness of UM cells. We also demonstrate the expression and secretion of DJ-1 oncoprotein by UM cells. We suggest a possible role for MUC18 and HMG-1 proteins in UM cell invasion. The secretion of DJ-1 by UM cells, and the ability to detect this protein in UM patients' sera implicate it as a potential noninvasive biomarker for this malignancy. © 2006 Wiley-Liss, Inc.

TITLE:Proteomics in cancer cell research: An analysis of therapy resistance 

[2-s2.0-3242668211] Refers [2-s2.0-0036280779] LEVEL: 3
Proteomics, the global analysis of expressed cellular proteins, provides powerful tools for the detailed comparison of proteins from normal and neoplastic tissue. In particular, cancer cell culture models are suited for applying proteomics techniques, such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry, to identify specific protein expression profiles and/or proteins that may be associated with a defined phenotype of the cancer cells. As an instance of such an application of proteomics techniques, the detailed proteome analyses of different drug-resistant and thermoresistant cancer cell lines will be discussed. Finally, the potential roles of newly identified factors in a distinct biological mechanism have to be proven by functional studies. This experimental validation strategy will be discussed for two different factors identified by 2D-PAGE analyses of drug-resistant carcinoma cell lines, the "transporter associated with antigen presentation 1" (TAP1) and 14-3-3 ?. © 2004 Elsevier GmbH. All rights reserved.

TITLE:Expanding the protein catalogue in the proteome reference map of human breast cancer cells 

[2-s2.0-33646264528] Refers [2-s2.0-0036280779] LEVEL: 3
In this report we present a catalogue of 162 proteins (including isoforms and variants) identified in a prototype of proteomic map of breast cancer cells. This work represents the prosecution of previous studies describing the protein complement of breast cancer cells of the line 8701-BC, which has been well characterized for several parameters, providing to be a useful model for the study of breast cancer-associated candidate biomarkers. In particular, 110 spots were identified ex novo by PMF, or validated following previous gel matching identification method; 30 were identified by N-terminal microsequencing and the remaining by gel matching with maps available from our former work. As a consequence of the expanded number of proteins, we have updated our previous classification extending the number of protein groups from 4 to 13. In order to facilitate comparative proteome studies of different kinds of breast cancers, in this report we provide the whole complement of proteins so far identified and grouped into the new classification. A consistent number of them were not described before in other proteomic maps of breast cancer cells or tissues, and therefore they represent a valuable contribution for breast cancer protein databases and for future application in basic and clinical researches. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

TITLE:Protein profiling in respiratory disease: Techniques and impact 

[2-s2.0-12344288221] Refers [2-s2.0-0036280779] LEVEL: 3
Multifactorial diseases such as respiratory disease call for a global analysis of such disorders. Recent advances in protein profiling techniques may allow for early diagnosis of respiratory disease, which is crucial for intervention and treatment. In order to reduce false-positive rates, clinical diagnosis requires a degree of sensitivity and specificity to be an effective screening tool. Protein profiles identified by ProteinChip® (Ciphergen Biosystems) technology coupled with mass spectrometry affords a global analysis of clinical samples and is beginning to reach acceptable levels of sensitivity and specificity. Combining the profile with another diagnostic tool enhances the effectiveness of protein profiles to classify disease. Although current efforts have centered on serum protein profiling, the local environment of the lung may be better reflected in proteins of bronchoalveolar lavage or sputum. Identification of biomarkers of disease by protein profiling analyses may lead to an understanding of the mechanisms of this disease and contribute to the discovery of new therapeutics for the prevention and treatment of disease. Advancing these analyses are techniques such as ProteinChip mass spectrometry, laser capture microdissection, tissue microarrays and fluorescently labeled antibody bead arrays, which enable the direct global analysis of complex mixtures. Effective high-throughput and ease of use of clinical testing will arrive with improvements in bioinformatics and decreases in instrumentation costs. © Future Drugs Ltd. All rights reserved.

TITLE:Identification of porcine oocyte proteins that are associated with somatic cell nuclei after co-incubation 

[2-s2.0-4544223880] Refers [2-s2.0-0036280779] LEVEL: 3
Relatively little is known with respect to the oocyte proteins that are involved in nuclear reprogramming of somatic cells in mammals. The aim of the present study was to use a cell-free incubation system between porcine oocyte proteins and somatic cell nuclei and to identify oocyte proteins that remain associated with these somatic cell nuclei. In two separate experiments, porcine oocytes were either labeled with biotin to label total proteins at the germinal vesicle stage or metaphase II stage or they were labeled with 0.1 mM 35S-methionine either during the first 6 h or 22-28 h of in vitro maturation to characterize protein synthesis during two distinct phases. To determine which oocyte proteins associate with somatic nuclei, labeled proteins were incubated in a collecting buffer and energy-regenerating system with isolated ovarian epithelial-like cell nuclei. After incubation, the nuclei were subjected to a novel affinity-binding system to recover biotin-labeled oocyte proteins or two-dimensional SDS-PAGE for separation and visualization of radiolabeled proteins. Proteins of interest were sent for identification using either matrix-assisted laser desorption/ionization time of flight or liquid chromatography-tandem mass spectrometry. Of the proteins that remain associated with isolated nuclei after incubation, 4 were identified using the affinity-binding system and 24 were identified using mass spectrometry and the two-dimensional gel interface. This study has identified porcine oocyte proteins that associate with somatic cell nuclei in a cell-free system using proteomics techniques, providing a novel way to identify oocyte proteins potentially functionally involved in nuclear reprogramming.

TITLE:Identification of Ī±-enolase as an autoantigen in lung cancer: Its overexpression is associated with clinical outcomes 

[2-s2.0-33750285970] Refers [2-s2.0-0036280779] LEVEL: 3
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TITLE:Towards a lung adenocarcinoma proteome map: Studies with SP-C/c-raf transgenic mice 

[2-s2.0-33744476697] Refers [2-s2.0-0036280779] LEVEL: 3
We report mapping of proteins of adenocarcinomas of the lung as a result of overexpression of the oncogenically activated N-terminal deletion mutant c-raf-1 BxB through usage of the human SP-C promotor. Proteins from non-transgenic controls and tumors were extracted with a lysis buffer containing 5 mol/L urea, 2 mol/L thiourea, 40 mmol/L Tris, 4% CHAPS, 100 mmol/L DTT, 0.5% BioLyte 3-10, separated by 2-DE and studied by image analysis. On average, 300-600 protein spots per gel were excised and analyzed by MALDI-TOF and -TOF/TOF MS. More than 1000 of the CBB-stained proteins were identified and traced back to 100 different gene products, including many of their isoforms. We observed significant changes in the expression of proteins involved in cellular defense or glycolysis, and this included glutathione S-transferase, peroxiredoxin 6, and ?-enolase, among others. Proteins associated with lung tumor growth and/or metastasis, i.e., lung carbonyl reductase, differed in expression, as did tumor-associated expression of cell adhesion and membrane-bound proteins such as vinculin. This map provides valuable insight into expression of pulmonary proteins associated with lung adenocarcinomas, some of which may be of utility as diagnostic markers in clinical trials. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

TITLE:Ubiquitous mitochondrial creatine kinase downregulated in oral squamous cell carcinoma 

[2-s2.0-33644842788] Refers [2-s2.0-0036280779] LEVEL: 3
In this study, we performed two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time of fly mass spectrometry to identify the protein(s) associated with the development of oral squamous cell carcinomas (OSCCs) by comparing patterns of OSCC-derived cell lines with normal oral keratinocytes (NOKs), and found that downregulation of ubiquitous mitochondrial creatine kinase (CKMTI) could be a good candidate. Decreased levels of CKMTI mRNA and protein were detected in all OSCC-derived cell lines examined (n = 9) when compared to those in primary normal oral keratinocytes. Although no sequence variation in the coding region of the CKMTI gene with the exception of a nonsense mutation in exon 8 was identified in these cell lines, we found a frequent hypermethylation in the CpG island region. CKMTI expression was restored by experimental demethylation. In addition, when we transfected CKMTI into the cell lines, they showed an apoptotic phenotype but no invasiveness. In clinical samples, high frequencies of CKMTI downregulation were detected by immunohistochemistry (19 of 52 (37%)) and quantitative real-time RT-PCR (21 of 50 (42%)). Furthermore, the CKMTI expression status was significantly correlated with tumour differentiation (P<0.0001). These results suggest that the CKMTI gene is frequently inactivated during oral carcinogenesis and that an epigenetic mechanism may regulate loss of expression, which may lead to block apoptosis. © 2006 Cancer Research UK.

TITLE:Proteomics in primary brain tumors 

[2-s2.0-3242698155] Refers [2-s2.0-0036280779] LEVEL: 3
Genomic damage present in tumors may lead to abnormal or altered expression of proteins. Some of the findings of genetic explorations of brain tumors have had their impact on making the diagnosis or are important with respect to therapeutic decisions. The identification of individual proteins or clusters of proteins expressed in neoplastic tissues potentially may have an even more important relevance for making the diagnosis, prognosis and treatment outcome. Diverse posttranslational modifications of proteins may be linked to neoplastic lineage or stage. For the rapidly developing fields of proteomics and its integration with genomics and transcriptomics (by now called operomics) the application of bioinformatics is crucial. This review addresses the nascent field of proteomics and its diagnostic potential in the feld of primary brain tumors. Various technical approaches to separate and identify proteins are described, including the most recent developments in the analytical proteomic technology.

TITLE:Serum amyloid A protein: A potential biomarker correlated with clinical stage of lung cancer 

[2-s2.0-34147220127] Refers [2-s2.0-0036280779] LEVEL: 3
Objective To identify serum diagnosis or progression biomarkers in patients with lung cancer using protein chip profiling analysis. Method Profiling analysis was performed on 450 sera collected from 213 patients with lung cancer, 19 with pneumonia, 16 with pulmonary tuberculosis, 65 with laryngeal carcinoma, 55 with laryngopharyngeal carcinoma patients, and 82 normal individuals. A new strategy was developed to identify the biomarkers on chip by trypsin pre-digestion. Results Profiling analysis demonstrated that an ll.okDa protein was significantly elevated in lung cancer patients, compared with the control groups (P<0.001). The level and percentage of 11.6kDa protein progressively increased with the clinical stages I-IV and were also higher in patients with squamous cell carcinoma than in other subtypes. This biomarker could be decreased after operation or chemotherapy. On the other hand, 11.6kDa protein was also increased in 50% benign diseases of lung and 13% of other cancer controls. After trypsin pre-digestion, a set of new peptide biomarkers was noticed to appear only in the samples containing a 11.6kDa peak. Further identification showed that 2177Da was a fragment of serum amyloid A (SAA, MW 11.6kDa). Two of the new peaks, 1550Da and 1611Da, were defined from the same protein by database searching. This result was further confirmed by partial purification of 11.6kDa protein and MS analysis. Conclusion SAA is a useful biomarker to monitor the progression of lung cancer and can directly identify some biomarkers on chip. Copyright © 2007 by China CDC.

TITLE:Protein pattern difference in the colon cancer cell lines examined by two-dimensional differential in-gel electrophoresis and mass spectrometry 

[2-s2.0-33751402104] Refers [2-s2.0-0036280779] LEVEL: 3
Purpose. The pivotal metastatic processes of colorectal cancer (CRC) have yet to be fully investigated by a comprehensive all-inclusive protein analysis. We used two-dimensional differential in-gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) to investigate the protein pattern changes during the metastasis of CRC. Two CRC cell lines were investigated: SW480 derived from the primary lesion and SW620 derived from lymph node metastasis in the same patient. Methods. The two cell lines were compared using 2D-DIGE with a maleimide CyDye fluorescent protein labeling technique, which has an enhanced sensitivity for many proteins at a low concentration. A comprehensive proteomics analysis was performed by the dual-labeling method using Cy3 and Cy5 and by LC/MS/MS. In addition, an in vivo experiment of metastasis using nude mice was performed by the injection of the two cell lines into the spleen. Results. Among approximately 1 500 proteins, we detected 9 protein spots with definitively significant changes between the two cell lines. Three out of the nine proteins were validated by a Western blot analysis. Alpha-enolase and triosephosphate isomerase were significantly upregulated in SW620 in comparison to SW480. Annexin A2 (annexin II) was significantly downregulated in SW620 compared to SW480. Neither liver metastasis nor peritoneal dissemination was established in the metastatic experiment using SW480 but some liver and peritoneal metastases occurred in the experiment using SW620. An in vivo metastatic experiment using SW620 showed the expressions of alpha-enolase and triosephosphate isomerase to increase in the liver metastases in comparison to those in the splenic implanted lesion. The expressions of triosephosphate isomerase increased in the peritoneal lesions in comparison to those in the splenic implanted lesion. Conclusions. 2D-DIGE and LC/MS/MS techniques identified nine proteins that increased significantly more in SW620 than in SW480. The finding of our in vivo metastatic experiment suggests that alpha-enolase and triosephosphate isomerase, at least in part, may be associated with the metastatic process of these two cell lines. © 2006 Springer-Verlag Tokyo.

TITLE:Antibody-directed targeting of angiostatin's receptor annexin II inhibits Lewis Lung Carcinoma tumor growth via blocking of plasminogen activation: Possible biochemical mechanism of angiostatin's action 

[2-s2.0-33750811184] Refers [2-s2.0-0036280779] LEVEL: 3
Angiostatin, the N-terminal four kringles (K1-4) of parent molecule plasminogen, is reported to block Lewis Lung Carcinoma (LLC) tumor growth and metastasis. However, angiostatin's mechanism of action is unclear. We earlier reported that angiostatin binds to cell surface annexin II through the lysine-binding domain (kringles 1-4) [Tuszynski, G.P., Sharma, M., Rothman, V.L., Sharma, M.C., 2002. Angiostatin binds to tyrosine kinase substrate annexin II through the lysine-binding domain in endothelial cells. Microvasc. Res. 64:448-462.]). We now show that annexin II on the cell surface of LLC cells regulates conversion of plasminogen to plasmin. Activation of plasminogen to plasmin is time-dependent, with the linear activation lasting up to 120 min. Monoclonal antibodies to annexin II reduced plasminogen activation by 92.6%, suggesting a specific role of annexin II in plasmin generation. Angiostatin also reduced plasmin generation by 81.6%, suggesting that angiostatin may be competing with plasminogen through lysine-binding domain. ?-Aminocaproic acid, a lysine analogue, effectively blocked plasminogen activation indicating that, indeed, the lysine-binding site of the kringles domain is required for activation. These data suggest that annexin II may be a receptor target for angiostatin's action. Therefore, we tested the effect of high affinity monoclonal antibody to annexin II in mouse model of LLC. A single dose of antibody treatment inhibited LLC tumor growth almost 70% with concomitant inhibition of circulating plasmin generation and its proteolytic activity. Taken together, it is possible that inhibition of LLC tumor growth and metastasis reported by angiostatin therapy may be due to blocking of annexin-II-dependent plasmin generation. Plasmin is known to influence angiogenic, invasive and metastatic capability of tumors. © 2006 Elsevier Inc. All rights reserved.

TITLE:Annexin-1 downregulation in thyroid cancer correlates to the degree of tumor differentiation 

[2-s2.0-33745769758] Refers [2-s2.0-0036280779] LEVEL: 3
We investigated the expression of annexin-1 (ANXA1) in thyroid carcinoma cell lines and in thyroid cancers with a different degree of differentiation. The highest level of ANXA1 expression examined by Western blotting was detected in the papillary carcinoma cells (NPA) and in the follicular cells (WRO). On the other hand, the most undifferentiated thyroid carcinoma cells (ARO and FRO) presented the lowest level of ANXA1 expression. In surgical tissue specimens from 32 patients with thyroid cancers, we found high immunoreactivity for ANXA1 in papillary (PTC) and follicular (FTC) thyroid cancers while in undifferentiated thyroid cancers (UTC) the expression of the protein was barely detectable. Control thyroid tissue resulted positive for ANXA1. In summary, 70% of UTC examined weakly expressed ANXA1, whereas 65% of PTC or FTC specimens tested showed high expression of the protein. Thus ANXA1 expression may correlate with the tumorigenesis suggesting that the protein may represent an effective differentiation marker in thyroid cancer. ©2006 Landes Bioscience.

TITLE:Biomarker discovery by tissue microdissection and ProteinChipĀ® array analysis 

[2-s2.0-0038354751] Refers [2-s2.0-0036280779] LEVEL: 3
In recent years, the search for new cancer biomarkers received a strong impulse from genomic and proteomic high throughput techniques. Biomarkers or biomarker patterns should enable scientists or medical staff to make a more reliable early diagnosis of certain human diseases, especially malignant tumors, and to make prediction of their progression, which could further contribute to a more differentiated, individually orientated tumor therapy. However, until now relevant markers have been established for only a few tumor diseases. One of the most promising proteomic tools for the detection of new cancer biomarkers is the ProteinChip® System technology. This mass spectrometry based system (SELDI; Surface Enhanced Laser Desorption and Ionization) appears to have a high potential for biomarker discovery and may serve in future as a clinical diagnostic assay platform. Herein, we will review the current scientific literature dealing with cancer biomarker discovery by users of the ProteinChip System. Special emphasis is given to the investigation of microdissected tumor material. Further, we will discuss bioinformatic tools, suitable for the localisation and assessment of potential biomarkers.

TITLE:Expression of annexin A1 in esophageal and esophagogastric junction adenocarcinomas: Association with poor outcome 

[2-s2.0-33748028823] Refers [2-s2.0-0036280779] LEVEL: 3
Purpose: Annexin A1 (ANXA1) is a calcium-binding protein involved in arachidonic acid metabolism and epidermal growth factor receptor tyrosine kinase pathway. ANXA1 has been implicated in early squamous cell carcinogenesis of esophagus and correlates with degree of tumor differentiation. However, the role of ANXA1 in esophageal adenocarcinoma is unclear. Our goal was to evaluate ANXA1 expression and determine its prognostic significance in adenocarcinoma of the esophagus and esophagogastric junction. Experimental Design: This study included 104 consecutive patients with primary resected esophageal and esophagogastric junction adenocarcinomas (11 stage I, 24 stage II, 53 stage III, and 16 stage IV). ANXA1 protein expression in each tumor was assessed by immunohistochemical staining of tissue microarrays. ANAX1 expression level was classified as high (>25% of tumor cells with cytoplasmic staining), low (<25% of tumor cells with cytoplasmic staining), or negative; and was correlated with clinicopathologic features and patients' outcomes. Results: High ANXA1 expression was present in 39% (41 of 104) of tumors and was associated with higher pathologic T stage (P = 0.03) and distant metastasis (P = 0.04). High ANXA1 expression correlated with increased recurrence rate (P = 0.004) and decreased overall survival (P - 0.003) in univariate analysis. In multivariate analysis, ANXA1 expression and pN stage significantly correlated with recurrence rate (P - 0.008 and P < 0.001, respectively) and overall survival (P = 0.02 and P < 0.001, respectively) independent of T stage. Conclusion: Our results indicate that high ANXA1 expression is frequent in esophageal and esophagogastric junction adenocarcinomas, correlates with more advanced pathologic T stage and the presence of distant metastasis, and is an independent prognostic factor for patient survival. © 2006 American Association for Cancer Research.

TITLE:The role of annexin II in angiogenesis and tumor progression: A potential therapeutic target 

[2-s2.0-36949012954] Refers [2-s2.0-0036280779] LEVEL: 3
It is well established that human tumors overproduce plasmin a serine protease that is known to promote angiogenesis, tumor growth and metastasis. However, the mechanism by which endothelial or tumor cells regulate the proteolytic activity of plasmin is not well understood. Cell surface receptors regulate activation of plasminogen to plasmin and its proteolytic activity. Annexin II is one of the well studied receptors for plasminogen and tPA, which binds to plasminogen and converts it to plasmin. Plasmin is a highly reactive enzyme which is physiologically involved in fibrinolysis. Since the proteolytic activity of plasmin is very tightly regulated, uncontrolled production of plasmin can degrade extracelluar matrix (ECM) and basement membrane (BM) of the surrounding blood vessels. Thus plasmin plays an important role in neoangiogenesis and cancer invasion and metastasis. Therefore, the receptor which regulates plasmin generation may be an attractive target for the development of anti-cancer/anti-metastatic agents. Angiostatin (AS), internal fragment of plasminogen, has been reported to inhibit human tumor growth and metastasis. We have shown that AS binds to endothelial/cancer cell surface annexin II with high affinity and interferes with plasmin generation suggesting that the role of plasmin/ plasminogen system may be more complex than we previously thought. In this review we provide a comprehensive analysis of the literature in context of the role of annexin II in angiogenesis, tumor progression and metastasis. Compelling evidence from the literature and our own findings suggest that annexin II may be a potential target for the development of effective therapeutic strategies for the treatment of cancer and its induced metastasis. © 2007 Bentham Science Publishers Ltd.

TITLE:Proteomic profiling of proteins dysregulted in Chinese esophageal squamous cell carcinoma 

[2-s2.0-34447536140] Refers [2-s2.0-0036280779] LEVEL: 3
Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer death in China. In the present study, proteins in tumors and adjacent normal esophageal tissues from 41 patients with ESCC were extracted, and two-dimensional electrophoresis (2-DE) was performed using the pH 3-10 and 4-7 immobilized pH gradient strips. The protein spots expressed differentially between tumors and normal tissues were identified by matrix-assisted laser desorption/ionization and liquid chromatography electrospray/ionization ion trap mass spectrometry. A total of 22 proteins differentially expressed between ESCC and normal esophageal tissues were identified, in which 17 proteins were upregulated and 5 downregulated in tumors. Biological functions of these proteins are related to cell signal transduction, cell proliferation, cell motility, glycolysis, regulation of transcription, oxidative stress processes, and protein folding. Some of the proteins obtained were confirmed by Western blotting and immunohistochemical staining. We showed that high expression of calreticulin and 78-kDa glucose-regulated protein (GRP78) were correlated with poor prognosis by Kaplan-Meier analysis and log rank analysis. Zinc finger protein 410, annexin V, similar to the ubiquitin-conjugating enzyme E2 variant 1 isoform c, mutant hemoglobin beta chain, TPM4-ALK fusion oncoprotein type 2, similar to heat shock congnate 71-kDa protein, GRP78, and pyruvate kinase M2 (M2-PK) were for the first time observed to be dysregulated in human ESCC tissues. The proteins here identified will contribute to the understanding of the tumorigenesis and progression of Chinese ESCC and may potentially provide useful markers for diagnosis or targets for therapeutic intervention and drug development. © 2007 Springer-Verlag.

TITLE:Antitumor activity of CTFB, a novel anticancer agent, is associated with the down-regulation of nuclear factor-ĪŗB expression and proteasome activation in head and neck squamous carcinoma cell lines 

[2-s2.0-34250744214] Refers [2-s2.0-0036280779] LEVEL: 3
This study aimed to characterize the antitumor activity of 5-Chloro-N-{2-[2-(4-chloro-phenyl)-3-methyl-butoxy]-5-trifluoromethyl-phenyl} -2-hydroxy-benzamide (CTFB), a novel anticancer agent, in head and neck cancer cell lines, FaDu, SCC-25 and cisplatin-resistant CAL-27. CTFB was generated as a result of an extensive medicinal chemistry effort on a lead compound series discovered in a high-throughput screen for inducers of apoptosis. All cell lines showed significant growth delay in response to CTFB treatment at a concentration of 1 Mmol/L with 17.16 ± 2.08%, 10.92 ± 1.22%, and 27.03 ± 1.86% of cells surviving at 120 h in FaDu, CAL-27, and SCC-25, respectively. To define proteins involved in the mechanism of action of CTFB, we determined differences in the proteome profile of cell lines before and after treatment with CTFB using two-dimensional difference gel electrophoresis followed by computational image analysis and mass spectrometry. Eight proteins were found to be regulated by CTFB in all cell lines. All these proteins are involved in cytoskeleton formation and function and/or in cell cycle regulation. We showed that CTFB-induced cell growth delay was accompanied by cell cycle arrest at the G0-G1 phase that was associated with the up-regulation of p21/ WAF1 and p27/Kip1 expression and the down-regulation of cyclin D1. Furthermore, we showed that activity of CTFB depended on the down-regulation of nuclear factor-?B (NF-?B) and NF-?B p65 phosphorylated at Ser536. The level of proteasome activity correlated with the response to CTFB treatment, and the down-regulation of NF-?B is accompanied by enhanced proteasome activity in all investigated head and neck cancer cell lines. In this report, we show that CTFB reveals multiple effects that lead to delayed cell growth. Our data suggest that this compound should be studied further in the treatment of head and neck cancer. Copyright © 2007 American Association for Cancer Research.

TITLE:Calumenin, a multiple EF-hands Ca2+-binding protein, interacts with ryanodine receptor-1 in rabbit skeletal sarcoplasmic reticulum 

[2-s2.0-33645108259] Refers [2-s2.0-0036280779] LEVEL: 3
Calumenin is a multiple EF-hand Ca2+-binding protein located in endo/sarcoplasmic reticulum of mammalian tissues. In the present study, we cloned two rabbit calumenin isoforms (rabbit calumenin-1 and -2, GenBank Accession Nos. AY225335 and AY225336, respectively) by RT-PCR. Both isoforms contain a 19 aa N-terminal signal sequence, 6 EF-hand domains, and a C-terminal ER/SR retrieval signal, HDEF. Both calumenin isoforms exist in rabbit cardiac and skeletal muscles, but calumenin-2 is the main isoform in skeletal muscle. Presence of calumenin in rabbit sarcoplasmic reticulum (SR) was identified by Western blot analysis. GST-pull down and co-immunoprecipitation experiments showed that ryanodine receptor 1 (RyR1) interacted with calumenin-2 in millimolar Ca2+concentration range. Experiments of gradual EF-hand deletions suggest that the second EF-hand domain is essential for calumenin binding to RyR1. Adenovirus-mediated overexpression of calumenin-2 in C2C12 myotubes led to increased caffeine-induced Ca2+ release, but decreased depolarization-induced Ca2+ release. Taken together, we propose that calumenin-2 in the SR lumen can directly regulate the RyR1 activity in Ca2+-dependent manner. © 2006 Elsevier Inc. All rights reserved.

TITLE:Extracellular calcium sensing and signalling 

[2-s2.0-0038125588] Refers [2-s2.0-0034742490] LEVEL: 3
Ca2+ is well established as an intracellular second messenger. However, the molecular identification of a detector for extracellular Ca2+ - the extracellular Ca2+-sensing receptor - has opened up the possibility that Ca2+ might also function as a messenger outside cells. Information about the local extracellular Ca2+ concentration is conveyed to the interior of many cell types through this unique G-protein-coupled receptor. Here, we describe new emerging concepts concerning the signalling function of extracellular Ca2+, with particular emphasis on the extracellular Ca2+-sensing receptor.

TITLE:Endoplasmic reticulum Ca2+ homeostasis and neuronal death 

[2-s2.0-1842831135] Refers [2-s2.0-0034742490] LEVEL: 3
The endoplasmic reticulum (ER) is a universal signalling organelle, which regulates a wide range of neuronal functional responses. Calcium release from the ER underlies various forms of intracellular Ca2+ signalling by either amplifying Ca2+ entry through voltage-gated Ca2+ channels by Ca2+-induced Ca2+ release (CICR) or by producing local or global cytosolic calcium fluctuations following stimulation of metabotropic receptors through inositol-1,4,5-trisphosphate-induced Ca 2+ release (HCR). The ER Ca2+ store emerges as a single interconnected pool, thus allowing for a long-range Ca2+ signalling via intra-ER tunnels. The fluctuations of intra-ER free Ca2+ concentration regulate the activity of numerous ER resident proteins responsible for post-translational protein folding and modification. Disruption of ER Ca2+ homeostasis results in the developing of ER stress response, which in turn controls neuronal survival. Altered ER Ca2+ handling may be involved in pathogenesis of various neurodegenerative diseases including brain ischemia and Alzheimer dementia.

TITLE:Mitochondrial potassium channels and uncoupling proteins in synaptic plasticity and neuronal cell death 

[2-s2.0-0037427415] Refers [2-s2.0-0034742490] LEVEL: 3
The function of the nervous system relies upon synaptic transmission, a process in which a neurotransmitter released from pre-synaptic terminals of one neuron (in response to membrane depolarization and calcium influx) activates post-synaptic receptors on dendrites of another neuron. Synapses are subjected to repeated bouts of oxidative and metabolic stress as the result of changing ion gradients and ATP usage. Mitochondria play central roles in meeting the demands of synapses for ATP and in regulating calcium homeostasis, and mitochondrial dysfunction can cause dysfunction and degeneration of synapses, and can trigger cell death. We have identified two types of mitochondrial proteins that serve the function of protecting synapses and neurons against dysfunction and death. Mitochondrial ATP-sensitive potassium (MitoKATP) channels modulate inner membrane potential and oxyradical production; mitochondrial potassium fluxes can affect cytochrome c release and caspase activation and may determine whether neurons live or die in experimental models of stroke and Alzheimer's disease. Uncoupling proteins (UCPs) are a family of mitochondrial membrane proteins that uncouple electron transport from ATP production by transporting protons across the inner membrane. Neurons express at least three UCPs including the widely expressed UCP-2 and the neuron-specific UCP-4 and UCP-5 (BMCP-1). We have found that UCP-4 protects neurons against apoptosis by a mechanism involving suppression of oxyradical production and stabilization of cellular calcium homeostasis. The expression of UCP-4 is itself regulated by changes in energy metabolism. In addition to their roles in neuronal cell survival and death, MitoKATP channels and UCPs may play roles in regulating neuronal differentiation during development and synaptic plasticity in the adult. © 2003 Elsevier Science (USA). All rights reserved.

TITLE:The endoplasmic reticulum and neuronal calcium signalling 

[2-s2.0-0036878801] Refers [2-s2.0-0034742490] LEVEL: 3
The endoplasmic reticulum (ER) is a multifunctional signalling organelle regulating a wide range of neuronal functional responses. The ER is intimately involved in intracellular Ca2+ signalling, producing local or global cytosolic calcium fluctuations via Ca2+-induced Ca2+ release (CICR) or inositol-1,4,5-trisphosphate-induced Ca2+ release (IICR). The CICR and IICR are controlled by two subsets of Ca2+ release channels residing in the ER membrane, the Ca2+-gated Ca2+ release channels, generally known as ryanodine receptors (RyRs) and InsP3-gated Ca2+ release channels, referred to as InsP3-receptors (InsP3Rs). Both types of Ca2+ release channels are expressed abundantly in nerve cells and their activation triggers cytoplasmic Ca2+ signals important for synaptic transmission and plasticity. The RyRs and InsP3Rs show heterogeneous localisation in distinct cellular sub-compartments, conferring thus specificity in local Ca2+ signals. At the same time, the ER Ca2+ store emerges as a single interconnected pool fenced by the endomembrane. The continuity of the ER Ca2+ store could play an important role in various aspects of neuronal signalling. For example, Ca2+ ions may diffuse within the ER lumen with comparative ease, endowing this organelle with the capacity for "Ca2+ tunnelling". Thus, continuous intra-ER Ca2+ highways may be very important for the rapid replenishment of parts of the pool subjected to excessive stimulation (e.g. in small compartments within dendritic spines), the facilitated removal of localised Ca2+ loads, and finally in conveying Ca2+ signals from the site of entry towards the cell interior and nucleus. © 2002 Elsevier Science Ltd. All rights reserved.

TITLE:Ca2+ regulation and gene expression in normal brain aging 

[2-s2.0-4544262624] Refers [2-s2.0-0034742490] LEVEL: 3
Understanding the cellular mechanisms that characterize the functional changes of the aged brain is an ongoing and formidable challenge for the neuroscience community. Evidence now links changes in Ca2+ influx and homeostasis with perturbations induced by the aging process in the function of the main intracellular organelles involved in Ca2+ regulation: the endoplasmic reticulum and mitochondria. New perspectives are also offered by recent gene microarray studies, illustrating the multifactorial nature of the aging process.

TITLE:Calcium-binding Protein 1 Is an Inhibitor of Agonist-evoked, Inositol 1,4,5-Trisphosphate-mediated Calcium Signaling 

[2-s2.0-0346422451] Refers [2-s2.0-0034742490] LEVEL: 3
Intracellular calcium signals are responsible for initiating a spectrum of physiological responses. The caldendrins/calcium-binding proteins (CaBPs) represent mammal-specific members of the CaM superfamily. CaBPs display a restricted pattern of expression in neuronal/retinal tissues, suggesting a specialized role in Ca2+ signaling in these cell types. Recently, it was reported that a splice variant of CaBP1 functionally interacts with inositol 1,4,5-trisphosphate (InsP3) receptors to elicit channel activation in the absence of InsP3 (Yang, J., McBride, S., Mak, D.-O. D., Vardi, N., Palczewski, K., Haeseleer, F., and Foskett, J. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711-7716). These data indicate a new mode of InsP3 receptor modulation and hence control of intracellular Ca 2+ concentration ([Ca2+]i) in neuronal tissues. We have analyzed the biochemistry of the long form splice variant of CaBP1 (L-CaBP1) and show that, in vitro, a recombinant form of the protein is able to bind Ca2+ with high affinity and undergo a conformational change. We also describe the localization of endogenous and overexpressed L-CaBP1 in the model neuroendocrine PC12 cell system, where it was associated with the plasma membrane and Golgi complex in a myristoylation-dependent manner. Furthermore, we show that overexpressed L-CaBP1 is able to substantially suppress rises in [Ca2+]i in response to physiological agonists acting on purinergic receptors and that this inhibition is due in large part to blockade of release from intracellular Ca2+ stores. The related protein neuronal calcium sensor-1 was without effect on the [Ca2+] i responses to agonist stimulation. Measurement of [Ca 2+]i within the ER of permeabilized PC12 cells demonstrated that L-CaBP1 directly inhibited InsP3-mediated Ca 2+ release. Expression of L-CaBP1 also inhibited histamine-induced [Ca2+]i oscillations in HeLa cells. Together, these data suggest that L-CaBP1 is able to specifically regulate InsP3 receptor-mediated alterations in [Ca2+]i during agonist stimulation.

TITLE:Presence and functional significance of presynaptic ryanodine receptors 

[2-s2.0-0038573741] Refers [2-s2.0-0034742490] LEVEL: 3
Ca2+-induced Ca2+ release (CICR) mediated by sarcoplasmic reticulum resident ryanodine receptors (RyRs) has been well described in cardiac, skeletal and smooth muscle. In brain, RyRs are localised primarily to endoplasmic reticulum (ER) and have been demonstrated in postsynaptic entities, astrocytes and oligodendrocytes where they regulate intracellular Ca2+ concentration ([Ca2+]i), membrane potential and the activity of a variety of second messenger systems. Recently, the contribution of presynaptic RyRs and CICR to functions of central and peripheral presynaptic terminals, including neurotransmitter release, has received increased attention. However, there is no general agreement that RyRs are localised to presynaptic terminals, nor is it clear that RyRs regulate a large enough pool of intracellular Ca2+ to be physiologically significant. Here, we review direct and indirect evidence that on balance favours the notion that ER and RyRs are found in presynaptic terminals and are physiologically significant. In so doing, it became obvious that some of the controversy originates from issues related to (i) the ability to demonstrate conclusively the physical presence of ER and RyRs, (ii) whether the biophysical properties of RyRs are such that they can contribute physiologically to regulation of presynaptic [Ca2+]i, (iii) how ER Ca2+ load and feedback gain of CICR contributes to the ability to detect functionally relevant RyRs, (iv) the distance that Ca2+ diffuses from plasma membranes to RyRs to trigger CICR and from RyRs to the Active Zone to enhance vesicle release, and (v) the experimental conditions used. The recognition that ER Ca2+ stores are able to modulate local Ca2+ levels and neurotransmitter release in presynaptic terminals will aid in the understanding of the cellular mechanisms controlling neuronal function. © 2003 Elsevier Science Ltd. All rights reserved.

TITLE:NADH Oxidase Activity of Rat Cardiac Sarcoplasmic Reticulum Regulates Calcium-Induced Calcium Release 

[2-s2.0-1542373629] Refers [2-s2.0-0034742490] LEVEL: 3
NADH and Ca2+ have important regulatory functions in cardiomyocytes related to excitation-contraction coupling and ATP production. To elucidate elements of these functions, we examined the effect of NADH on sarcoplasmic reticulum (SR) Ca2+ release and the mechanisms of this regulation. Physiological concentrations of cytosolic NADH inhibited ryanodine receptor type 2 (RyR2)-mediated Ca2+-induced Ca2+ release (CICR) from SR membranes (IC50=120 ?mol/L) and significantly lowered single channel open probability. In permeabilized single ventricular cardiomyocytes, NADH significantly inhibited the amplitude and frequency of spontaneous Ca2+ release. Blockers of electron transport prevented the inhibitory effect of NADH on CICR in isolated membranes and permeabilized cells, as well as on the activity of RyR2 channels reconstituted in lipid bilayer. An endogenous NADH oxidase activity from rat heart copurified with SR enriched with RyR2. A significant contribution by mitochondria was excluded as NADH oxidation by SR exhibited >9-fold higher catalytic activity (8.8 ?mol/mg protein per minute) in the absence of exogenous mitochondrial complex I (ubiquinone) or complex III (cytochrome c) electron acceptors, but was inhibited by rotenone and pyridaben (IC50=2 to 3 nmol/L), antimycin A (IC50=13 nmol/L), and diphenyleneiodonium (IC 50=28 ?mol/L). Cardiac junctional SR treated with [ 3H](trifluoromethyl)diazirinyl-pyridaben specifically labeled a single 23-kDa PSST-like protein. These data indicate that NADH oxidation is tightly linked to, and essential for, negative regulation of the RyR2 complex and is a likely component of an important physiological negative-feedback mechanism coupling SR Ca2+ fluxes and mitochondrial energy production.

TITLE:Ca2+ Syntillas, Miniature Ca2+ Release Events in Terminals of Hypothalamic Neurons, Are Increased in Frequency by Depolarization in the Absence of Ca2+ Influx 

[2-s2.0-10744220681] Refers [2-s2.0-0034742490] LEVEL: 3
Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of ? 250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of -80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals.

TITLE:Correlated calcium uptake and release by mitochondria and endoplasmic reticulum of CA3 hippocampal dendrites after afferent synaptic stimulation 

[2-s2.0-0037115108] Refers [2-s2.0-0034742490] LEVEL: 3
Mitochondria and endoplasmic reticulum (ER) are important modulators of intracellular calcium signaling pathways, but the role of these organelles in shaping synaptic calcium transients in dendrites of pyramidal neurons remains speculative. We have measured directly the concentrations of total Ca (bound plus free) within intracellular compartments of proximal dendrites of CA3 hippocampal neurons at times after synaptic stimulation corresponding to the peak of the cytoplasmic free Ca2+ transient (1 sec), to just after its decay (30 sec), and to well after its return to prestimulus levels (180 sec). Electron probe microanalysis of cryosections from rapidly frozen slice cultures has revealed that afferent mossy fiber stimulation evokes large, rapid elevations in the concentration of total mitochondrial Ca ([Ca]mito) in depolarized dendrites. A single tetanus (50 Hz/1 sec) elevated [Ca]mito more than fivefold above characteristically low basal levels within 1 sec of stimulation and >10-fold by 30 sec after stimulation. This strong Ca accumulation was reversible, because [Ca]mito had recovered by 180 sec after the tetanus. Ca sequestered within mitochondria was localized to small inclusions that were distributed heterogeneously within, and probably among, individual mitochondria. By 30 sec after stimulation an active subpopulation of ER cisterns had accumulated more Ca than had mitochondria despite a ? 1 sec delay before the onset of accumulation. Active ER cisterns retained their Ca load much longer (>3 min) than mitochondria. The complementary time courses of mitochondrial versus ER Ca2+ uptake and release suggest that these organelles participate in a choreographed interplay, each shaping dendritic Ca2+ signals within characteristic regimes of cytosolic Ca2+ concentration and time.

TITLE:Ca2+ signaling in mouse cortical neurons studied by two-photon imaging and photoreleased inositol triphosphate 

[2-s2.0-0037289779] Refers [2-s2.0-0034742490] LEVEL: 3
IP3-mediated Ca2+ release is a crucial neuronal signaling mechanism that has not been extensively characterized in the mammalian cerebral cortex. We used two-photon, video-rate microscopy to image Ca2+ signals evoked by photoreleased IP3 in pyramidal neurons of mouse prefrontal cortex. Ca2+ responses to photoreleased IP3 varied greatly between different neurons; however, within IP3-responsive neurons, the soma invariably showed highest sensitivity, with signals increasing nonlinearly with [IP3]. Responses to paired photorelease displayed inhibition, whereas IP3-evoked Ca2+ liberation was potentiated by Ca2+ entry during action potentials and vice versa. IP3-mediated Ca2+ signals strongly inhibited spike firing through activation of K+ membrane conductance. Metabotropic signaling via the phosphoinositide pathway thus serves as a powerful and sustained modulator of excitability in cortical neurons and displays complex reciprocal interactions between electrical and chemical signals.

TITLE:Monitoring of free calcium in the neuronal endoplasmic reticulum: An overview of modern approaches 

[2-s2.0-0037207302] Refers [2-s2.0-0034742490] LEVEL: 3
The concentration of free calcium within the lumen of the endoplasmic reticulum ([Ca2+]L) fluctuates between 100 and 1000 ?M. High [Ca2+]L provides an electro-driving force for Ca2+ release and supports high Ca2+ diffusion rate within the endoplasmic reticulum lumen. Fluctuations in [Ca2+]L also regulate numerous chaperones, responsible for postranslational protein processing. Thus, [Ca2+]L integrates various signalling events and establishes a link between fast signalling, associated with the endoplasmic reticulum Ca2+release/uptake, and long-lasting adaptive responses relying primarily on the regulation of protein synthesis. This paper overviews modern approaches for the direct monitoring of [Ca2+]L which rely on three classes of low-affinity Ca2+ probes: ER-targeted aequorin, synthetic fluorescent Ca2+ dyes and GFP-based ER-targeted Ca2+ probes. These techniques, especially as applied to neurones, may substantially widen our appreciation of the endoplasmic reticulum as a universal signalling organelle. © 2002 Elsevier Science B.V. All rights reserved.

TITLE:The ryanodine receptors Ca2+ release channels: Cellular redox sensors? 

[2-s2.0-21344435250] Refers [2-s2.0-0034742490] LEVEL: 3
The release of Ca2+ from intracellular stores mediated by ryanodine receptors (RyR) Ca2+ release channels is essential for striated muscle contraction and contributes to diverse neuronal functions including synaptic plasticity. Through Ca2+-induced Ca 2+-release, RyR can amplify and propagate Ca2+ signals initially generated by Ca2+ entry into cardiac muscle cells or neurons. In contrast, RyR activation in skeletal muscle is under membrane potential control and does not require Ca2+ entry. Non-physiological or endogenous redox molecules can change RyR function via modification of a few RyR cysteine residues. This critical review will address the functional effects of RyR redox modification on Ca2+ release in skeletal muscle and cardiac muscle as well as in the activation of signaling cascades and transcriptional regulators required for synaptic plasticity in neurons. Specifically, the effects of endogenous redox-active agents, which induce S-nitrosylation or S-glutathionylation of particular channel cysteine residues, on the properties of muscle RyRs will be discussed. The effects of endogenous redox RyR modifications on cardiac preconditioning will be analyzed as well. In the hippocampus, sequential activation of ERKs and CREB is a requisite for Ca2+-dependent gene expression associated with long lasting synaptic plasticity. Results showing that reactive oxygen/nitrogen species modify RyR channels from neurons and the RyR-mediated sequential activation of neuronal ERKs and CREB produced by hydrogen peroxide and other stimuli will be also discussed. © 2005 IUBMB.

TITLE:Mitochondria, synaptic plasticity, and schizophrenia 

[2-s2.0-2342562466] Refers [2-s2.0-0034742490] LEVEL: 3
The conceptualization of schizophrenia as a disorder of connectivity, i.e., of neuronal/synaptic plasticity, suggests abnormal synaptic modeling and neuronal signaling, possibly as a consequence of flawed interactions with the environment, as at least a secondary mechanism underlying the pathophysiology of this disorder. Indeed, deficits in episodic memory and malfunction of hippocampal circuitry, as well as anomalies of axonal sprouting and synapse formation, are all suggestive of diminished neuronal plasticity in schizophrenia. Evidence supports a dysfunction of mitochondria in schizophrenia, including mitochondrial hypoplasia, and a dysfunction of the oxidative phosphorylation system, as well as altered mitochondrial-related gene expression. Mitochondrial dysfunction leads to alterations in ATP production and cytoplasmatic calcium concentrations, as well as reactive oxygen species and nitric oxide production. All of the latter processes have been well established as leading to altered synaptic strength or plasticity. Moreover, mitochondria have been shown to play a role in plasticity of neuronal polarity, and studies in the visual cortex show an association between mitochondria and synaptogenesis. Finally, mitochondrial gene upregulation has been observed following synaptic and neuronal activity. This review proposes that mitochondrial dysfunction in schizophrenia could cause, or arise from, anomalies in processes of plasticity in this disorder. Copyright 2004, Elsevier Inc. All rights reserved.

TITLE:Presynaptic calcium stores modulate afferent release in vestibular hair cells 

[2-s2.0-0041632265] Refers [2-s2.0-0034742490] LEVEL: 3
Hair cells, the mechanoreceptors of the acoustic and vestibular system, are presynaptic to primary afferent neurons of the eighth nerve and excite neural activity by the release of glutamate. In the present work, the role played by intracellular Ca2+ stores in afferent transmission was investigated, at the presynaptic level, by monitoring changes in the intracellular Ca2+ concentration ([Ca2+]i) in vestibular hair cells, and, the postsynaptic level, by recording from single posterior canal afferent fibers. Application of 1-10 mM caffeine to hair cells potentiated Ca2+ responses evoked by depolarization at selected Ca2+ hot spots, and also induced a graded increase in cell membrane capacitance (?Cm), signaling exocytosis of the transmitter. Ca2+ signals evoked by caffeine peaked in a region located ?10 ?m from the base of the hair cell. [Ca2+]i increases, similarly localized, were observed after 500 msec depolarizations, but not with 50 msec depolarizations, suggesting the occurrence of calcium-induced calcium release (CICR) from the same stores. Both Ca2+ and ?Cm responses were inhibited after incubation with ryanodine (40 ?M) for 8 - 10 min. Consistent with these results, afferent transmission was potentiated by caffeine and inhibited by ryanodine both at the level of action potentials and of miniature EPSPs (mEPSPs). Neither caffeine nor ryanodine affected the shape and amplitude of mEPSPs, indicating that both drugs acted at the presynaptic level. These results strongly suggest that endogenous modulators of the CICR process will affect afferent activity elicited by mechanical stimuli in the physiological frequency range.

TITLE:Neuronal endoplasmic reticulum acts as a single functional Ca2+ store shared by ryanodine and inositol-1,4,5-trisphosphate receptors as revealed by intra-ER [Ca2+] recordings in single rat sensory neurones 

[2-s2.0-0042166005] Refers [2-s2.0-0034742490] LEVEL: 3
We addressed the fundamentally important question of functional continuity of endoplasmic reticulum (ER) Ca2+ store in nerve cells. In cultured rat dorsal root ganglion neurones we measured dynamic changes in free Ca2+ concentration within the ER lumen ([Ca2+]L) in response to activation of inositol-1,4,5-trisphosphate receptors (InsP3Rs) and ryanodine receptors (RyRs). We found that both receptors co-exist in these neurones and their activation results in Ca2+ release from the ER as judged by a decrease in [Ca2+]L. Depletion of Ca2+ stores following an inhibition of sarco(endoplasmic)reticulum Ca2+-ATPase by thapsigargin or cyclopiazonic acid completely eliminated Ca2+ release via both InsP3Rs and RyRs. Similarly, when the store was depleted by continuous activation of InsP3Rs, activation of RyRs (by caffeine or 0.5 ?M ryanodine) failed to produce Ca2+ release, and vice versa, when the stores were depleted by activators of RyRs, the InsP3-induced Ca2+ release disappeared. We conclude that in mammalian neurones InsP3Rs and RyRs share the common continuous Ca2+ pool associated with ER.

TITLE:Xestospongin C empties the ER calcium store but does not inhibit InsP3-induced Ca2+ release in cultured dorsal root ganglia neurones 

[2-s2.0-0036629423] Refers [2-s2.0-0034742490] LEVEL: 3
The action of Xestospongin C (XeC) on calcium concentration in the cytosol ([Ca2+]i) and within the lumen of endoplasmic reticulum (ER) ([Ca2+]L) was studied using cultured dorsal root ganglia (DRG) neurones. Application of 2.5 ?M of XeC triggered a slow [Ca2+]i transient as measured by Fura-2 video-imaging. The kinetics and amplitude of XeC-induced [Ca2+]i response was similar to that triggered by 1 ?M thapsigargin (TG). The [Ca2+]L was monitored in cells loaded with low-affinity Ca2+ indicator Mag-Fura-2. The cytosolic portion of Mag-Fura-2 was removed by permeabilisation of the plasmalemma with saponin. Application of XeC to these permeabilised neurones resulted in a slow depletion of the ER Ca2+ store. XeC, however, failed to inhibit inositol 1,4,5-trisphosphate (InsP3)-induced [Ca2+]L responses. We conclude that XeC is a potent inhibitor of sarco(endo)plasmic reticulum calcium ATPase, and it cannot be regarded as a specific inhibitor of InsP3 receptors in cultured DRG neurones. © 2002 Elsevier Science Ltd. All rights reserved.

TITLE:Microdomains for dopamine volume neurotransmission in primate prefrontal cortex 

[2-s2.0-2942534133] Refers [2-s2.0-0034742490] LEVEL: 3
The explicit yet enigmatic involvement of dopamine in cortical physiology is in part volumetric (beyond the synapse), as is apparently the action of neuroleptics targeting dopamine receptors. The notion that nonsynaptic neuronal membranes would translate extracellular dopamine into receptor-specific spatiotemporal downstream signaling, similar to the chemical synapse, is intriguing. Here, we report that dopamine D5 (but not D1 or D2) receptors in the perisomatic plasma membrane of prefrontal cortical neurons form discrete and exclusively extrasynaptic microdomains with inositol 1,4,5-trisphosphate-gated calcium stores of subsurface cisterns and mitochondria. These findings introduce a novel dopaminoceptive substratum in the brain and a unique D5 receptor-specific signaling paradigm.

TITLE:Heme proteins and nitric oxide (NO): The neglected, eloquent chemistry in NO redox signaling and regulation 

[2-s2.0-0038236742] Refers [2-s2.0-0034742490] LEVEL: 3
The role of nitric oxide (NO) in cellular physiology and signaling has been an important aspect in biomedical science over the last decade. As NO is a small uncharged radical, the chemistry of NO within the redox environment of the cell dictates the majority of its biological effects. The mechanisms that have received the most attention from a biological perspective involve reactions with oxygen and superoxide, despite the rich literature of metal-NO chemistry. However, NO and its related species participate in important chemistry with metalloproteins. In addition to the well known direct interactions of NO with heme proteins such as soluble guanylate cyclase and oxyhemoglobin, there is much important, but often underappreciated, chemistry between other nitrogen oxides and heme/metal proteins. Here the basic chemistry of nitrosylation and the interactions of NO and other nitrogen oxides with metal-oxo species such as found in peroxidases and monoxygenases are discussed.

TITLE:A reassessment of the effects of luminal [Ca2+] on inositol 1,4,5-trisphosphate-induced Ca2+ release from internal stores 

[2-s2.0-0141960206] Refers [2-s2.0-0034742490] LEVEL: 3
Inositol 1,4,5-trisphosphate (InSP3)-induced Ca2+ release from intracellular stores displays complex kinetic behavior. While it well established that cytosolic [Ca2+] can modulate release by acting on the InsP3 receptor directly, the role of the filling state of internal Ca2+ stores in modulating Ca2+ release remains unclear. Here we have reevaluated this topic using a technique that permits rapid and reversible changes in free [Ca2+] in internal stores of living intact cells without altering cytoplasmic [Ca2+], InsP3 receptors, or sarcoendoplasmic reticulum Ca2+ ATPases (SERCAs). N,N,N?,N?-Tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), a membrane-permeant, low affinity Ca2+ chelator was used to manipulate [Ca2+] in intracellular stores, while [Ca 2+] changes within the store were monitored directly with the low-affinity Ca2+ indicator, mag-fura-2, in intact BHK-21 cells. 200 ?M TPEN caused a rapid drop in luminal free [Ca2+] and significantly reduced the extent of the response to stimulation with 100 nM bradykinin, a calcium-mobilizing agonist. The same effect was observed when intact cells were pretreated with 1,2-bis(2-amino-phenoxy)ethane-N,N,N? ,N?-tetraacetic acid(acetoxy-methyl ester) (BAPTA-AM) to buffer cytoplasmic [Ca2+] changes. Although inhibition of Ca2+ uptake using the SERCA inhibitor tBHQ permitted significantly larger release of Ca2+ from stores, TPEN still attenuated the release in the presence of tBHQ in BAPTA-AM-loaded cells. These results demonstrate that the filling state of stores modulates the magnitude of InsP3-induced Ca 2+ release by additional mechanism(s) that are independent of regulation by cytoplasmic [Ca2+] or effects on SERCA pumps.

TITLE:Long distance communication between muscarinic receptors and Ca2+ release channels revealed by carbachol uncaging in cell-attached patch pipette 

[2-s2.0-0037743397] Refers [2-s2.0-0034742490] LEVEL: 3
We have investigated the characteristics of cytosolic Ca2+ signals induced by muscarinic receptor activation of pancreatic acinar cells that reside within intact pancreatic tissue. We show that these cells exhibit global Ca2+ waves and local apical Ca2+ spikes. This is the first evidence for local Ca2+ signaling in undissociated pancreatic tissue. The mechanism of formation of localized Ca2+ signals was examined using a novel approach involving photolysis of caged carbachol inside a patch pipette attached to the basal surface of an acinar unit. This local activation of basal muscarinic receptors elicited local cytosolic Ca2+ spikes in the apical pole more than 15 ?m away from the site of stimulation. In some experiments, local basal receptor activation elicited a Ca2+ wave that started in the apical pole and then spread toward the base. Currently, there are two competing hypotheses for preferential apical Ca2+ signaling. One invokes the need for structural proximity of the cholinergic receptors and the Ca2+ release channels in the apical pole, whereas the other postulates long distance communication between basal receptors and the channels. Our intrapipette uncaging experiments provide definitive evidence for long distance communication between basal muscarinic receptors and apical Ca2+ release channels.

TITLE:The role of the endoplasmic reticulum Ca2+ store in the plasticity of central neurons 

[2-s2.0-31844450573] Refers [2-s2.0-0034742490] LEVEL: 3
The smooth endoplasmic reticulum (SER) is a well-characterized buffer and source of Ca2+ in both axonal and dendritic compartments of neurons. Ca2+ release from the SER can be evoked by stimulation of the ryanodine receptor or the inositol (1,4,5)-trisphosphate [Ins(1,4,5)P 3] receptor. Both receptors can couple to the activation of neurotransmitter-gated receptors and voltage-gated Ca2+ channels on the plasma membrane, thus enabling the SER to discriminate between different types of neuronal activity. In axonal terminals, Ca2+-induced Ca 2+ release (CICR) mediates spontaneous, evoked and facilitated neurotransmission. Store release might also regulate the mobilization and recycling of synaptic vesicles. In the dendritic compartment, the distribution of Ins(1,4,5)P3 receptors and ryanodine receptors influences the intracellular encoding of neuronal activity. Thus, the functionality of the Ca2+ store can affect both the polarity and the spatial extent of Ca2+-dependent shifts in synaptic efficacy. In hippocampal neurons, for example, CICR in the spine heads underlies homosynaptic plasticity, whereas heterosynaptic plasticity is mediated by Ins(1,4,5)P3-dependent Ca2+ signalling. Purkinje neurons primarily express Ins(1,4,5)P 3 receptors in the spine heads, and long-term depression of synaptic efficacy is crucially dependent on Ins(1,4,5)P3. © 2005 Elsevier Ltd. All rights reserved.

TITLE:Neurosecretion competence. A comprehensive gene expression program identified in PC12 cells 

[2-s2.0-0037184069] Refers [2-s2.0-0034742490] LEVEL: 3
The phenotype of neurosecretory cells is characterized by clear vesicles and dense granules, both discharged by regulated exocytosis. However, these organelles are lacking completely in a few neurosecretion-incompetent clones of the pheochromocytoma PC12 line, in which other specific features are maintained (incompetent clones). In view of the heterogeneity of PC12 cells, a differential characterization of the incompetent phenotype based on the comparison of a single incompetent and a single wild-type clone would have been inconclusive. Therefore, we have compared two pairs of PC12 clones, studying in parallel the transcript levels of 4,200 genes and 19,000 express sequence tags (ESTs) by high density oligonucleotide arrays. After accurate data processing for quality control and filtration, a total of 755 transcripts, corresponding to 448 genes and 307 ESTs, was found consistently changed, with 46% up-regulated and 54% down-regulated in incompetent versus wild-type clones. Many but not all neurosecretion genes were profoundly down-regulated in incompetent cells. Expression of endocytosis genes was normal, whereas that of many nuclear and transcription factors, including some previously shown to play key roles in neurogenesis, was profoundly changed. Additional differences appeared in genes involved in signaling and metabolism. Taken together these results demonstrate for the first time that expression of neurosecretory vesicles and granules is part of a complex gene expression program that includes many other features that so far have not been recognized.

TITLE:Calcium and glial cell death 

[2-s2.0-24644442777] Refers [2-s2.0-0034742490] LEVEL: 3
Calcium (Ca2+) homeostasis is crucial for development and survival of virtually all types of cells including glia of the central nervous system (CNS). Astrocytes, oligodendrocytes and microglia, the major glial cell types in the CNS, are endowed with a rather sophisticated array of Ca2+-permeable receptors and channels, as well as store-operated channels and pumps, all of which determine Ca2+ homeostasis. In addition, glial cells detect functional activity in neighbouring neurons and respond to it by means of Ca2+ signals that can modulate synaptic interactions. Like in neurons, Ca2+ overload resulting from dysregulation of channels and pumps can be deleterious to glia. In this review, we summarize recent advances in the understanding Ca2+ homeostasis in glial cells, the consequences of its alteration in cell demise as well as in neurological and psychiatric disorders that experience glial cell loss. © 2005 Elsevier Ltd. All rights reserved.

TITLE:Total calcium ultrastructure: Advances in excitable cells 

[2-s2.0-0034961436] Refers [2-s2.0-0034742490] LEVEL: 3
This is a review which is written on the basis of a cell calcium lecture delivered on 22 July 2000 at the European Research Meeting 'Calcium as a molecule of cellular integration'. © 2001 Harcourt Publishers Ltd.

TITLE:Modulation of agonist-induced Ca2+ release by SR Ca2+ load: Direct SR and cytosolic Ca2+ measurements in rat uterine myocytes 

[2-s2.0-12444318634] Refers [2-s2.0-0034742490] LEVEL: 3
Release of Ca2+ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca2+ release is considered to be dependent on the Ca2+ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca2+ load on the amplitude of [Ca2+]i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca2+ level ([Ca2+]L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca2+ (10 mM) extracellular solution leads to a substantial increase in [Ca2+]L (SR overload). However, despite increased SR Ca2+ content this did not potentiate ATP-induced [Ca2+]i transients. Repetitive applications of ATP in the absence of extracellular Ca2+, as well as prolonged incubation in Ca2+-free solution without agonist, depleted the [Ca2+]L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca2+ release. ATP-induced [Ca2+]i transients were completely abolished when SR Ca2+ content was decreased below 80% of its normal value indicating a steep dependence of the IP3-mediated Ca2+ release on the Ca2+ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca2+ load below its normal resting level substantially reduces the IP3-mediated Ca2+ release, while Ca2+ overload of the SR has no impact on such release. © 2004 Elsevier Ltd. All rights reserved.

TITLE:The extracellular calcium-sensing receptor and cell-cell signaling in epithelia 

[2-s2.0-1442350442] Refers [2-s2.0-0034742490] LEVEL: 3
In multicellular organisms, cells are crowded together in organized communities, surrounded by an interstitial fluid of extremely limited volume. Local communication between adjacent cells is known to occur through gap junctions in cells that are physically connected, or through the release of paracrine signaling molecules (e.g. ATP, glutamate, nitric oxide) that diffuse to their target receptors through the extracellular microenvironment. Recent evidence hints that calcium ions may possibly be added to the list of paracrine messengers that allow cells to communicate with one another. Local fluctuations in extracellular [Ca2+] can be generated as a consequence of intracellular Ca2+ signaling events, owing to the activation of Ca2+ influx and efflux pathways at the plasma membrane. In intact tissues, where the interstitial volumes between cells are much smaller than the cells themselves, this can result in significant alterations in external [Ca2+]. This article will explore emerging evidence that these extracellular [Ca2+] changes can be detected by the extracellular calcium-sensing receptor (CaR) on adjacent cells, forming the basis for a paracrine signaling system. Such a mechanism could potentially provide CaR-expressing cells with the means to sense the Ca2+ signaling status of their neighbors, and expand the utility of the intracellular Ca2+ signal to a domain outside the cell. © 2003 Elsevier Ltd. All rights reserved.

TITLE:Intracellular calcium store filling by an L-type calcium current in the basolateral amygdala at subthreshold membrane potentials 

[2-s2.0-12744253486] Refers [2-s2.0-0034742490] LEVEL: 3
The long-term changes that underlie learning and memory are activated by rises in intracellular Ca2+ that activate a number of signalling pathways and trigger changes in gene transcription. Ca2+ rises due to influx via L-type voltage-dependent Ca2+ channels (L-VDCCs) and release from intracellular Ca2+ stores have been consistently implicated in the biochemical cascades that underlie the final changes in memory formation. Here, we show that pyramidal neurones in the basolateral amygdala express an L-VDCC that is active at resting membrane potentials. Subthreshold depolarization of neurones either by current injection or summating synaptic potentials led to a sustained rise in cytosolic Ca2+ that was blocked by the dihydropyridine nicardipine. Activation of metabotropic receptors released Ca2+ from intracellular Ca2+ stores. At hyperpolarized potentials, metabotropic-evoked store release ran down with repeated stimulation. Depolarization of cells to - 50 mV, or maintaining them at the resting membrane potential, restored release from intracellular Ca2+ stores, an effect that was blocked by nicardipine. These results show that Ca2+ influx via a low-voltage-activated L-type Ca2+ current refills inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ stores, and maintains Ca2+ release and wave generation by metabotropic receptor activation. © The Physiological Society 2004.

TITLE:Metabotropic receptor-mediated Ca2+ signaling elevates mitochondrial Ca2+ and stimulates oxidative metabolism in hippocampal slice cultures 

[2-s2.0-0042316858] Refers [2-s2.0-0034742490] LEVEL: 3
Metabotropic receptors modulate numerous cellular processes by intracellular Ca2+ signaling, but less is known about their role in regulating mitochondrial metabolic function within the CNS. In this study, we demonstrate in area CA3 of rat organotypic hippocampal slice cultures that glutamatergic, serotonergic, and muscarinic metabotropic receptor ligands, namely trans-azetidine-2,4-dicarboxylic acid, ? -methyl-5-hydroxytryptamine, and carbachol, transiently increase mitochondrial Ca2+ concentration ([Ca2+]m) as recorded by changes in Rhod-2 fluorescence, stimulate mitochondrial oxidative metabolism as revealed by elevations in NAD(P)H fluorescence, and induce K+ outward currents as monitored by rapid increases in extracellular K+ concentration ([K+]o). Carbachol (1-1,000 ?M) elevated NAD(P)H fluorescence by ?14%?F/F0 and increased [K +]o by ?4.3 mM in a dose-dependent manner. Carbachol-induced responses persisted in Ca2+-free solution and blockade of ionotropic glutamatergic and nicotinic receptors. Under similar conditions caffeine, known to cause Ca2+-induced Ca2+ release (CICR), also evoked elevations in [Ca2+]m, NAD(P) H fluorescence and [K+]o that, in contrast to carbachol-induced responses, displayed oscillations. After depletion of intracellular Ca2+ stores by carbachol in Ca2+-free solution, re-application of 1.6 mM Ca2+-containing solution triggered marked elevations in [Ca2+]m, NAD(P)H fluorescence and [K+]o. These data indicate that metabotropic transmission effectively regulates mitochondrial oxidative metabolism via diverse receptor types in hippocampal cells and that inonitol 1,4,5-trisphosphate-induced Ca2+ release (IICR) or CICR or capacitative Ca2+ entry might suffice in stimulating oxidative metabolism by elevating [Ca2+]m. Thus activation of metabotropic receptors might significantly contribute to generation of ATP within neurons and glial cells.

TITLE:Neuronal survival in the balance: Are endoplasmic reticulum membrane proteins the fulcrum? 

[2-s2.0-0036877969] Refers [2-s2.0-0034742490] LEVEL: 3
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TITLE:Differential signaling in presynaptic neurotransmitter release 

[2-s2.0-18544373178] Refers [2-s2.0-0034742490] LEVEL: 3
Neuronal communication is tightly regulated by presynaptic signaling, thereby temporarily and locally secreting one or more transmitters in order to exert propagation or modulation of network activity. In the last 2 decades our insight into the molecular regulation of presynaptic transmitter vesicle traffic and fusion has exponentionally grown due to the identification of specific functional interactions between presynaptic proteins involved in these processes. In addition, a plethora of extracellular and intracellular messengers regulate neurotransmitter release, occasionally leading to short- or long-term adaptations of the synapse to altered environmental signals. Important in this respect is the ability of various nerve terminals to diverge their output by differentiation in secretion of co-localized transmitters. This divergence in presynaptic signaling may converge in the postsynaptic target neuron or spread to neighbouring cells. In this review differential presynaptic signaling mechanisms will be related to their potential divergent roles in transmitter release. © Birkha?user Verlag, 2005.

TITLE:Ultra rapid calcium events in electrically stimulated frog nerve terminals 

[2-s2.0-0034811240] Refers [2-s2.0-0034742490] LEVEL: 3
Fast calcium events occurring in cytoplasmic organelles after a single electrical stimulus were investigated by electron spectroscopic imaging (an electron microscope technique that reveals total calcium with high sensitivity and spatial resolution) in quick frozen presynaptic terminals of the frog neuromuscular junction. In resting preparations synaptic vesicles showed a prominent calcium signal whereas mitochondria were mostly negative and only some of the cisternae of the endoplasmic reticulum were clearly positive. In preparations quick frozen 10 ms after the application to the nerve of a single, supramaximal electric stimulus, no obvious change was observed in synaptic vesicles, while calcium levels rose to high values in the endoplasmic reticulum cisternae and in the matrix of mitochondria. Voltage-induced influx of Ca2+ within synaptic terminals appears therefore to induce an extremely rapid uptake into selected organelles. The possible physiological role of this response is discussed. © 2001 Academic Press.

TITLE:Calcium microdomains and gene expression in neurons and skeletal muscle cells 

[2-s2.0-33750517974] Refers [2-s2.0-0034742490] LEVEL: 3
Neurons generate particular calcium microdomains in response to different stimuli. Calcium microdomains have a central role in a variety of neuronal functions. In particular, calcium microdomains participate in long-lasting synaptic plasticity-a neuronal response presumably correlated with cognitive brain functions that requires expression of new gene products. Stimulation of skeletal muscle generates - with few milliseconds delay - calcium microdomains that have a central role in the ensuing muscle contraction. In addition, recent evidence indicates that sustained stimulation of skeletal muscle cells in culture generates calcium microdomains, which stimulate gene expression but not muscle contraction. The mechanisms whereby calcium microdomains activate signaling cascades that lead to the transcription of genes known to participate in specific cellular responses are the central topic of this review. Thus, we will discuss here the signaling pathways and molecular mechanisms, which via activation of particular calcium-dependent transcription factors regulate the expression of specific genes or set of genes in neurons or skeletal muscle cells. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Ischemia-induced increase in long-term potentiation is warded off by specific calpain inhibitor PD150606 

[2-s2.0-4644239251] Refers [2-s2.0-0034742490] LEVEL: 3
In the present study, the effect of specific, membrane-permeable calpain inhibitor, PD150606, was analysed on synaptic efficacy in in vitro brain slices experiments after ischemic insult of rats in vivo, and on cell viability in a glutamate excitotoxicity test in mouse cell culture. Bilateral common carotid artery ligation (BCCL) for 24 h markedly increased calpain activity and enhanced LTP induction in rat hippocampus, although the CA1 layer significantly shrank. The enhancement of LTP could be diminished by short-term application of PD150606 (40 ?M) into the perfusion solution. Intracerebroventricular administration of PD150606 (100 ?M) parallel with ischemic insult prevented LTP and effectively inhibited hippocampal calpain activity. Intracerebroventricularly applied PD150606 inhibited the CA1 layer shrinkage after common carotid ligation. High level of exogenous glutamate caused marked decrease of cell viability in mouse cerebellar granule cell cultures, which could be partly warded off by 20 ?M PD150606. Our data witness that calpain action is intricately involved in the regulation of synaptic efficacy. © 2004 Elsevier B.V. All rigths reserved.

TITLE:Muscarinic and nicotinic ACh receptor activation differentially mobilize Ca2+ in rat intracardiac ganglion neurons 

[2-s2.0-0141565095] Refers [2-s2.0-0034742490] LEVEL: 3
The origin of intracellular Ca2+ concentration ([Ca 2+]i) transients stimulated by nicotinic (nAChR) and muscarinic (mAChR) receptor activation was investigated in fura-2-loaded neonatal rat intracardiac neurons. ACh evoked [Ca2+]i increases that were reduced to ?60% of control in the presence of either atropine (1 ?M) or mecamylamine (3 ?M) and to <20% in the presence of both antagonists. Removal of external Ca2+ reduced ACh-induced responses to 58% of control, which was unchanged in the presence of mecamylamine but reduced to 5% of control by atropine. The nAChR-induced [Ca2+]i response was reduced to 50% by 10 ?M ryanodine, whereas the mAChR-induced response was unaffected by ryanodine, suggesting that Ca2+ release from ryanodine-sensitive Ca2+ stores may only contribute to the nAChR-induced [Ca2+]i responses. Perforated-patch whole cell recording at -60 mV shows that the rise in [Ca2+]i is concomitant with slow outward currents on mAChR activation and with rapid inward currents after nAChR activation. In conclusion, different signaling pathways mediate the rise in [Ca 2+]i and membrane currents evoked by ACh binding to nicotinic and muscarinic receptors in rat intracardiac neurons.

TITLE:Cross talk between Ca2+ and redox signalling cascades in muscle and neurons through the combined activation of ryanodine receptors/Ca 2+ release channels 

[2-s2.0-33745234143] Refers [2-s2.0-0034742490] LEVEL: 3
Calcium release mediated by the ryanodine receptors (RyR) Ca2+ release channels is required for muscle contraction and contributes to neuronal plasticity. In particular, Ca2+ activation of RyR-mediated Ca 2+ release can amplify and propagate Ca2+ signals initially generated by Ca2+ entry into cells. Redox modulation of RyR function by a variety of non-physiological or endogenous redox molecules has been reported. The effects of RyR redox modification on Ca2+ release in skeletal muscle as well as the activation of signalling cascades and transcription factors in neurons will be reviewed here. Specifically, the different effects of S-nitrosylation or S-glutathionylation of RyR cysteines by endogenous redox-active agents on the properties of skeletal muscle RyRs will be discussed. Results will be presented indicating that these cysteine modifications change the activity of skeletal muscle RyRs, modify their behaviour towards both activators and inhibitors and affect their interactions with FKBP12 and calmodulin. In the hippocampus, sequential activation of ERK1/2 and CREB is a requisite for Ca2+-dependent gene expression associated with long-lasting synaptic plasticity. The effects of reactive oxygen/nitrogen species on RyR channels from neurons and RyR-mediated sequential activation of neuronal ERK1/2 and CREB produced by hydrogen peroxide and other stimuli will be discussed as well. © 2005 The Royal Society.

TITLE:Hypoxic regulation of Ca2+ signaling in cultured rat astrocytes 

[2-s2.0-11144298991] Refers [2-s2.0-0034742490] LEVEL: 3
Acute hypoxia modulates various cell processes, such as cell excitability, through the regulation of ion channel activity. Given the central role of Ca2+ signaling in the physiological functioning of astrocytes, we have investigated how acute hypoxia regulates such signaling, and compared results with those evoked by bradykinin (BK), an agonist whose ability to liberate Ca2+ from intracellular stores is well documented. In Ca2+-free perfusate, BK evoked rises of [Ca2+]i in all cells examined. Hypoxia produced smaller rises of [Ca2+] i in most cells, but always suppressed subsequent rises of [Ca 2+]i induced by BK. Thapsigargin pre-treatment of cells prevented any rise of [Ca2+]i evoked by either BK or hypoxia. Restoration of Ca2+ to the perfusate following a period of acute hypoxia always evoked capacitative Ca2+ entry. During mitochondrial inhibition (due to exposure to carbonyl cyanide p-trifluromethoxyphenyl hydrazone (FCCP) and oligomycin), rises in [Ca 2+]i (observed in Ca2+-free perfusate) evoked by hypoxia or by BK, were significantly enhanced, and hypoxia always evoked responses. Our data indicate that hypoxia triggers Ca2+ release from endoplasmic reticulum stores, efficiently buffered by mitochondria. Such liberation of Ca2+ is sufficient to trigger capacitative Ca 2+ entry. These findings indicate that the local O2 level is a key determinant of astrocyte Ca2+ signaling, likely modulating Ca2+-dependent astrocyte functions in the central nervous system. © 2004 Wiley-Liss, Inc.

TITLE:Rapidly exchanging Ca2+ stores: Ubiquitous partners of surface channels in neurons 

[2-s2.0-0036667276] Refers [2-s2.0-0034742490] LEVEL: 3
Neuronal rapidly exchanging Ca2+ stores coincide with the endoplasmic reticulum and possess ample, nonrandom distribution, dual receptor channels, IP3 and ryanodine receptors, and heterogeneous membranes. Because of these properties, the stores are able to reinforce and expand the Ca2+ signals generated at the surface, working as partners of voltage- and receptor-gated channels.

TITLE:Dynamic distribution of endoplasmic reticulum in hippocampal neuron dendritic spines 

[2-s2.0-26944455925] Refers [2-s2.0-0034742490] LEVEL: 3
The role of the endoplasmic reticulum (ER) localized in dendritic spines has become a subject of intense interest because of its potential functions in local protein synthesis and signal transduction. Although it is recognized from electron microscopic studies that not all spines contain ER, little is know of its dynamic regulation or turnover. Here, we report a surprising degree of turnover of ER within spines. Using confocal microscopy imaging we observed continuity of spine-ER with dendritic ER in hippocampal primary neurons. Over 24 h, less than 50% of spine ER was stable. Despite this high degree of turn over, we identified a significant subset of spines that maintained ER for at least 4 days. These results indicate that within a single neuron, the organelle composition of a spine is unexpectedly dynamic and may explain aspects of the spine-to-spine variation in calcium spike magnitude and localized protein synthesis and trafficking. © Federation of European Neuroscience Societies.

TITLE:A role for reactive oxygen/nitrogen species and iron on neuronal synaptic plasticity 

[2-s2.0-33846197505] Refers [2-s2.0-0034742490] LEVEL: 3
A great body of experimental evidence collected over many years indicates that calcium has a central role in a variety of neuronal functions. In particular, calcium participates in synaptic plasticity, a neuronal process presumably correlated with cognitive brain functions such as learning and memory. In contrast, only recently, evidence has begun to emerge supporting a physiological role of reactive oxygen (ROS) and nitrogen (RNS) species in synaptic plasticity. This subject will be the central topic of this review. The authors also present recent results showing that, in hippocampal neurons, ROS/RNS, including ROS generated by iron through the Fenton reaction, stimulate ryanodine receptor-mediated calcium release, and how the resulting calcium signals activate the signaling cascades that lead to the transcription of genes known to participate in synaptic plasticity. They discuss the possible participation of ryanodine receptors jointly stimulated by calcium and ROS/RNS in the normal signaling cascades needed for synaptic plasticity, and how too much ROS production may contribute to neurodegeneration via excessive calcium release. In addition, the dual role of iron as a necessary, but potentially toxic, element for normal neuronal function is discussed. © Mary Ann Liebert, Inc.

TITLE:Protection from neuronal damage evoked by a motivational excitation is a driving force of intentional actions 

[2-s2.0-27544487587] Refers [2-s2.0-0034742490] LEVEL: 3
Motivation may be understood as an organism's subjective attitude to its current physiological state, which somehow modulates generation of actions until the organism attains an optimal state. How does this subjective attitude arise and how does it modulate generation of actions? Diverse lines of evidence suggest that elemental motivational states (hunger, thirst, fear, drug-dependence, etc.) arise as the result of metabolic disturbances and are related to transient injury, while rewards (food, water, avoidance, drugs, etc.) are associated with the recovery of specific neurons. Just as motivation and the very life of an organism depend on homeostasis, i.e., maintenance of optimum performance, so a neuron's behavior depends on neuronal (i.e., ion) homeostasis. During motivational excitation, the conventional properties of a neuron, such as maintenance of membrane potential and spike generation, are disturbed. Instrumental actions may originate as a consequence of the compensational recovery of neuronal excitability after the excitotoxic damage induced by a motivation. When the extent of neuronal actions is proportional to a metabolic disturbance, the neuron theoretically may choose a beneficial behavior even, if at each instant, it acts by chance. Homeostasis supposedly may be directed to anticipating compensation of the factors that lead to a disturbance of the homeostasis and, as a result, participates in the plasticity of motivational behavior. Following this line of thought, I suggest that voluntary actions arise from the interaction between endogenous compensational mechanisms and excitotoxic damage of specific neurons, and thus anticipate the exogenous compensation evoked by a reward. © 2005 Elsevier B.V. All rights reserved.

TITLE:Mutation in slowmo causes defects in Drosophila larval locomotor behaviour 

[2-s2.0-9744280501] Refers [2-s2.0-0034742490] LEVEL: 3
We have identified a mutant slowmotion phenotype in first instar larval peristaltic behaviour of Drosophila. By the end of embryogenesis and during early first instar phases, slowmo mutant animals show a marked decrease in locomotory behaviour, resulting from both a reduction in number and rate of peristaltic contractions. Inhibition of neurotransmitter release, using targeted expression of tetanus toxin light chain (TeTxLC), in the slowmo neurons marked by an enhancer-trap results in a similar phenotype of largely absent or uncoordinated contractions. Cloning of the slowmo gene identifies a product related to a family of proteins of unknown function. We show that Slowmo is associated with mitochondria, indicative of it being a mitochondrial protein, and that during embryogenesis and early larval development is restricted to the nervous system in a subset of cells. The enhancer-trap marks a cellular component of the CNS that is seemingly required to regulate peristaltic movement. © Springer-Verlag 2003.

TITLE:Mechanism of acetylcholine-induced calcium signaling during neuronal differentiation of P19 embryonal carcinoma cells in vitro 

[2-s2.0-38349193811] Refers [2-s2.0-0034742490] LEVEL: 3
Muscarinic (mAChRs) and nicotinic acetylcholine receptors (nAChRs) are involved in various physiological processes, including neuronal development. We provide evidence for expression of functional nicotinic and muscarinic receptors during differentiation of P19 carcinoma embryonic cells, as an in vitro model of early neurogenesis. We have detected expression and activity of ?2-?7, ?2, ?4 nAChR and M1-M5 mAChR subtypes during neuronal differentiation. Nicotinic ?3 and ?2 mRNA transcription was induced by addition of retinoic acid to P19 cells. Gene expression of ?2, ?4-?7, ?4 nAChR subunits decreased during initial differentiation and increased again when P19 cells underwent final maturation. Receptor response in terms of nicotinic agonist-evoked Ca2+ flux was observed in embryonic and neuronal-differentiated cells. Muscarinic receptor response, merely present in undifferentiated P19 cells, increased during neuronal differentiation. The nAChR-induced elevation of intracellular calcium ([Ca2+]i) response in undifferentiated cells was due to Ca2+ influx. In differentiated P19 neurons the nAChR-induced [Ca2+]i response was reduced following pretreatment with ryanodine, while the mAChR-induced response was unaffected indicating the contribution of Ca2+ release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca2+ responses. The presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca2+ waves during initial neuronal differentiation. © 2007 Elsevier Ltd. All rights reserved.

TITLE:Calcium, iron and neuronal function 

[2-s2.0-34248549227] Refers [2-s2.0-0034742490] LEVEL: 3
Calcium and iron play dual roles in neuronal function: they are both essential but when present in excess they cause neuronal damage and may even induce neuronal death. Calcium signals are required for synaptic plasticity, a neuronal process that entails gene expression and which is presumably the cellular counterpart of cognitive brain functions such as learning and memory. Neuronal activity generates cytoplasmic and nuclear calcium signals that in turn stimulate pathways that promote the transcription of genes known to participate in synaptic plasticity. In addition, evidence discussed in this article shows that iron deficiency causes learning and memory impairments that persist following iron repletion, indicating that iron is necessary for normal development of cognitive functions. Recent results from our group indicate that iron is required for long-term potentiation in hippocampal CA1 neurons and that iron stimulates ryanodine receptor-mediated calcium release through ROS produced via the Fenton reaction leading to stimulation of the ERK signaling pathway. These combined results support a coordinated action between iron and calcium in synaptic plasticity and raise the possibility that elevated iron levels may contribute to neuronal degeneration through excessive intracellular calcium increase caused by iron-induced oxidative stress. © 2007 IUBMB.

TITLE:Calcium signaling in diabetic neuropathy 

[2-s2.0-15044351905] Refers [2-s2.0-0034742490] LEVEL: 3
Diabetic neuropathy is a frequent complication of diabetes mellitus, for which no adequate clinical treatment is currently available. One of the main reasons for the absence of effective treatment of this disease is that information on how metabolic, vascular, and other abnormalities involved in the pathogenesis of diabetic neuropathy lead to dysfunction of nerve cells and pathways remains insufficient. Recent studies demonstrated that substantial abnormalities of calcium homeostasis in input neurons of the somatosensory nociceptive system are associated with many symptoms of diabetic neuropathy. Although proof of the causal linkage between calcium abnormalities and neuropathic complications is not conclusive, current research in neuroscience mostly indicates that such a linkage exists. Practically all known modifications of synaptic transmission in both central and peripheral nervous systems result from calcium-dependent modifications of the molecular players involved in this transmission. This is why the main goal of our review is to analyze in detail the fundamental cellular and molecular calcium-regulating mechanisms that are deteriorated in diabetes. As an important end-point of the proposed review, the capability of a widely used calcium channel blocker, nimodipine, to correct cytosolic and endoplasmic reticulum calcium abnormalities in neurons of the dorsal root ganglia and spinal dorsal horn and possible curative value of this agent in diabetic neuropathy are discussed. © 2004 Springer Science+Business Media, Inc.

TITLE:Role of Ca2+,Mg2+-ATPases in diabetes-induced alterations in calcium homeostasis in input neurons of the nociceptive system 

[2-s2.0-9144257932] Refers [2-s2.0-0034742490] LEVEL: 3
In a rat model of streptozotocin (STZ)-induced diabetes, we earlier showed that under these conditions the concentration of free cytosolic Ca2+ in input neurons of the nociceptive system increases, Ca2+ signals are prolonged, while Ca2+ release from intracellular calcium stores decreases. The aim of our study was to test the hypothesis that changes in the activities of Ca2+,Mg2+-ATPases of the endoplasmic reticulum (SERCA) and plasmalemma (PMCA) could be responsible for diabetes-induced disorders of calcium homeostasis in nociceptive neurons. We measured the Ca2+,Mg2+-ATPase activities in microsomal fractions obtained from tissues of the dorsal root ganglia (DRG) and spinal dorsal horn (DH) of control rats and rats with experimentally induced diabetes. The integral specific Ca2+,Mg2+-ATPase activity in microsomes from diabetic rats was lower than that in the control group. The activity of SERCA in samples of DRG and DH of diabetic rats was reduced by 50 ± 8 and 48 ± 12%, respectively, as compared with the control (P<0.01). At the same time, the activity of PMCA decreased by 63 ± 6% in DRG and by 60 ± 9% in DH samples (P<0.01). We conclude that diabetic polyneuropathy is associated with the reduction of the rate of recovery of the Ca2+ level in the cytosol of DRG and DH neurons due to down-regulation of the SERCA and PMCA activities. © 2004 Springer+Business Media, Inc.

TITLE:The role of calcium and intracellular calcium stores in the mechanisms of neuronal ischemia/reperfusion injury 

[2-s2.0-0346306319] Refers [2-s2.0-0034742490] LEVEL: 3
The mechanisms of neuronal ischemia/reperfusion injury are not yet fully understood. Several processes may contribute to cell death in brain ischemia/reperfusion damage. In addition to the traditional hypothesis of cytotoxic high levels of cytosolic calcium, experiments suggest that mitochondria and endoplasmic reticulum (ER) play an important role in the different modes of neuronal death. We discuss here the role of the ER in triggering apoptotic/necrotic infarction of neural cells. The ER is the site of complex processes, such as calcium storage, synthesis and folding of proteins and cell response to stress. ER function can be altered under conditions associated with oxidative stress, energetic failure, calcium store depletion and in many acute diseases in the brain including ischemia/reperfusion. Complex responses have evolved in response to these disturbances of ER function, which have a great impact to neural viability. Because of intimate connection of ER to mitochondria, alterations of the ER/mitochondria couple may be an alternative pathway, which control neuronal loss after acute injuries and chronic pathological states of the brain. Understanding of the mechanisms leading to the cellular dysfunction may lead to recognition of protection strategies of neurons.

TITLE:Melanin pigmentation in mammalian skin and its hormonal regulation 

[2-s2.0-4644237315] Refers [2-s2.0-12244267707] LEVEL: 3
Cutaneous melanin pigment plays a critical role in camouflage, mimicry, social communication, and protection against harmful effects of solar radiation. Melanogenesis is under complex regulatory control by multiple agents interacting via pathways activated by receptor-dependent and -independent mechanisms, in hormonal, auto-, para-, or intracrine fashion. Because of the multidirectional nature and heterogeneous character of the melanogenesis modifying agents, its controlling factors are not organized into simple linear sequences, but they interphase instead in a multidimensional network, with extensive functional overlapping with connections arranged both in series and in parallel. The most important positive regulator of melanogenesis is the MCI receptor with its ligands melanocortins and ACTH, whereas among the negative regulators agouti protein stands out, determining intensity of melanogenesis and also the type of melanin synthesized. Within the context of the skin as a stress organ, melanogenic activity serves as a unique molecular sensor and transducer of noxious signals and as regulator of local homeostasis. In keeping with these multiple roles, melanogenesis is controlled by a highly structured system, active since early embryogenesis and capable of superselective functional regulation that may reach down to the cellular level represented by single melanocytes. Indeed, the significance of melanogenesis extends beyond the mere assignment of a color trait.

TITLE:Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions 

[2-s2.0-12144291702] Refers [2-s2.0-12244267707] LEVEL: 3
Proteins secreted by activated platelets can adhere to the vessel wall and promote the development of atherosclerosis and thrombosis. Despite this biologic significance, however, the complement of proteins comprising the platelet releasate is largely unknown. Using a proteomics approach, we have identified more than 300 proteins released by human platelets following thrombin activation. Many of the proteins identified were not previously attributed to platelets, including secretogranin III, a potential monocyte chemoattractant precursor; cyclophilin A, a vascular smooth muscle cell growth factor; calumenin, an inhibitor of the vitamin K epoxide reductase-warfarin interaction, as well as proteins of unknown function that map to expressed sequence tags. Secretogranin III, cyclophilin A, and calumenin were confirmed to localize in platelets and to be released upon activation. Furthermore, while absent in normal vasculature, they were identified in human atherosclerotic lesions. Therefore, these and other proteins released from platelets may contribute to atherosclerosis and to the thrombosis that complicates the disease. Moreover, as soluble extracellular proteins, they may prove suitable as novel therapeutic targets. © 2004 by The American Society of Hematology.

TITLE:Genetics: SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans 

[2-s2.0-29144485743] Refers [2-s2.0-12244267707] LEVEL: 3
Lighter variations of pigmentation in humans are associated with diminished number, size, and density of melanosomes, the pigmented organelles of melanocytes. Here we show that zebrafish golden mutants share these melanosomal changes and that golden encodes a putative cation exchanger slc24a5 (nckx5) that localizes to an intracellular membrane, likely the melanosome or its precursor. The human ortholog is highly similar in sequence and functional in zebrafish. The evolutionarily conserved ancestral allele of a human coding polymorphism predominates in African and East Asian populations. In contrast, the variant allele is nearly fixed in European populations, is associated with a substantial reduction in regional heterozygosity, and correlates with lighter skin pigmentation in admixed populations, suggesting a key role for the SLC24A5 gene in human pigmentation.

TITLE:Toward a protein profile of Escherichia coli: Comparison to its transcription profile 

[2-s2.0-0041923788] Refers [2-s2.0-12244267707] LEVEL: 3
High-pressure liquid chromatography-tandem mass spectrometry was used to obtain a protein profile of Escherichia coli strain MG1655 grown in minimal medium with glycerol as the carbon source. By using cell lysate from only 3 × 108 cells, at least four different tryptic peptides were detected for each of 404 proteins in a short 4-h experiment. At least one peptide with a high reliability score was detected for 986 proteins. Because membrane proteins were under-represented, a second experiment was performed with a preparation enriched in membranes. An additional 161 proteins were detected, of which from half to two-thirds were membrane proteins. Overall, 1,147 different E. coli proteins were identified, almost 4 times as many as had been identified previously by using other tools. The protein list was compared with the transcription profile obtained on Affymetrix GeneChips. Expression of 1,113 (97%) of the genes whose protein products were found was detected at the mRNA level. The arithmetic mean mRNA signal intensity for these genes was 3-fold higher than that for all 4,300 protein-coding genes of E. coli. Thus, GeneChip data confirmed the high reliability of the protein list, which contains about one-fourth of the proteins of E. coli. Detection of even those membrane proteins and proteins of undefined function that are encoded by the same operons (transcriptional units) encoding proteins on the list remained low.

TITLE:A proteomic analysis of lysosomal integral membrane proteins reveals the diverse composition of the organelle 

[2-s2.0-14944385370] Refers [2-s2.0-12244267707] LEVEL: 3
Lysosomes are endocytic subcellular compartments that contribute to the degradation and recycling of cellular material. Using highly purified rat liver tritosomes (Triton WR1339-filled lysosomes) and an ion exchange chromatography/LC-tandem MS-based protein/peptide separation and identification procedure, we characterized the major integral membrane protein complement of this organelle. While many of the 215 proteins we identified have been previously associated with lysosomes and endosomes, others have been associated with the endoplasmic reticulum, Golgi, cytosol, plasma membrane, and lipid rafts. At least 20 proteins were identified as unknown cDNAs that have no orthologues of known function, and 35 proteins were identified that function in protein and vesicle trafficking. This latter group includes multiple Rab and SNARE proteins as well as ubiquitin. Defining the roles of these proteins in the lysosomal membrane will assist in elucidating novel lysosomal functions involved in cellular homeostasis and pathways that are affected in various disease processes. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:Regulation of Tyrosinase Processing and Trafficking by Organellar pH and by Proteasome Activity 

[2-s2.0-10744223486] Refers [2-s2.0-12244267707] LEVEL: 3
Pigmentation of the hair, skin, and eyes of mammals results from a number of melanocyte-specific proteins that are required for the biosynthesis of melanin. Those proteins comprise the structural and enzymatic components of melanosomes, the membrane-bound organelles in which melanin is synthesized and deposited. Tyrosinase (TYR) is absolutely required for melanogenesis, but other melanosomal proteins, such as TYRP1, DCT, and gp100, also play important roles in regulating mammalian pigmentation. However, pigmentation does not always correlate with the expression of TYR mRNA/protein, and thus its function is also regulated at the post-translational level. Thus, TYR does not necessarily exist in a catalytically active state, and its post-translational activation could be an important control point for regulating melanin synthesis. In this study, we used a multidisciplinary approach to examine the processing and sorting of TYR through the endoplasmic reticulum (ER), Golgi apparatus, coated vesicles, endosomes and early melanosomes because those organelles hold the key to understanding the trafficking of TYR to melanosomes and thus the regulation of melanogenesis. In pigmented cells, TYR is trafficked through those organelles rapidly, but in amelanotic cells, TYR is retained within the ER and is eventually degraded by proteasomes. We now show that TYR can be released from the ER in the presence of protonophore or proton pump inhibitors which increase the pH of intracellular organelles, after which TYR is transported correctly to the Golgi, and then to melanosomes via the endosomal sorting system. The expression of TYRP1, which facilitates TYR processing in the ER, is down-regulated in the amelanotic cells; this is analogous to a hypopigmentary disease known as oculocutaneous albinism type 3 and further impairs melanin production. The sum of these results shows that organellar pH, proteasome activity, and down-regulation of TYRP1 expression all contribute to the lack of pigmentation in TYR-positive amelanotic melanoma cells.

TITLE:The Seiji Memorial Lecture - The melanosome: An ideal model to study cellular differentiation 

[2-s2.0-0037902561] Refers [2-s2.0-12244267707] LEVEL: 3
Melanosomes provide an intriguing model for study at many levels. In part this is due to their unique structure and function, but also in part to their involvement in pigmentary diseases and as a model to study basic cellular mechanisms of organelle biogenesis. Recent studies have elucidated the full proteome of the melanosome and the metabolic and molecular lesions involved in a number of pigmentary diseases have been resolved. This paper summarizes recent advances in the field in these areas.

TITLE:Biogenesis of pigment granules: A sensitive way to regulate melanocyte function 

[2-s2.0-11144314304] Refers [2-s2.0-12244267707] LEVEL: 3
Pigmentation not only provides a wide range of cosmetic coloration to the skin, hair and eyes, but also provides the underlying tissue significant protection from ultraviolet (UV) damage, which can lead to photoaging and photocarcinogenesis. The melanin pigment is synthesized and deposited within a unique, membrane-bound organelle termed the melanosome. Recent advances in molecular biology and biochemistry have allowed a greater appreciation of how melanocytes generate this organelle and how its biogenesis, structure and function is regulated by the environment. Melanosomes serve as ideal models for the study of organelle biogenesis, protein trafficking, organelle movement and cell-cell interactions that occur during the transfer of melanosomes to keratinocytes. Our understanding of the mechanisms behind a wide range of human pigmentary diseases have grown remarkably as melanosomes have been unraveled.

TITLE:AP-3-dependent mechanisms control the targeting of a chloride channel (ClC-3) in neuronal and non-neuronal cells 

[2-s2.0-2942614762] Refers [2-s2.0-12244267707] LEVEL: 3
Adaptor protein (AP)-2 and AP-3-dependent mechanisms control the sorting of membrane proteins into synaptic vesicles. Mouse models deficient in AP-3, mocha, develop a neurological phenotype of which the central feature is an alteration of the luminal synaptic vesicle composition. This is caused by a severe reduction of vesicular levels of the zinc transporter 3 (ZnT3). It is presently unknown whether this mocha defect is restricted to ZnT3 or encompasses other synaptic vesicle proteins capable of modifying synaptic vesicle contents, such as transporters or channels. In this study, we identified a chloride channel, ClC-3, whose level in synaptic vesicles and hippocampal mossy fiber terminals was reduced in the context of the mocha AP-3 deficiency. In PC-12 cells, ClC-3 was present in transferrin receptor-positive endosomes, where it was targeted to synaptic-like microvesicles (SLMV) by a mechanism sensitive to brefeldin A, a signature of the AP-3-dependent route of SLMV biogenesis. ClC-3 was packed in SLMV along with the AP-3-targeted synaptic vesicle protein ZnT3. Co-segregation of ClC-3 and ZnT3 to common intracellular compartments was functionally significant as revealed by increased vesicular zinc transport with increased ClC3 expression. Our work has identified a synaptic vesicle protein in which trafficking to synaptic vesicles is regulated by AP-3. In addition, our findings indicate that ClC-3 and ZnT3 reside in a common vesicle population where they functionally interact to determine vesicle luminal composition.

TITLE:The Silver locus product Pmel17/gp100/Silv/ME20: Controversial in name and in function 

[2-s2.0-25444456658] Refers [2-s2.0-12244267707] LEVEL: 3
Mouse coat color mutants have led to the identification of more than 120 genes that encode proteins involved in all aspects of pigmentation, from the regulation of melanocyte development and differentiation to the transcriptional activation of pigment genes, from the enzymatic formation of pigment to the control of melanosome biogenesis and movement [Bennett and Lamoreux (2003) Pigment Cell Res. 16, 333]. One of the more perplexing of the identified mouse pigment genes is encoded at the Silver locus, first identified by Dunn and Thigpen [(193O) J. Heredity 21, 495] as responsible for a recessive coat color dilution that worsened with age on black backgrounds. The product of the Silver gene has since been discovered numerous times in different contexts, including the initial search for the tyrosinase gene, the characterization of major melanosome constituents in various species, and the identification of tumor-associated antigens from melanoma patients. Each discoverer provided a distinct name: Pmel17, gp100, gp95, gp85, ME20, RPE1, SILV and MMP115 among others. Although all its functions are unlikely to have yet been fully described, the protein clearly plays a central role in the biogenesis of the early stages of the pigment organelle, the melanosome, in birds, and mammals. As such, we will refer to the protein in this review simply as pre-melanosomal protein (Pmel). This review will summarize the structural and functional aspects of Pmel and its role in melanosome biogenesis. © 2005 The Authors. Journal compilation © 2005 Blackwell Munksgaard.

TITLE:The role of the endosomal/lysosomal system in amyloid-beta production and the pathophysiology of Alzheimer's disease: Reexamining the spatial paradox from a lysosomal perspective 

[2-s2.0-1842532328] Refers [2-s2.0-12244267707] LEVEL: 3
One of the hallmarks of Alzheimer's disease is the cerebral deposition of plaques composed of a 37-43 amino acid amyloid-beta (A?) peptide. A? is produced by the sequential proteolytic cleavage of an integral-membrane protein, amyloid ?-protein precursor (A?PP), first by ?-secretase (BACE), and then by ?-secretase, a complex containing presenilin and Nicastrin. Although these cleavages were originally documented to occur in the endosomal/lysosomal system, other lines of evidence suggest that the responsible proteins and activity reside in the ER or Golgi. This lack of intracellular co-localization of enzyme and substrate has been referred to as the spatial paradox of Alzheimer's disease. Here we will review the biology of the lysosome and the literature supporting the endosomal/lysosomal production of A?. We will also examine some of the data supporting A? production in the biosynthetic compartments and demonstrate its compatibility with an endosomal/lysosomal model. Finally, we will discuss the possible role of the acidic environment of the lysosome in the amyloidogenic process, and review the evidence for intracellular amyloidogenesis preceding amyloid plaque formation.

TITLE:Tyrosinase processing and intracellular trafficking is disrupted in mouse primary melanocytes carrying the underwhite (uw) mutation. A model for oculocutaneous albinism (OCA) type 4 

[2-s2.0-0041885144] Refers [2-s2.0-12244267707] LEVEL: 3
Oculocutaneous albinism (OCA) type 4 is a newly identified human autosomal recessive hypopigmentary disorder that disrupts pigmentation in the skin, hair and eyes. Three other forms of OCA have been previously characterized, each resulting from the aberrant processing and/or sorting of tyrosinase, the enzyme critical to pigment production in mammals. The disruption of tyrosinase trafficking occurs at the level of the endoplasmic reticulum (ER) in OCA1 and OCA3, but at the post-Golgi level in OCA2. The gene responsible for OCA4 is the human homologue of the mouse underwhite (uw) gene, which encodes the membrane-associated transporter protein (MATP). To characterize OCA4, we investigated the processing and sorting of melanogenic proteins in primary melanocytes derived from uw/uw mice and from wild-type mice. OCA4 melanocytes were found to be constantly secreted into the medium dark vesicles that contain tyrosinase and two other melanogenic enzymes, Tyrp1 (tyrosinase-related protein 1) and Dct (DOPAchrome tautomerase); this secretory process is not seen in wild-type melanocytes. Although tyrosinase was synthesized at comparable rates in wild-type and in uw-mutant melanocytes, tyrosinase activity in uw-mutant melanocytes was only about 20% of that found in wild-type melanocytes, and was enriched only about threefold in melanosomes compared with the ninefold enrichment in wild-type melanocytes. OCA4 melanocytes showed a marked difference from wild-type melanocytes in that tyrosinase was abnormally secreted from the cells, a process similar to that seen in OCA2 melanocytes, which results from a mutation of the pink-eyed dilution (P) gene. The P protein and MATP have 12 transmembrane regions and are predicted to function as transporters. Ultrastructural analysis shows that the vesicles secreted from OCA4 melanocytes are mostly early stage melanosomes. Taken together, our results show that in OCA4 melanocytes, tyrosinase processing and intracellular trafficking to the melanosome is disrupted and the enzyme is abnormally secreted from the cells in immature melanosomes, which disrupts the normal maturation process of those organelles. This mechanism explains the hypopigmentary phenotype of these cells and provides new insights into the involvement of transporters in the normal physiology of melanocytes.

TITLE:Epitope mapping of the melanosomal matrix protein gp100 (PMEL17). Rapid processing in the endoplasmic reticulum and glycosylation in the early Golgi compartment 

[2-s2.0-3142546894] Refers [2-s2.0-12244267707] LEVEL: 3
Melanosomes, specific organelles produced only by melanocytes, undergo a unique maturation process that involves their transition form amorphous rounded vesicles to fibrillar ellipsoid organelles, during which they move from the perinuclear to the distal areas of the cells. This depends upon the trafficking and processing of gp100 (also known as Pmel17 and the silver protein), a protein of great interest, because it elicits immune responses in melanoma patients but in which specific function(s) remains elusive. In this study, we have used biochemical and immunochemical approaches to more critically assess the synthesis, processing, glycosylation, and trafficking of gp100. We now report that gp100 is processed and sorted in a manner distinct from other melanosomal proteins (such as tyrosinase, Tyrp1 and Dct) and is predominantly delivered directly to immature melanosomes following its rapid processing in the endoplasmic reticulum and cis-Golgi. Following its arrival, gp100 is cleaved at the amino and at the carboxyl termini in a series of specific steps that result in the reorganization of immature melanosomes to the fibrillar mature melanosomes. Once this structural reorganization occurs, melanogenic enzymes begin to be targeted to the melanosomes, which are then competent to synthesize melanin pigment.

TITLE:Fatty acids regulate pigmentation via proteasomal degradation of tyrosinase: A new aspect of ubiquitin-proteasome function 

[2-s2.0-2442506956] Refers [2-s2.0-12244267707] LEVEL: 3
Fatty acids are common components of biological membranes that are known to play important roles in intracellular signaling. We report here a novel mechanism by which fatty acids regulate the degradation of tyrosinase, a critical enzyme associated with melanin biosynthesis in melanocytes and melanoma cells. Linoleic acid (unsaturated fatty acid, C18:2) accelerated the spontaneous degradation of tyrosinase, whereas palmitic acid (saturated fatty acid, C16:0) retarded the proteolysis. The linoleic acid-induced acceleration of tyrosinase degradation could be abrogated by inhibitors of proteasomes, the multicatalytic proteinase complexes that selectively degrade intracellular ubiquitinated proteins. Linoleic acid increased the ubiquitination of many cellular proteins, whereas palmitic acid decreased such ubiquitination, as compared with untreated controls, when a proteasome inhibitor was used to stabilize ubiquitinated proteins. Immunoprecipitation analysis also revealed that treatment with fatty acids modulated the ubiquitination of tyrosinase, i.e. linoleic acid increased the amount of ubiquitinated tyrosinase whereas, in contrast, palmitic acid decreased it. Furthermore, confocal immunomicroscopy showed that the colocalization of ubiquitin and tyrosinase was facilitated by linoleic acid and diminished by palmitic acid. Taken together, these data support the view that fatty acids regulate the ubiquitination of tyrosinase and are responsible for modulating the proteasomal degradation of tyrosinase. In broader terms, the function of the ubiquitin-proteasome pathway might be regulated physiologically, at least in part, by fatty acids within cellular membranes.

TITLE:"Subcellular proteomics" of neuromelanin granules isolated from the human brain 

[2-s2.0-24944450803] Refers [2-s2.0-12244267707] LEVEL: 3
"Subcellular proteomics" is currently the most effective approach to characterize subcellular compartments. Based on the powerful combination of subcellular fractionation and protein identification by LC-MS/MS we were able for the first time to 1) isolate intact neuromelanin granules from the human brain and 2) establish the first protein profile of these granules. This compartment containing neuromelanin (NM) is primarily located in the primate's substantia nigra, one of the main brain regions that severely degenerates in Parkinson disease. We used mechanic tissue disaggregation, discontinuous sucrose gradient centrifugation, cell disruption, and organelle separation to isolate NM granules from human substantia nigra. Using transmission electron microscopy we demonstrated that the morphological characteristics of the isolated NM granules are similar to those described in human brain tissue. Fundamentally we found numerous proteins definitely demonstrating a close relationship of NM-containing granules with lysosomes or lysosome-related organelles originating from the endosome-lysosome lineage. Intriguingly we further revealed the presence of endoplasmic reticulum-derived chaperones, especially the transmembrane protein calnexin, which recently has been located in lysosome-related melanosomes and has been suggested to be a melanogenic chaperone. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:MART-1 is required for the function of the melanosomal matrix protein PMEL17/GP100 and the maturation of melanosomes 

[2-s2.0-17144379660] Refers [2-s2.0-12244267707] LEVEL: 3
More than 125 genes that regulate pigmentation have been identified to date. Of those, MART-1 has been widely studied as a melanoma-specific antigen and as a melanosome-specific marker. Whereas the functions of other melanosomal proteins, such as tyrosinase, tyrosinase-related protein-1, dopachrome tautomerase, and Pmel17, are known, the function of MART-1 in melanogenesis, is unclear. A role for MART-1 in pigmentation is expected because its expression pattern and subcellular distribution is quite similar to the other melanosomal proteins and usually correlates with melanin content. We investigated the function of MART-1 using a multidisciplinary approach, including the use of siRNA to inhibit MART-1 function and the use of transfection to re-express MART-1 in MART-1-negative cells. We show that MART-1 forms a complex with Pmel17 and affects its expression, stability, trafficking, and the processing which is required for melanosome structure and maturation. We conclude that MART-1 is indispensable for Pmel17 function and thus plays an important role in regulating mammalian pigmentation.

TITLE:Identification of protein associations in organelles, using mass spectrometry-based proteomics 

[2-s2.0-3042687234] Refers [2-s2.0-12244267707] LEVEL: 3
Recent literature that highlights the power of using mass spectrometry (MS) for protein identification from preparations of highly purified organelles and other large subcellular structures is covered in this review with an emphasis on techniques that preserve the integrity of the functional protein complexes. Recent advances in distinguishing contaminant proteins from "bonafide" organelle-localized proteins and the affinity capture of protein complexes are reviewed, as well as bioinformatic strategies to predict protein organellar localization and to integrate protein-protein interaction maps obtained from MS-affinity capture methods with data obtained from other techniques. Those developments demonstrate that a revolution in cellular biology, fueled by technical advances in MS-based proteomic techniques, is well underway. © 2004 Wiley Periodicals, Inc.

TITLE:Comparison of structural and chemical properties of black and red human hair melanosomes 

[2-s2.0-14744281656] Refers [2-s2.0-12244267707] LEVEL: 3
Melanosomes in black and red human hair are isolated and characterized by various chemical and physical techniques. Different yields of 4-amino-hydroxyphenolanaline by HI hydrolysis (a marker for pheomelanin) and pyrrole-2,3,5-tricarboxylic acid by KMnO4/H+ oxidation (a marker for eumelanin) indicate that the melanosomes in black hair are eumelanosomes, whereas those in red hair are mainly pheomelanosomes. Atomic force microscopy reveals that eumelanosomes and pheomelanosomes have ellipsoidal and spherical shapes, respectively. Eumelanosomes maintain structural integrity upon extraction from the keratin matrix, whereas pheomelanosomes tend to fall apart. The black-hair eumelanosomes have an average of 14.6 ± 0.5% amino acids content, which is attributed to the internal proteins entrapped in the melanosomes granules. The red-hair melanosomes contain more than 44% of amino acid content even after extensive proteolytic digestion. This high content of amino acids and the poorly reserved integrity of red-hair melanosomes suggest that some proteins are possibly covalently bonded with the melanin constituents in addition to those that are entrapped inside the melanin species. Soluene solubilization assay indicates the absorbance of melanin per gram of sample, adjusted for the amino acid content, is a factor of 2.9 greater for the black-hair melanosomes than the red-hair melanosomes. Metal analysis reveals significant amounts of diverse heavy metal ions bound to the two types of melanosomes. The amount of Cu(II) and Zn(II) are similar but Fe(III) content is four times higher in the red-hair melanosomes. 13C solid-state nuclear magnetic resonance spectra and infrared spectra are presented and are shown to be powerful techniques for discerning differences in the amino acid contents, the 5,6-dihydroxyindole-2-carboxylic acid:5,6-dihydroxyindole ratio, and the degree of cross-linking in the pigment. Excellent agreement is observed between these spectral results and the chemical degradation data. © 2005 American Society for Photobiology.

TITLE:The relationship between Na+/H+ exchanger expression and tyrosinase activity in human melanocytes 

[2-s2.0-3242685938] Refers [2-s2.0-12244267707] LEVEL: 3
The activity of melanosome-associated tyrosinase in human melanocytes differs based on racial skin type. In melanocytes from Black skin, tyrosinase activity is high while in White melanocytes the activity of the enzyme is low. Recent studies suggest that low tyrosinase activity in White melanocytes may be due to an acidic pH environment within the melanosome. Because sodium/hydrogen (Na+/H+) exchangers (NHEs) are known to regulate intracellular pH, melanocytes were treated with NHE inhibitors to determine what effect this inhibition might have on tyrosinase activity. Treatment of Black melanocytes with ethyl-isopropyl amiloride (EIPA) caused a rapid dose-dependent inhibition of tyrosinase activity. This inhibition was not due to either direct enzyme inhibition or to a decrease in tyrosinase abundance. In contrast, treatment of White melanocytes with EIPA, cimetidine, or clonidine resulted in little inhibition of tyrosinase activity. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis showed that both Black and White melanocytes expressed mRNA and protein for NHE-1, NHE-3, NHE-5, NHE-6, and NHE-7. Immunohistochemical analysis showed that NHE-7 and NHE-3 co-localized with the melanosomal protein, Tyrosinase Related Protein-1 (TRP-1). In addition, the vesicular proton pump, vesicular ATPase (V-ATPase), was found to be present in both White and Black melanosomes, indicating that organelles from both racial skin types are capable of being acidified. The results suggest that one or more NHEs may help regulate melanosome pH and tyrosinase activity in human melanocytes. © 2004 Elsevier Inc. All rights reserved.

TITLE:Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes 

[2-s2.0-33750979817] Refers [2-s2.0-12244267707] LEVEL: 3
Melanin, which is responsible for virtually all visible skin, hair, and eye pigmentation in humans, is synthesized, deposited, and distributed in subcellular organelles termed melanosomes. A comprehensive determination of the protein composition of this organelle has been obstructed by the melanin present. Here, we report a novel method of removing melanin that includes in-solution digestion and immobilized metal affinity chromatography (IMAC). Together with in-gel digestion, this method has allowed us to characterize melanosome proteomes at various developmental stages by tandem mass spectrometry. Comparative profiling and functional characterization of the melanosome proteomes identified ?1500 proteins in melanosomes of all stages, with ?600 in any given stage. These proteins include 16 homologous to mouse coat color genes and many associated with human pigmentary diseases. Approximately 100 proteins shared by melanosomes from pigmented and nonpigmented melanocytes define the essential melanosome proteome. Proteins validated by confirming their intracellular localization include PEDF (pigment-epithelium derived factor) and SLC24A5 (sodium/potassium/calcium exchanger 5, NCKX5). The sharing of proteins between melanosomes and other lysosome-related organelles suggests a common evolutionary rigin. This work represents a model for the study of the biogenesis of lysosome-related organelles. © 2006 American Chemical Society.

TITLE:Melanosome biogenesis: Shedding light on the origin of an obscure organelle 

[2-s2.0-1542504222] Refers [2-s2.0-12244267707] LEVEL: 3
Melanosomes are specialized intracellular organelles in which melanin pigments are synthesized and stored. The ontogenesis of these morphologically unique organelles, as well as their relationship to 'conventional' organelles of the secretory and endocytic pathways, has for decades been a matter of study - and controversy. Recent work by the groups of Michael Marks and Grac?a Raposo has uncovered the molecular mechanism that results in the formation of the lumenal striations characteristic of melanosome precursor organelles.

TITLE:The ocular albinism type 1 (OA1) protein and the evidence for an intracellular signal transduction system involved in melanosome biogenesis 

[2-s2.0-22744435972] Refers [2-s2.0-12244267707] LEVEL: 3
Ocular albinism type 1 is an X-linked disorder characterized by severe reduction of visual acuity, retinal hypopigmentation, foveal hypoplasia, optic misrouting and the presence of giant melanosomes (macromelanosomes) in skin melanocytes and retinal pigment epithelium. The protein product of the 1 OA1 gene is a pigment cell specific membrane glycoprotein, displaying structural and functional features of G protein-coupled receptors (GPCRs). F However, in contrast to all other previously characterized GPCRs, OA1 is not localized to the plasma membrane, but is targeted to intracellular organelles, namely late endosomes/lysosomes and melanosomes. These unique characteristics suggest that OA1 represents the first example described so far of an exclusively intracellular GPCR and regulates melanosome biogenesis by transducing signals from the organelle lumen to the cytosol. These findings support previous hypotheses that GPCR-mediated signaling might also operate at the internal membranes in mammalian cells. Copyright © Blackwell Munksgaard 2005.

TITLE:The potentials of MS-based subproteomic approaches in medical science: The case of lysosomes and breast cancer 

[2-s2.0-7544243714] Refers [2-s2.0-12244267707] LEVEL: 3
Because of the great number of women who are diagnosed with breast cancer each year, and though this disease presents the lowest mortality rate among cancers, breast cancer remains a major public health problem. As for any cancer, the tumorigenic and metastatic processes are still hardly understood, and the biochemical markers that allow either a precise monitoring of the disease or the classification of the numerous forms of breast cancer remain too scarce. Therefore, great hopes are put on the development of high-throughput genomic and proteomic technologies. Such comprehensive techniques should help in understanding the processes and in defining steps of the disease by depicting specific genes or protein profiles. Because techniques dedicated to the current proteomic challenges are continuously improving, the probability of the discovery of new potential protein biomarkers is rapidly increasing. In addition, the identification of such markers should be eased by lowering the sample complexity; e.g., by sample fractionation, either according to specific physico-chemical properties of the proteins, or by focusing on definite subcellular compartments. In particular, proteins of the lysosomal compartment have been shown to be prone to alterations in their localization, expression, or post-translational modifications (PTMs) during the cancer process. Some of them, such as the aspartic protease cathepsin D (CatD), have even been proven as participating actively in the disease progression. The present review aims at giving an overview of the implication of the lysosome in breast cancer, and at showing how subproteomics and the constantly refining MS-based proteomic techniques may help in making breast cancer research progress, and thus, hopefully, in improving disease treatment. © 2004 Wiley Periodicals, Inc.

TITLE:The repeat domain of the melanosomal matrix protein PMEL17/GP100 is required for the formation of organellar fibers 

[2-s2.0-33746323972] Refers [2-s2.0-12244267707] LEVEL: 3
Over 125 pigmentation-related genes have been identified to date. Of those, PMEL17/GP100 has been widely studied as a melanoma-specific antigen as well as a protein required for the formation of fibrils in melanosomes. PMEL17 is synthesized, glycosylated, processed, and delivered to melanosomes, allowing them to mature from amorphous round vesicles to elongated fibrillar structures. In contrast to other melanosomal proteins such as TYR and TYRP1, the processing and sorting of PMEL17 is highly complex. Monoclonal antibody HMB45 is commonly used for melanoma detection, but has the added advantage that it specifically reacts with sialylated PMEL17 in the fibrillar matrix in melanosomes. In this study, we generated mutant forms of PMEL17 to clarify the subdomain of PMEL17 required for formation of the fibrillar matrix, a process critical to pigmentation. The internal proline/serine/threonine-rich repeat domain (called the RPT domain) of PMEL17 undergoes variable proteolytic cleavage. Deletion of the RPT domain abolished its recognition by HMB45 and its capacity to form fibrils. Truncation of the C-terminal domain did not significantly affect the processing or trafficking of PMEL17, but, in contrast, deletion of the N-terminal domain abrogated both. We conclude that the RPT domain is essential for its function in generating the fibrillar matrix of melanosomes and that the luminal domain is necessary for its correct processing and trafficking to those organelles.

TITLE:Functional genomics tools for the analysis of zebrafish pigment 

[2-s2.0-4644239351] Refers [2-s2.0-12244267707] LEVEL: 3
Genetic model organisms are increasingly valuable in the post-genomics era to provide a basis for comparative analysis of the human genome. For higher order processes of vertebrate pigment cell biology and development, the mouse has historically been the model of choice. A complementary organism, the zebrafish (Danio rerio), shares many of the signaling and biological processes of vertebrates, e.g. neural crest development. The zebrafish has a number of characteristics that make it an especially valuable model for the study of pigment cell biology and disease. Large-scale genetic screens have identified a collection of pigmentation mutants that have already made valuable contributions to pigment research. An increasing repertoire of genomic resources such as an expressed sequence tag-based Gene Index (The Institute for Genomic Research) and improving methods of mutagenesis, transgenesis, and gene targeting make zebrafish a particularly attractive model. Morpholino phosphorodiamidate oligonucleotide (MO) 'knockdown' of pigment gene expression provides a nonconventional antisense tool for the analysis of genes involved in pigment cell biology and disease. In addition, an ongoing, reverse-genetic, MO-based screen for the rapid identification of gene function promises to be a valuable complement to other high-throughput microarray and proteomic approaches for understanding pigment cell biology. Novel reagents for zebrafish transgenesis, such as the Sleeping Beauty transposon system, continue to improve the capacity for genetic analysis in this system and ensure that the zebrafish will be a valuable genetic model for understanding a variety of biological processes and human diseases for years to come.

TITLE:Dual loss of ER export and endocytic signals with altered melanosome morphology in the silver mutation of Pmel17 

[2-s2.0-33746617878] Refers [2-s2.0-12244267707] LEVEL: 3
Pmel17 is a pigment cell-specific integral membrane protein that participates in the formation of the intralumenal fibrils upon which melanins are deposited in melanosomes. The Pmel17 cytoplasmic domain is truncated by the mouse silver mutation, which is associated with coat hypopigmentation in certain strain backgrounds. Here, we show that the truncation interferes with at least two steps in Pmel17 intracellular transport, resulting in defects in melanosome biogenesis. Human Pmel17 engineered with the truncation found in the mouse silver mutant (hPmel17si) is inefficiently exported from the endoplasmic reticulum (ER). Localization and metabolic pulse-chase analyses with site-directed mutants and chimeric proteins show that this effect is due to the loss of a conserved C-terminal valine that serves as an ER exit signal. hPmel17si that exits the ER accumulates abnormally at the plasma membrane due to the loss of a di-leucine-based endocytic signal. The combined effects of reduced ER export and endocytosis significantly deplete Pmel17 within endocytic compartments and delay proteolytic maturation required for premelanosome-like fibrillogenesis. The ER export delay and cell surface retention are also observed for endogenous Pmel17si in melanocytes from silver mice, within which Pmel17 accumulation in premelanosomes is dramatically reduced. Mature melanosomes in these cells are larger, rounder, more highly pigmented, and less striated than in control melanocytes. These data reveal a dual sorting defect in a natural mutant of Pmel17 and support a requirement of endocytic trafficking in Pmel17 fibril formation. © 2006 by The American Society for Cell Biology.

TITLE:Proteomic analysis of mature melanosomes from the retinal pigmented epithelium 

[2-s2.0-33644845391] Refers [2-s2.0-12244267707] LEVEL: 3
The protein content of melanosomes in the retinal pigment epithelium (RPE) was analyzed by mass spectrometry. More than 100 proteins were found to be common to two out of three variations of sample preparation. Some proteins normally associated with other organelles were detected. Several lysosomal enzymes were detected, with the presence of cathepsin D confirmed by immunoelectron microscopy, thus supporting the previously suggested notion that melanosomes may contribute to the degradation of ingested photoreceptor outer segment disks. © 2006 American Chemical Society.

TITLE:Proteomics of the human brain: Sub-proteomes might hold the key to handle brain complexity 

[2-s2.0-33746102556] Refers [2-s2.0-12244267707] LEVEL: 3
Proteomics is a promising approach, which provides information about the expression of proteins and increasingly finds application in life science and disease research. Meanwhile, proteomics has proven to be applicable even on post mortem human brain tissue and has opened a new area in neuroproteomics. Thereby, neuroproteomics is usually employed to generate large protein profiles of brain tissue, which mostly reflect the expression of highly abundant proteins. As a complementary approach, the focus on sub-proteomes would enhance more specific insight into brain function. Sub-proteomes are accessible via several strategies, including affinity pull-down approaches, immunoprecipitation or subcellular fractionation. The extraordinary potential of subcellular proteomics to reveal even minute differences in the protein constitution of related cellular organelles is exemplified by a recent global description of neuromelanin granules from the human brain, which could be identified as pigmented lysosome-related organelles. © Springer-Verlag 2006.

TITLE:Different approaches for assaying melanosome transfer 

[2-s2.0-25444465430] Refers [2-s2.0-12244267707] LEVEL: 3
Many approaches have been tried to establish assays for melanosome transfer to keratinocytes. In this report, we describe and summarize various novel attempts to label melanosomes in search of a reliable, specific, reproducible and quantitative assay system. We tried to fluorescently label melanosomes by transfection of GFP-labeled melanosomal proteins and by incubation of melanocytes with fluorescent melanin intermediates or homologues. In most cases a weak cytoplasmic fluorescence was perceived, which was probably because of incorrect sorting or deficient incorporation of the fluorescent protein and different localization. We were able to label melanosomes via incorporation of 14C-thiouracil into melanin. Consequently, we tried to develop an assay to separate keratinocytes with transferred radioactivity from melanocytes after co-culture. Differential trypsinization and different magnetic bead separation techniques were tested with unsatisfactory results. An attempt was also made to incorporate fluorescent thiouracil, since this would allow cells to be separated by FACS. In conclusion, different methods to measure pigment transfer between donor melanocytes and acceptor keratinocytes were thoroughly examined. This information could give other researchers a head start in the search for a melanosome transfer assay with said qualities to better understand pigment transfer. Copyright © Blackwell Munksgaard 2005.

TITLE:Co-localization of Ī²1,6-branched oligosaccharides and coarse melanin in macrophage-melanoma fusion hybrids and human melanoma cells in vitro 

[2-s2.0-2642575686] Refers [2-s2.0-12244267707] LEVEL: 3
Fusion hybrids between normal macrophages and Cloudman S91 melanoma cells were shown earlier to have increased metastatic potential, along with high expression of ?1,6-N-acetylglucosaminyltransferase V and ?1, 6-branched oligosaccharides. Curiously, hybrids, but not parental melanoma cells, also produced 'coarse melanin' - autophagic vesicles with multiple melanosomes. As ?1,6-branched oligosaccharides were known to be associated with metastasis, and coarse melanin had been described in invasive human melanomas, we looked for potential relationships between the two. Using lectin- and immunohistochemistry, we analyzed cell lines producing coarse melanin for ?1,6-branched oligosaccharides: gp100/pmel-17 (a melanosomal structural component) and CD63 (a late endosome/lysosome component associated with melanoma and certain other human cancers). Cell lines used in this study were (i) hybrid 94-H48, a highly metastatic, macrophage-melanoma experimental fusion hybrid; (ii) 6neo mouse melanoma cells, the weakly metastatic, parental fusion partner; and (iii) SKmel-23, a human melanoma cell line derived from a metastasis. Coarse melanin granules were prominent both in hybrids and in SKmel-23 cells, and co-localized with stains for ?1,6-branched oligosaccharides, gp100/pmel 17, and CD63. This is the first report of this phenotype being expressed in vitro, although co-expression of ?1, 6-branched oligosaccharides and coarse melanin was recently shown to be a common and pervasive characteristic in archival specimens of human melanomas, and was most prominent in metastases. The results suggest that pathways of melanogenesis in melanoma may differ significantly from those in normal melanocytes. In vitro expression of this phenotype provides new biological systems for more detailed analyses of its genesis and regulation at the molecular genetic level.

TITLE:Organelle Transport: A Park-and-Ride System for Melanosomes 

[2-s2.0-0345526419] Refers [2-s2.0-12244267707] LEVEL: 3
Organisms that are able to change the color of their skin do so by regulating the intracellular distribution of pigment-containing vesicles. A recent study has shed new light on the coordination in cells of the molecular motors that mediate the transport of these vesicles on microtubules and actin.

TITLE:Human skin pigmentation: Melanocytes modulate skin color in response to stress 

[2-s2.0-33947616725] Refers [2-s2.0-12244267707] LEVEL: 3
All organisms, from simple invertebrates to complex human beings, exist in different colors and patterns, which arise from the unique distribution of pigments throughout the body. Pigmentation is highly heritable, being regulated by genetic, environmental, and endocrine factors that modulate the amount, type, and distribution of melanins in the skin, hair, and eyes. In addition to its roles in camouflage, heat regulation, and cosmetic variation, melanin protects against UV radiation and thus is an important defense system in human skin against harmful factors. Being the largest organ of the body that is always under the influence of internal and external factors, the skin often reacts to those agents by modifying the constitutive pigmentation pattern. The focus of this review is to provide an updated overview of important physiological and biological factors that increase pigmentation and the mechanisms by which they do so. We consider endocrine factors that induce temporary (e.g., during pregnancy) or permanent (e.g., during aging) changes in skin color, environmental factors (e.g., UV), certain drugs, and chemical compounds, etc. Understanding the mechanisms by which different factors and compounds induce melanogenesis is of great interest pharmaceutically (as therapy for pigmentary diseases) and cosmeceutically (e.g., to design tanning products with potential to reduce skin cancer risk). © FASEB.

TITLE:Adeno-associated virus interactions with B23/Nucleophosmin: Identification of sub-nucleolar virion regions 

[2-s2.0-33751414730] Refers [2-s2.0-12244267707] LEVEL: 3
Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four replication proteins (Rep78, 68, 52 and 40) encoded by AAV are pleiotropic effectors of virus integration, replication, transcription and virion assembly. Using Rep68 column chromatography and mass spectrometry, we have identified the nucleolar, B23/Nucleophosmin (NPM) protein as an Rep-interacting partner. Rep-NPM interactions were verified by co-immunofluorescence and chemical cross-linking studies. We have found that there is demonstrable, but limited co-localization between Rep and NPM in co-infected cells. In contrast, there was significant co-localization between NPM and AAV Cap proteins. In vitro experiments using purified MBPRep78 and NPM show that NPM stimulates MBPRep78 interactions with the AAV ITR as well as endonuclease activity. These studies suggest that NPM plays a role in AAV amplification affecting Rep function and virion assembly. © 2006 Elsevier Inc. All rights reserved.

TITLE:Lysosomal membranes from Beige mice contain higher than normal levels of endoplasmic reticulum proteins 

[2-s2.0-33846570350] Refers [2-s2.0-12244267707] LEVEL: 3
Chediak-Higashi syndrome is characterized by dysfunctional giant organelles of common origin, that is, lysosomes, melanosomes, and platelet dense bodies. Its defective gene LYST encodes a large molecular weight protein whose function is unknown. The Beige mouse also defective in Lyst is a good model of the human disease. Purified lysosomes from Beige and normal black mouse livers were used to carry out a proteomics study. Two-dimensional gel electrophoretic separation of soluble lysosomal proteins of Beige and normal mice revealed no major differences. The cleavable isotope-coded affinity tag (cICAT) technique was used to compare the composition of Beige and normal lysosomal membrane proteins. While the levels of common proteins, that is, Lamp1, Lamp2, and Niemann-Pick type C1, were decreased in Beige mice, there was an increase in the levels of endoplasmic reticulum (ER) resident proteins, for example, cytochrome P450, NADPH-cytochrome P450 oxidoreductase, and flavin-containing monooxygenase. Confocal microscopy confirmed that another ER protein, calnexin, colocalizes with Lamp1 on membranes of giant lysosomes from fibroblasts of Chediak-Higashi syndrome patient. Our results suggest that LYST may play a role in either preventing inappropriate incorporation of proteins into the lysosomal membrane or in membrane recycling/maturation. © 2007 American Chemical Society.

TITLE:Subcellular proteomics reveals neuromelanin granules to be a lysosome-related organelle 

[2-s2.0-33744937070] Refers [2-s2.0-12244267707] LEVEL: 3
The powerful combination of subcellular fractionation and protein identification by electrospray ionization tandem mass spectrometry (ESI-MS/MS) pioneered the molecular elucidation of neuromelanin (NM) granules. We recently isolated NM granules from the human brain and succeeded in the establishment of the first protein profile of this compartment. NM granules are pigmented organelles, which are mainly found in the catecholaminergic neurons of the human substantia nigra (SN) pars compacta and the locus coeruleus. These granules contain the insoluble pigment NM, which is regarded as the most important iron storage system in these neurons. A global examination of NM granules, however, has so far been hampered due to the lack of a pigmented brain stem in rodents, the absence of an appropriate experimental system and their scarcity in the human brain. 'Subcellular proteomics', which increasingly emerges as the method of choice to characterize cellular compartments and to elucidate their biogenesis, has recently been shown to be an adequate approach to tackle a thorough description of NM granules. Thereby, NM granules could be described as a 'lysosome-related organelle'. This indicates a genetic program underlying a biogenesis of NM rather than its autoxidative formation. © Springer-Verlag 2006.

TITLE:Pmel17: Controversial indeed but critical to melanocyte function [2] 

[2-s2.0-33646698641] Refers [2-s2.0-12244267707] LEVEL: 3
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TITLE:Synthetic curcuminoids modulate the arachidonic acid metabolism of human platelet 12-lipoxygenase and reduce sprout formation of human endothelial cells 

[2-s2.0-33745087445] Refers [2-s2.0-12244267707] LEVEL: 3
Platelet 12-lipoxygenase (P-12-LOX) is overexpressed in different types of cancers, including prostate cancer, and the level of expression is correlated with the grade of this cancer. Arachidonic acid is metabolized by 12-LOX to 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], and this biologically active metabolite is involved in prostate cancer progression by modulating cell proliferation in multiple cancer-related pathways inducing angiogenesis and metastasis. Thus, inhibition of P-12-LOX can reduce these two processes. Several lipoxygenase inhibitors are known, including plant and mammalian lipoxygenases, but only a few of them are known inhibitors of P-12-LOX. Curcumin is one of these lipoxygenase inhibitors. Using a homology model of the three-dimensional structure of human P-12-LOX, we did computational docking of synthetic curcuminoids (curcumin derivatives) to identify inhibitors superior to curcumin. Docking of the known inhibitors curcumin and NDGA to P-12-LOX was used to optimize the docking protocol for the system in study. Over 75% of the compounds of interest were successfully docked into the active site of P-12-LOX, many of them sharing similar binding modes. Curcuminoids that did not dock into the active site did not inhibit P-12-LOX. From a set of the curcuminoids that were successfully docked and selected for testing, two were found to inhibit human lipoxygenase better than curcuminin. False-positive curcuminoids showed high LogP (theoretical) values, indicating poor water solubility, a possible reason for lack of inhibitory activity or/and nonrealistic binding. Additionally, the curcuminoids inhibiting P-12-LOX were tested for their ability to reduce sprout formation of endothelial cells (in vitro model of angiogenesis). We found that only curcuminoids inhibiting human P-12-LOX and the known inhibitor NDGA reduced sprout formation. Only limited inhibition of sprout formation at ? IC50 concentrations has been seen. At IC50, a substantial amount of 12-HETE can be produced by lipoxygenase, providing a stimulus for angiogenic sprouting of endothelial cells. Increasing the concentration of lipoxygenase inhibitors above IC50, thus decreasing the concentration of 12(S)-HETE produced, greatly reduced sprout formation for all inhibitors tested. This universal event for all tested lipoxygenase inhibitors suggests that the inhibition of sprout formation was most likely due to the inhibition of human P-12-LOX but not other cancer-related pathways. Copyright © 2006 American Association for Cancer Research.

TITLE:Human melanocyte biology, toxicology, and pathology 

[2-s2.0-27644584480] Refers [2-s2.0-12244267707] LEVEL: 3
The human melanocytes of the skin, hair, eyes, inner ears, and covering of the brain provide physiologic functions important in organ development and maintenance. Melanocytes develop from embryonic neural crest progenitors and share certain traits with other neural crest derivatives found in the adrenal medulla and peripheral nervous system. The distinctive metabolic feature of melanocytes is the synthesis of melanin pigments from tyrosine and cysteine precursors involving over 100 gene products. These complex biochemical mechanisms create inherent liabilities for melanocytic cells if intracellular systems necessary for compartmentalization, detoxification, or repair are compromised. Melanocyte disorders may involve pigmentation, sensory functions, autoimmunity, or malignancy. Environmental factors such as ultraviolet radiation and chemical exposures, combined with heritable traits, represent the principal hazards associated with melanocyte disorders. Copyright © Taylor & Francis Inc.

TITLE:Ouabain decreases sarco(endo)plasmic reticulum calcium ATPase activity in rat hearts by a process involving protein oxidation 

[2-s2.0-33845420039] Refers [2-s2.0-12244267707] LEVEL: 3
The effect of cardiac glycosides to increase cardiac inotropy by altering Ca2+ cycling is well known but still poorly understood. The studies described in this report focus on defining the effects of ouabain signaling on sarcoplasmic reticulum Ca2+-ATPase function. Rat cardiac myocytes treated with 50 ?M ouabain demonstrated substantial increases in systolic and diastolic Ca2+ concentrations. The recovery time constant for the Ca2+ transient, ?Ca2+, was significantly prolonged by ouabain. Exposure to 10 ?M H2O2, which causes an increase in intracellular reactive oxygen species similar to that of 50 ?M ouabain, caused a similar increase in ?Ca2+. Concurrent exposure to 10 mM N-acetylcysteine or an aqueous extract from green tea (50 mg/ml) both prevented the increases in ?Ca2+ as well as the changes in systolic or diastolic Ca2+ concentrations. We also observed that 50 ?M ouabain induced increases in developed pressure in addition to diastolic dysfunction in the isolated perfused rat heart. Coadministration of ouabain with N-acetylcysteine prevented these increases. Analysis of sarcoplasmic reticulum Ca2+-ATPase protein revealed increases in both the oxidation and nitrotyrosine content in the ouabain-treated hearts. Liquid chromatography-mass spectrometric analysis confirmed that the sarcoplasmic reticulum Ca 2+-ATPase protein from ouabain-treated hearts had modifications consistent with oxidative and nitrosative stress. These data suggest that ouabain induces oxidative changes of the sarcoplasmic reticulum Ca 2+-ATPase structure and function that may, in turn, produce some of the associated changes in Ca2+ cycling and physiological function. Copyright © 2006 the American Physiological Society.

TITLE:In Situ Hybridization: An Informative Technique for Pigment Cell Researchers 

[2-s2.0-0842348923] Refers [2-s2.0-12244267707] LEVEL: 3
Many cellular events are regulated at the transcriptional level. Recent technical advances such as DNA microarray have made it possible to determine mRNA profiles of cultured cells or tissues. However, since it is still impossible to completely simulate the in vivo environment in culture conditions, mRNA profiles of cultured cells are not perfect representatives of original cells. Furthermore, for cells that exist at lower densities, mRNA profiling using tissue samples would be difficult. By using tissue in situ hybridization, mRNA levels of genes in tissues can be determined at cellular resolution. Although throughput of tissue in situ hybridization is not high enough for mRNA profiling, it may be sufficient to investigate temporal/spatial expression profiles of genes that are known to be important or found to be interesting in high-throughput transcriptome/proteome analyses. Recent technical advances have made it easier for everybody to perform tissue in situ hybridization using normal experimental instruments with sufficient sensitivity to detect most genes. Although this technique has been utilized mainly in developmental biology, it will be fully advantageous when combined with high-throughput comprehensive transcriptome/proteome analyses.

TITLE:The differing embryonic origins of retinal and uveal (iris/ciliary body and choroid) melanosomes are mirrored by their phospholipid composition 

[2-s2.0-33846125559] Refers [2-s2.0-12244267707] LEVEL: 3
The phospholipids present in uveal (iris/ciliary body and choroid) and retinal bovine ocular melanosomes were identified using mass spectrometry. Similar phospholipid content is found for the two types of uveal melanosome, with sphingomyelin being the major species. Significant differences are found between the uveal and retinal melanosome. Glycerophosphoethanolamine (GPEtn) is the major species in the retinal pigment epithelium (RPE); 93% of the GPEtn contain polyunsaturated fatty acids, notably docosahexanoic acid and arachidonic acid, in the sn-2 position. RPE melanosomes also contain detectable quantities of glycerophosphoserine and glycerophosphate; these species were not detected in the uveal samples. While the structural and functional roles of melanosomal lipids largely remain to be determined, these different lipid compositions reported herein offer new insights into the roles of melanosomes in the different ocular tissues. © 2007 The Authors.

TITLE:Identification of novel proteins induced by estradiol, 4-hydroxytamoxifen and acolbifene in T47D breast cancer cells 

[2-s2.0-33750011457] Refers [2-s2.0-12244267707] LEVEL: 3
Tamoxifen is currently used as adjuvant therapy for estrogen receptor (ER) positive breast cancer patients and as a chemopreventative agent. Although ER is a predictive marker for tamoxifen response, ER status fails to predict tamoxifen response in a significant number of patients highlighting the need to identify new pathways for tamoxifen sensitivity/resistance. To identify novel proteins induced by tamoxifen in breast cancer cells sensitive to tamoxifen growth inhibition, two-dimensional (2D) gel electrophoresis was used to profile proteins in T47D breast cancer cells. Six proteins were identified that were differentially regulated by 17?-estradiol, 4-hydroxytamoxifen and the pure antagonist acolbifene (EM-652); calreticulin, synapse associated protein 1 (SYAP1), CD2 antigen binding protein 2 (CD2BP2), nucleosome assembly protein 1 like 1 (NAP1L1), d-3-phosphoglycerate dehydrogenase (3-PHGDH) and pyridoxine 5? phosphate oxidase (PNPO). At the mRNA level, these ligands differentially regulated expression of mRNAs encoding the identified proteins in T47D and MCF7 cells but had no effect on mRNA in ER?-negative MDA-MB-231 breast cancer cells. These novel SERM-regulated proteins may participate in new or existing pathways for sensitivity or resistance to SERMs. © 2006 Elsevier Inc. All rights reserved.

TITLE:Reply to Valencia et al. [3] 

[2-s2.0-33646673333] Refers [2-s2.0-12244267707] LEVEL: 3
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TITLE:An unconventional dileucine-based motif and a novel cytosolic motif are required for the lysosomal and melanosomal targeting of OA1 

[2-s2.0-33745193071] Refers [2-s2.0-12244267707] LEVEL: 3
The protein product of the gene responsible for ocular albinism type 1, named OA1, is a pigment-cell-specific membrane glycoprotein, displaying features of G-protein-coupled receptors, yet exclusively localized to late endosomes, lysosomes and melanosomes. To dissect the signals responsible for the intracellular localization of OA1, we generated chimeric proteins consisting of the cytosolic domains of OA1 fused to the lumenal and transmembrane domains of LAMP1; in addition, we generated missense and deletion mutants of full-length OA1. Using this approach, we identified two separate sorting signals that are both necessary and sufficient for intracellular retention, as well as lysosomal and melanosomal localization, in melanocytic and non-melanocytic cells. These sorting signals are an unconventional dileucine motif within the third cytosolic loop and a novel motif, characterized by a tryptophan-glutamic acid doublet, within the C-terminal tail. Both motifs must be mutated to promote the plasma membrane localization of OA1, suggesting that they can independently drive its intracellular targeting. In addition, both motifs act similarly as lysosomal sorting signals in non-melanocytic cells, but appear to carry different specificities in melanocytic cells. Our findings indicate that OA1 contains multiple unconventional signals responsible for its lysosomal and melanosomal localization, and reveal a remarkable and unforeseen complexity in the regulation of polytopic protein sorting to specialized secretory organelles.

TITLE:GRP78/BiP is involved in ouabain-induced endocytosis of the Na/K-ATPase in LLC-PK1 cells 

[2-s2.0-21344440546] Refers [2-s2.0-12244267707] LEVEL: 3
We have demonstrated that ouabain causes dose- and time-dependent decreases both in 86Rb+ uptake and plasmalemmal Na/K-ATPase content of LLC-PK1 cells, which is related to ouabain-induced endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells through a clathrin-dependent mechanism. GRP78/BiP is a resident protein of the endoplasmic reticulum (ER) and acts as a molecular chaperone. Recently, several studies have shown that GRP78/BiP is also expressed on the cell surface and forms heterogeneous, high molecular weight complexes with other proteins. To identify the proteins that are possibly involved in ouabain-induced endocytosis of the Na/K-ATPase in LLC-PK1 cells, we separated and identified endosomal proteins by 2D gel electrophoresis and MS/MS from both control and ouabain-treated LLC-PK1 cells. GRP78/BiP was identified by MS/MS as one of the several up-regulated proteins and confirmed by Western Blot. By using a cell surface protein biotinylation technique to isolate the cell surface membrane proteins, we found that GRP78/BiP is also expressed on the cell surface of LLC-PK1 cells, and surface-expressed GRP78/BiP is down regulated in a time-dependent manner in response to ouabain. By comparing the cellular redistributions, our data suggest that both the Na/K-ATPase ?-1 subunit and GRP78/BiP follow the same redistribution pattern in response to ouabain.

TITLE:Targets of the cyclin-dependent kinase Cdk1 

[2-s2.0-0242300176] Refers [2-s2.0-0036008093] LEVEL: 3
The events of cell reproduction are governed by oscillations in the activities of cyclin-dependent kinases (Cdks). Cdks control the cell cycle by catalysing the transfer of phosphate from ATP to specific protein substrates. Despite their importance in cell-cycle control, few Cdk substrates have been identified. Here, we screened a budding yeast proteomic library for proteins that are directly phosphorylated by Cdk1 in whole-cell extracts. We identified about 200 Cdk1 substrates, several of which are phosphorylated in vivo in a Cdk1-dependent manner. The identities of these substrates reveal that Cdk1 employs a global regulatory strategy involving phosphorylation of other regulatory molecules as well as phosphorylation of the molecular machines that drive cell-cycle events. Detailed analysis of these substrates is likely to yield important insights into cell-cycle regulation.

TITLE:Phosphospecific proteolysis for mapping sites of protein phosphorylation 

[2-s2.0-0042861519] Refers [2-s2.0-0036008093] LEVEL: 3
Protein phosphorylation is a dominant mechanism of information transfer in cells, and a major goal of current proteomic efforts is to generate a system-level map describing all the sites of protein phosphorylation. Recent efforts have focused on developing technologies for enriching and quantifying phosphopeptides. Identification of the sites of phosphorylation typically relies on tandem mass spectrometry to sequence individual peptides. Here we describe an approach for phosphopeptide mapping that makes it possible to interrogate a protein sequence directly with a protease that recognizes sites of phosphorylation. The key to this approach is the selective chemical transformation of phosphoserine and phosphothreonine residues into lysine analogs (aminoethylcysteine and ?-methylaminoethylcysteine, respectively). Aminoethylcysteine-modified peptides are then cleaved with a lysine-specific protease to map sites of phosphorylation. A blocking step enables single-site cleavage, and adaptation of this reaction to the solid phase facilitates phosphopeptide enrichment and modification in one step.

TITLE:Interaction of cortactin and N-WASp with Arp2/3 complex 

[2-s2.0-0037031144] Refers [2-s2.0-0036008093] LEVEL: 3
Background: Dynamic actin assembly is required for diverse cellular processes and often involves activation of Arp2/3 complex. Cortactin and N-WASp activate Arp2/3 complex, alone or in concert. Both cortactin and N-WASp contain an acidic (A) domain that is required for Arp2/3 complex binding. Results: We investigated how cortactin and the constitutively active VCA domain of N-WASp interact with Arp2/3 complex. Structural studies showed that cortactin is a thin, elongated monomer. Chemical crosslinking studies demonstrated selective interaction of the Arp2/3 binding NTA domain of cortactin (cortactin NTA) with the Arp3 subunit and VCA with Arp3, Arp2, and ARPC1/ p40. Cortactin NTA and VCA crosslinking to the Arp3 subunit were mutually exclusive; however, cortactin NTA did not inhibit VCA crosslinking to Arp2 or ARPC1/ p40, nor did it inhibit activation of Atp2/3 complex by VCA. We conducted an experiment in which a saturating concentration of cortactin NTA modestly lowered the binding affinity of VCA for Atp2/3; the results of this experiment provided further evidence for ternary complex formation. Consistent with a common binding site on Arp3, a saturating concentration of VCA abolished binding of cortactin to Arp2/3 complex. Conclusions: Under certain circumstances, cortactin and N-WASp can bind simultaneously to Arp2/3 complex, accounting for their synergy in activation of actin assembly. The interaction of cortactin NTA with Arp2/3 complex does not inhibit Arp2/3 activation by N-WASp, despite competition for a common binding site located on the Arp3 subunit. These results suggest a model in which cortactin may bridge Arp2/3 complex to actin filaments via Arp3 and N-WASp activates Atp2/3 complex by binding Atp2 and/or ARPC1/p40.

TITLE:Regulation of F-actin-dependent processes by the Abl family of tyrosine kinases 

[2-s2.0-0038445642] Refers [2-s2.0-0036008093] LEVEL: 3
The F-actin cytoskeleton is a fundamental component of all eukaryotic cells. It provides force and stability and plays an integral role in a diverse array of cellular processes. The spatiotemporal regulation of F-actin dynamics is essential for proper biological output. The basic molecular machinery underlying the assembly and disassembly of filamentous actin is conserved in all eukaryotic cells. Additionally, protein tyrosine kinases, found only in multicellular eukaryotes, provide links between extracellular signals and F-actin-dependent cellular processes. Among the tyrosine kinases, c-Abl and its relative Arg are unique in binding directly to F-actin. Recent results have demonstrated a role for c-Abl in membrane ruffling, cell spreading, cell migration, and neurite extension in response to growth factor and extracellular matrix signals. c-Abl appears to regulate the assembly of F-actin polymers into different structures, depending on the extracellular signal. Interestingly, c-Abl contains nuclear import and export signals, and the nuclear c-Abl inhibits differentiation and promotes apoptosis in response to genotoxic stress. The modular structure and the nuclear-cytoplasmic shuttling of c-Abl suggest that it integrates multiple signals to coordinate F-actin dynamics with the cellular decision to differentiate or to die.

TITLE:The emerging power of chemical genetics 

[2-s2.0-0036532116] Refers [2-s2.0-0036008093] LEVEL: 3
Chemical genetic methods allow signal transduction pathways to be probed in a domain-specific manner. This subtle perturbation of function, when combined with classical genetic and biochemical data, allows for a better understanding of protein function. This in turn is leading to elucidation of pharmacological maps of signaling pathways. Recent studies have focused on diverse pathways, including the initiation of actin polymerization, oncogenic tyrosine kinase control of cell transformation, and molecular motor involvement in adaptation of sensory cells of the inner ear.

TITLE:Src family kinases: Potential targets for the treatment of human cancer and leukemia 

[2-s2.0-0141538143] Refers [2-s2.0-0036008093] LEVEL: 3
The inherited or acquired deregulation of protein kinase activity has been implicated in the pathogenesis of many human diseases, including cancer. Therefore, the inhibition of kinases has been proposed to be a promising strategy in the context of anti-cancer treatment. Many other kinases have been selected as drug discovery targets based on the prevalence of mutations, over-expression and unscheduled activation in human cancer. Of the various protein kinases chosen, Src family kinases are amongst the most extensively studied kinase oncogenes in academia and industry. This review focuses on our current understanding of the deregulation and role of Src family kinases in human cancer and leukemia. Recent data implicate the action of c-Src in cancer metastasis, mediated by up-regulation of various protease systems (calpain, uPA) as well as disruption of E-cadherin signalling. Moreover, novel roles of various Src family members in the development of human leukemia have been found. New insights into downstream signalling mechanisms, including the activation of STAT3, PDK1 and Akt, further corroborate the importance of Src family kinases in tumorigenesis and chemoresistance. Despite our rather clear understanding of Src family kinases as pro-oncogenes no Src family kinase inhibitor has entered a clinical trial so far. This review will discuss prerequisites to be fulfilled for clinically targeting c-Src and its homologues using small molecule drugs.

TITLE:Novel chemical genetic approaches to the discovery of signal transduction inhibitors 

[2-s2.0-0037101904] Refers [2-s2.0-0036008093] LEVEL: 3
Concurrent advances in both high-throughput chemistry and genomics have given rise to the field of chemical genetics as a discipline for elucidating and validating drug targets, and generating novel therapeutics. Indeed, chemical genetic approaches to drug discovery have now been applied to several important drug target classes, especially those involved in signal transduction. Chemical genetics is distinct from the broader term 'chemogenomics' which is defined as the description of all possible drugs against all possible targets (reviewed in [1]). This review covers several 'orthogonal' chemical genetic approaches and focuses on a unique analog sensitive kinase technology and its applications to kinase drug discovery.

TITLE:Two Cyclin-Dependent Kinases Promote RNA Polymerase II Transcription and Formation of the Scaffold Complex 

[2-s2.0-0842347413] Refers [2-s2.0-0036008093] LEVEL: 3
Three cyclin-dependent kinases, CDK7, -8, and -9, are specifically involved in transcription by RNA polymerase II (Pol II) and target the Pol II C-terminal domain (CTD). The role of CDK7 and CDK8 kinase activity in transcription has been unclear, with CDK7 shown to have variable effects on transcription and CDK8 suggested to repress transcription and/or to target other gene-specific factors. Using a chemical genetics approach, the Saccharomyces cerevisiae homologs of these kinases, Kin28 and Srb10, were engineered to respond to a specific inhibitor and the inhibitor was used to test the role of these kinases in transcription in vivo and in vitro. In vitro, these kinases can both promote transcription, with up to 70% of transcription abolished when both kinases are inhibited together. Similarly, in vivo inhibition of both kinases together gives the strongest decrease in transcription, as measured by chromatin immunoprecipitation of Pol II. Kin28 and Srb10 also have overlapping roles in promoting ATP-dependent dissociation of the preinitiation complex (PIC) into the Scaffold complex. Using the engineered kinases and an ATP analog, specific kinase substrates within the PIC were identified. In addition to the previously known substrate, the Pol II CTD, it was found that Kin28 phosphorylates two subunits of Mediator and Srb10 targets two subunits of TFIID for phosphorylation.

TITLE:LIME: A New Membrane Raft-associated Adaptor Protein Involved in CD4 and CD8 Coreceptor Signaling 

[2-s2.0-10744225431] Refers [2-s2.0-0036008093] LEVEL: 3
Lymphocyte membrane rafts contain molecules critical for immunoreceptor signaling. Here, we report identification of a new raft-associated adaptor protein LIME (Lck-interacting molecule) expressed predominantly in T lymphocytes. LIME becomes tyrosine phosphorylated after cross-linking of the CD4 or CD8 coreceptors. Phospho-LIME associates with the Src family kinase Lck and its negative regulator, Csk. Ectopic expression of LIME in Jurkat T cells results in an increase of Csk in lipid rafts, increased phosphorylation of Lck and higher Ca2+ response to CD3 stimulation. Thus, LIME appears to be involved in regulation of T cell activation by coreceptors.

TITLE:Identification of novel ERK2 substrates through use of an engineered kinase and ATP analogs 

[2-s2.0-0038351827] Refers [2-s2.0-0036008093] LEVEL: 3
The mitogen-activated protein kinases are key regulators of cellular organization and function. To understand the mechanisms(s) by which these ubiquitous kinases affect specific cellular changes, it is necessary to identify their diverse and numerous substrates in different cell contexts and compartments. As a first step in achieving this goal, we engineered a mutant ERK2 in which a bulky amino acid residue in the ATP binding site (glutamine 103) is changed to glycine, allowing this mutant to utilize an analog of ATP (cyclopentyl ATP) that cannot be used by wild-type ERK2 or other cellular kinases. The mutation did not inhibit ERK2 kinase activity or substrate specificity in vitro or in vivo. This method allowed us to detect only ERK2-specific phosphorylations within a mixture of proteins. Using this ERK2 mutant/analog pair to phosphorylate ERK2-associated proteins in COS-1 cells, we identified the ubiquitin ligase EDD (E3 identified by differential display) and the nucleoporin Tpr (translocated promoter region) as two novel substrates of ERK2, in addition to the known ERK2 substrate Rskl. To further validate the method, we present data that confirm that ERK2 phosphorylates EDD in vitro and in vivo. These results not only identify two novel ERK2 substrates but also provide a framework for the future identification of numerous cellular targets of this important signaling cascade.

TITLE:Chemical genetics 

[2-s2.0-33745726644] Refers [2-s2.0-0036008093] LEVEL: 3
The field of chemical genetics is dynamic and rapidly growing. Chemical genetics has now grown into a major wing of chemical biology and a standard tool of many researchers. The proof of principle stage of chemical genetics has long passed and many are seeking to optimize and expand upon the existing tools. The increasingly diverse libraries being generated will provide a rich source of probes for future studies.

TITLE:Mek1 Kinase Activity Functions Downstream of RED1 in the Regulation of Meiotic Double Strand Break Repair in Budding Yeast 

[2-s2.0-0347990577] Refers [2-s2.0-0036008093] LEVEL: 3
Three meiosis-specific chromosomal components in budding yeast, Mek1, Red1, and Hop1, are required for recombination, proper segregation of homologs, and the meiotic recombination checkpoint. Mek1 is a protein kinase. Mutations that increase the size of the ATP binding pocket of Mek1 (mek1-as1) sensitize the kinase to specific small molecule inhibitors. Experiments using mek1-as1 demonstrate that the requirement for Mek1 kinase activity coincides with the formation of double strand breaks (DSBs) and that this activity is necessary after DSB formation to prevent repair by DMC1-independent pathways. Contrary to previous reports, Red1 is not a substrate for Mek1. Instead, RED1 is required for wild-type levels of Mekl kinase activity. In addition, activation of Mek1 requires HOP1, the formation of Red1/Hop1 complexes and a functional Mek1 FHA domain. The requirement for RED1 to produce active kinase can be bypassed by a mek1 mutation that creates a constitutively active Mekl kinase. We propose that Redl is phosphorylated by a kinase other than MEK1 and that phosphothreonines on Red1 then interact with the Mek1 FHA domain to recruit the kinase to sites of DSBs where Mek1 is activated to prevent DMC1-independent DSB repair.

TITLE:PTP-1B is an essential positive regulator of platelet integrin signaling 

[2-s2.0-24144482771] Refers [2-s2.0-0036008093] LEVEL: 3
Outside-in integrin ?IIb?3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process. In resting platelets, c-Src forms a complex with ?IIb?3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to ?IIb?3 triggers PTP-1B recruitment to the ?IIb?3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from ?IIb?3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B-deficient platelets are defective in outside-in ?IIb?3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in ?IIb?3 signaling in platelets. © The Rockefeller University Press.

TITLE:Combining chemical genetics and proteomics to identify protein kinase substrates 

[2-s2.0-29144455854] Refers [2-s2.0-0036008093] LEVEL: 3
Phosphorylation is a ubiquitous protein modification important for regulating nearly every aspect of cellular biology. Protein kinases are highly conserved and constitute one of the largest gene families. Identifying the substrates of a kinase is essential for understanding its cellular role, but doing so remains a difficult task. We have developed a high-throughput method to identify substrates of yeast protein kinases that employs a collection of yeast strains each expressing a single epitope-tagged protein and a chemical genetic strategy that permits kinase reactions to be performed in native, whole-cell extracts. Using this method, we screened 4,250 strains expressing epitope-tagged proteins and identified 24 candidate substrates of the Pho85-Pcl1 cyclin-dependent kinase, including the known substrate Rvs167. The power of this method to identify true kinase substrates is strongly supported by functional overlap and colocalization of candidate substrates and the kinase, as well as by the specificity of Pho85-Pcl1 for some of the substrates compared with another Pho85-cyclin kinase complex. This method is readily adaptable to other yeast kinases. © 2005 by The National Academy of Sciences of the USA.

TITLE:Advances in chemical genetics 

[2-s2.0-25844519058] Refers [2-s2.0-0036008093] LEVEL: 3
Chemical genetics is an emerging approach for studying biological systems using chemical tools. This strategy aims to reveal the macromolecules responsible for regulating biological systems; thus, the approach shares much in common with genetics. In both strategies, one must (a) develop an assay that reports on a biological process of interest, (b) perturb this process systematically (with mutations or small molecules), and (c) determine the target of each perturbation to reveal macromolecules (i.e., proteins and genes) regulating the process of interest. In this review, we discuss advances and challenges in this field that have emerged over the past four years. Several technologies have converged, raising the hope that it may be possible to systematically apply chemical probes to biological processes. Copyright © 2005 by Annual Reviews. All rights reserved.

TITLE:c-Abl phosphorylates Dok1 to promote filopodia during cell spreading 

[2-s2.0-2542478972] Refers [2-s2.0-0036008093] LEVEL: 3
Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/ SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.

TITLE:Chemical rescue of a mutant enzyme in living cells 

[2-s2.0-33644684486] Refers [2-s2.0-0036008093] LEVEL: 3
The restoration of catalytic activity to mutant enzymes by small molecules is well established for in vitro systems. Here, we show that the protein tyrosine kinase Src arginine-388?alanine (R388A) mutant can be rescued in live cells with the use of the small molecule imidazole. Cellular rescue of a viral Src homolog was rapid and reversible and conferred predicted oncogenic properties. Using chemical rescue in combination with mass spectrometry, we confirmed six known Src kinase, substrates and identified several new protein targets. Chemical rescue data suggest that cellular Src is active under basal conditions. Rescue of R388A cellular Src provided insights into the mitogen-activated protein kinase pathway. This chemical rescue approach will likely have many applications in cell signaling.

TITLE:Inhibitor scaffolds as new allele specific kinase substrates 

[2-s2.0-0037120875] Refers [2-s2.0-0036008093] LEVEL: 3
The elucidation of protein kinase signaling networks is challenging due to the large size of the protein kinase superfamily (>500 human kinases). Here we describe a new class of orthogonal triphosphate substrate analogues for the direct labeling of analogue-specific kinase protein targets. These analogues were constructed as derivatives of the Src family kinase inhibitor PP1 and were designed based on the crystal structures of PP1 bound to HCK and N6-(benzyl)-ADP bound to c-Src (T338G). 3-Benzylpyrazolo-pyrimidine triphosphate (3-benzyl-PPTP) proved to be a substrate for a mutant of the MAP kinase p38 (p38-T106G/A157L/L167A). 3-Benzyl-PPTP was preferred by v-Src (T338G) (kcat/KM = 3.2 x 106 min-1 M-1) over ATP or the previously described ATP analogue, N6 (benzyl) ATP. For the kinase CDK2 (F80G)/ cyclin E, 3-benzyl-PPTP demonstrated catalytic efficiency (kcat/KM = 2.6 x 104 min-1 M-1) comparable to ATP (kcat/KM = 5.0 x 104 min-1 M-1) largely due to a significantly better KM (6.4 ?M vs 530 ?M). In kinase protein substrate labeling experiments both 3-benzyl-PPTP and 3-phenyl-PPTP prove to be over 4 times more orthogonal than N6-(benzyl)-ATP with respect to the wild-type kinases found in murine spleenocyte cell lysates. These experiments also demonstrate that [?-32p]-3-benzyl-PPTP is an excellent phosphodonor for labeling the direct protein substrates of CDK2 (F80G)/E in murine spleenocyte cell lysates, even while competing with cellular levels (4 mM) of unlabeled ATP. The fact that this new more highly orthogonal nucleotide is accepted by three widely divergent kinases studied here suggests that it is likely to be generalizable across the entire kinase superfamily.

TITLE:Structural Basis for the Specific Recognition of RET by the Dok1 Phosphotyrosine Binding Domain 

[2-s2.0-10744231760] Refers [2-s2.0-0036008093] LEVEL: 3
Dok1 is a common substrate of activated protein-tyrosine kinases. It is rapidly tyrosine-phosphorylated in response to receptor tyrosine activation and interacts with ras GTPase-activating protein and Nck, leading to inhibition of ras signaling pathway activation and the c-Jun N-terminal kinase (JNK) and c-Jun activation, respectively. In chronic myelogenous leukemia cells, it has shown constitutive phosphorylation. The N-terminal phosphotyrosine binding (PTB) domain of Dok1 can recognize and bind specifically to phosphotyrosine-containing motifs of receptors. Here we report the crystal structure of the Dok1 PTB domain alone and in complex with a phosphopeptide derived from RET receptor tyrosine kinase. The structure consists of a ?-sandwich composed of two nearly orthogonal, 7-stranded, antiparallel ?-sheets, and it is capped at one side by a C-terminal ?-helix. The RET phosphopeptide binds to Dok1 via a surface groove formed between strand ?5 and the C-terminal ?-helix of the PTB domain. The structures reveal the molecular basis for the specific recognition of RET by the Dok1 PTB domain. We also show that Dok1 does not recognize peptide sequences from TrkA and IL-4, which are recognized by Shc and IRS1, respectively.

TITLE:Dexamethasone up-regulates the inhibitory adaptor protein Dok-1 and suppresses downstream activation of the mitogen-activated protein kinase pathway in antigen-stimulated RBL-2H3 mast cells 

[2-s2.0-14944347295] Refers [2-s2.0-0036008093] LEVEL: 3
The glucocorticoid dexamethasone suppresses antigen-induced degranulation, cytokine production, and intermediate signaling events in RBL-2H3 mast cells, although the exact mechanisms are uncertain. By microarray analysis, we discovered that expression of the inhibitory adaptor protein, downstream of tyrosine kinase (Dok)-1, was up-regulated 4-fold in dexamethasone-treated RBL-2H3 cells. The up-regulation was apparent with as little as 1 to 10 nM dexamethasone. Treatment with dexamethasone also enhanced tyrosine phosphorylation of Dok-1, augmented recruitment of Ras GTPase-activating protein (RasGAP) by Dok-1, and inhibited activation of the mitogen-activated protein (MAP) kinase pathway in antigen-stimulated cells. The same effects were obtained by transient overexpression of Dok-1 but not by overexpression of Dok-1 that was mutated in RasGAP-binding domain. The negative regulatory role of Dok-1 was further validated by the expression of small interfering RNA directed against Dok-1, which enhanced activation of MAP kinase and subsequent release of arachidonic acid and tumor necrosis factor-?. These findings identify Dok-1 as mediator of the antiallergic actions of dexamethasone and as a negative regulator of the MAP kinase pathway and downstream release of inflammatory mediators.

TITLE:Functional interaction of RasGAP-binding proteins Dok-1 and Dok-2 with the Tec protein tyrosine kinase 

[2-s2.0-1542427147] Refers [2-s2.0-0036008093] LEVEL: 3
The Dok adaptor family of proteins binding to RasGAP, consisting of Dok-1 and Dok-2, are critical regulators in cell proliferation. These molecules are partners and/or substrates of different protein tyrosine kinases considered as oncoproteins. Here, we show that Dok-1 and Dok-2 are the major tyrosine-phosphorylated proteins associated to Tec, a protein tyrosine kinase expressed in T cells. Furthermore, we evaluate the effect of Dok-1 or Dok-2 on Tec-mediated signalling pathways in T cells. Here, we provide evidence that Dok-1 and Dok-2 proteins are involved in a negative feedback regulation of Tec via a downregulation of its tyrosine phosphorylation and downstream signalling pathways including the Ras pathway. Either Dok-1 or Dok-2 therefore represents a mean of potent retrograde control for protein tyrosine kinase signalling, and then possibly of tumor development.

TITLE:Dok-R mediates attenuation of epidermal growth factor-dependent mitogen-activated protein kinase and Akt activation through processive recruitment of c-Src and Csk 

[2-s2.0-17644396027] Refers [2-s2.0-0036008093] LEVEL: 3
Dok-R has previously been shown to associate with the epidermal growth factor receptor (EGFR) and become tyrosine phosphorylated in response to EGF stimulation. The recruitment of Dok-R to the EGFR, which is mediated through its phosphotyrosine binding (PTB) domain, results in attenuation of mitogen-activated protein kinase (MAPK) activation. Dok-R's ability to attenuate EGF-driven MAPK activation is independent of its ability to recruit rasGAP, a known attenuator of MAPK activity, suggesting an alternate Dok-R-mediated pathway. Herein, we have determined the structural determinants within Dok-R that are required for its ability to attenuate EGF signaling and to associate with c-Src and with the Src family kinase (SFK)-inhibitory kinase, Csk. We demonstrate that Dok-R associates constitutively witli c-Src through an SH3-dependent interaction and that this association is essential to Dok-R's ability to attenuate c-Src activity and diminish MAPK and Akt/PKB activity. We further illustrate that EGF-dependent phosphorylation of Dok-R requires SFK activity and, more specifically, that SFK-dependent phosphorylation of tyrosine 402 on Dok-R facilitates the inducible recruitment of Csk. We propose that recruitment of Csk to Dok-R serves to bring Csk to c-Src and down-regulate its activity, resulting in a concomitant attenuation of MAPK and Akt/PKB activity. Furthermore, we demonstrate that Dok-R can abrogate c-Src's ability to protect the breast cancer cell line SKBR3 from anoikis and that an association with c-Src and Csk is required for this activity. Collectively these results demonstrate that Dok-R acts as an EGFR-recruited scaffolding molecule that processively assembles c-Src and Csk to attenuate signaling from the EGFR. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

TITLE:DOK1 mediates SHP-2 binding to the Ī±VĪ²3 integrin and thereby regulates insulin-like growth factor I signaling in cultured vascular smooth muscle cells 

[2-s2.0-13544270759] Refers [2-s2.0-0036008093] LEVEL: 3
Recruitment of the Src homology 2 domain tyrosine phosphatase (SHP-2) to the phosphorylated ?3 subunit of the ?V?3 integrin is required for insulin-like growth factor I (IGF-I)-stimulated cell migration and proliferation in vascular smooth muscle cells. Because SHP-2 does not bind directly to ?3, we attempted to identify a linker protein that could mediate SHP-2/?3 association. DOK1 is a member of insulin receptor substrate protein family that binds ?3 and contains YXXL/I motifs that are potential binding sites for SHP-2. Our results show that IGF-I induces DOK1 binding to ?3 and to SHP-2. Pre-incubation of cells with synthetic peptides that blocked either DOK1/?3 or DOK1/SHP-2 association inhibited SHP-2 recruitment to ?3. Expression of a DOK1 mutant that does not bind to ?3 also disrupts SHP-2/?3 association. As a result of SHP-2/?3 disruption, IGF-I dependent phosphorylation of Akt and p44/p42 mitogen-activated protein kinase and its ability to stimulate cell migration and proliferation were significantly impaired. These results demonstrate that DOK1 mediates SHP-2/?3 association in response to IGF-I thereby mediating the effect of integrin ligand occupancy on IGF-IR-linked signaling in smooth muscle cells.

TITLE:Dok-1 tyrosine residues at 336 and 340 are essential for the negative regulation of Ras-Erk signalling, but dispensable for rasGAP-binding 

[2-s2.0-3042774135] Refers [2-s2.0-0036008093] LEVEL: 3
Dok-1 is a common substrate of many protein tyrosine kinases (PTKs). It recruits rasGAP and other SH2-containing proteins and negatively regulates Ras-Erk signalling downstream of PTKs. However, the mechanisms of its inhibitory effect are yet unclear. Here, a series of C-terminal deletion mutants of Dok-1 delineated the core domain for the inhibition of Erk from 334 to 346 amino acid, which contains two SH2-binding motifs having Tyr-336 or Tyr-340. The Dok-1 mutants having tyrosine-to-phenylalanine (YF) substitution(s) at Tyr-336 and/or Tyr-340 lost their inhibitory effect on Ras and Erk downstream of Src-like PTK, Lyn or Fyn, whereas the rasGAP-binding of each mutant remained intact. However, the Dok-1 mutant having YF substitutions at the rasGAP-binding sites (Tyr-295 and Tyr-361) also showed incapability of Ras and Erk inhibition. Moreover, the Dok-1 mutant having YF substitutions at Tyr-336 and Tyr-340 showed an impaired inhibitory effect on v-Abl-induced transformation of NIH-3T3 cells. These results demonstrate that Tyr-336 and Tyr-340 of Dok-1 are dispensable for rasGAP-binding but essential for inhibition of Ras-Erk signalling and cellular transformation downstream of PTKs. Thus, Dok-1 probably recruits as yet unidentified molecule(s), which, in concert with rasGAP, negatively regulate Ras-Erk signalling. © Blackwell Publishing Limited.

TITLE:Engineering a "methionine clamp" into Src family kinases enhances specificity toward unnatural ATP analogues 

[2-s2.0-0038047895] Refers [2-s2.0-0036008093] LEVEL: 3
A single alanine or glycine mutation in the ATP binding site of a protein kinase allows unique use of an unnatural analogue of ATP (N6-(benzyl) ATP) as a phosphodonor, which is not accepted by wild-type kinases. Addition of [?32P] N6-(benzyl) ATP to a cell lysate containing an ATP analog-specific kinase allele (as1 allele) results in the exclusive radiolabeling of bona fide substrates of the mutant kinase. Here we report efforts to engineer kinase alleles that have enhanced selectivity for ATP analogues and decreased catalytic activity with ATP, thus increasing the signal-to-noise ratio of substrate labeling. Two conserved leucine residues that contact each face of the adenine ring of ATP were mutated to methionine. The introduction of this "methionine clamp" resulted in Src and Fyn kinase alleles that have markedly improved specificity for unnatural N6-substituted ATP analogues over the natural substrate, ATP. This preference for unnatural nucleotides is reflected in more efficient labeling of protein substrates in cell extracts using the new analogue-specific v-Src allele. Kinase alleles with enhanced selectivity for unnatural ATP analogues should greatly facilitate the ultimate goal of labeling kinase substrates in intact cells, where concentrations of ATP and other competing nucleotides are high.

TITLE:Divergent roles of c-Src in controlling platelet-derived growth factor-dependent signaling in fibroblasts 

[2-s2.0-27644454764] Refers [2-s2.0-0036008093] LEVEL: 3
The vast complexity of platelet-derived growth factor (PDGF)-induced downstream signaling pathways is well known, but the precise roles of critical players still elude us due to our lack of specific and temporal control over their activities. Accordingly, although Src family members are some of the better characterized effectors of PDGF? signaling, considerable controversy still surrounds their precise functions. To address these questions and limitations, we applied a chemical-genetic approach to study the role of c-Src at the cellular level, in defined signaling cascades; we also uncovered novel phosphorylation targets and defined its influence on transcriptional events. The spectacular control of c-Src on actin reorganization and chemotaxis was delineated by global substrate labeling and transcriptional analysis, revealing multiple cytoskeletal proteins and chemotaxis promoting genes to be under c-Src control. Additionally, this tool revealed the contrasting roles of c-Src in controlling DNA synthesis, where it transmits conflicting inputs via the phosphatidylinositol 3 kinase and Ras pathways. Finally, this study reveals a mechanism by which Src family kinases may control PDGF-mediated responses both at transcriptional and translational levels. © 2005 by The American Society for Cell Biology.

TITLE:Engineering the serine/threonine protein kinase Raf-1 to utilise an orthogonal analogue of ATP substituted at the N6 position 

[2-s2.0-9144232533] Refers [2-s2.0-0036008093] LEVEL: 3
One key area of protein kinase research is the identification of cognate substrates. The search for substrates is hampered by problems in unambiguously assigning substrates to a particular kinase in vitro and in vivo. One solution to this impasse is to engineer the kinase of interest to accept an ATP analogue which is orthogonal (unable to fit into the ATP binding site) for the wild-type enzyme and the majority of other kinases. The acceptance of structurally modified, ?-32P-labelled, nucleotide analogue by active site-modified kinase can provide a unique handle by which the direct substrates of any particular kinase can be displayed in crude mixtures or cell lysates. We have taken this approach with the serine/threonine kinase Raf-1, which plays an essential role in the transduction of stimuli through the Ras?Raf? MEK?ERK/MAP kinase cascade. This cascade plays essential roles in proliferation, differentiation and apoptosis. Here we detail the mutagenesis strategy for the ATP binding pocket of Raf-1, such that it can utilise an N 6-substituted ATP analogue. We show that these mutations do not alter the substrate specificity and signal transduction through Raf-1. We screen a library of analogues to identify which are orthogonal for Raf-1, and show that mutant Raf-1 can utilise the orthogonal analogue N6(2- phenethyl) ATP in vitro to phosphorylate its currently only accepted substrate MEK. Importantly we show that our approach can be used to tag putative direct substrates of Raf-1 kinase with 32P-N6(2-phenethyl) ATP in cell lysates. © 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

TITLE:Phospho-proteomic analysis of cellular signaling 

[2-s2.0-33745933821] Refers [2-s2.0-0036008093] LEVEL: 3
Reversible protein phosphorylation plays an important role in the regulation of many different processes, such as cell growth, differentiation, migration, metabolism, and apoptosis. Identification of differentially phosphorylated proteins by means of phospho-proteomic analysis provides insight into signal transduction pathways that are activated in response to, for example, growth factor stimulation or toxicant-induced apoptosis. This review summarizes recent advances made in the field of phospho-proteomics and provides examples of how phospho-proteomic techniques can be combined to quantitatively investigate the dynamic changes in protein phosphorylation in time. By linking experimental data to clinical data (e.g., disease progression or response to therapy) new disease markers could be identified, which could then be validated for applications in disease diagnosis and progression or prediction of a response to drugs. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

TITLE:Dok-1 independently attenuates Ras/mitogen-activated protein kinase and Src/c-Myc pathways to inhibit platelet-derived growth factor-induced mitogenesis 

[2-s2.0-33645221824] Refers [2-s2.0-0036008093] LEVEL: 3
The Dok adaptor proteins play key regulatory roles in receptor and non-receptor kinase-initiated signaling pathways. Dok-1, the prototype member of this family, negatively regulates cell proliferation elicited by numerous growth factors, including platelet-derived growth factor (PDGF). However, how Dok-1 exerts its negative effect on mitogenesis has remained elusive. Using Dok-1 knockout cells and Dok-1 mutants deficient in binding to specific Dok-1-interacting proteins, we show that Dok-1 interferes with PDGF-stimulated c-myc induction and Ras/mitogen-activated protein kinase (MAPK) activation by tethering different signaling components to the cell membrane. Specifically, Dok-1 attenuates PDGF-elicited c-myc induction by recruiting Csk to active Src kinases, whereupon their activities and consequent c-myc induction are diminished. On the other hand, Dok-1 negatively regulates PDGF-induced MAPK activation by acting on Ras-GAP and at least one other Dok-1-interacting protein. Importantly, we demonstrate that Dok-1's actions on both of these signaling pathways contribute to its inhibitory effect on mitogenesis. Our data suggest a mechanistic basis for the inhibitory effect of Dok-1 on growth factor-induced mitogenesis and its role as a tumor suppressor. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

TITLE:Derivatization of phosphopeptides with mercapto- and amino-functionalized conjugate groups by phosphate elimination and subsequent Michael addition 

[2-s2.0-24144459552] Refers [2-s2.0-0036008093] LEVEL: 3
Kinetics of the ?-elimination of the phosphate group from H-Tyr-Ser(PO3H2)-Phe-OH and H-Tyr-Thr(PO3H 2)-Phe-OH and subsequent addition of thiols and amines to the dehydroalaninyl and ?-methyldehydroalaninyl residues formed, were followed by RP HPLC under alkaline conditions in the absence and presence of Ba 2+ ions. By this reaction sequence, the phosphoserinyl peptide was conjugated with mono-N-(2-mercaptoethyl)amide of 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid (4), a mercapto-functionalized pentapeptide, H-His-Gly-Gly-His-Gly-NH(CH2)4SH, and an amino-functionalized fluorescent dye, 5-dimethylaminonaphthalene-1-[N-(5- aminopentyl)]sulfonamide (dansyl cadaverine). The ?-methyldehydroalanine residue was, in turn, observed to be a poor Michael acceptor. © The Royal Society of Chemistry 2005.

TITLE:Chemical kinomics - A target gene family approach in chemical biology 

[2-s2.0-13344249686] Refers [2-s2.0-0036008093] LEVEL: 3
Traditionally, protein kinases have been regarded as non-druggable targets, instead they play a central role in physiological and pathophysiological processes. This changed when STI571, an inhibitor of the Bcr-Abl kinase, known as Gleevec, reached the market as the first designer drug. Ever since, kinase-directed research and development (R&D) expanded rapidly, leading to more than 45 clinically relevant kinase inhibitors. At a comparable pace the kinase-based technologies matured, cumulating in the development of sophisticated chemogenetic and chemoproteomic tools, which are referred to as chemical kinomics. © 2004 Elsevier Ltd. All rights reserved.

TITLE:Frameshift mutation in the Dok1 gene in chronic lymphocytic leukemia 

[2-s2.0-1842589307] Refers [2-s2.0-0036008093] LEVEL: 3
B-cell chronic lymphocytic leukemia (B-CLL) is a malignant disease characterized by an accumulation of monoclonal CD5 + mature B cells, with a high percentage of cells arrested in the G0/G1 phase of the cell cycle, and a particular resistance toward apoptosis-inducing agents. Dok1 (downstream of tyrosine kinases) is an abundant Ras-GTPase-activating protein (Ras-GAP)-associated tyrosine kinase substrate, which negatively regulates cell proliferation, downregulates MAP kinase activation and promotes cell migration. The gene encoding Dok1 maps to human chromosome 2p13, a region previously found to be rearranged in B-CLL. We have screened the Dok1 gene for mutations from 46 individuals with B-CLL using heteroduplex analysis. A four-nucleotide GGCC deletion in the coding region was found in the leukemia cells from one patient. This mutation causes a frameshift leading to protein truncation at the carboxyl-terminus, with the acquisition of a novel amino-acid sequence. In contrast to the wild-type Dok1 protein, which has cytoplasmic/ membrane localization, the mutant Dok1 is a nuclear protein containing a functional bipartite nuclear localization signal. Whereas overexpression of wild-type Dok1 inhibited PDGF-induced MAP kinase activation, this inhibition was not observed with the mutant Dok1. Furthermore the mutant Dok1 forms lieterodimers with Dok1 wild type and the association can be enhanced by Lck-mediated tyrosine-phosphorylation. This is the first example of a Dok1 mutation in B-CLL and the data suggest that Dok1 might play a role in leukemogenesis.

TITLE:Direct methods for modulating protein function 

[2-s2.0-0037344734] Refers [2-s2.0-0036008093] LEVEL: 3
During the past few years, the drug discovery process has shifted from a chemistry- to a biology-driven paradigm. Genome sciences have made a significant contribution to this shift, leading to a plethora of potential drug targets that are mainly proteins. Genetic methods will continue to be used to characterize proteins, but more direct methods are needed to determine the suitability of these protein targets for pharmacological intervention. In addition to the use of antibodies and aptamers, technologies focusing on the direct modulation of protein activity, including chemical genetics, analog-sensitive enzyme alleles and chromophore-assisted laser inactivation, should significantly contribute to the field of post-genomic drug discovery and development.

TITLE:High-Level Expression of Dok-1 in Neurons of the Primate Prefrontal Cortex and Hippocampus 

[2-s2.0-0347360091] Refers [2-s2.0-0036008093] LEVEL: 3
The docking protein p62Dok-1 (Dok-1) has a central role in cell signaling mediated by a wide range of protein tyrosine kinases, including intrinsic membrane kinases, such as the insulin-like growth factor-1 (IGF-1) receptor. To elucidate potential IGF signaling mechanisms, we used DNA array technology to investigate novel kinase targets expressed in the primate dorsolateral prefrontal cortex (DLPFC). Dok-1 transcripts were among the most abundant found in this structure. Because Dok-1 expression has not been characterized in brain, we evaluated its expression pattern using immunoblotting, in situ hybridization, and immunohistochemistry in the rhesus monkey prefrontal cortex and hippocampal formation. Dok-1 antibodies identified a 62-kDa band in lysates from the DLPFC, consistent with the known size for Dok-1. In situ hybridization showed that Dok-1 mRNA was expressed in all layers of the DLPFC and in all neuronal subregions of the hippocampal formation. Immunohistochemical analysis showed Dok-1 immunoreactivity concentrated in pyramidal neurons of cortical layers IV-V and throughout Ammon's horn and in granule neurons of the dentate gyrus. Dok-1 expression was also identified in endothelial cells of cerebral blood vessels. These expression patterns are very similar to those of the IGF-1 receptor and suggest that Dok-1 could be among the downstream targets of IGF signaling in areas of the primate brain involved in learning and memory.

TITLE:Conformational restraint is a critical determinant of unnatural nucleotide recognition by protein kinases 

[2-s2.0-0037020764] Refers [2-s2.0-0036008093] LEVEL: 3
This report describes the synthesis of N4-(benzyl) AICAR triphosphate, a conformationally restrained analogue of N4-(benzyl) ribavirin triphosphate. Both of these nucleotides were evaluated as phosphodonors for wild-type p38 MAP kinase and T106G p38 MAP kinase, a designed mutant with expanded nucleotide specificity. The conformationally restrained nucleotide, N4-(benzyl) AICAR triphosphate, is orthogonal to (not accepted as a substrate by) wild-type p38 MAP kinase, in contrast to N4-(benzyl) ribavirin triphosphate. Furthermore, N4-(benzyl) AICAR triphosphate, is accepted as a substrate by T106G p38 MAP kinase, in contrast to N4-(benzyl) ribavirin triphosphate. We hypothesize that the presence of an internal hydrogen bond in N4-(benzyl) AICAR and its absence in N4-(benzyl) ribavirin triphosphate is the main determinant for their differing structure-activity relationships. © 2002 Elsevier Science Ltd. All rights reserved.

TITLE:Using chemical genetics and ATP analogues to dissect protein kinase function 

[2-s2.0-34347394374] Refers [2-s2.0-0036008093] LEVEL: 3
Protein kinases catalyze the transfer of the ?-phosphate of ATP to a protein substrate and thereby profoundly alter the properties of the phosphorylated protein. The identification of the substrates of protein kinases has proven to be a very difficult task because of the multitude of structurally related protein kinases present in cells, their apparent redundancy of function, and the lack of absolute specificity of small-molecule inhibitors. Here, we review approaches that utilize chemical genetics to determine the functions and substrates of protein kinases, focusing on the design of ATP analogues and protein kinase binding site mutants. © 2007 by American Chemical Society.

TITLE:Orthogonal chemical genetic approaches for unraveling signaling pathways 

[2-s2.0-23044450708] Refers [2-s2.0-0036008093] LEVEL: 3
While chemical genetic approach uses small molecules to probe protein functions in cells or organisms, orthogonal chemical genetics refers to strategies that utilize reengineered protein-small molecule interfaces, to alter specificities, in order to probe their functions. The advantage of orthogonal chemical genetics is that the changes at the interfaces are generally so minute that it goes undetected by natural processes, and thus depicts a true physiological picture of biological phenomenon. This review highlights the recent advances in the area of orthogonal chemical genetics, especially those designed to probe signaling processes. Dynamic protein-protein and enzyme-substrate interactions following stimuli form the foundation of signal transduction. These processes not only break spatial and temporal boundaries between interacting proteins, but also impart distinct regulatory properties by creating functional diversity at the interfaces. Functional and temporal modulation of these dynamic interactions by specific chemical probes provides extremely powerful tools to initiate, ablate, decouple and deconvolute different components of a signaling pathway at multiple stages. Not surprisingly, multiple receptor-ligand reengineering approaches have been developed in the last decade to selectively manipulate these transient interactions with the aim of unraveling signaling events. However, given the diversity of protein-protein interactions and novel chemical genetic probes developed to perturb these processes, a short review cannot do adequate justice to all aspects of signaling. For this reason, this review focuses on some orthogonal chemical-genetic strategies that are developed to study signaling processes involving enzyme-substrate interactions. © 2005 IUBMB.

TITLE:Unraveling kinase signaling pathways with chemical genetic and chemical proteomic approaches 

[2-s2.0-25444501580] Refers [2-s2.0-0036008093] LEVEL: 3
Protein kinases are involved in a wide variety of physiological and pathological processes. Small kinase inhibitor molecules are frequently used to dissect signaling pathways or to counteract oncogenic events. However, in many cases the precise role of a given inhibitor is not well understood. Besides a known primary target, potential secondary targets might be involved in the biological response to the drug. We describe recent advances in chemical genetics and chemical proteomics that allow a new comprehensive analysis of kinase signaling pathways. One approach consists of engineering the kinase pocket to design inhibitor sensitive and resistant alleles. A second strategy is to modify the kinase pocket to specifically accept radio-labeled ATP analogs, allowing the identification of direct downstream substrates after transphosphorylation. The third method takes advantage of recently improved inhibitor affinity chromatography to identify inhibitor targets by mass spectrometry. Ultimately, these new technologies will help define the actions of kinase inhibitors useful for human disease treatment. ©2005 Landes Bioscience.

TITLE:Use of a chemical genetic technique to identify myosin IIB as a substrate of the Abl-related gene (Arg) tyrosine kinase 

[2-s2.0-35348923794] Refers [2-s2.0-0036008093] LEVEL: 3
Abl family kinases have been implicated in the regulation of cell morphogenesis and migration, but the molecular mechanisms through which they operate are not fully elucidated. We applied the bump-hole technique, pioneered by Shokat and colleagues, to identify direct substrates of Abl and the Abl-related gene (Arg) kinases. This technique required the engineering of Abl/Arg to utilize an unnatural ATP analogue as a phospho-donor. Mutation of T334A and T361A in Abl and Arg, respectively, altered their nucleotide specificity and allowed them to utilize N6-benzyl-ATP as a phospho-donor. These mutations did not affect the catalytic activity or protein substrate specificity of Abl and Arg. An unexpected high level of background labeling necessitated further optimization of this approach. Dialysis, pretreatment with a broad-spectrum Ser/Thr kinase inhibitor, K-252a, and purification of phosphotyrosine-containing proteins allowed for definitive identification of putative substrates. Using mass spectrometry, we identified eight putative substrates. One of these putative substrates, myosin IIB, can be phosphorylated in vivo by Arg. Our results indicate that the bump-hole technique can be used to identify Abl family kinase substrates and suggests that myosin IIB may be regulated by tyrosine phosphorylation. © 2007 American Chemical Society.

TITLE:Identification of ChChd3 as a novel substrate of the cAMP-dependent protein kinase (PKA) using an analog-sensitive catalytic subunit 

[2-s2.0-34447540362] Refers [2-s2.0-0036008093] LEVEL: 3
Due to the numerous kinases in the cell, many with overlapping substrates, it is difficult to find novel substrates for a specific kinase. To identify novel substrates of cAMP-dependent protein kinase (PKA), the PKA catalytic subunit was engineered to accept bulky N6-substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src (1). Methionine 120 was mutated to glycine in the ATP-binding pocket of the catalytic subunit. To express the stable mutant C-subunit in Escherichia coli required co-expression with PDK1. This mutant protein was active and fully phosphorylated on Thr197 and Ser338. Based on its kinetic properties, the engineered C-subunit preferred N6(benzyl)-ATP and N 6(phenethyl)-ATP over other ATP analogs, but still retained a 30 ?M Km for ATP. This mutant recombinant C-subunit was used to identify three novel PKA substrates. One protein, a novel mitochondrial ChChd protein, ChChd3, was identified, suggesting that PKA may regulate mitochondria proteins. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:A single amino-acid change in ERK1/2 makes the enzyme susceptible to PP1 derivatives 

[2-s2.0-30944438639] Refers [2-s2.0-0036008093] LEVEL: 3
We generated extracellular signal-regulated kinase 1/2 (ERK1/2) mutants by introducing a single amino-acid substitution in subdomain V of the catalytic domain and then examined the susceptibility of these mutants to PP1 derivatives originally designed as Src inhibitors. Substituting smaller amino acids (alanine [Ala (A)] or glycine [Gly (G)]) for glutamine [Gln (Q)] in subdomain V drastically increased the susceptibility of ERK1/2 to 1-naphthyl PP1 (1NA-PP1). Wild-type ERK1/2 was resistant to 1NA-PP1 inhibition. ERK1(Q122A) and ERK2(Q103A) were inhibited by 1NA-PP1 at IC50 values of 1.7 ± 0.13 and 2.1 ± 0.18 ?M, respectively. ERK1(Q122G) and ERK2(Q103G) were inhibited by 1NA-PP1 with IC50 values of 3.6 ± 0.26 and 18 ± 2.2 ?M, respectively. Other derivatives of PP1 (1-naphthylmethyl PP1 and 2-naphthylmethyl PP1) did not significantly inhibit ERK1/2 and its various mutants. In addition, these ERK1/2 mutants were activated by TPA when they were expressed in mammalian cells. These results suggest that the Gln residue of subdomain V is important in determining the susceptibility of ERK1/2 to 1NA-PP1 without significant changes in their enzymatic characteristics. © 2006 Elsevier Inc. All rights reserved.

TITLE:Identifying specific kinase substrates through engineered kinases and ATP analogs 

[2-s2.0-1542345542] Refers [2-s2.0-0036008093] LEVEL: 3
Intracellular signaling by protein kinases controls many aspects of cellular biochemistry and physiology. Determining the direct substrates of protein kinases is important in understanding how these signaling enzymes exert their effect on cellular functions. One of the recent developments in this area takes advantage of the similarity in the ATP binding domains of protein kinases, where a few conserved amino acids containing large side chains come in close contact with the N-6 position of bound ATP. Mutation of one or more of these residues generates a "pocket" in the ATP binding site that allows the mutant kinase, but not other cellular kinases, to utilize analogs of ATP with bulky substituents synthesized onto the N-6 position. The use of such a mutated kinase and radiolabeled ATP analogs allows for the specific labeling of direct substrates of the kinase within a mixture of cellular proteins. We have recently reported the generation of "pocket" mutants of extracellular regulated kinase 2 (ERK2) and their use in the identification of two novel substrates of ERK2. In this report, we discuss the generation and characterization of ERK2 mutants that utilize analogs of ATP and describe the methodology used to identify ERK2-associated substrates. We also describe the direct labeling of ERK2 substrates in cell lysates. These methodologies can be adapted for use with other protein kinases to increase the understanding of intracellular signal transduction. © 2003 Elsevier Inc. © 2003 Elsevier Inc. All rights reserved.

TITLE:Molecular physiology of the SERCA and SPCA pumps 

[2-s2.0-0036877135] Refers [2-s2.0-0035815727] LEVEL: 3
Intracellular Ca2+-transport ATPases exert a pivotal role in the endoplasmic reticulum and in the compartments of the cellular secretory pathway by maintaining a sufficiently high lumenal Ca2+ (and Mn2+) concentration in these compartments required for an impressive number of vastly different cell functions. At the same time this lumenal Ca2+ represents a store of releasable activator Ca2+ controlling an equally impressive number of cytosolic functions. This review mainly focuses on the different Ca2+-transport ATPases found in the intracellular compartments of mainly animal non-muscle cells: the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps. Although it is not our intention to treat the ATPases of the specialized sarcoplasmic reticulum in depth, we can hardly ignore the SERCA1 pump of fast-twitch skeletal muscle since its structure and function is by far the best understood and it can serve as a guide to understand the other members of the family. In a second part of this review we describe the relatively novel family of secretory pathway Ca2+/Mn2+ ATPases (SPCA), which in eukaryotic cells are primarily found in the Golgi compartment. © 2002 Elsevier Science Ltd. All rights reserved.

TITLE:Functional expression in yeast of the human secretory pathway Ca2+, Mn2+-ATPase defective in Hailey-Hailey disease 

[2-s2.0-0037155166] Refers [2-s2.0-0035815727] LEVEL: 3
The discovery and biochemical characterization of the secretory pathway Ca2+-ATPase, PMR1, in Saccharomyces cerevisiae, has paved the way for identification of PMR1 homologues in many species including rat, Caenorhabditis elegans, and Homo sapiens. In yeast, PMR1 has been shown to function as a high affinity Ca2+/Mn2+ pump and has been localized to the Golgi compartment where it is important for protein sorting, processing, and glycosylation. However, little is known about PMR1 homologues in higher organisms. Loss of one functional allele of the human gene, hSPCA1, has been linked to Hailey-Hailey disease, characterized by skin ulceration and improper keratinocyte adhesion. We demonstrate that expression of hSPCA1 in yeast fully complements pmr1 phenotypes of hypersensitivity to Ca2+ chelators and Mn2+ toxicity. Similar to PMR1, epitope-tagged hSPCA1 also resides in the Golgi when expressed in yeast or in chinese hamster ovary cells. 45Ca2+ transport by hSPCA1 into isolated yeast Golgi vesicles shows an apparent Ca2+ affinity of 0.26 ?M, is inhibitable by Mn2+, but is thapsigargin-insensitive. In contrast, heterologous expression of vertebrate sarcoplasmic reticulum and plasma membrane Ca2+-ATPases in yeast complement the Ca2+ - but not Mn2+-related phenotypes of the pmr1-null strain, suggesting that high affinity Mn2+ transport is a unique feature of the secretory pathway Ca2+-ATPases.

TITLE:Emerging themes in manganese transport, biochemistry and pathogenesis in bacteria 

[2-s2.0-0038240674] Refers [2-s2.0-0035815727] LEVEL: 3
Though an essential trace element, manganese is generally accorded little importance in biology other than as a cofactor for some free radical detoxifying enzymes and in the photosynthetic photosystem II. Only a handful of other Mn2+-dependent enzymes are known. Recent data, primarily in bacteria, suggest that Mn2+-dependent processes may have significantly greater physiological importance. Two major classes of prokaryotic Mn2+ uptake systems have now been described, one homologous to eukaryotic Nramp transporters and one a member of the ABC-type ATPase superfamily. Each is highly selective for Mn2+ over Fe2+ or other transition metal divalent cations, and each can accumulate millimolar amounts of intracellular Mn2+ even when environmental Mn2+ is scarce. In Salmonella enterica serovar Typhimurium, simultaneous mutation of both types of transporter results in avirulence, implying that one or more Mn2+-dependent enzymes is essential for pathogenesis. This review summarizes current literature on Mn2+ transport, primarily in the Bacteria but with relevant comparisons to the Archaea and Eukaryota. Mn2+-dependent enzymes are then discussed along with some speculations as to their role(s) in cellular physiology, again primarily in Bacteria. It is of particular interest that most of the enzymes which interconvert phosphoglycerate, pyruvate, and oxaloacetate intermediates are either strictly Mn2+-dependent or highly stimulated by Mn2+. This suggests that Mn2+ may play an important role in central carbon metabolism. Further studies will be required, however, to determine whether these or other actions of Mn2+ within the cell are the relevant factors in pathogenesis. © 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

TITLE:Human Keratinocyte ATP2C1 Localizes to the Golgi and Controls Golgi Ca 2+ Stores 

[2-s2.0-0141991200] Refers [2-s2.0-0035815727] LEVEL: 3
Hailey-Hailey disease (MIM16960) is a blistering skin disease caused by mutations in the Ca2+ ATPase ATP2C1. We found that the abnormal Ca2+ signaling seen in Hailey-Hailey disease keratinocytes correlates with decreased protein levels of ATP2C1. Human ATP2C1 protein approximated 115 kDa in size. The ATP2C1 is localized to the Golgi apparatus in human keratinocytes, similar to its localization in yeast and Caenorhabditis elegans. To test whether the ATP2C1 controls Golgi Ca2+ stores, we measured intraorganelle Ca2+ concentrations using specifically targeted aequorins. Whereas normal keratinocytes display Golgi Ca2+ levels comparable to other epithelial cells, Hailey-Hailey disease keratinocyte Golgi Ca2+ refill is slower, and the maximum Ca2+ concentration reached is significantly lower. These findings were replicated in vivo, because clinically normal Hailey-Hailey disease epidermis contained lower Ca2+ stores and displayed an abnormal Ca2+ gradient. In this report we localize the ATP2C1, demonstrate its physiologic relevance in mammalian cells, and measure intraorganelle Golgi Ca2+ in keratinocytes.

TITLE:Signalling specificity in GPCR-dependent Ca2+ signalling 

[2-s2.0-0037343205] Refers [2-s2.0-0035815727] LEVEL: 3
Cells use signalling networks to translate with high fidelity extracellular signals into specific cellular functions. Signalling networks are often composed of multiple signalling pathways that act in concert to regulate a particular cellular function. In the centre of the networks are the receptors that receive and transduce the signals. A versatile family of receptors that detect a remarkable variety of signals are the G protein-coupled receptors (GPCRs). Virtually all cells express several GPCRs that use the same biochemical machinery to transduce their signals. Considering the specificity and fidelity of signal transduction, a central question in cell signalling is how signalling specificity is achieved, in particular among GPCRs that use the same biochemical machinery. Ca2+ signalling is particularly suitable to address such questions, since [Ca2+]i can be recorded with excellent spatial and temporal resolutions in living cells and tissues and now in living animals. Ca2+ is a unique second messenger in that both biochemical and biophysical components form the Ca2+ signalling complex to regulate its concentration. Both components act in concert to generate repetitive [Ca2+]i oscillations that can be either localized or in the form of global, propagating Ca2+ waves. Most of the key proteins that form Ca2+ signalling complexes are known and their activities are reasonably well understood on the biochemical and biophysical levels. We review here the information gained from studying Ca2+ signalling by GPCRs to gain further understanding of the mechanisms used to generate cellular signalling specificity. © 2002 Elsevier Science Inc. All rights reserved.

TITLE:Baseline Cytosolic Ca2+ Oscillations Derived from a Non-endoplasmic Reticulum Ca2+ Store 

[2-s2.0-0035914318] Refers [2-s2.0-0035815727] LEVEL: 3
Cytosolic Ca2+ oscillations can be due to cycles of release and re-uptake of internally stored Ca2+. To investigate the nature of these Ca2+ stores, we expressed the Pmr1 Ca2+ pump of Caenorhabditis elegans in COS-1 cells and pretreated the cells with thapsigargin to prevent Ca2+ uptake by the sarco(endo)plasmic reticulum Ca2+-ATPase. Pmr1 co-localized with the Golgi-specific 58K protein and was targeted to a Ca2+ store that was less leaky for Ca2+ than the endoplasmic reticulum and whose inositol trisphosphate receptors were less sensitive to inositol trisphosphate and ATP than those in the endoplasmic reticulum. ATP-stimulated Pmr1-overexpressing cells responded after a latency to extracellular Ca2+ with a regenerative Ca 2+ signal, which could be prevented by caffeine. They also produced very stable ilimaquinone-sensitive baseline Ca2+ spikes, even in the presence of thapsigargin. Such responses never occurred in non-transfected cells or in cells that overexpressed the type-1 sarco(endo)plasmic reticulum Ca2+-ATPase. Abortive Ca2+ spikes also occurred in histamine-stimulated untransfected HeLa cells pretreated with thapsigargin, and they too were inhibited by ilimaquinone. We conclude that the Pmr1-induced Ca2+ store, which probably corresponds to the Golgi compartment, can play a crucial role in setting up baseline Ca2+ spiking.

TITLE:Phenotypes of SERCA and PMCA knockout mice 

[2-s2.0-4444349979] Refers [2-s2.0-0035815727] LEVEL: 3
P-type Ca2+-ATPases of the sarco(endo)plasmic reticulum (SERCAs) and plasma membrane (PMCAs) are responsible for maintaining the Ca2+ gradients across cellular membranes that are required for regulation of Ca 2+-mediated signaling and other biological processes. Gene-targeting studies of SERCA isoforms 1, 2, and 3 and PMCA isoforms 1, 2, and 4 have confirmed some of the general functions proposed for these pumps, such as a major role in excitation-contraction coupling for SERCA1 and SERCA2 and housekeeping functions for PMCA1 and SERCA2, but have also revealed some unexpected phenotypes. These include squamous cell cancer and plasticity in the regulation of Ca2+-mediated exocytosis in SERCA2 heterozygous mutant mice, modulation of Ca2+ signaling in SERCA3-deficient mice, deafness and balance disorders in PMCA2 null mice, and male infertility in PMCA4 null mice. These unique phenotypes provide new information about the cellular functions of these pumps, the requirement of their activities for higher order physiological processes, and the pathophysiological consequences of pump dysfunction. © 2004 Elsevier Inc. All rights reserved.

TITLE:Subcellular distribution of the inositol 1,4,5-trisphosphate receptors: Functional relevance and molecular determinants 

[2-s2.0-1242298679] Refers [2-s2.0-0035815727] LEVEL: 3
The inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel that is for the largest part expressed in the endoplasmic reticulum. Its precise subcellular localization is an important factor for the correct initiation and propagation of Ca2+ signals. The relative position of the IP3Rs, and thus of the IP3-sensitive Ca2+ stores, to mitochondria, nucleus or plasma membrane determines in many cases the physiological consequences of IP3-induced Ca2+ release. Most cell types express more than one IP3R isoform and their subcellular distribution is cell-type dependent. Moreover, it was recently demonstrated that depending on the physiological status of the cell redistribution of IP3Rs and/or of IP3-sensitive Ca2+ stores could occur. This indicates that the cell must be able to regulate not only IP3R expression but also its distribution. The various proteins potentially determining IP3R localization and redistribution will therefore be discussed. © 2003 Elsevier SAS. All rights reserved.

TITLE:Hailey-Hailey disease: Molecular and clinical characterization of novel mutations in the ATP2C1 gene 

[2-s2.0-0036178232] Refers [2-s2.0-0035815727] LEVEL: 3
Hailey-Hailey disease is an autosomal dominant skin disorder characterized by suprabasal cell separation (acantholysis) of the epidermis. Mutations in ATP2C1, the gene encoding a novel, P-type Ca2+-transport ATPase, were recently found to cause Hailey-Hailey disease. In this study, we used conformation-sensitive gel electrophoresis to screen all 28 translated exons of ATP2C1 in 24 Hailey-Hailey disease families and three sporadic cases with the disorder. We identified 22 different mutations, 18 of which have not previously been reported, in 25 probands. The novel mutations comprise three nonsense, six insertion/deletion, three splice-site, and six missense mutations and are distributed throughout the ATP2C1 gene. Six mutations were found in multiple families investigated here or in our previous study. Haplotype analysis revealed that two of these are recurrent mutations that have not been inherited from a common ancestor. Comparison between genotype and phenotype in 23 families failed to yield any clear correlation between the nature of the mutation and clinical features of Hailey-Hailey disease. The extensive interfamilial and intrafamilial phenotypic variability observed suggests that modifying genes and/or environmental factors may greatly influence the clinical features of this disease.

TITLE:Effect of Hailey-Hailey Disease Mutations on the Function of a New Variant of Human Secretory Pathway Ca2+/Mn2+-ATPase (hSPCA1) 

[2-s2.0-0042354668] Refers [2-s2.0-0035815727] LEVEL: 3
ATP2C1, encoding the human secretory pathway Ca2+/Mn 2+ ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey Disease (HHD), an autosomal dominant skin disorder characterized by persistent blisters and erosions. To investigate the underlying cause of HHD, we have analyzed the changes in expression level and function of hSPCA1 caused by mutations found in HHD patients. Mutations were introduced into hSPCA1d, a novel splice variant expressed in keratinocytes, described here for the first time. Encoded by the full-length of optional exons 27 and 28, hSPCA1d was longer than previously identified splice variants. The protein competitively transported Ca2+ and Mn2+ with equally high affinity into the Golgi of COS-1 cells. Ca2+- and Mn2+-dependent phosphoenzyme intermediate formation in forward (ATP-fuelled) and reverse (Pi-fuelled) directions was also demonstrated. HHD mutant proteins L341P, C344Y, C411R, T570I, and G789R showed low levels of expression, despite normal levels of mRNA and correct targeting to the Golgi, suggesting instability or abnormal folding of the mutated hSPCA1 polypeptides. P201L had little effect on the enzymatic cycle, whereas I580V caused a block in the E 1?P ? E2-P conformational transition. D742Y and G309C were devoid of Ca2+- and Mn2+-dependent phosphoenzyme formation from ATP. The capacity to phosphorylate from P i was retained in these mutants but with a loss of sensitivity to both Ca2+ and Mn2+ in D742Y and a preferential loss of sensitivity to Mn2+ in G309C. These results highlight the crucial role played by Asp-742 in the architecture of the hSPCA1 ion-binding site and reveal a role for Gly-309 in Mn2+ transport selectivity.

TITLE:The contribution of the SPCA1 Ca2+ pump to the Ca2+ accumulation in the Golgi apparatus of HeLa cells assessed via RNA-mediated interference 

[2-s2.0-0037870343] Refers [2-s2.0-0035815727] LEVEL: 3
The secretory-pathway Ca2+-ATPase SPCA1 is a thapsigargin-insensitive intracellular Ca2+ pump found mostly in the Golgi compartment. We have explored the contribution of this Ca2+ pump to cytosolic Ca2+ signaling in HeLa cells by using RNA-mediated interference to disrupt its expression. Removal of SPCA1 was confirmed by immunofluorescence with specific anti-SPCA1 antibodies. Measurements of the free Ca2+ concentration in the lumen of the Golgi apparatus by specifically targeting the Ca2+-sensitive luminescent protein aequorin to this organelle revealed that endogenous SPCA1 was responsible for Ca2+ uptake in a subfraction of the Golgi apparatus. HeLa cells lacking SPCA1 could still set up baseline Ca2+ spiking when stimulated with histamine, indicating that the SPCA1-containing Ca2+ store was not absolutely needed to set up these oscillations. However, baseline Ca2+ oscillations occurred less frequently than in control cells, pointing to a contribution of SPCA1 in the shaping of the cytosolic Ca2+ signal in HeLa cells. © 2003 Elsevier Science (USA). All rights reserved.

TITLE:Physiological functions of plasma membrane and intracellular Ca2+ pumps revealed by analysis of null mutants 

[2-s2.0-0038239926] Refers [2-s2.0-0035815727] LEVEL: 3
It is known that plasma membrane Ca2+-transporting ATPases (PMCAs) extrude Ca2+ from the cell and that sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) and secretory pathway Ca2+-ATPases (SPCAs) sequester Ca2+ in intracellular organelles; however, the specific physiological functions of individual isoforms are less well understood. This information is beginning to emerge from studies of mice and humans carrying null mutations in the corresponding genes. Mice with targeted or spontaneous mutations in plasma membrane Ca2+-ATPase isoform 2 (PMCA2) are profoundly deaf and have a balance defect due to the loss of PMCA2 in sensory hair cells of the inner ear. In humans, mutations in SERCA1 (ATP2A1) cause Brody disease, an impairment of skeletal muscle relaxation; loss of one copy of the SERCA2 (ATP2A2) gene causes Darier disease, a skin disorder; and loss of one copy of the SPCA1 (ATP2C1) gene causes Hailey-Hailey disease, another skin disorder. In the mouse, SERCA2 null mutants do not survive to birth, and heterozygous SERCA2 mutants have impaired cardiac performance and a high incidence of squamous cell cancers. SERCA3 null mutants survive and appear healthy, but endothelium-dependent relaxation of vascular smooth muscle is impaired and Ca2+ signaling is altered in pancreatic ? cells. The diversity of phenotypes indicates that the various Ca2+-transporting ATPase isoforms serve very different physiological functions.

TITLE:Ca2+ uptake and release properties of a thapsigargin-insensitive nonmitochondrial Ca2+ store in A7r5 and 16HBE14o- cells 

[2-s2.0-0036489420] Refers [2-s2.0-0035815727] LEVEL: 3
In a previous study we overexpressed the thapsigargin (tg)-insensitive Pmr1 Ca2+ pump of the Golgi apparatus of Caenorhabditis elegans in COS-1 cells and studied the properties of the Ca2+ store into which it was integrated. Here we assessed the properties of an endogenous tg-insensitive nonmitochondrial Ca2+ store in A7r5 and 16HBE14o- cells, which express a mammalian homologue of Pmr1. The tg-insensitive Ca2+ store was considerably less leaky for Ca2+ than the sarco(endo)-plasmic-reticulum Ca2+-ATPase (SERCA)-containing Ca2+ store. Moreover like for the worm Pmrl Ca2+ pump expressed in COS-1 cells, Ca2+ accumulation into the endogenous tg-insensitive store showed a 2 orders of magnitude lower sensitivity to cyclopiazonic acid than the SERCA-mediated transport. 2,5-Di-(tert-butyl)-1,4-benzohydroquinone was only a very weak inhibitor of the tg-insensitive Ca2+ uptake in A7r5 and 16HBE14o-cells and in COS-1 cells overexpressing the worm Pmr1. Inositol 1,4,5-trisphosphate released 11% of the Ca2+ accumulated in permeabilized A7r5 cells pretreated with tg with an EC50 that was 5 times higher than for the SERCA-containing Ca2+ store but failed to release Ca2+ in 16HBE14o- cells. In the presence of tg, 15% of intact A7r5 cells responded to 10 ?m arginine-vasopressin with a small rise in cytosolic Ca2+ concentration after a long latency. In conclusion, A7r5 and 16HBE14o- cells express a Pmr1-containing Ca2+ store with properties that differ substantially from the SERCA-containing Ca2+ store.

TITLE:Inositol trisphosphate producing agonists do not mobilize the thapsigargin-insensitive part of the endoplasmic-reticulum and Golgi Ca2+ store 

[2-s2.0-0942287905] Refers [2-s2.0-0035815727] LEVEL: 3
Non-mitochondrial intracellular Ca2+ stores contain both thapsigargin-sensitive sarco(endo)plasmic-reticulum Ca2+-ATPases (SERCA) and thapsigargin-insensitive secretory-pathway Ca2+-ATPases (SPCA1). We now have studied the Ca2+-release properties of the compartments associated with these pumps in intact, i.e. non-permeabilized, cells of different origin (HeLa, keratinocytes, 16HBE14o-, COS-1, A7r5) and with different approaches (45Ca2+ fluxes, Ca2+ imaging and measurements of the free luminal [Ca2+] in the endoplasmic-reticulum and the Golgi apparatus using targeted aequorin). Application of an extracellular agonist in the absence of thapsigargin induced in all cells a Ca2+ release from both the endoplasmic-reticulum and the Golgi apparatus. The agonists were not able to release Ca2+ in the presence of 10?M thapsigargin, except in COS-1 cells overexpressing SPCA1, where this pump not only appeared in the Golgi compartment but also overflowed into the agonist-sensitive part of the endoplasmic-reticulum. We conclude that the subcompartments of the endoplasmic-reticulum and of the Golgi complex that endogenously express SPCA1 are insensitive to agonist stimulation. © 2003 Elsevier Ltd. All rights reserved.

TITLE:Similar Ca2+-signaling properties in keratinocytes and COS-1 cells overexpressing the secretory-pathway Ca2+-ATPase SPCA1 

[2-s2.0-10744221254] Refers [2-s2.0-0035815727] LEVEL: 3
Mutations in the ubiquitously expressed secretory-pathway Ca2+-ATPase (SPCA1) Ca2+ pump result in Hailey-Hailey disease, which almost exclusively affects the epidermal part of the skin. We have studied Ca2+ signaling in human keratinocytes by measuring the free Ca2+ concentration in the cytoplasm and in the lumen of both the Golgi apparatus and the endoplasmic reticulum. These signals were compared with those recorded in SPCA1-overexpressing and control COS-1 cells. Both the sarco(endo plasmic-reticulum Ca2+-ATPase (SERCA) and SPCA1 can mediate Ca2+ uptake into the Golgi stacks. Our results indicate that keratinocytes mainly used the SPCA1 Ca2+ pump to load the Golgi complex with Ca2+ whereas the SERCA Ca2+ pump was mainly used in control COS-1 cells. Cytosolic Ca2+ signals in keratinocytes induced by extracellular ATP or capacitative Ca2+ entry were characterized by an unusually long latency reflecting extra Ca2+ buffering by an SPCA1-containing Ca2+ store, similarly as in SPCA1-overexpressing COS-1 cells. Removal of extracellular Ca2+ elicited spontaneous cytosolic Ca2+ transients in keratinocytes, similarly as in SPCA1-overexpressing COS-1 cells. With respect to Ca2+ signaling keratinocytes and SPCA1-overexpressing COS-1 cells therefore behaved similarly but differed from control COS-1 cells. The relatively large contribution of the SPCA1 pumps for loading the Golgi stores with Ca2+ in keratinocytes may, at least partially, explain why mutations in the SPCA1 gene preferentially affect the skin in Hailey-Hailey patients. © 2003 Elsevier Science Ltd. All rights reserved.

TITLE:SPCA1 pumps and Hailey-Hailey disease 

[2-s2.0-4444333900] Refers [2-s2.0-0035815727] LEVEL: 3
Both the endoplasmic reticulum and the Golgi apparatus are agonist-sensitive intracellular Ca2+ stores. The Golgi apparatus has Ca2+-release channels and a Ca2+-uptake mechanism consisting of sarco(endo)plasmic-reticulum Ca2+-ATPases (SERCA) and secretory-pathway Ca2+-ATPases (SPCA). SPCA1 has been shown to transport both Ca2+ and Mn2+ in the Golgi lumen and therefore plays an important role in the cytosolic and intra-Golgi Ca 2+ and Mn2+ homeostasis. Human genetic studies have provided new information on the physiological role of SPCA1. Loss of one functional copy of the SPCA1 (ATP2C1) gene causes Hailey-Hailey disease, a skin disorder arising in the adult age with recurrent vesicles and erosions in the flexural areas. Here, we review recent experimental evidence showing that the Golgi apparatus plays a much more important role in intracellular ion homeostasis than previously anticipated. © 2004 Elsevier Inc. All rights reserved.

TITLE:Functional expression of heterologous proteins in yeast: Insights into Ca2+ signaling and Ca2+-transporting ATPases 

[2-s2.0-4143052387] Refers [2-s2.0-0035815727] LEVEL: 3
The baker's yeast Saccharomyces cerevisiae is a well-developed, versatile, and widely used model organism. It offers a compact and fully sequenced genome, tractable genetics, simple and inexpensive culturing conditions, and, importantly, a conservation of basic cellular machinery and signal transducing pathways with higher eukaryotes. In this review, we describe recent technical advances in the heterologous expression of proteins in yeast and illustrate their application to the study of the Ca2+ homeostasis machinery, with particular emphasis on Ca2+-transporting ATPases. Putative Ca2+-ATPases in the newly sequenced genomes of organisms such as parasites, plants, and vertebrates have been investigated by functional complementation of an engineered yeast strain lacking endogenous Ca2+ pumps. High-throughput screens of mutant phenotypes to identify side chains critical for ion transport and selectivity have facilitated structure-function analysis, and genomewide approaches may be used to dissect cellular pathways involved in Ca2+ transport and trafficking. The utility of the yeast system is demonstrated by rapid advances in the study of the emerging family of Golgi/secretory pathway Ca2+,Mn2+-ATPases (SPCA). Functional expression of human SPCA1 in yeast has provided insight into the physiology, novel biochemical characteristics, and subcellular localization of this pump. Haploinsufficiency of SPCA1 leads to Hailey-Hailey disease (HDD), a debilitating blistering disorder of the skin. Missense mutations, identified in patients with HHD, may be conveniently assessed in yeast for loss-of-function phenotypes associated with the disease.

TITLE:Ca2+ signals in Pmr1-GFP-expressing COS-1 cells with functional endoplasmic reticulum 

[2-s2.0-0036298516] Refers [2-s2.0-0035815727] LEVEL: 3
We studied the role of the Pmr1-containing Ca2+ store in COS-1 cells endowed with a functional endoplasmic reticulum. Transfected cells could be recognized by using a green-fluorescent-protein (GFP)-tagged form of Pmr1. Pmr1-GFP fluorescence showed a typical juxtanuclear Golgi-like distribution. Pmr1-GFP-containing cells with functional endoplasmic reticulum responded to 100?M ATP with baseline Ca2+ spiking, while non-transfected cells produced an initial Ca2+ peak followed by a long-lasting plateau. The Ca2+ signal often appeared after a long latency in Pmr1-GFP-expressing cells. ATP-stimulated Pmr1-GFP-expressing cells with functional endoplasmic reticulum responded after a latency period to extracellular Ca2+ with a regenerative Ca2+ signal, while non-transfected control cells responded with an immediate slow rise in free cytosolic Ca2+ concentration. These results demonstrate the importance of the Pmr1-containing Ca2+ store in generating or modifying cellular Ca2+ signals. © 2002 Elsevier Science (USA). All rights reserved.

TITLE:Local Ca2+ signals in cellular signalling 

[2-s2.0-1942421346] Refers [2-s2.0-0035815727] LEVEL: 3
Local Ca2+ rises and propagated Ca2+ signals represent different patterns that are differentially decoded for fine tuning cellular signalling. This Ca2+ concentration plasticity is absolutely required to allow adaptation to different needs of the cells ranging from contraction or increased learning to proliferation and cell death. A wide diversity of molecular structures and specific location of Ca2+ signalling molecules confer spatial and temporal versatility to the Ca2+ changes allowing specific cellular responses to be elicited. Various types of local Ca2+ signals have been described. Ca2+ spikes correspond to Ca2+ signals spanning several micrometers but displaying limited propagation into a cell leading to regulation of cellular functions in one particular zone of this cell. This is of particular relevance in cells presenting distinct morphological specializations, i.e. apical versus basal sites or dendritic versus somatic/axonal sites. More stereotyped elementary Ca2+ events (denominated Ca2+ sparks or Ca2+ puffs depending on the type of endoplasmic reticulum Ca2+ release channel involved) are highly confined and non-propagated Ca2+ rises which are observed in the close neighbouring of the Ca2+ channels. These elementary Ca2+ events play a major role in controlling cellular excitability. Elementary Ca2+ events involve Ca2+ release channels such as the ryanodine receptors (RyRs) and the inositol 1,4,5-trisphosphate receptors (InsP3Rs). The molecular bases underlying the various local Ca2+ release events will be discussed by reviewing the channels and particularly the different isoforms of RyRs and InsP3Rs and their role in inducing localized Ca2+ responses. These calcium release events are controlled by various second messengers and are regulated by Ca2+ channel-associated proteins, intra-luminal Ca2+ content of the endoplasmic reticulum (ER) and other Ca2+ organelles. We will discuss on how the control of local cellular Ca2+ content may account for cellular functions in physiological and physiopathological conditions. © 2004 Bentham Science Publishers Ltd.

TITLE:Actin Reorganization Is Abnormal and Cellular ATP Is Decreased in Hailey-Hailey Keratinocytes 

[2-s2.0-0141956485] Refers [2-s2.0-0035815727] LEVEL: 3
Actin reorganization and the formation of adherens junctions are necessary for normal cell-to-cell adhesion in keratinocytes. Hailey-Hailey disease (HHD) is blistering skin disease, resulting from mutations in the Ca2+ ATPase ATP2C1, which controls Ca2+ concentrations in the cytoplasm and Golgi of human keratinocytes. Because actin reorganization is among the first responses to raised cytoplasmic Ca2+, we examined Ca2+-induced actin reorganization in normal and HHD keratinocytes. Even though HHD keratinocytes display raised baseline cytoplasmic Ca2+, we found that actin reorganization in response to Ca2+ was impaired in HHD keratinocytes. Defects in actin reorganization were linked to a marked decrease in cellular ATP in HHD keratinocytes, which persists, in vivo, in HHD epidermis. Defective actin reorganization was reproduced in normal keratinocytes in which the intracellular ATP concentration had been lowered pharmacologically. ATP concentrations in undifferentiated keratinocytes markedly declined after extracellular Ca2+ was increased, but then recovered to a new baseline that was approximately 150% of the previous baseline. In contrast, ATP concentrations in HHD keratinocytes did not change in response to increased extracellular Ca2+. This report provides new insights into how the ATP2C1-controlled ATP metabolism mediates Ca2+-induced cell-to-cell adhesion in normal keratinocytes. In addition, these findings implicate inadequate ATP stores as an additional cause in the pathogenesis of HHD and suggest novel therapeutic options.

TITLE:The secretory pathway Ca2+/Mn2+-ATPase 2 is a Golgi-localized pump with high affinity for Ca2+ ions 

[2-s2.0-20744450586] Refers [2-s2.0-0035815727] LEVEL: 3
Accumulation of Ca2+ into the Golgi apparatus is mediated by sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) and by secretory pathway Ca2+-ATPases (SPCAs). Mammals and birds express in addition to the housekeeping SPCA1 (human gene name ATP2C1, cytogenetic position 3q22.1) a homologous SPCA2 isoform (human gene name ATP2C2, cytogenetic position 16q24.1). We show here that both genes present an identical exon/intron layout. We confirmed that hSPCA2 has the ability to transport Ca2+, demonstrated its Mn2+-transporting activity, showed its Ca 2+- and Mn2+-dependent phosphoprotein intermediate formation, and documented the insensitivity of these functional activities to thapsigargin inhibition. The mRNA encoding hSPCA2 showed a limited tissue expression pattern mainly confined to the gastrointestinal and respiratory tract, prostate, thyroid, salivary, and mammary glands. Immunocytochemical localization in human colon sections presented a typical apical juxtanuclear Golgi-like staining. The expression in COS-1 cells allowed the direct demonstration of 45Ca2+ (K0.5 = 0.27 ?M) or 54Mn2+ transport into an A23187-releasable compartment. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:Secretory pathway Ca2+-ATPase (SPCA1) Ca2+ pumps, not SERCAs, regulate complex [Ca2+]i signals in human spermatozoa 

[2-s2.0-18844413331] Refers [2-s2.0-0035815727] LEVEL: 3
The sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors thapsigargin (0.1-1 ?M) and cyclopiazonic acid (10 ?M), failed to affect resting [Ca2+]i in human spermatozoa. Slow progesterone-induced [Ca2+]i oscillations in human spermatozoa, which involve cyclic emptying-refilling of an intracellular Ca2+ store were also insensitive to these inhibitors. Non-selective doses of thapsigargin (5-30 ?M, 50-300 times the saturating dose for SERCA inhibition), caused elevation of resting [Ca2+]i and partial, dose-dependent disruption of oscillations. A 10-40 ?M concentration of bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)methane (bis-phenol), which inhibits both thapsigargin-sensitive and -insensitive microsomal Ca2+ ATPases, caused elevation of resting [Ca2+]i and inhibition of [Ca2+]i oscillations at doses consistent with inhibition of thapsigargin-resistant, microsomal ATPase and liberation of stored Ca2+. Low doses of bis-phenol had marked effects on [Ca2+]i oscillation kinetics. Application of the drug to cells previously stimulated with progesterone had effects very similar to those observed when it was applied to unstimulated cells, suggesting that the sustained Ca2+ influx induced by progesterone is not mediated via mobilisation of Ca2+ stores. Western blotting for human sperm proteins showed expression of secretory pathway Ca2+ ATPase (SPCA1). Immunolocalisation studies revealed expression of SPCA1 in all cells in an area behind the nucleus, extending into the midpiece. Staining for SERCA, carried out in parallel, detected no expression with either technique. We conclude that: (1) intracellular Ca2+ store(s) and store-dependent [Ca2+]i oscillations in human spermatozoa rely primarily on a thapsigargin/ cyclopiazonic acid-insensitive Ca2+ pump, which is not a SERCA as characterised in somatic cells; (2) effects of high-dose thapsigargin on spermatozoa primarily reflect non-specific actions on non-SERCAs and; (3) secretory pathway Ca2+ ATPases contribute at least part of this non-SERCA Ca2+ pump activity.

TITLE:A novel isoform of the secretory pathway Ca2+,Mn 2+-ATPase, hSPCA2, has unusual properties and is expressed in the brain 

[2-s2.0-15744398885] Refers [2-s2.0-0035815727] LEVEL: 3
Unlike lower eukaryotes, mammalian genomes have a second gene, ATP2C2, encoding a putative member of the family of secretory pathway Ca 2+,Mn2+-ATPases, SPCA2. Human SPCA2 shares 64% amino acid identity with the protein defective in Hailey Hailey disease, hSPCA1. We show that human SPCA2 (hSPCA2) has a more limited tissue distribution than hSPCA1, with prominent protein expression in brain and testis. In primary neuronal cells, endogenous SPCA2 has a highly punctate distribution that overlaps with vesicles derived from the trans-Golgi network and is thus different from the compact perinuclear distribution of hSPCA1 seen in keratinocytes and nonpolarized cells. Heterologous expression in a yeast strain lacking endogenous Ca2+ pumps reveals further functional differences from hSPCA1. Although the Mn2+-specific phenotype of hSPCA2 is similar to that of hSPCA1, Ca2+ ions are transported with much poorer affinity, resulting in only weak complementation of Ca2+-specific yeast phenotypes. These observations suggest that SPCA2 may have a more specialized role in mammalian cells, possibly in cellular detoxification of Mn2+ ions, similar to that in yeast. We point to the close links between manganese neurotoxicity and Parkinsonism that would predict an important physiological role for SPCA2 in the brain. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:Characterization of Cos-7 cells overexpressing the rat secretory pathway Ca2+-ATPase 

[2-s2.0-0347988217] Refers [2-s2.0-0035815727] LEVEL: 3
On the basis of sequence similarities to the yeast PMR1 and hSPCA gene, the rat alternatively spliced mRNA has been suggested to be a Golgi secretory pathway Ca2+-ATPase (SPCA). Data in this report lend further support for this hypothesis in that sucrose gradient fractionation of rat liver microsomes resulted in SPCA comigrating with the Golgi calcium binding protein CALNUC, which was well resolved from the endoplasmic reticulum marker calreticulin. Also, in PC-12 cells, antibody to SPCA colocalized with an antibody to the Golgi marker ?-mannosidase II. To study the biological effects of SPCA expression, we performed stable overexpression of SPCA in COS-7 cells. Seven clones were selected for further comparison with COS-7 cells containing an empty expression vector. Overexpression of SPCA resulted in a significant reduction of plasma membrane Ca2+-ATPase, sarco(endo)plasmic reticulum Ca2+-ATPase, and calreticulin expression in these clones. In contrast, the expression of the Golgi calcium-binding protein CALNUC increased significantly. The phosphoenzyme intermediate formed using membranes from clone G11/5 was calcium dependent, significantly more intense than in COS-7 cells, and not affected by La 3+ treatment. Calcium uptake by G11/5 microsomes was ATP dependent and significantly greater than in microsomes from parent COS-7 cells. The overexpression of SPCA significantly increased the growth rate of these cells compared with COS-7 cells containing only the empty vector. These data demonstrate that overexpression of the rat SPCA results in significant changes in the expression of calcium transport and storage proteins in COS-7 cells.

TITLE:Functional coupling between the caffeine/ryanodine-sensitive Ca2+ store and mitochondria in rat aortic smooth muscle cells 

[2-s2.0-0035878801] Refers [2-s2.0-0035815727] LEVEL: 3
We investigated the role of mitochondria in the agonist-induced and/or caffeine-induced Ca2+ transients in rat aortic smooth muscle cells. We explored the possibility that proliferation modulates the coupling between mitochondria and endoplasmic reticulum, Ca2+ transients induced by either ATP or caffeine were measured in presence or absence of drugs interfering with mitochondrial activity in freshly dissociated cells (day 1) and in subconfluent primary culture (day 12). We found that the mitochondrial inhibitors, rotenone or carbonyl cyanide m-chlorophenylhydrazone, as well as the permeability transition pore inhibitor, cyclosporin A, had no effect on the ATP-induced Ca2+ transient at either day 1 or day 12, but prevented caffeine-induced cytosolic Ca2+ increase at day 12 but not at day 1. Close connections between ryanodine receptors and mitochondria were observed at both day 1 and 12. Thapsigargin (TG) prevented ATP- and caffeine-induced Ca2+ transients at day 1. At day 12, where only 50 % of the cells were sensitive to caffeine, TG did not prevent the caffeine-induced Ca2+ transient, and prevented ATP-induced Ca2+ transient in only half of the cells. Together, these data demonstrate that rat aortic smooth muscle cells at day 1 have an ATP- and caffeine-sensitive pool, which is functionally independent but physically closely linked to mitochondria and totally inhibited by TG. At day 12, we propose the existence of two cell populations: half contains IP3 receptors and TG-sensitive Ca2+ pumps only; the other half contains, in addition to the IP3-sensitive pool independent from mitochondria, a caffeine-sensitive pool. This latter pool is linked to mitochondria through the permeability transition pore and is refilled by both TG-sensitive and insensitive mechanisms.

TITLE:The isoform- and location-dependence of the functioning of the plasma membrane calcium pump 

[2-s2.0-0036960028] Refers [2-s2.0-0035815727] LEVEL: 3
The plasma membrane is a specialised multi-component structure with inter- and intracellular signalling functions. Ca2+ plays a crucial role incellular physiology, and an ATP-driven plasma membrane calcium pump (PMCA) plays the greatest role in the maintenance of a low free Ca2+ concentration in the cytoplasm. The enzyme is coded by four separate genes (PMCA 1-4), and, due to alternative splicing, more than 20 variants can exist. PMCA 1 and 4 isoforms are present in almost all tissues, whereas PMCA 2 and 3 are found in more specialised cell types. The variants differ primarily in their regulatory regions, thus the modulation of calcium pump activity strongly depends on the isoform and the membrane composition. The unique function of PMCA isoforms was confirmed using the practical experimental models - a rat pheochromocytoma cell line, a human neuroblastoma cell line, or, more recently, knockout mice. In addition, based on the finding that PMCA could interact with several specific signaling proteins, it was concluded that its location in defined sites of the cell membrane could be a prerequisite for efficient intercellular communication.

TITLE:Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments 

[2-s2.0-5544273772] Refers [2-s2.0-0035815727] LEVEL: 3
Extracellular agonists mobilize Ca2+ from SERCA-comprising intracellular Ca2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca2+ content of the stores. The latencies and initial rates of Ca2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca2+ response occurred before the lumenal [Ca2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca2+] upon agonist stimulation, but the kinetics of the Ca2+ release are different. © 2004 Elsevier Ltd. All rights reserved.

TITLE:Expression of a P-type Ca2+-transport ATPase in Bacillus subtilis during sporulation 

[2-s2.0-0036671494] Refers [2-s2.0-0035815727] LEVEL: 3
The open reading frame designated yloB in the genomic sequence of Bacillus subtilfis encodes a putative protein that is most similar to the typically eukaryotic type IIA family of P-type ion-motive ATPases, including the endo(sarco)plasmic reticulum (SERCA) and PMR1 Ca2+-transporters, located respectively in the SERCA and the Golgi apparatus. The overall amino acid sequence is more similar to that of the Pmr1s than to the SERCAs, whereas the inverse is seen for the 10 amino acids that form the two Ca2+-binding sites in SERCA. Sporulating but not vegetative B. subtilis cells express the predicted protein, as shown by Western blotting and by the formation of a Ca2+-dependent phosphorylated intermediate. Half-maximal activation of phosphointermediate formation occurred at 2.5 ?M Ca2+. Insertion mutation of the yloB gene did not affect the growth of vegetative cells, did not prevent the formation of viable spores, and did not significantly affect 45Ca accumulation during sporulation. However, spores from knockouts were less resistant to heat and showed a slower rate of germination. It is concluded that the P-type Ca2+-transport ATPase from B. subtilis is not essential for survival, but assists in the formation of resistant spores. The evolutionary relationship of the transporter to the eukaryotic P-type Ca2+-transport ATPases is discussed. © 2002 Elsevier Science Ltd. All rights reserved.

TITLE:Dissection of the functional differences between human secretory pathway Ca2+/Mn2+-ATPase (SPCA) 1 and 2 isoenzymes by steady-state and transient kinetic analyses 

[2-s2.0-33645633094] Refers [2-s2.0-0035815727] LEVEL: 3
Human secretory pathway Ca2+/Mn2+-ATPase (SPCA) 2 encoded by ATP2C2 is only expressed in a limited number of tissues, unlike the ubiquitously expressed SPCA1 pump (encoded by ATP2C1, the gene defective in Hailey-Hailey disease). It has not been determined whether there are significant functional differences between SPCA1 and SPCA2 pump enzymes. Therefore, steady-state and transient kinetic approaches were used to characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human SPCA2 enzyme upon heterologous expression in HEK-293 cells. The catalytic turnover rate of SPCA2 was found enhanced relative to SPCA1 pumps. SPCA2 displayed a very high apparent affinity for cytosolic Ca2+ (K0.5 = 0.025 ?M) in activation of the phosphorylation activity but still 2.5-fold lower than that of SPCA1d. Our kinetic analysis traced both differences to the increased rate characterizing the E1?P(Ca) to E2-P transition of SPCA2. Moreover, the reduced rate of the E 2 to E1 transition seems to contribute in determining the lower apparent Ca2+ affinity and the increased sensitivity to thapsigargin inhibition, relative to SPCA1d. SPCA2 also displayed a reduced apparent affinity for inorganic phosphate, which could be explained by the observed enhanced rate of the E2-P dephosphorylation. The insensitivity to modulation by pH and K+ concentration of the constitutively enhanced E2-P dephosphorylation of SPCA2 is similar to SPCA1d and possibly represents a novel SPCA-specific feature, which is not shared by sarco(endo)plasmic reticulum Ca2+-ATPases. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:Functional comparison between secretory pathway Ca2+/Mn 2+-ATPase (SPCA) 1 and sarcoplasmic reticulum Ca2+-ATPase (SERCA) 1 isoforms by steady-state and transienf kinetic analyses 

[2-s2.0-28244490516] Refers [2-s2.0-0035815727] LEVEL: 3
Steady-state and transient kinetic studies were performed to functionally analyze the overall and partial reactions of the Ca2+ transport cycle of the human secretory pathway Ca2+/Mn2+-ATPase 1 (SPCA1) isoforms: SPCA1a, SPCA1b, SPCA1c, and SPCA1d (encoded by ATP2C1, the gene defective in Hailey-Hailey disease) upon heterologous expression in mammalian cells. The expression levels of SPCA1 isoforms were 200-350-fold higher than in control cells except for SPCA1c, whose low expression level appears to be the effect of rapid degradation because of protein misfolding. Relative to SERCA1a, the active SPCA1a, SPCA1b, and SPCA1d enzymes displayed extremely high apparent affinities for cytosolic Ca2+ in activation of the overall ATPase and phosphorylation activities. The maximal turnover rates of the ATPase activity for SPCA1 isoforms were 4.7-6.4-fold lower than that of SERCA1a (lowest for the shortest SPCA1a isoform). The kinetic analysis traced these differences to a decreased rate of the E1 ? P(Ca) to E2-P transition. The apparent affinity for inorganic phosphate was reduced in the SPCA1 enzymes. This could be accounted for by an enhanced rate of the E2-P hydrolysis, which showed constitutive activation, lacking the SERCA1a-specific dependence on pH and K+. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:Differential capacity for high-affinity manganese uptake contributes to differences between barley genotypes in tolerance to low manganese availability 

[2-s2.0-33644837857] Refers [2-s2.0-0035815727] LEVEL: 3
There is considerable variability among barley (Hordeum vulgare) genotypes in their ability to grow in soils containing a low level of plant available manganese (Mn). The physiological basis for the tolerance to low Mn availability is unknown. In this work, Mn2+ influx and compartmentation in roots of the Mn-efficient genotype Vanessa and the Mn-inefficient genotype Antonia were investigated. Two separate Mn transport systems, mediating high-affinity Mn2+ influx at concentrations up to 130 nM and low-affinity Mn 2+ influx at higher concentrations, were identified in both genotypes. The two genotypes differed only in high-affinity kinetics with the Mn-efficient genotype Vanessa having almost 4 times higher Vmax than the inefficient Antonia, but similar Km values. Online inductively coupled plasma-mass spectrometry measurements verified that the observed differences in high-affinity influx resulted in a higher Mn net uptake of Vanessa compared to Antonia. Further evidence for the importance of the differences in high-affinity uptake kinetics for Mn acquisition was obtained in a hydroponic system with mixed cultivation of the two genotypes at a continuously low Mn concentration (10-50 nM) similar to that occurring in soil solution. Under these conditions, Vanessa had a competitive advantage and contained 55% to 75% more Mn in the shoots than did Antonia. Subcellular compartmentation analysis of roots based on 54Mn2+ efflux established that up to 93% and 83% of all Mn was presentin the vacuole in Vanessa and Antonia, respectively. It is concluded that differential capacity for high-affinity Mn influx contributes to differences between barley genotypes in Mn efficiency. © 2005 American Society of Plant Biologists.

TITLE:Calcium in the Golgi apparatus 

[2-s2.0-33947216270] Refers [2-s2.0-0035815727] LEVEL: 3
The secretory-pathway Ca2+-ATPases (SPCAs) represent a recently recognized family of phosphorylation-type ATPases that supply the lumen of the Golgi apparatus with Ca2+ and Mn2+ needed for the normal functioning of this structure. Mutations of the human SPCA1 gene (ATP2C1) cause Hailey-Hailey disease, an autosomal dominant skin disorder in which keratinocytes in the suprabasal layer of the epidermis detach. We will first review the physiology of the SPCAs and then discuss how mutated SPCA1 proteins can lead to an epidermal disorder. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Keratinocytes cultured from patients with Hailey-Hailey disease and Darier disease display distinct patterns of calcium regulation 

[2-s2.0-22944483801] Refers [2-s2.0-0035815727] LEVEL: 3
Background: Hailey-Hailey disease (HHD) (OMIM 16960) and Darier disease (DD) (OMIM 124200) are dominantly inherited acantholytic skin diseases, respectively, caused by mutations in the genes encoding the Golgi secretory pathway Ca2+-ATPase (SPCA1, ATP2C1) and the sarco/endoplasmic reticulum Ca2+-ATPase type 2 (SERCA2, ATP2A2) genes. Objectives: To investigate calcium regulation in keratinocytes cultured from patients with HHD and DD by measuring intracellular calcium resting levels and the cellular responses to ATP and thapsigargin. Methods The study was carried out using keratinocyte cultures established from four patients with HHD and four with DD. Calcium concentrations were measured with fluorescence ratio imaging using fura-2 loading. Results: Control and HHD keratinocytes displayed approximately the same Ca2+ levels in resting phase, while DD keratinocytes showed elevated Ca2+ levels. Application of ATP caused less pronounced elevation of intracellular calcium concentration ([Ca2+]i) in both HHD and DD keratinocytes than in control cells. HHD keratinocytes did not lower their [Ca2+]i as efficiently as control keratinocytes after treatment with thapsigargin. In addition, DD keratinocytes were practically incapable of lowering their [Ca2+]i after treatment with thapsigargin. Conclusions: The results demonstrate that the defects in SPCA1 and SERCA2 calcium ATPases result in distinct patterns of calcium metabolism. This is also supported by the different clinical features of the diseases. © 2005 British Association of Dermatologists.

TITLE:H2O2 directly activates inositol 1,4,5-trisphosphate receptors in endothelial cells 

[2-s2.0-27644495213] Refers [2-s2.0-0035815727] LEVEL: 3
The mechanisms of H2O2-induced Ca2+ release from intracellular stores were investigated in human umbilical vein endothelial cells. It was found that U73122, the selective inhibitor of phospholipase C, could not inhibit the H2O2-induced cytosolic Ca2+ mobilization. No elevation of inositol 1,4,5-trisphosphate (IP3) was detected in cells exposed to H 2O2. By loading mag-Fura-2, a Ca2+ indicator, into intracellular store, the H2O2-induced Ca2+ release from intracellular calcium store was directly observed in the permeabilized cells in a dose-dependent manner. This release can be completely blocked by heparin, a well-known antagonist of IP3 receptor, indicating a direct activation of IP3 receptor on endoplasmic reticulum (ER) membrane by H2O2. It was also found that H2O2 could still induce a relatively small Ca2+ release from internal stores after the Ca2+-ATPaSe on ER membrane and the Ca2+ uptake to mitochondria were simultaneously inhibited by thapsigargin and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The later observation suggests that a thapsigargin-insensitive non-mitochondrial intracellular Ca2+ store might be also involved in H 2O2-induced Ca2+ mobilization. © W. S. Maney & Son Ltd.

TITLE:Cytosolic Ca2+ signals depending on the functional state of the Golgi in HeLa cells 

[2-s2.0-26044439336] Refers [2-s2.0-0035815727] LEVEL: 3
The Golgi apparatus is, like the endoplasmic reticulum, an inositol-1,4,5-trisphosphate-sensitive Ca2+ store, but its role in setting up Ca2+ signals is not well understood. We have now measured histamine-induced Ca2+ signals in HeLa cells pretreated with brefeldin A, a fungal metabolite that leads to the fragmentation and subsequent disappearance of the Golgi apparatus by its reabsorption within the endoplasmic reticulum. Ca2+ responses in which the free cytoplasmic Ca2+ concentration returned to resting levels during the histamine stimulation (mainly baseline Ca2+ oscillations or a single Ca2+ peak) occurred more often in brefeldin A pretreated cells, resulting in a lower Ca2+ plateau in population measurements. The latencies before the onset of the Ca2+ signals were longer after brefeldin A pretreatment. These results suggest that the integrity of the Golgi apparatus contributes to the shaping of intracellular Ca2+ signals. © 2005 Published by Elsevier Ltd.

TITLE:Effect of selenium-supplement on the calcium signaling in human endothelial cells 

[2-s2.0-24344446442] Refers [2-s2.0-0035815727] LEVEL: 3
Intracellular Ca2+ signaling controls many cellular functions. Understanding its regulation by selenoproteins is essential for understanding the role of selenoproteins in regulating cell functions. The activity of thioredoxin reductase (TrxR), thioredoxin (Trx) content, and the activity of glutathione peroxidase (GPx) in the human endothelial cells cultured in selenium-supplemented medium (refer as Se+ cells) was found 70%, 40%, and 20% higher, respectively than those in the cells cultured in normal medium (refer as Se0 cells). The intracellular Ca2+ signaling initiated by inositol 1,4,5-trisphosphate (IP3), histamine, thapsigargin (TG), carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone (FCCP), and cyclosporin A (CsA) was investigated in both Se+ and Se0 cells. It was interestingly found that the higher activity of selenoproteins reduced the sensitivity of IP3 receptor to the IP 3-triggered Ca2+ release from intracellular stores, but enhanced activation of the receptor-coupled phospholipase C in histamine-stimulated Se+ cells by showing much more generation of IP3 and higher elevation of cytosolic Ca2+. The higher selenoprotein activity also reduced susceptibility of the uniporter to the mitochondrial uncoupler, susceptibility of the permeability transition pore (PTP) to its inhibitor, and the vulnerability of endoplasmic reticulum (ER) Ca2+-ATPase to its inhibitor in selenium-supplementing cells. The results suggest that cell calcium signaling is subjected to thiol-redox regulation by selenoproteins. © 2005 Wiley-Liss, Inc.

TITLE:Caenorhabditis elegans PMR1, a P-type calcium ATPase, is important for calcium/manganese homeostasis and oxidative stress response 

[2-s2.0-12744273945] Refers [2-s2.0-0035815727] LEVEL: 3
The Caenorhabditis elegans PMR1, a P-type Ca2+/Mn2+ ATPase, is expressed in hypodermal seam cells, intestinal cells and spermatheca; localized in Golgi complex. Knock down of pmr-1 as well as overexpression of truncated Caenorhabditis elegans PMR1, which mimics dominant mutations observed in human Hailey-Hailey disease, renders the worm highly sensitive to EGTA and Mn2+. Interestingly, pmr-1 knock down not only causes animals to become resistant to oxidative stress but also suppresses high reactive oxygen species sensitivity of smf-3 RNA-mediated interference and daf-16 worms. These findings suggest that C. elegans PMR1 has important roles in Ca2+ and Mn2+ homeostasis and oxidative stress response. © 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

TITLE:Differential thapsigargin-sensitivities and interaction of Ca2+ stores in human SH-SY5Y neuroblastoma cells 

[2-s2.0-2442509611] Refers [2-s2.0-0035815727] LEVEL: 3
In human SH-SY5Y neuroblastoma cells, two distinct intracellular Ca 2+ stores, a KCl-/caffeine-sensitive and a carbachol-/IP 3-sensitive store, were demonstrated previously. In this study, responses of these two intracellular Ca2+ stores to thapsigargin were characterized. Ca2+-release from these stores was evoked either by high K+ (100 mM KCl) or by 1 mM carbachol, and changes in the intracellular Ca2+ level were monitored using Fura-2 fluorimetry. A sequential stimulation protocol (KCl?carbachol or vice versa) allowed evaluation of the individual contribution of different Ca2+ stores to the evoked intracellular Ca2+ ([Ca2+]i)- transients and the dynamic interaction between them. Thapsigargin (0.05 nM-20 ?M) alone induced a [Ca2+]i-transient. Both the carbachol- and the KCl-evoked [Ca2+]i-transients were inhibited by thapsigargin, but with very different sensitivities. Thapsigargin inhibited the carbachol-evoked [Ca2+]i-transients with (IC50=0.353 nM) or without (IC50=0.448 nM) a KCl-prestimulation, but an additional small component, with a much lower sensitivity (IC50=4814 nM), was observed in the absence of a KCl-prestimulation. In contrast, the KCl-evoked [Ca2+] i-transients displayed only one component with a very low sensitivity to thapsigargin in both absence (IC50=3343 nM) and presence (IC 50=6858 nM) of a carbachol-prestimulation. These findings suggest that the sarco-/endoplasmic reticular Ca2+ ATPases associated with the KCl-/caffeine- and carbachol-/IP3-sensitive intracellular Ca 2+ stores differ from each other, either in types or in their post-translational modification. Such difference might play important role in the regulation of neuronal Ca2+ homeostasis. © 2004 Elsevier B.V. All rights reserved.

TITLE:Proposed function of the accumulation of plasma membrane-type Ca 2+-ATPase mRNA in resting cysts of the ciliate Sterkiella histriomuscorum 

[2-s2.0-13744257791] Refers [2-s2.0-0035815727] LEVEL: 3
From an mRNA differential-display analysis of the encystment-excystment cycle of the ciliate Sterkiella histriomuscorum, we have isolated an expressed sequence tag encoding a plasma membrane-type Ca2+-ATPase (PMCA). PMCAs are located either in the plasma membranes or in the membranes of intracellular organelles, and their function is to pump calcium either out of the cell or into the intracellular calcium stores, respectively. The S. histriomuscorum macronuclear PMCA gene (ShPMCA) and its corresponding cDNA were cloned; it is the first member of the Ca2+-ATPase family identified in Sterkiella. The predicted protein of 1,065 amino acids exhibits 37% identity with PMCAs of diverse organisms. A phylogenetic analysis showed its relatedness to homologs of two alveolates: the ciliate Paramecium tetraurelia and the apicomplexan Toxoplasma gondii. Overexpression of the protein ShPMCA failed to rescue the wild-type phenotype of three Ca2+-ATPase-defective mutant strains of Saccharomyces cerevisiae; this failure contrasts with the reported ability of the PMCAs of parasites to complement defects in yeast. ShPMCA mRNA is markedly accumulated during encystment and in resting cysts, suggesting a function during excystment. To address the possibility of a signaling role for calcium at excystment, the capacity of calcium to induce excystment was examined.

TITLE:The role of endothelial cell Ca2+ store release in the regulation of microvascular permeability in vivo 

[2-s2.0-3543136456] Refers [2-s2.0-0035815727] LEVEL: 3
Microvascular permeability is regulated by changes in intracellular calcium concentration. The mechanism by which this increase in calcium determines permeability under normal conditions and during stimulation with agonists remains to be elucidated. In order to determine whether calcium release from intracellular stores could contribute towards the regulation of vascular permeability, hydraulic conductivity (Lp) was measured in frog mesenteric microvessels during stimulation of the endothelial cells of these vessels with agonists that release calcium from the intracellular stores. ATP (which acts through activation of inositol 1,4,5-trisphosphate (IP3) receptors) increased Lp in the absence of calcium influx across the plasma membrane 2.3 ± 0.3 fold (mean ± S.E.M., P < 0.01, n = 8), which was less than the increase in the presence of calcium influx (3.1 ± 1.1 fold). Caffeine (which acts through activation of ryanodine receptors) also increased Lp in the absence of calcium influx across the plasma membrane 3.8 ± 1.0 fold (P < 0.01, n = 9), but by at least as much as it does in the presence of calcium influx (2.8 ± 0.5 fold). It is surprising that there was a strong positive correlation between the size of the response during store release and the baseline permeability (r = 0.91 for ATP, r = 0.75 for caffeine). This suggests that the filling state of the stores may regulate the baseline permeability of the microvessels. © The Physiological Society 2004.

TITLE:Toward comparative genomics of calcium transporters 

[2-s2.0-0036965618] Refers [2-s2.0-0035815727] LEVEL: 3
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TITLE:Liver SPCA, a Secretory Pathway Ca2+-ATPase, Is Predominantly Localized in Plasma Membrane 

[2-s2.0-0842285316] Refers [2-s2.0-0035815727] LEVEL: 3
Ca2+-ATPases play an essential role in calcium homeostasis. In mammals, three families of these enzymes are known: plasma membrane Ca 2+-ATPaSe, sarco-endoplasmic reticulum Ca2+-ATPase, and SPCA (Secretory Pathway Ca2+-ATPase) which is the least studied. Here we describe subcellular localization of rat liver SPCA revealed with the help of membrane fractionation and immunoblotting. A fragment of SPCA1 comprising its second cytoplasmic loop was expressed in E. coli and purified for subsequent production of polyclonal antibodies. The antibodies specifically recognize SPCA in immunoblots of membrane proteins from several rat tissues indicating its ubiquitous distribution and the highest content in kidney. Subcellular compartments of rat liver were separated by well known technique of sucrose density gradient centrifugation to yield fractions enriched with plasma membranes, mitochondria, Golgi complex, endoplasmic reticulum, and nuclei. Immunoblotting of the fractions with anti-SPCA antibodies showed that SPCA1 is present in all fractions except nuclei, in varyous concentrations. The most intense signal was observed in the fraction enriched with plasma membrane. This result indicates that, at least in liver, mammalian SPCA is not a Golgi-resident enzyme, as it was shown for its yeast homologue pmrl. Indeed, requirements and mechanisms of calcium homeostasis in mammals and yeast may differ significantly. A hypothesis suggests that mammalian SPCA is a component of recycling membranes of the secretory pathway.

TITLE:Endoplasmic reticulum: A primary target in various acute disorders and degenerative diseases of the brain 

[2-s2.0-0043037253] Refers [2-s2.0-0037025272] LEVEL: 3
Changes in neuronal calcium activity in the various subcellular compartments have divergent effects on affected cells. In the cytoplasm and mitochondria, where calcium activity is normally low, a prolonged excessive rise in free calcium levels is believed to be toxic, in the endoplasmic reticulum (ER), in contrast, calcium activity is relatively high and severe stress is caused by a depletion of ER calcium stores. Besides its role in cellular calcium signaling, the ER is the site where membrane and secretory proteins are folded and processed. These calcium-dependent processes are fundamental to normal cell functioning. Under conditions of ER dysfunction unfolded proteins accumulate in the ER lumen, a signal responsible for activation of the unfolded protein response (UPR) and the ER-associated degradation (ERAD). UPR is characterized by activation of two ER-resident kinases, PKR-like ER kinase (PERK) and IRE1. PERK induces phosphorylation of the eukaryotic initiation factor (eIF2?), resulting in a shut-down of translation at the initiation step. This stress response is needed to block new synthesis of proteins that cannot be correctly folded, and thus to protect cells from the effect of unfolded proteins which tend to form toxic aggregates. IRE1, on the other hand, is turned after activation into an endonuclease that cuts out a sequence of 26 bases from the coding region of xbp1 mRNA. Processed xbp1 mRNA is translated into the respective protein, an active transcription factor specific for ER stress genes such as grp78. In acute disorders and degenerative diseases, the ER calcium pool is a primary target of toxic metabolites or intermediates, such as oxygen free radicals, produced during the pathological process. Affected neurons need to activate the entire UPR to cope with the severe form of stress induced by ER dysfunction. This stress response is however hindered under conditions where protein synthesis is suppressed to such an extent that processed xbp1 mRNA is not translated into the processed XBP1 protein (XBP1proc). Furthermore, activation of ERAD is important for the degradation of unfolded proteins through the ubiquitin/proteasomal pathway, which is impaired in acute disorders and degenerative diseases, resulting in further ER stress. ER functioning is thus impaired in two different ways: first by the direct action of toxic intermediates, produced in the course of the pathological process, hindering vital ER reactions, and second by the inability of cells to fully activate UPR and ERAD, leaving them unable to withstand the severe form of stress induced by ER dysfunction. © 2003 Elsevier Ltd. All rights reserved.

TITLE:Oxidative damage to the endoplasmic reticulum is implicated in ischemic neuronal cell death 

[2-s2.0-0141925706] Refers [2-s2.0-0037025272] LEVEL: 3
The endoplasmic reticulum (ER), which plays important roles in apoptosis, is susceptible to oxidative stress. Because reactive oxygen species (ROS) are robustly produced in the ischemic brain, ER damage by ROS may be implicated in ischemic neuronal cell death. We induced global brain ischemia on wild-type and copper/zinc superoxide dismutase (SOD1) transgenic rats and compared ER stress and neuronal damage. Phosphorylated forms of eukaryotic initiation factor 2? (eIF2?) and RNA-dependent protein kinase-like ER eIF2? kinase (PERK), both of which play active roles in apoptosis, were increased in hippocampal CA1 neurons after ischemia but to a lesser degree in the transgenic animals. This finding, together with the finding that the transgenic animals showed decreased neuronal degeneration, indicates that oxidative ER damage is involved in ischemic neuronal cell death. To elucidate the mechanisms of ER damage by ROS, we analyzed glucose-regulated protein 78 (GRP78) binding with PERK and oxidative ER protein modification. The proteins were oxidatively modified and stagnated in the ER lumen, and GRP78 was detached from PERK by ischemia, all of which were attenuated by SOD1 overexpression. We propose that ROS attack and modify ER proteins and elicit ER stress response, which results in neuronal cell death.

TITLE:Induction of GRP78 by ischemic preconditioning reduces endoplasmic reticulum stress and prevents delayed neuronal cell death 

[2-s2.0-0041568574] Refers [2-s2.0-0037025272] LEVEL: 3
Although the endoplasmic reticulum (ER) is implicated in neuronal degeneration in some situations, its role in delayed neuronal cell death (DND) after ischemia remains uncertain. The authors speculated that ER stress is involved in DND, that it is reduced by ischemic preconditioning, and that ER stress reduction by preconditioning is due to ER molecular chaperone induction. The phosphorylation status of eukaryotic initiation factor 2? (eIF2?) and RNA-dependent protein kinase-like ER eIF2?. kinase (PERK) was investigated in the rat hippocampus after ischemia with and without preconditioning. PERK is phosphorylated by ER stress, which phosphorylates eIF2?. To investigate the role of ER molecular chaperones in preconditioning, the authors examined GRP78 and GRP94 expression, both of which are ER chaperones that inhibit PERK phosphorylation, and compared their induction and ischemic tolerance time windows. Phosphorylation of eIF2?. and PERK was confirmed after severe ischemia but was inhibited by preconditioning. After preconditioning, GRP78 was increased in the brain with a peak at 2 days, which corresponded with the ischemic tolerance time window. Immunoprecipitation and double staining demonstrated involvement of GRP78 in prevention of PERK phosphorylation. These results suggest that GRP78 induced by preconditioning may reduce ER stress and eventual DND after ischemia.

TITLE:Neuronal ageing from an intraneuronal perspective: Roles of endoplasmic reticulum and mitochondria 

[2-s2.0-0043037254] Refers [2-s2.0-0037025272] LEVEL: 3
The nature of brain ageing and the age-dependent decline in cognitive functions remains poorly understood. Physiological brain ageing is characterised by mild mental dysfunctions, whereas age-dependent neurodegeneration, as illustrated by Alzheimer disease (AD), results rapidly in severe dementia. These two states of the aged brain, the physiological and the pathological, are fundamentally different as the latter stems from significant neuronal loss, whereas the former develops without significant neuronal demise. In this paper, we review the changes in neuronal Ca2+ homeostasis that occur during brain ageing, and conclude that normal, physiological ageing is characterised mainly by a decrease of neuronal homeostatic reserve, defined as the capacity to respond effectively to functional and metabolic stressors, but does not reach the trigger required to induce neuronal death. In contrast, during neurodegenerative states, Ca2+ homeostasis is affected early during the pathological process and result in significant neuronal demise. We also review recent evidence suggesting that the endoplasmic reticulum (ER) might play an important role in controlling the balance between healthy and pathological neuronal ageing. © 2003 Elsevier Ltd. All rights reserved.

TITLE:Amplification of receptor signalling by Ca2+ entry-mediated translocation and activation of PLCĪ³2 in B lymphocytes 

[2-s2.0-0141848670] Refers [2-s2.0-0037025272] LEVEL: 3
In non-excitable cells, receptor-activated Ca2+ signalling comprises initial transient responses followed by a Ca2+ entry-dependent sustained and/or oscillatory phase. Here, we describe the molecular mechanism underlying the second phase linked to signal amplification. An in vivo inositol 1,4,5-trisphosphate (IP3) sensor revealed that in B lymphocytes, receptor-activated and store-operated Ca2+ entry greatly enhanced IP3 production, which terminated in phospholipase C?2 (PLC?2)-deficient cells. Association between receptor-activated TRPC3 Ca2+ channels and PLC?2, which cooperate in potentiating Ca2+ responses, was demonstrated by co-immunoprecipitation. PLC?2-deficient cells displayed diminished Ca2+ entry-induced Ca2+ responses. However, this defect was canceled by suppressing IP3-induced Ca2+ release, implying that IP3 and IP3 receptors mediate the second Ca2+ phase. Furthermore, confocal visualization of PLC?2 mutants demonstrated that Ca2+ entry evoked a C2 domain-mediated PLC?2 translocation towards the plasma membrane in a lipase-independent manner to activate PLC?2. Strikingly, Ca2+ entry-activated PLC?2 maintained Ca2+ oscillation and extracellular signal-regulated kinase activation downstream of protein kinase C. We suggest that coupling of Ca 2+ entry with PLC?2 translocation and activation controls the amplification and co-ordination of receptor signalling.

TITLE:Mitochondrial Ca2+ uptake requires sustained Ca2+ release from the endoplasmic reticulum 

[2-s2.0-0038689310] Refers [2-s2.0-0037025272] LEVEL: 3
We analyzed the role of inositol 1,4,5-trisphosphate-induced Ca2+ release from the endoplasmic reticulum (ER) (i) in powering mitochondrial Ca2+ uptake and (ii) in maintaining a sustained elevation of cytosolic Ca2+ concentration ([Ca2+]c). For this purpose, we expressed in HeLa cells aequorin-based Ca2+-sensitive probes targeted to different intracellular compartments and studied the effect of two agonists: histamine, acting on endogenous H1 receptors, and glutamate, acting on co-transfected metabotropic glutamate receptor (mGluR1a), which rapidly inactivates through protein kinase C-dependent phosphorylation and thus causes transient inositol 1,4,5-trisphosphate production. Glutamate induced a transient [Ca2+]c rise and drop in ER luminal [Ca2+] ([Ca2+]er), and then the ER refilled with [Ca2+]c at resting values. With histamine, [Ca2+]c after the initial peak stabilized at a sustained plateau, and [Ca2+]er decreased to a low steady-state value. In mitochondria, histamine evoked a much larger mitochondrial Ca2+ response than glutamate (?15 versus ?65 ?M). Protein kinase C inhibition, partly relieving mGluR1a desensitization, reestablished both the [Ca2+]c plateau and the sustained ER Ca2+ release and markedly increased the mitochondrial Ca2+ response. Conversely, mitochondrial Ca2+ uptake evoked by histamine was drastically reduced by very transient (?2-s) agonist applications. These data indicate that efficient mitochondrial Ca2+ uptake depends on the preservation of high Ca2+ microdomains at the mouth of ER Ca2+ release sites close to mitochondria. This in turn depends on continuous Ca2+ release balanced by Ca2+ reuptake into the ER and maintained by Ca2+ influx from the extracellular space.

TITLE:Use of microarray analysis to unveil transcription factor and gene networks contributing to Ī² cell dysfunction and apoptosis 

[2-s2.0-1942507406] Refers [2-s2.0-0037025272] LEVEL: 3
The ? cell fate following immune-mediated damage depends on an intricate pattern of dozens of genes up- or downregulated in parallel and/or sequentially. We are utilizing microarray analysis to clarify the pattern of gene expression in primary rat ? cells exposed to the proapoptotic cytokines, IL-1? and/or IFN-?. The picture emerging from these experiments is that ? cells are not passive bystanders of their own destruction. On the contrary, ? cells respond to damage by activating diverse networks of transcription factors and genes that may either lead to apoptosis or preserve viability. Of note, cytokine-exposed ? cells produce and release chemokines that may contribute to the homing and activation of T cells and macrophages during insulitis. Several of the effects of cytokines depend on the activation of the transcription factor, NF-?B. NF-?B blocking prevents cytokine-induced ? cell death, and characterization of NF-?B-dependent genes by microarray analysis indicated that this transcription factor controls diverse networks of transcription factors and effector genes that are relevant for maintenance of ? cell differentiated status, cytosolic and ER calcium homeostasls, attraction of mononuclear cells, and apoptosis. Identification of this and additional "transcription factor networks" is being pursued by cluster analysis of gene expression in insulin-producing cells exposed to cytokines for different time periods. Identification of complex gene patterns poses a formidable challenge, but is now technically feasible. These accumulating evidences may finally unveil the molecular mechanisms regulating the ? cell "decision" to undergo or not apoptosis in early T1DM.

TITLE:Oxidative injury to the endoplasmic reticulum in mouse brains after transient focal ischemia 

[2-s2.0-1542300773] Refers [2-s2.0-0037025272] LEVEL: 3
Oxidative damage to the endoplasmic reticulum (ER) could be involved in ischemic neuronal cell death because this organelle is susceptible to reactive oxygen species. Using wild-type mice and copper/zinc-superoxide dismutase (SOD1) transgenic mice, we induced focal cerebral ischemia and compared neuronal degeneration and ER stress, that is, phosphorylation of eukaryotic initiation factor 2? (eIF2?) and RNA-dependent protein kinase-like ER eIF2? kinase (PERK). We found that neurons with severe and prolonged phosphorylation of eIF2? and PERK underwent later degeneration, and that this was partially prevented by SOD1 overexpression. Signals for superoxide production and phospho-PERK were colocalized, which further indicates a pivotal role for superoxide in ER damage. We investigated the molecular mechanisms of oxidative ER stress and found that detachment of glucose-regulated protein 78 from PERK was the key step. We conclude that ER damage is involved in oxidative neuronal injury in the brain after ischemia/reperfusion. © 2004 Elsevier Inc. All rights reserved.

TITLE:Mechanisms of ATP-induced calcium signaling and growth arrest in human prostate cancer cells 

[2-s2.0-0038082168] Refers [2-s2.0-0037025272] LEVEL: 3
This study investigates the calcium mechanisms involved in growth arrest induced by extracellular ATP in DU-145 androgen-independent human prostate cancer cells. Exposure of DU-145 cells to 100 ?M ATP produced an increase in cytoplasmic calcium concentration ([Ca2+]i), due to a mobilization of calcium from the endoplasmic reticulum stores and to subsequent capacitative calcium entry (CCE). We have shown that this [Ca2+]i increase occurs after stimulation by ATP of the phospholipase C (PLC) pathway. For the first time, we have identified the inositol 1,4,5-trisphosphate receptor (IP3R) isoforms expressed in this cell line and have demonstrated a participation of protein kinase C in CCE. Using fluorescence imaging, we have shown that a long-term treatment with ATP leads to a decrease in the intraluminal endoplasmic reticulum calcium concentration as well as in the amount of releasable Ca2+. Modulating extracellular free calcium concentrations indicated that variations in [Ca2+]i did not affect the ATP-induced growth arrest of DU-145 cells. However, treating cells with 1 nM thapsigargin (TG) to deplete intracellular calcium pools prevented the growth arrest induced by ATP. Altogether, these results indicate that growth arrest induced in DU-145 cells by extracellular ATP is not correlated with an increase in [Ca2+]i but rather with a decrease in intracellular calcium pool content. © 2003 Elsevier Science Ltd. All rights reserved.

TITLE:Fast neurotransmitter release triggered by Ca influx through AMPA-type glutamate receptors 

[2-s2.0-33749867164] Refers [2-s2.0-0037025272] LEVEL: 3
Feedback inhibition at reciprocal synapses between A17 amacrine cells and rod bipolar cells (RBCs) shapes light-evoked responses in the retina. Glutamate-mediated excitation of A17 cells elicits GABA (?-aminobutyric acid)-mediated inhibitory feedback onto RBCs, but the mechanisms that underlie GABA release from the dendrites of A17 cells are unknown. If, as observed at all other synapses studied, voltage-gated calcium channels (VGCCs) couple membrane depolarization to neurotransmitter release, feedforward excitatory postsynaptic potentials could spread through A17 dendrites to elicit 'surround' feedback inhibitory transmission at neighbouring synapses. Here we show, however, that GABA release from A17 cells in the rat retina does not depend on VGCCs or membrane depolarization. Instead, calcium-permeable AMPA (?-amino-3- hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs), activated by glutamate released from RBCs, provide the calcium influx necessary to trigger GABA release from A17 cells. The AMPAR-mediated calcium signal is amplified by calcium-induced calcium release (CICR) from intracellular calcium stores. These results describe a fast synapse that operates independently of VGCCs and membrane depolarization and reveal a previously unknown form of feedback inhibition within a neural circuit. © 2006 Nature Publishing Group.

TITLE:A novel Ca2+-induced Ca2+ release mechanism in A7r5 cells regulated by calmodulin-like proteins 

[2-s2.0-0042847424] Refers [2-s2.0-0037025272] LEVEL: 3
Intracellular Ca2+ release is involved in setting up Ca2+ signals in all eukaryotic cells. Here we report that an increase in free Ca2+ concentration triggered the release of up to 41 ± 3% of the intracellular Ca2+ stores in permeabilized A7r5 (embryonic rat aorta) cells with an EC50 of 700 nM. This type of Ca2+-induced Ca2+ release (CICR) was neither mediated by inositol 1,4,5-trisphosphate receptors nor by ryanodine receptors, because it was not blocked by heparin, 2-aminoethoxydiphenyl borate, xestospongin C, ruthenium red, or ryanodine. ATP dose-dependently stimulated the CICR mechanism, whereas 10 mM MgCL2 abolished it. CICR was not affected by exogenously added calmodulin (CaM), but CaM1234, a Ca2+insensitive CaM mutant, strongly inhibited the CICR mechanism. Other proteins of the CaM-like neuronal Ca2+-sensor protein family such as Ca2+-binding protein 1 and neuronal Ca2+ sensor-1 were equally potent for inhibiting the CICR. Removal of endogenous CaM, using a CaM-binding peptide derived from the ryanodine receptor type-1 (amino acids 3614-3643) prevented subsequent activation of the CICR mechanism. A similar CICR mechanism was also found in 16HBE14o- (human bronchial mucosa) cells. We conclude that A7r5 and 16HBE14o- cells express a novel type of CICR mechanism that is silent in normal resting conditions due to inhibition by CaM but becomes activated by a Ca2+-dependent dissociation of CaM. This CICR mechanism, which may be regulated by members of the family of neuronal Ca2+-sensor proteins, may provide an additional route for Ca2+ release that could allow amplification of small Ca2+ signals.

TITLE:PERK (eIF2Ī± kinase) is required to activate the stress-activated MAPKs and induce the expression of immediate-early genes upon disruption of ER calcium homoeostasis 

[2-s2.0-30044438461] Refers [2-s2.0-0037025272] LEVEL: 3
The eIF2a (eukaryotic initiation factor-2?) kinase PERK (double-stranded RNA-activated protein kinase-like ER kinase) is essential for the normal function of highly secretory cells in the pancreas and skeletal system, as well as the UPR (unfolded protein response) in mammalian cells. To delineate the regulatory machinery underlying PERK-dependent stress-responses, gene profiling was employed to assess global changes in gene expression in PERK-deficient MEFs (mouse embryonic fibroblasts). Several IE (immediate-early) genes, including c-myc, c-jun, egr-1 (early growth response factor-1 ), and fra-1 (fos-related antigen-1), displayed PERK-dependent expression in MEFs upon disruption of calcium homoeostasis by inhibiting the ER (endoplasmic reticulum) transmembrane SERCA (sarcoplasmic/ER Ca2+-ATPase) calcium pump. Induction of c-myc and egr-1 by other reagents that elicit the UPR, however, showed variable dependence upon PERK. Induction of c-myc expression by thapsigargin was shown to be linked to key signalling enzymes including PLC (phospholipase C), PI3K (phosphatidylinositol 3-kinase) and p38 MAPK (mitogen-activated protein kinase). Analysis of the phosphorylated status of major components in MAPK signalling pathways indicated that thapsigargin and DTT (dithiothreitol) but not tunicamycin could trigger the PERK-dependent activation of JNK (c-Jun N-terminal kinase) and p38 MAPK. However, activation of JNK and p38 MAPK by non-ER stress stimuli including UV irradiation, anisomycin, and TNF-? (tumour necrosis factor-?) was found to be independent of PERK. PERK plays a particularly important role in mediating the global cellular response to ER stress that is elicited by the depletion of calcium from the ER. We suggest that this specificity of PERK function in the UPR is an extension of the normal physiological function of PERK to act as a calcium sensor in the ER. © 2006 Biochemical Society.

TITLE:The effects of HCl and CaCl2 injections on intracellular calcium and pH in voltage-clamped snail (Helix aspersa) neurons 

[2-s2.0-0036795337] Refers [2-s2.0-0037025272] LEVEL: 3
To investigate the mechanisms by which low intracellular pH influences calcium signaling, I have injected HCl, and in some experiments CaCl2, into snail neurons while recording intracellular pH (pHi) and calcium concentration ([Ca2+]i) with ion-sensitive microelectrodes. Unlike fluorescent indicators, these do not increase buffering. Slow injections of HCl (changing pHi by 0.1-0.2 pH units min -1) first decreased [Ca2+]i while pHi was still close to normal, but then increased [Ca2+]i when pHi fell below 6.8-7. As pHi recovered after such an injection, [Ca2+]i started to fall but then increased transiently before returning to its preinjection level. Both the acid-induced decrease and the recovery-induced increase in [Ca2+]i were abolished by cyclopiazonic acid, which empties calcium stores. Caffeine with or without ryanodine lowered [Ca2+]i and converted the acid-induced fall in [Ca2+]i to an increase. Injection of ortho-vanadate increased steady-state [Ca2+]i and its response to acidification, which was again blocked by CPA. The normal initial response to 10 mM caffeine, a transient increase in [Ca2+]i, did not occur with pHi below 7.1. When HCl was injected during a series of short CaCl2 injections, the [Ca2+]i transients (recorded as changes in the potential (VCa) of the Ca2+-sensitive microelectrode), were reduced by only 20% for a 1 pH unit acidification, as was the rate of recovery after each injection. Calcium transients induced by brief depolarizations, however, were reduced by 60% by a similar acidification. These results suggest that low pHi has little effect on the plasma membrane calcium pump (PMCA) but important effects on the calcium stores, including blocking their response to caffeine. Acidosis inhibits spontaneous calcium release via the RYR, and leads to increased store content which is unloaded when pHi returns to normal. Spontaneous release is enhanced by the rise in [Ca2+]i caused by inhibiting the PMCA.

TITLE:Endoplasmic reticulum calcium transport ATPase expression during differentiation of colon cancer and leukaemia cells 

[2-s2.0-4444349975] Refers [2-s2.0-0037025272] LEVEL: 3
The calcium homeostasis of the endoplasmic reticulum (ER) is connected to a multitude of cell functions involved in intracellular signal transduction, control of proliferation, programmed cell death, or the synthesis of mature proteins. Calcium is accumulated in the ER by various biochemically distinct sarco/endoplasmic reticulum calcium transport ATPase isoenzymes (SERCA isoforms). Experimental data indicate that the SERCA composition of some carcinoma and leukaemia cell types undergoes significant changes during differentiation, and that this is accompanied by modifications of SERCA-dependent calcium accumulation in the ER. Because ER calcium homeostasis can also influence cell differentiation, we propose that the modulation of the expression of various SERCA isoforms, and in particular, the induction of the expression of SERCA3-type proteins, is an integral part of the differentiation program of some cancer and leukaemia cell types. The SERCA content of the ER may constitute a new parameter by which the calcium homeostatic characteristics of the organelle are adjusted. The cross-talk between ER calcium homeostasis and cell differentiation may have some implications for the better understanding of the signalling defects involved in the acquisition and maintenance of the malignant phenotype. © 2004 Published by Elsevier Inc.

TITLE:Disruption of endoplasmic reticulum calcium stores is involved in neuronal death induced by glycolysis inhibition in cultured hippocampal neurons 

[2-s2.0-27844472724] Refers [2-s2.0-0037025272] LEVEL: 3
Disturbances in neuronal calcium homeostasis have been implicated in a variety of neuropathological conditions, including cerebral ischemia, hypoglycemia, and epilepsy, and possibly constitute part of the cell death process associated with chronic neurodegenerative disorders. We investigated if endoplasmic reticulum (ER) calcium stores participate in neuronal death triggered by moderate glycolysis inhibition induced by iodoacetate, an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, in cultured hippocampal neurons. Results show that exposure to iodoacetate leads to a slow partial decrease in cell survival, which is significantly prevented in the absence of Ca 2+ or in the presence of the calcium chelator BAPTA-AM. Treatment with caffeine and a low (1 ?M) concentration of ryanodine, which activates the ryanodine receptor (RyR), exacerbates neuronal death, whereas dantrolene and 25 ?M ryanodine, which antagonizes RyR, prevents damage. Xestospongin C (XeC), an antagonist of the inositol-3-phosphate (IP3) receptor (IP3R) also prevents neuronal damage. Inhibitors of the ER calcium ATPase (sarcoendoplasmic reticulum Ca2+ ATPase; SERCA) have no effect. The decrease in ATP levels induced by iodoacetate is potentiated by caffeine and prevented by dantrolene. Although only a slight increase in glutamate extracellular levels is observed 3.5 hr after iodoacetate exposure, the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, MK-801, efficiently prevents neuronal damage. Taken together, the data suggest that neuronal death induced during moderate glycolysis inhibition involves calcium influx through NMDA receptors and calcium release from intracellular ER stores. These results might be relevant to the understanding the mechanisms involved in neuronal damage related to aging and chronic neurodegenerative diseases, which have been associated with decreased glucose metabolism. © 2005 Wiley-Liss, Inc.

TITLE:Group I Metabotropic Receptor Antagonism Blocks Depletion of Calcium Stores and Reduces Potentiated Capacitative Calcium Entry in Strain-Injured Neurons and Astrocytes 

[2-s2.0-1642564272] Refers [2-s2.0-0037025272] LEVEL: 3
Antagonism of the group I metabotropic receptor subtype 1 (mGluR 1) with (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) has been shown to reduce deficits after in vivo or in vitro traumatic brain injury. We have previously demonstrated that AIDA prevents elevation of astrocyte IP 3 subsequent to injury-induced activation of mGluRs and phospholipase C. Since IP3 can cause release of intracellular Ca 2+ stores we tested the hypothesis that pre- or post-injury treatment with AIDA can affect (1) the depletion of Ca2+ stores which occurs soon after strain injury of cultured neurons and astrocytes and (2) the delayed potentiation of capacitative calcium entry in strain-injured neurons. Astrocyte or neuronal plus glial cultures were grown on Silastic membranes that were subjected to a 50-msec pulse of compressed gas, which caused membrane displacement and biaxial strain (stretch) injury of the adhering cells. Cells were treated 10 min before or immediately after injury with 100 ?M AIDA and the intracellular free Ca2+ ([Ca 2+]i) response to thapsigargin, which inhibits the ability of the stores to sequester Ca2+, was measured at 15 min or 3 h after injury. AIDA pre- or post-injury treatment prevented the depletion of intracellular calcium stores at 15 min post-injury in astrocytes and neurons and reduced the potentiated neuronal capacitative calcium influx 3 h after injury. Since Ca2+ and Ca2+ stores influence many factors, including neuronal excitability, plasticity, protein synthesis, and neuronal-glial interactions, prevention of Ca2+ store depletion and subsequent exaggerated capacitative calcium entry may be an important subcellular mechanism by which antagonism of mGluR1 receptors exert an injury-reducing effect. More globally, the results further emphasize the importance of altered signaling and calcium regulatory mechanisms in the immediate and delayed sequelae of traumatic brain injury.

TITLE:Acute effects of pulsed microwaves and 3-nitropropionic acid on neuronal ultrastructure in the rat caudate-putamen 

[2-s2.0-12844253203] Refers [2-s2.0-0037025272] LEVEL: 3
Ultrastructure of the medium sized "spiny" neuron in rat dorsal-lateral caudate-putamen was assessed after administration of 3-nitropropionic acid (3-NP) and exposure to pulsed microwaves. Sprague-Dawley male rats were given two daily intraperitoneal doses of 0 or 10 mg/kg 3-NP and 1.5 h after each dose were exposed to microwave radiation at a whole body averaged specific absorption rate (SAR) of 0 (sham exposure), 0.6, or 6 W/kg for 30 min. Microwave exposure consisted of 1.25 GHz radiation delivered as 5.9 ?s pulses with repetition frequency 10 Hz. Tissue samples taken 2-3 h after the second sham or microwave exposure showed no injury with light microscope methods. Blinded qualitative assessment of ultrastructure of randomly selected neurons from the same samples did reveal differences. Subsequent detailed, quantitative measurements showed that, when followed by sham exposure, administration of 3-NP significantly increased endoplasmic reticulum (ER) intracisternal width, ER area density, and nuclear envelope thickness. Microwave exposure at 6 W/kg alone also significantly increased these measures. Exposure of 3-NP treated animals at 6 W/kg significantly increased effects of 3-NP on ultrastructure. Although exposure at 0.6 W/kg alone did not affect ultrastructure measures, exposure of 3-NP treated animals at 0.6 W/kg reduced the effects of 3-NP. We concluded that 3-NP changed neuronal ultrastructure and that the microwave exposures used here changed neuronal ultrastructure in ways that depended on microwave SAR and neuron metabolic status. The apparent cancellation of 3-NP induced changes by exposure to pulsed microwaves at 0.6 W/kg indicated the possibility that such exposure can protect against the effects of mitochondrial toxins on the nervous system.

TITLE:Increased vulnerability of hippocampal neurons with age in culture: Temporal association with increases in NMDA receptor current, NR2A subunit expression and recruitment of L-type calcium channels 

[2-s2.0-34248233012] Refers [2-s2.0-0037025272] LEVEL: 3
Excessive glutamate (Glu) stimulation of the NMDA-R is a widely recognized trigger for Ca2+-mediated excitotoxicity. Primary neurons typically show a large increase in vulnerability to excitotoxicity with increasing days in vitro (DIV). This enhanced vulnerability has been associated with increased expression of the NR2B subunit or increased NMDA-R current, but the detailed age-courses of these variables in primary hippocampal neurons have not been compared in the same study. Further, it is not clear whether the NMDA-R is the only source of excess Ca2+. Here, we used primary hippocampal neurons to examine the age dependence of the increase in excitotoxic vulnerability with changes in NMDA-R current, and subunit expression. We also tested whether L-type voltage-gated Ca2+ channels (L-VGCCs) contribute to the enhanced vulnerability. The EC50 for Glu toxicity decreased by approximately 10-fold between 8-9 and 14-15 DIV, changing little thereafter. Parallel experiments found that during the same period both amplitude and duration of NMDA-R current increased dramatically; this was associated with an increase in protein expression of the NR1 and NR2A subunits, but not of the NR2B subunit. Compared to MK-801, ifenprodil, a selective NR2B antagonist, was less effective in protecting older than younger neurons from Glu insult. Conversely, nimodipine, an L-VGCC antagonist, protected older but not younger neurons. Our results indicate that enhanced excitotoxic vulnerability with age in culture was associated with a substantial increase in NMDA-R current, concomitant increases in NR2A and NR1 but not NR2B subunit expression, and with apparent recruitment of L-VGCCs into the excitotoxic process. © 2007 Elsevier B.V. All rights reserved.

TITLE:Calcium controls smooth muscle TRPC gene transcription via the CaMK/calcineurin-dependent pathways 

[2-s2.0-33846392020] Refers [2-s2.0-0037025272] LEVEL: 3
Transient receptor potential protein family C (TRPC) has been proposed as a candidate for channels involved in capacitative Ca2+ entry (CCE) mechanisms, but the modulation of their gene expression remains unexplored. In this study we show that guinea pig gallbladder smooth muscle contains mRNA encoding TRPC1, TRPC2, TRPC3, and TRPC4 proteins whose abundance depends on cytosolic Ca2+ level ([Ca2+]i). Thus lowering the levels of cellular calcium with the chelators EGTA and BAPTA AM results in a downregulation of TRPC1-TRPC4 gene and protein expression. In contrast, activation of Ca2+ influx through L-type Ca2+ channels and Ca2+ release from intracellular stores induced an increase in TRPC1-TRPC4 mRNA and protein abundance. Activation of Ca2+/ calmodulin-dependent kinases (CaMK) and phosphorylation of cAMP-response element binding protein accounts for the increase in TRPC mRNA transcription in response to L-type channel-mediated Ca2+ influx . In addition to this mechanism, activation of TRPC gene expression by intracellular Ca2+ release also involves calcineurin pathway. According to the proposed role for these channels, activation of CCE induced an increase in TRPC1 and TRPC3 mRNA abundance, which depends on the integrity of the calcineurin and CaMK pathways. These findings show for the first time an essential autoregulatory role of Ca2+ in Ca2+ homeostasis at the level of TRPC gene and protein expression. Copyright © 2007 the American Physiological Society.

TITLE:Mitochondria maintain maturation and secretion of lipoprotein lipase in the endoplasmic reticulum 

[2-s2.0-33646806797] Refers [2-s2.0-0037025272] LEVEL: 3
Considering the physiological Ca2+ dynamics within the ER (endoplasmic reticulum), it remains unclear how efficient protein folding is maintained in living cells. Thus, utilizing the strictly folding-dependent activity and secretion of LPL (lipoprotein lipase), we evaluated the impact of ER Ca2+ content and mitochondrial contribution to Ca 2+-dependent protein folding. Exhaustive ER Ca2+ depletion by inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases caused strong, but reversible, reduction of cell-associated and released activity of constitutive and adenovirus-encoded human LPL in CHO-K1 (Chinese-hamster ovary K1) and endothelial cells respectively, which was not due to decline of mRNA or intracellular protein levels. In contrast, stimulation with the IP3 (inositol 1,4,5-trisphosphate)-generating agonist histamine only moderately and transiently affected LPL maturation in endothelial cells that paralleled a basically preserved ER Ca2+ content. However, in the absence of extracellular Ca2+ or upon prevention of transmitochondrial Ca2+ flux, LPL maturation discontinued upon histamine stimulation. Collectively, these data indicate that Ca 2+-dependent protein folding in the ER is predominantly controlled by intraluminal Ca2+ and is largely maintained during physiological cell stimulation owing to efficient ER Ca2+ refilling. Since Ca 2+ entry and mitochondrial Ca2+ homoeostasis are crucial for continuous Ca2+-dependent protein maturation in the ER, their pathological alterations may result in dysfunctional protein folding. © 2006 Biochemical Society.

TITLE:Do we know the absolute values of intracellular free calcium concentration? 

[2-s2.0-0344307501] Refers [2-s2.0-0037025272] LEVEL: 3
More than 20 years ago, it was shown that the addition of EGTA increases the affinity of the plasma membrane Ca2+ pump for Ca2+ by an order of magnitude. The left-hand shift of Ca2+-dependencies in the presence of EGTA has been also documented in studies of the sarcoplasmic reticulum Ca2+ pump, mitochondrial Ca2+-transporter as well as Ca2+-binding by calmodulin and troponin C. These data allow us to hypothesise that this effect is caused by an admixture of di- and trivalent cations possessing high affinity for EGTA and interacting with Ca2+-transporting and binding proteins. Here, we propose that polyvalent cations affect the estimation of absolute values of free intracellular Ca2+ concentration. Indeed, EGTA sharply increases the apparent affinity of the fluorescent Ca2+ indicators quin-2 and fluo-3 for Ca2+. The impact of polyvalent cations on Ca2+ measurement was further confirmed by the study showing the high sensitivity of Ca2+-induced fluo-3 fluorescence to Mn2+, Fe2+, Cu2+, and CO2+ seen in the absence of EGTA. © 2003 Elsevier Ltd. All rights reserved.

TITLE:Patching the membranes up - New views on cellular healing 

[2-s2.0-0242438648] Refers [2-s2.0-0037025272] LEVEL: 3
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TITLE:Antisense therapy for cancer 

[2-s2.0-20344371233] Refers [2-s2.0-0038326942] LEVEL: 3
Improved understanding of the molecular mechanisms that mediate cancer progression and therapeutic resistance has identified many therapeutic gene targets that regulate apoptosis, proliferation and cell signalling. Antisense oligonucleotides offer one approach to target genes involved in cancer progression, especially those that are not amenable to small-molecule or antibody inhibition. Better chemical modifications of antisense oligonucleotides increase resistance to nuclease digestion, prolong tissue half-lives and improve scheduling. Indeed, recent clinical trials confirm the ability of this class of drugs to significantly suppress target-gene expression. The current status and future directions of several antisense drugs that have potential clinical use in cancer are reviewed.

TITLE:Intracellular Localization and Activity State of Tissue Transglutaminase Differentially Impacts Cell Death 

[2-s2.0-1542335670] Refers [2-s2.0-0038326942] LEVEL: 3
Tissue transglutaminase (tTG) is a unique member of the transglutaminase family as it is both a transamidating enzyme and a GTPase. In the cell tTG is mostly cytosolic, however it is also found in the nucleus and associated with the plasma membrane. tTG can be pro-apoptotic, however anti-apoptotic activities of the enzyme have also been reported. To determine how the intracellular localization and transamidating activity of tTG modulates its effects on apoptosis, HEK293 cells were transiently transfected with tTG or $(C277S$)tTG (which lacks transamidating activity) constructs that were targeted to different intracellular compartments. Apoptosis was induced by thapsigargin treatment, which results in increased intracellular calcium concentrations. Cytosolic tTG was pro-apoptotic, while nuclear localization of $(C277S$)tTG attenuated apoptosis. Membrane-targeted tTG had neither pro- nor anti-apoptotic functions. This finding indicates for the first time that intracellular localization is an important determinant of the effect of tTG on apoptosis. Previous studies have suggested that tTG may modulate retinoblastoma (Rb) protein, an important suppressor of apoptosis. tTG interacted with Rb and after induction of apoptosis, the interaction of nuclear-targeted $(C277S$)tTG with Rb was increased significantly concomitant with an attenuation of apoptosis. In contrast, the interaction of nuclear-targeted tTG with Rb was significantly decreased and apoptosis was not attenuated. These data suggest that tTG protects cells against apoptosis in response to stimuli that do not result in increased transamidating activity by translocating to the nucleus, and that complexing with Rb may be an important aspect of the protective effects of tTG.

TITLE:Expression of heat shock protein 27 in human renal cell carcinoma 

[2-s2.0-3543060077] Refers [2-s2.0-0038326942] LEVEL: 3
Heat shock protein 27 (HSP27, Swiss-Prot accession number P04792) is a component of the large and heterogeneous group of chaperone proteins, and its main functions are inhibition of apoptosis and prevention of aggregation of actin intermediate filament. Modified expression of HSP27 has been described in several cancers including testis, breast, and ovaric cancer. In the present work, 18 renal cell carcinoma (RCC) tissues and homologous normal kidney tissues have been investigated for HSP27 expression by combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) separation and Western blotting immunodetection. The results showed significant differences either in expression and in HSP27 isoform numbers in RCC compared to normal kidney. The average number of isoforms was 21 in RCC and 15 in normal tissues with 4.5-5.9 p/ range and 18-29 kDa Mr range. The overexpression was also observed by immunohistochemistry on tissue sections. Only two of RCC samples showed less isoforms than homologous normal samples. Two isoforms were not detected using anti-Ser82 phosphorylated HSP27 antibody, neither in normal nor in RCC samples. Five of all the immunodetected isoforms were confirmed by mass spectrometry as HSP27, but no evidence of post-translational modifications was pointed out. The numerous isoforms observed in RCC are not consistent with data reported in the literature so far, and they might be due to different post-translational modifications such as phosphorylation and S-thiolation. Since activation of HSP27 seems to be involved in tumor proliferation and drug resistance, it would be crucial to correlate the severity of disease with the different isoforms from RCC samples to generate diagnostic and prognostic markers.

TITLE:Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibit doxorubicin-induced apoptosis in human breast cancer cells 

[2-s2.0-4744351463] Refers [2-s2.0-0038326942] LEVEL: 3
Tissue transglutaminase (TGase) exhibits both a GTP binding/hydrolytic capability and an enzymatic transamidation activity. Increases in TGase expression and activation often occur in response to stimuli that promote cellular differentiation and apoptosis, yet the signaling mechanisms used by these stimuli to regulate TGase expression and activation and the role of TGase in these cellular processes are not well understood. Retinoic acid (RA) consistently induces TGase expression and activation, and it was shown recently that RA-induced TGase expression was inhibited in NIH3T3 mouse fibroblasts co-stimulated with epidermal growth factor (EGF). Here we investigate whether EGF also antagonized RA-induced TGase expression in breast cancer cells. We found that EGF stimulation affected TGase expression and activation very differently in these cancer cells. Not only did EGF fail to block RA-induced TGase expression, but also EGF alone was sufficient to potently up-regulate TGase expression and activation in SKBR3 cells, as well as MDAMB468 and BT-20 cells. Inhibiting phosphoinositide 3-kinase activity severely diminished the ability of EGF and RA to increase TGase protein levels, whereas a constitutively active form of phosphoinositide 3-kinase potentiated the induction of TGase expression by EGF in SKBR3 cells. Because EGF is an established antiapoptotic factor, we examined whether the protection afforded by EGF was dependent on its ability to up-regulate TGase activity in SKBR3 and BT-20 cells. Exposure of cells to a TGase inhibitor or expression of a dominant-negative form of TGase potently inhibited EGF-mediated protection from doxorubicin-induced apoptosis. Moreover, expression of exogenous TGase in SKBR3 cells mimicked the survival advantage of EGF, suggesting that TGase activation is necessary and sufficient for the antiapoptotic properties of EGF. These findings indicate for the first time that EGF can induce TGase expression and activation in human breast cancer cells and that this contributes to their oncogenic potential by promoting chemoresistance.

TITLE:Interactions of EGFR and caveolin-1 in human glioblastoma cells: Evidence that tyrosine phosphorylation regulates EGFR association with caveolae 

[2-s2.0-4644280871] Refers [2-s2.0-0038326942] LEVEL: 3
Epidermal growth factor receptor (EGFR) amplifleation and type III mutation (EGFRvIII), associated with constitutive tyrosine kinase activation and high malignancy, are commonly observed in glioblastoma tumors. The association of EGFR and EGFRvIII with caveolins was investigated in human glioblastoma cell lines, U87MG and U87MG-EGFRvIII. Caveolin-1 expression, determined by RT-PCR, real-time quantitative PCR and Western blot, was upregulated in glioblastoma cell lines (two-fold) and tumors (20-300-fold) compared to primary human astrocytes and nonmalignant brain tissue, respectively. U87MG-EGFRvIII expressed higher levels of caveolin-1 than U87MG. In contrast, the expression of caveolin-2 and -3 were downregulated in glioblastoma cells compared to astrocytes. A colocalization of EGFR, but not of EGFRvIII, with lipid rafts and caveolin-1 was observed by immunocytochemistry. Association of EGFR and EGFRvIII with caveolae, assessed in vitro by binding to caveolin scaffolding domain peptides and in vivo by immunocolocalization studies in cells and caveolae-enriched cellular fraction, was phosphorylation-dependent: ligand-induced phosphorylation of EGFR resulted in dissociation of EGFR from caveolae. In contrast, inhibition of the EGFRvIII constitutive tyrosine phosphorylation by AG1478 increased association of EGFRvIII with caveolin-1. AG1478 also increased caveolin-1 expression and reduced glioblastoma cell growth in a semi-solid agar. The evidence suggests that the phosphorylation-regulated sequestration of EGFR in caveolae may be involved in arresting constitutive or ligand-induced signaling through EGFR responsible for glial cell transformation.

TITLE:Increased Hsp27 after androgen ablation facilitates androgen-independent progression in prostate cancer via signal transducers and activators of transcription 3-mediated suppression of apoptosis 

[2-s2.0-28244456529] Refers [2-s2.0-0038326942] LEVEL: 3
One strategy to improve therapies in prostate cancer involves targeting cytoprotective genes activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. The objectives of this study were to define changes in Hsp27 levels after androgen ablation and to evaluate the functional relevance of these changes in AI progression. Using a tissue microarray of 232 specimens of hormone-nai?ve and post-hormone ablation-treated prostate cancer, we found that Hsp27 levels increase after androgen ablation to become highly expressed (>4-fold, P ? 0.01) in AI tumors. Hsp27 overexpression rendered LNCaP cells highly resistant to androgen withdrawal both in vitro and in vivo. Tumor volume and serum prostate-specific antigen levels increased 4.3- and 10-fold faster after castration when Hsp27 was overexpressed. Treatment of LNCaP tumor cells in vitro with Hsp27 antisense oligonucleotides (ASO) or short-interfering RNA suppressed Hsp27 levels in a dose-dependent and sequence-specific manner increased the apoptotic sub-G 0-G1 fraction and caspase-3 cleavage >2-fold, as well as decreased signal transducers and activators of transcription 3 (Stat3) levels and its downstream genes, c-fos and sPLA-2. The cytoprotection afforded by Hsp27 overexpression was attenuated by StatS knockdown using specific Stat3 ASO. Coimmunoprecipitation and immunofluorescence confirmed that Hsp27 interacts with Stat3 and that Stat3 levels correlated directly with Hsp27 levels. Hsp27 ASO treatment in athymic mice bearing LNCaP tumors significantly delayed LNCaP tumor growth after castration, decreasing mean tumor volume and serum prostate-specific antigen levels by 57% and 69%, respectively. These findings identify Hsp27 as a modulator of Stat3-regulated apoptosis after androgen ablation and as a potential therapeutic target in advanced prostate cancer. © 2005 American Association for Cancer Research.

TITLE:Proteins and protein pattern differences between glioma cell lines and glioblastoma multiforme 

[2-s2.0-21044434153] Refers [2-s2.0-0038326942] LEVEL: 3
Introduction: Research into the pathogenesis, molecular signaling, and treatment of glioblastoma multiforme (GBM) has traditionally been conducted using cell lines derived from malignant gliomas. We compared protein expression patterns between solid primary GBMs and GBM cell lines to identify proteins whose expression may be altered in cell culture. Methods: We cultured cell lines U87, U118, U251, and A172 and used tissue-selective microdissection of eight primary GBMs to obtain pure populations of tumor cells, which we studied using two-dimensional gel electrophoresis (2DGE) and examined using differential expression software. Select protein targets expressed differentially between GBM tumors and GBM cell lines were sequenced using tandem mass spectrometry. Results: Analysis of the primary GBM tumor samples (n = 8) and the GBM cell lines revealed reproducibly similar proteomic patterns for each group, which distinguished tumors from the cell lines. Gels contained up to 500 proteins that were consistently identified in the pH 4 to 7 range. Comparison of proteins identified in the GBM tumors and in the cell lines showed ?160 proteins that were gained and 60 proteins that were lost on culture. Using normalized intensity patterns from the 2DGE images, ANOVA tests were done and statistically significant spots were identified. Seven proteins found in the cell lines were significantly increased when compared with the GBM tumors (P < 0.05), whereas 10 proteins were significantly decreased from cell lines compared with the GBM tumors. Proteins identified included transcription factors, tumor suppressor genes, cytoskeletal proteins, and cellular metabolic proteins. Conclusion: Global protein and proteomic differences were identified between primary GBM tumor samples and GBM cell lines. The proteins identified by 2DGE analysis elucidate some of the selection pressures of in vitro culture, help accentuate the advantages and limitations of cell culture, and may aid comprehension of gliomagenesis and enhance development of new therapeutics. © 2005 American Association for Cancer Research.

TITLE:Francisella tularensis proteome: Low levels of ASB-14 facilitate the visualization of membrane proteins in total protein extracts 

[2-s2.0-26844577229] Refers [2-s2.0-0038326942] LEVEL: 3
Proteomic analysis of bacterial pathogens isolated from in vivo sources, such as infected tissues, provides many challenges not the least of which is the limited quantity of sample available for analysis. It is, therefore, highly desirable to develop a one-step cellular lysis and protein solubilization method that minimizes protein losses and allows the maximum possible coverage of the proteome. Here, we have used standard sample buffer constituents including urea, thiourea and DTT, but varied the detergent composition of the buffers in order to achieve the best quality of gels and the greatest spot resolution. We found that the most efficient solubilizing solution in this case consisted of 7 M urea, 2 M thiourea, 1% DTT, 0.5% amidosulfobetaine-14 (ASB-14) and 4% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Inclusion of low levels of ASB-14 in solutions allowed visualization of a subset of 24 new protein spots in the Live Vaccine Strain (LVS) of Francisella tularensis and 21 spots in a virulent A-strain of the pathogen. Further investigation showed that 15 of the 24 enriched LVS spots were membrane or membrane-associated proteins suggesting that the optimized lysis and solubilization solution aids in the detection of more hydrophobic proteins. This methodology is now being applied to the analysis of Francisella obtained from in vivo sources. © 2005 American Chemical Society.

TITLE:Differential protein expression in human gliomas and molecular insights 

[2-s2.0-20144388217] Refers [2-s2.0-0038326942] LEVEL: 3
Gliomas are the most common of the primary intracranial tumors with astrocytomas constituting about 40%. Using clinically and histologically assessed astrocytomas, we have studied their protein profiles using a two-dimensional gel electrophoresis-mass spectrometry approach and identified differentially expressed proteins which may be useful molecular indicators to understand these tumors. Examination of the protein profiles of 27 astrocytoma samples of different grades revealed 72 distinct, differentially expressed proteins belonging to various functional groups such as cytoskeleton and intermediate filament proteins, heat shock proteins (HSPs), enzymes and regulatory proteins. Based on the consistency of their differential expression, 29 distinct proteins could be short-listed and may have a role in the pathology of astrocytomas. Some were found to be differentially expressed in both Grade III and IV astrocytomas while others were associated with a particular grade. A notable observation was underexpression of Prohibitin, a potential tumor suppressor protein, Rho-GDP dissociation inhibitor, Rho-GDI, a regulator of Rho GTPases and HSPs as well as destabilization of glial fibrillary acidic protein, GFAP, major protein of the glial filaments, in Grade III malignant rumors. We attempt to explain glioma malignancy and progression in terms of their combined role. © 2005 WILEY-VCH Verlag GmbH & Co. KGaA.

TITLE:Proteomics and disease: Opportunities and challenges 

[2-s2.0-20444477520] Refers [2-s2.0-0038326942] LEVEL: 3
? Since the human genome was sequenced, there has been intense activity to understand the function of the 30 000 identified genes; attention has now turned to the products of genes - proteins. ? Proteomics is the large-scale study of the structure and function of proteins; it includes the rapidly evolving field of disease proteomics, which aims to identify proteins involved in human disease and to understand how their expression, structure and function cause illness. ? Proteomics has identified proteins that offer promise as diagnostic or prognostic markers, or as therapeutic targets in a range of illnesses, including cancer, immune rejection after transplantation, and infectious diseases such as tuberculosis and malaria; it has the potential to allow patient-tailored therapy. ? Some major challenges remain, both technical (eg, detecting "low-abundance" proteins, and maintaining sample stability) and in data management (eg, correlating changes in proteins with disease processes).

TITLE:Proteomic studies on low- and high-grade human brain astrocytomas 

[2-s2.0-20844447307] Refers [2-s2.0-0038326942] LEVEL: 3
Human brain astrocytomas range from the indolent low-grade to the highly infiltrating and aggressive high-grade form, also known as glioblastoma multiforme. The extensive heterogeneity of astrocytic tumors complicates their pathological classification. In this study, we compared the protein pattern of low-grade fibrillary astrocytomas to that of glioblastoma multiforme by 2D electrophoresis. The level of most proteins remains unchanged between the different grade tumors and only few differences are reproducibly observable. Fifteen differentially expressed proteins, as well as seventy conserved spots, were identified by mass spectrometry. Western and immnunohistochemical analysis confirmed the differential expression of the identified proteins. These data provide an initial reference map for brain gliomas. Among the proteins more highly expressed in glioblastoma multiforme, we found peroxiredoxin 1 and 6, the transcription factor BTF3, and ?-B-crystallin, whereas protein disulfide isomerase A3, the catalytic subunit of the cAMP-dependent protein kinase, and the glial fibrillary acidic protein are increased in low-grade astrocytomas. Our findings contribute to deepening our knowledge of the factors that characterize this class of tumors and, at the same time, can be applied toward the development of novel molecular biomakers potentially useful for an accurate classification of the grade of astrocytomas. © 2005 American Chemical Society.

TITLE:Phosphoinositide 3-kinase accelerates postoperative tumor growth by inhibiting apoptosis and enhancing resistance to chemotherapy-induced apoptosis: Novel role for an old enemy 

[2-s2.0-20444380697] Refers [2-s2.0-0038326942] LEVEL: 3
Tumor removal remains the principal treatment modality in the management of solid tumors. The process of tumor removal may potentiate the resurgent growth of residual neoplastic tissue. Herein, we describe a novel murine model in which flank tumor cytoreduction is followed by accelerated local tumor recurrence. This model held for primary and recurrent tumors generated using a panel of human and murine (LS174T, DU145, SW480, SW640, and 3LL) cell lines and replicated accelerated tumor growth following excisional surgery. In investigating this further, epithelial cells were purified from LS174T primary and corresponding recurrent tumors for comparison. Baseline as well as tumor necrosis factor apoptosis-inducing ligand (TRAIL)-induced apoptosis were significantly reduced in recurrent tumor epithelia. Primary and recurrent tumor gene expression profiles were then compared. This identified an increase and reduction in the expression of p110? and p85? class Ia phosphoinositide 3-kinase (PI3K) subunits in recurrent tumor epithelia. These changes were further confirmed at the protein level. The targeting of PI3K ex vivo, using LY294002, restored sensitivity to TRAIL in recurrent tumor epithelia. In vivo, adjuvant LY294002 prolonged survival and significantly attenuated recurrent tumor growth by greatly enhancing apoptosis levels. Hence, PI3K plays a role in generating the antiapoptotic and chemoresistant phenotype associated with accelerated local tumor recurrence. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:Proteome analysis of mouse primary astrocytes 

[2-s2.0-20544433351] Refers [2-s2.0-0038326942] LEVEL: 3
Astrocytes play a role in energy metabolism, neuronal homeostasis and release of neuronal growth factors and several neurotransmitters. They also relate to a variety of brain diseases and contribute to restore brain dysfunction. Although current research has revealed several roles for astrocytes, knowledge on astrocytic protein expression is limited and a systematic and comprehensive proteome study of astrocytes has not been reported so far. We applied a proteomics technique based on two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF) and unambiguously identified 301 spots corresponding to 191 individual proteins in primary mouse astrocytes. The identified proteins were from antioxidant, chaperone, cytoskeleton, nucleic acid binding, signaling, proteasomal, hypothetical and miscellaneous proteins. A reference database is provided and proteins were identified in astrocytes specifically and unambiguously for the first time. A reliable analytical tool independent of antibody availability and specificity along with tentative astrocytic marker proteins is described. © 2005 Elsevier Ltd. All rights reserved.

TITLE:Two isoforms of tissue transglutaminase mediate opposing cellular fates 

[2-s2.0-33845489411] Refers [2-s2.0-0038326942] LEVEL: 3
Opposing cellular responses are typically regulated by distinct sets of genes. However, tissue transglutaminase (TGase) provides an interesting example of a single gene product that has been implicated both in affording protection against cellular insults as well as in promoting cell death. Here, we shed some light on how these conflicting activities might be manifested by demonstrating that alternative transcripts of TGase differentially affect cell viability. We show that although the full-length TGase protein affords strong protection against cell death signals, a shorter version of TGase that is truncated at the 3? end, and thus called TGase-short (TGase-S), is cytotoxic. The apoptotic activity of TGase-S is not dependent on its transamidation activity because the mutation of a cysteine residue that is essential for catalyzing this reaction does not compromise the ability of TGase-S to induce cell death. Intriguingly, TGase-S undergoes inappropriate oligomer formation in cells before cell death, suggesting a novel mechanism for the apoptotic effects of this protein. © 2006 by The National Academy of Sciences of the USA.

TITLE:Reversal of drug resistance in breast cancer cells by transglutaminase 2 inhibition and nuclear factor-ĪŗB inactivation 

[2-s2.0-33845307265] Refers [2-s2.0-0038326942] LEVEL: 3
Induction of transglutaminase 2 (TGase 2) by epidermal growth factor (EGF) in human breast cancer cells increases their oncogenic potential and chemoresistance. The role of TGase 2 in the development of these tumor-related phenotypes remains to be elucidated, but it has been shown that expression of a dominant-negative form of TGase 2 reverses EGF-mediated chemoresistance in breast cancer cells. We examined several different breast cancer cell lines, representing both EGF receptor (EGFR)-positive and EGFR-negative breast cancers, and found that doxorubicin-resistant cells had a higher level of TGase 2 compared with doxorubicin-sensitive cells independent of the EGFR expression level. TGase 2 inhibition increased the chemosensitivity of drug-resistant cells, concomitant with a decrease in nuclear factor-?B (NF-?B) activity. Increasing the level of TGase 2 in drug-sensitive cells by transient transfection reduced the level of inhibitory subunit ? of NF-?B (I?B?) and increased NF-?B activity in these cells. Inhibition of TGase 2 in drug-resistant cells by RNA interference increased the levels of I?B?, and this correlated with a shift in the accumulation of NF-?B from the nucleus to the cytosol. We recently showed that TGase 2 activated NF-?B through polymerization and depletion of free I?B? during inflammation. Therefore, increased expression of TGase 2 and subsequent activation of NF-?B may contribute to drug resistance in breast cancer cells independently of EGF signaling. ©2006 American Association for Cancer Research.

TITLE:Biomarker discovery: A proteomic approach for brain cancer profiling 

[2-s2.0-33845633819] Refers [2-s2.0-0038326942] LEVEL: 3
Gliomas in the form of astrocytomas, anaplastic astrocytomas and glioblastomas are the most common brain tumors in humans. Early detection of these cancers is crucial for successful treatment. Proteomics promises the discovery of biomarkers and tumor markers for early detection and diagnosis. In the current study, a differential gel electrophoresis technology coupled with matrix-assisted laser desorption/ ionization-time of flight and liquid chromatography-tandem mass spectroscopy was used to investigate tumor-specific changes in the proteome of human brain cancer. Fifty human brain tissues comprising varying diagnostic groups (non-tumor, grade I, grade II, grade III and grade IV) were run in duplicate together with an internal pool sample on each gel. The proteins of interest were automatically picked, in-gel digested and mass spectrometry fingerprinted. Two hundred and eleven protein spots were identified successfully and were collapsed into 91 unique proteins. Approximately 20 of those 91 unique proteins had, to our knowledge, not been reported previously as differentially expressed in human brain cancer. Alb protein, peroxiredoxin 4 and SH3 domain-binding glutamic acid-rich-like protein 3 were upregulated in glioblastoma multiform versus non-tumor tissues. However, aldolase C fructose-biphosphate, creatine kinase, B chain dihydrolipoyl dehydrogenase, enolase 2, fumarate hydratase, HSP60, lactoylglutathione lyase, lucine aminopeptidase, Mu-crystallin homolog, NADH-UO 24, neurofilament triplet L protein, septin 2, stathmin and vacuolar ATP synthase subunit E were downregulated in glioblastoma multiform compared with non-tumor tissues. These differentially expressed proteins provided novel information on the differences existing between normal brain and gliomas, and thus might prove to be useful molecular indicators of diagnostic or prognostic value. © 2007 Japanese Cancer Association.

TITLE:Protein markers of ischemic insult in brain endothelial cells identified using 2D gel electrophoresis and ICAT-based quantitative proteomics 

[2-s2.0-33846580480] Refers [2-s2.0-0038326942] LEVEL: 3
The blood-brain barrier (BBB) is formed by endothelial cells of cerebral microvessels sealed by tight junctions. Ischemic brain injury is known to initiate a series of biochemical and molecular processes that lead to the disruption of the BBB, development of vascular inflammation, and subsequent neurovascular remodeling including angiogenesis. Molecular effectors of these changes are multiple and are regulated in a dynamic fashion. The current study was designed to analyze changes in cellular and secreted proteins in rat brain endothelial cells (BEC) exposed to ischemic insult in vitro using two complementary quantitative proteomic approaches: two-dimensional gel electrophoresis (2DE) and isotope-coded affinity tag (ICAT)-based proteomics. We show a comprehensive qualitative and quantitative comparison between the two proteomic methods applied to the same experimental system with respect to their reproducibility, specificity, and the type of proteins identified. In total, >160 proteins showed differential expression in response to the ischemic insult, with 38 identified by 2DE and 138 by ICAT. Only 15 proteins were commonly identified. ICAT showed superior reproducibility over 2DE and was more suitable for detecting small, large, basic, hydrophobic, and secreted proteins than 2DE. However, positive identification of proteins by MS/MS was more reliably done using a 2DE-based method compared to ICAT. Changes in proteins involved in nucleic acid, protein, and carbohydrate metabolism, signal transduction, cell structure, adhesion and motility, immunity and defense, cell cycle, and apoptosis were observed. The functional significance of observed protein changes was evaluated through a multifaceted protein classification and validation process, which included literature mining and comparative evaluation of protein changes in analogous in vitro and in vivo ischemia models. The comparative analyses of protein changes between the in vitro and in vivo models demonstrated a significant correlative relationship, emphasizing the 'translational' value of in vitro endothelial models in neurovascular research. © 2007 American Chemical Society.

TITLE:Cellular functions of vaults and their involvement in multidrug resistance 

[2-s2.0-33747288211] Refers [2-s2.0-0038326942] LEVEL: 3
Vaults are evolutionary highly conserved ribonucleoprotein (RNP) particles with a hollow barrel-like structure. They are 41 × 73 nm in size and are composed of multiple copies of three proteins and small untranslated RNA (vRNA). The main component of vaults represents the 110 kDa major vault protein (MVP), whereas the two minor vault proteins comprise the 193 kDa vault poly(ADP-ribose) polymerase (VPARP) and the 240 kDa telomerase-associated protein-1 (TEP1). Vaults are abundantly present in the cytoplasm of eukaryotic cells and they were found to be associated with cytoskeletal elements as well as occasionally with the nuclear envelope. Vaults and MVP have been associated with several cellular processes which are also involved in cancer development like cell motility and differentiation. Due to the over-expression of MVP (also termed lung resistance-related protein or LRP) in several P-glycoprotein (P-gp)-negative chemoresistant cancer cell lines, vaults have been linked to multidrug resistance (MDR). Accordingly, high levels of MVP were found in tissues chronically exposed to xenobiotics. In addition, the expression of MVP correlated with the degree of malignancy in certain cancer types, suggesting a direct involvement in tumor development and/or progression. Based on the finding that MVP binds several phosphatases and kinases including PTEN, SHP-2 as well as Erk, evidence is accumulating that MVP might be involved in the regulation of important cell signalling pathways including the PI3K/Akt and the MAPK pathways. In this review we summarize the current knowledge concerning the vault particle and discuss its possible cellular functions, focusing on the role of vaults in chemotherapy resistance. © 2006 Bentham Science Publishers Ltd.

TITLE:Insulin-like growth factor binding protein-4 (IGFBP-4) is a novel anti-angiogenic and anti-tumorigenic mediator secreted by dibutyryl cyclic AMP (dB-cAMP)-differentiated glioblastoma cells 

[2-s2.0-33646531617] Refers [2-s2.0-0038326942] LEVEL: 3
cAMP has been shown to reverse the transformed phenotype of various cancer cells. Human glioblastoma U87MG cells exposed to 500 ?M dB-cAMP for 6 days showed reduced proliferation, attenuated invasiveness, and inability to induce angiogenic responses in human brain endothelial cells (HBECs) grown in Matrigel?. VEGF was the principal mediator of angiogenic actions of U87MG conditioned media (CM), since VEGF neutralizing antibody completely inhibited U87MG-induced angiogenic responses and no detectable levels of IGF, bFGF, and PlGF were found in U87MG CM. VEGF release was induced (?20%) in dB-cAMP-treated U87MG cells, suggesting a simultaneous induction of anti-angiogenic mediators. Down-stream effectors of dB-cAMP actions in U87MG were investigated by microarray gene expression analysis. Detected increases in differentiation genes, staniocalcin-1 and Wnt-5a, and angiogenesis-related genes, PAI-1, SPARC, IGFBP-4, IGFBP-7, PAPP-A, and PRSS-11 in dB-cAMP-treated U87MG cells were validated by real-time PCR, Western blot, and/or ELISA. A subsequent series of experiments identified IGFBP-4 as the principal anti-angiogenic mediator secreted by glioblastoma cells in response to dB-cAMP. Human recombinant IGFBP-4 inhibited the angiogenic response of HBEC induced by U87MG CM, whereas anti-human IGFBP-4 antibody restored the pro-angiogenic activity of dB-cAMP-treated U87MG CM. Since neither U87MG nor HBEC cells secreted detectable levels of IGF-I, and there are no known cellular IGFBP-4 receptors, the anti-angiogenic effect of IGFBP-4 was likely IGF-I-independent and indirect. IGFBP-4 also antagonized angiogenic effects of VEGF165, PlGF, and bFGF, and reduced U87MG colony formation in soft-agar. IGFBP-4 is a novel dB-cAMP-induced anti-angiogenic and anti-tumorigenic mediator that may be a promising candidate for glioblastoma therapy. © 2006 Wiley-Liss, Inc.

TITLE:Solution structure of a two-repeat fragment of major vault protein 

[2-s2.0-30744462797] Refers [2-s2.0-0038326942] LEVEL: 3
Major vault protein (MVP) is the main constituent of vaults, large ribonucleoprotein particles implicated in resistance to cancer therapy and correlated with poor survival prognosis. Here, we report the structure of the main repeat element in human MVP. The ?55 amino acid residue MVP domain has a unique, novel fold that consists of a three-stranded antiparallel ?-sheet. The solution NMR structure of a two-domain fragment reveals the interdomain contacts and relative orientations of the two MVP domains. We use these results to model the assembly of 672 MVP domains from 96 MVP molecules into the ribs of the 13 MDa vault structure. The unique features include a thin, skin-like structure with polar residues on both the cytoplasmic and internal surface, and a pole-to-pole arrangement of MVP molecules. These studies provide a starting point for understanding the self-assembly of MVP into vaults and their interactions with other proteins. Chemical shift perturbation studies identified the binding site of vault poly(ADP-ribose) polymerase, another component of vault particles, indicating that MVP domains form a new class of interaction-mediating modules. Crown Copyright © 2005 Published by Elsevier Ltd. All rights reserved.

TITLE:Comparative status of activated ERK1/2 and PARP cleavage in human gliomas 

[2-s2.0-16444380429] Refers [2-s2.0-0038326942] LEVEL: 3
Gliomas are the most common form of cerebral tumors. Understanding molecular features of glioma will eventually allow for targeted intervention and more promising approaches for treating gliomas. The present study is therefore carried out to check the levels of activated ERK1/2 with respect to phospho-tyrosine and cleavage of poly ADP-ribose polymerase (PARP). Recent experiments support that extracellular signal regulated kinase (ERK), a mitogen activated protein (MAP) kinase might have a critical role in cell proliferation. PARP is a DNA-repair enzyme activated by DNA strand breaks. Overactivation of PARP after cellular insult lead to cell death caused by rapid depletion of cellular ATP. Three glioblastoma multiforme (GBM) and two astrocytoma biopsies (core tumor) and peripheral tissues were analyzed for the expression of p-ERK172 and PARP. Results indicate higher p-ERK1/2 in GBM. Cleaved fragments of PARP (89 kDa) were found to be more in core tumor tissue samples as compared to peripheral tumor tissues of both astrocytoma and GBM.

TITLE:Genomic and proteomic analyses of vascular endothelial growth factor and insulin-like growth factor-binding protein 3 lung adenocarcinomas 

[2-s2.0-2442530968] Refers [2-s2.0-0038326942] LEVEL: 3
Vascular endothelial growth factor (VEGF) is regulated by the hypoxia-inducible factor 1 (HIF1) pathway and is implicated in tumor progression and patient survival in many types of cancer. Insulin-like growth factor-binding protein 3 (IGFBP3) is also regulated by HIF1 but works in a completely different manner by modulating the activities of insulin-like growth factors and inducing apoptosis. In this study, 2-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) was used to analyze the protein expression profiles of VEGF and IGFBP3 isoforms in 93 lung adenocarcinomas and 10 uninvolved lung samples. The same samples were examined for messenger RNA (mRNA) expression with use of oligonucleotide arrays. Correlation analysis in the lung adenocarcinomas between mRNA expression levels of VEGF and all 4966 other genes was used to identify other biologic processes that may be associated with increased VEGF expression. Two-dimensional gel separations revealed 7 VEGF protein isoforms and 5 isoforms of IGFBP3. VEGF and IGFBP3 mRNA were found to be overexpressed in bronchial-derived lung adenocarcinomas (P < 0.0001), and expression was decreased in well-differentiated lung adenocarcinomas (P < 0.0002). There was a significant correlation (P < 0.01) between VEGF and IGFBP3 mRNA in lung adenocarcinomas; however, no correlation was detected in uninvolved lung samples. Forty genes were identified as the most significantly associated with VEGF expression (r > 0.38, P < 0.001), 17 of which were also associated with IGFBP3, and 12 were known to be induced through the HIF1 pathway. Among other highly correlated genes, several, including bradykinin receptor B2, suggest additional cellular processes that were not previously known to be associated with VEGF expression in lung adenocarcinoma.

TITLE:Identification of glioma neovascularization-related proteins by using MALDI-FTMS and Nano-LC fractionation to microdissected tumor vessels 

[2-s2.0-34547103513] Refers [2-s2.0-0038326942] LEVEL: 3
The identification of angiogenesis-related proteins is important for the development of new antiangiogenic therapies, and such proteins are potential new biomarkers for gliomas. The aim of this study was to identify proteins that are exclusively present in glioma neovasculature and not in the vasculature of normal brain. We combined advanced proteomics techniques to compare the expression profiles of microdissected blood vessels from glioma with blood vessels of normal control brain samples. We measured the enzymatic generated peptide profiles from these microdissected samples by MALDI-FTMS. Subsequently, the samples were fractionated by nano-LC prior to MALDI-TOF/TOF. This combined approach enabled us to identify four proteins that appeared to be exclusively expressed in the glioma blood vessels. Two of these proteins, fibronectin and colligin 2, were validated on tissue sections using specific antibodies. We found that both proteins are present in active angiogenesis in glioma, other neoplasms, and reactive conditions in which neoangiogenesis takes place. This work proves that gel-free mass spectrometric techniques can be used on relatively small numbers of cells generated by microdissection procedures to successfully identify differentially expressed proteins. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:Proteomic investigation of glioblastoma cell lines treated with wild-type p53 and cytotoxic chemotherapy demonstrates an association between galectin-1 and p53 expression 

[2-s2.0-33847399649] Refers [2-s2.0-0038326942] LEVEL: 3
Global protein analysis of treated and untreated glioblastoma cell lines was performed. Proteomic analysis revealed the identity of proteins that were significantly modulated by the treatment with wild-type TP53 and the cytotoxic chemotherapy SN38. In particular, galectin-1 was found to be negatively regulated by transfection with TP53 and further down-regulated by SN38. Expression level changes were confirmed by Western blot. Subsequent analysis of several high-grade glioma cell lines demonstrated very high levels of galectin-1, regardless if the cell lines contained mutant or wild-type TP53. High expression of galectin-1 in a human orthotopic murine tumor model was also detected by immunohistochemistry and revealed a consistent pattern of preferential expression in peripheral or leading tumor edges. Further examination of galectin-1 expression through microarray analysis in tumor materials from patients confirmed galectin-1 as a valuable biomarker and possible therapeutic target. These results demonstrate the utility of using proteomic approaches to interrogate and identify potential useful targets for cancer therapy by evaluating specific tumor responses, either positive or negative, to various therapies. © 2007 American Chemical Society.

TITLE:Genome-wide allelic imbalance analysis of pediatric gliomas by single nucleotide polymorphic allele array 

[2-s2.0-33845774308] Refers [2-s2.0-0038326942] LEVEL: 3
Using single nucleotide polymorphic (SNP) allele arrays, we analyzed 28 pediatric gliomas consisting of 14 high-grade gliomas and 14 low-grade gliomas. Most of the low-grade gliomas had no detectable loss of heterozygosity (LOH) in any of the 11,562 SNP loci; exceptions were two gangliogliomas (3q and 9p), one astrocytoma (6q), and two subependymal giant cell astrocytomas (16p and 21q). On the other hand, all high-grade gliomas had various degrees of LOH affecting 52 to 2,168 SNP loci on various chromosomes. LOH occurred most frequently in regions located at 4q (54%), 6q (46%), 9p (38%), 10q (38%), 11p (38%), 12 (38%), 13q (69%), 14q (54%), 17 (38%), 18p (46%), and 19q (38%). We also detected amplifications of epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor ? (PDGFR?) in a few of the 13 cases of glioblastoma multiforme analyzed. Interestingly, the amplified EGFR and PDGFR? were located within regions of LOH. SNP loci with LOH and copy number changes were validated by sequencing and quantitative PCR, respectively. Our results indicate that, in some pediatric glioblastoma multiforme, one allele each of EGFR and PDGFR? was lost but the remaining allele was amplified. This may represent a new molecular mechanism underlying tumor progression. ©2006 American Association for Cancer Research.

TITLE:Therapeutic aspects of chaperones/heat-shock proteins in neuro-oncology 

[2-s2.0-33745121223] Refers [2-s2.0-0038326942] LEVEL: 3
Tumors of the CNS frequently have devastating consequences in terms of cognitive and motor function, personality and mortality. Despite decades of work, current therapies have done little to alter the course of these deadly diseases. The discovery that chaperones/heat-shock proteins play an important role in tumor biology and immunology have sparked much interest in utilizing these proteins as targets of therapeutics, or as therapeutics themselves, in the treatments of a variety of cancers. Neuro-oncology has only recently taken notice of these entities, and the purpose of this review is to provide a background, an update and a view to the future for the roles of chaperones/heat-shock proteins in the treatment of brain tumors. © 2006 Future Drugs Ltd.

TITLE:Effects of tissue transglutaminase on Ī² -amyloid1-42- induced apoptosis 

[2-s2.0-33744489315] Refers [2-s2.0-0038326942] LEVEL: 3
Tissue transglutaminase (TGase) has been implicated in both cell survival and apoptosis. Here we investigate the role of TGase in ?-amyloid-induced neurotoxicity using retinoic acid (RA)-differentiated, neuronal SH-SY5Y cells. We show that ?-amyloid-induced cell death was reduced in RA-differentiated SH-SY5Y cells treated with the TGase inhibitor monodansyl cadaverine. Expression of wild-type TGase enhanced ?-amyloid1-42-induced apoptosis, whereas transamidation-defective TGase did not. These effects were specific for ?-amyloid-treated cells, as TGase reversed the neurotoxic effects caused by hydrogen peroxide treatment. Enhancement of ?-amyloid1-42- induced cell death by TGase was accompanied by marked increases in TGase activity in the membrane fractions and translocation of TGase to the cell surface. Overall, these findings suggest that the ability of TGase to exhibit pro-survival versus pro-apoptotic activity is linked to its cellular localization, with ?-amyloid-induced recruitment of TGase to the cell surface accentuating neuronal toxicity and apoptosis. © 2006 Springer Science+Business Media, Inc.

TITLE:Cytosolic activation of cathepsins mediates parvovirus H-1-induced killing of cisplatin and TRAIL-resistant glioma cells 

[2-s2.0-34247170947] Refers [2-s2.0-0038326942] LEVEL: 3
Gliomas are often resistant to the induction of apoptotic cell death as a result of the development of survival mechanisms during astrocyte malignant transformation. In particular, the overexpression of Bcl-2-family members interferes with apoptosis initiation by DNA-damaging agents (e.g., cisplatin) or soluble death ligands (e.g., TRAIL). Using low-passage-number cultures of glioma cells, we have shown that parvovirus H-1 is able to induce deatli in cells resistant to TRAIL, cisplatin, or both, even when Bcl-2 is overexpressed. Parvovirus H-1 triggers cell death through both the accumulation of lysosomal cathepsins B and L in the cytosol of infected cells and the redaction of the levels of cystatin B and C, two cathepsin inhibitors. The impairment of either of these effects protects glioma cells from the viral lytic effect. In normal human astrocytes, parvovirus H-1 fails to induce a killing mechanism. In vivo, parvovirus H-1 infection of rat glioma cells intracranially implanted into recipient animals triggers cathepsin B activation as well. This report identifies for the first time cellular effectors of the killing activity of parvovirus H-1 against malignant brain cells and opens up a therapeutic approach which circumvents their frequent resistance to other death inducers. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

TITLE:Protein expression in experimental malignant glioma varies over time and is altered by radiotherapy treatment 

[2-s2.0-33745061390] Refers [2-s2.0-0038326942] LEVEL: 3
Radiotherapy is one of the mainstays of glioblastoma (GBM) treatment. This study aims to investigate and characterise differences in protein expression patterns in brain tumour tissue following radiotherapy, in order to gain a more detailed understanding of the biological effects. Rat BT4C glioma cells were implanted into the brain of two groups of 12 BDIX-rats. One group received radiotherapy (12 Gy single fraction). Protein expression in normal and tumour brain tissue, collected at four different time points after irradiation, were analysed using surface enhanced laser desorption/ionisation - time of flight - mass spectrometry (SELDI-TOF-MS). Mass spectrometric data were analysed by principal component analysis (PCA) and partial least squares (PLS). Using these multivariate projection methods we detected differences between tumours and normal tissue, radiation treatment-induced changes and temporal effects. 77 peaks whose intensity significantly changed after radiotherapy were discovered. The prompt changes in the protein expression following irradiation might help elucidate biological events induced by radiation. The combination of SELDI-TOF-MS with PCA and PLS seems to be well suited for studying these changes. In a further perspective these findings may prove to be useful in the development of new GBM treatment approaches. © 2006 Cancer Research UK.

TITLE:Expression and Activity of Transglutaminase II in Spontaneous Tumours of Dogs and Cats 

[2-s2.0-33645861961] Refers [2-s2.0-0038326942] LEVEL: 3
Tissue transglutaminase II (TGase II) is a dual function protein with both transamidating and guanidine triphosphate (GTP)-binding capabilities. Previous studies have implicated TGase as a pro-apoptotic molecule; however, our recent findings indicate that TGase II may act as a survival factor in various cell types. The purpose of this study was to survey TGase II expression in normal tissue and spontaneous tumours of dogs and cats, by Western blotting and immunohistochemistry. Bladder, liver and adrenal gland exhibited prominent expression of TGase II while other tissues, including mammary gland, displayed limited expression and activity. TGase II GTP-binding in normal tissues was proportional to the level of expression in all tissues examined. Normal mammary tissue and that showing benign hyperplasia did not express TGase II. However, 11/25 (44%) of canine mammary carcinomas and 10/12 (83%) of feline mammary carcinomas strongly expressed TGase II in either a stromal, cellular or combined pattern. The pattern of expression was not related to the classification of mammary carcinoma (solid, tubulopapillary, complex or anaplastic), except that two anaplastic canine mammary carcinomas showed prominent TGase II expression. Two canine mammary carcinoma cell lines showed prominent TGase expression, and when the TGase activity was inhibited, the cells became more sensitive to doxorubicin-induced cell death. Thus, TGase II was significantly expressed in mammary cancers from dogs and cats and immunoreactivity of TGase II was similar to that reported in humans beings. The pro-survival effect of TGase II in canine mammary carcinoma cell lines was similar to that previously reported in humans patients. © 2005 Elsevier Ltd. All rights reserved.

TITLE:Pharmacological modulation of sarcoplasmic reticulum function in smooth muscle 

[2-s2.0-10644264370] Refers [2-s2.0-0038519661] LEVEL: 3
The sarco/endoplasmic reticulum (SR/EM) is the primary storage and release site of intracellular calcium. (Ca2+) in many excitable cells. The SR is a tabular network, which in smooth muscle (SM) cells distributes close to cellular periphery (superficial SR) and in deeper aspects of the cell (deep SR). Recent attention has focused on the regulation of cell function by the superficial SR, which can act as a buffer and also as a regulator of membrane channels and transporters. Ca2+ is released from the SR via two types of ionic channels [ryanodine- and inositol 1,4,5-trisphosphate-gated], whereas accumulation from the cytoplasm occurs exclusively by an energy-dependent sarco-endoplasmic reticulum Ca2+-ATPase pump (SERCA). Within the SR, Ca2+ is bound to various storage proteins. Emerging evidence also suggests that the perinuclear portion of the SR may play an important role in nuclear transcription. In this review, we detail the pharmacology of agents that alter the functions of Ca2+ release channels and of SERCA. We describe their use and selectivity and indicate the concentrations used in investigating various SM preparations. Important aspects of cell regulation and excitation-contractile activity coupling in SM have been uncovered through the use of such activators and inhibitors of processes that determine SR function. Likewise, they were instrumental in the recent finding of an interaction of the SR with other cellular organelles such as mitochondria. Thus, an appreciation of the pharmacology and selectivity of agents that interfere with SR function in SM has greatly assisted in unveiling the multifaceted nature of the SR.

TITLE:Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte. Induction of [Ca2+]i oscillations and cyclical transitions in flagellar beating 

[2-s2.0-8544221933] Refers [2-s2.0-0038519661] LEVEL: 3
Progesterone is present at micromolar concentrations in the cumulus matrix, which surrounds mammalian oocytes. Exposure of human spermatozoa to a concentration gradient of progesterone (0-3 ?M) to simulate approach to the oocyte induced a slowly developing increase in [Ca2+] upon which, in many cells, slow oscillations were superimposed. [Ca2+]i oscillations often started at very low progesterone (<10 nM), and their frequency did not change during the subsequent rise in concentration. Oscillations also occurred, but in a much smaller proportion of cells, in response to stepped application of progesterone (3 ?M). When progesterone was removed, [Ca2+]i oscillations often persisted or quickly resumed. Superfusion with low-Ca2+ bathing medium (no added Ca 2+) did not prevent [Ca2+]i oscillations, but they could be abolished by addition of EGTA or La3+. Inhibitors of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases or inositol trisphosphate signaling had no effect on [Ca2+]i oscillations, but pharmacological manipulation of ryanodine receptors affected both their frequency and amplitude. Staining of live spermatozoa with BODIPY FL-X ryanodine showed localization of ryanodine binding primarily to the caudal part of the head and mid-piece. [Ca2+]i oscillations did not induce acrosome reaction, but in cells generating oscillations, the flagellar beat mode alternated in synchrony with the oscillation cycle. Flagellar bending and lateral movement of the sperm head during [Ca 2+]i peaks were markedly increased compared with during [Ca2+]i troughs. This alternating pattern of activity is likely to facilitate zona penetration. These observations show that progesterone initiates unusual and complex store-mediated [Ca2+]i signaling in human spermatozoa and identify a previously unrecognized effect of progesterone in regulating sperm "behavior" during fertilization.

TITLE:Null mutation in the gene encoding plasma membrane Ca2+-ATPase isoform 2 impairs calcium transport into milk 

[2-s2.0-5644246145] Refers [2-s2.0-0038519661] LEVEL: 3
The means by which calcium is transported into the milk produced by mammary glands is a poorly understood process. One hypothesis is that it occurs during exocytosis of secretory products via the Golgi pathway, consistent with the observation that the SPCA1 Ca2+-ATPase, which is expressed in the Golgi, is induced in lactating mammary tissue. However, massive up-regulation of the PHCA2bw plasma membrane Ca2+-ATPase also occurs during lactation and is more strongly correlated with increases in milk calcium, suggesting that calcium may be secreted directly via this pump. To examine the physiological role of PHCA2bw in lactation we compared lactating PHCA2-null mice to heterozygous and wild-type mice. Relative expression levels of individual milk proteins were unaffected by genotype. However, milk from PMCA2-null mice had 60% less calcium than milk from heterozygous and wild-type mice, the total milk protein concentration was lower, and an indirect measure of milk production (litter weights) suggested that the PHCA2-null mice produce significantly less milk. In contrast, lactose was higher in milk from PMCA2-null mice during early lactation, but by day 12 of lactation there were no differences in milk lactose between the three genotypes. These data demonstrate that the activity of PMCA2bw is required for secretion of much of the calcium in milk. This major secretory function represents a novel biological role for the plasma membrane Ca 2+-ATPases, which are generally regarded as premier regulators of intracellular Ca2+.

TITLE:Calcium pump disorders of the skin 

[2-s2.0-7444270483] Refers [2-s2.0-0038519661] LEVEL: 3
The causes of Darier disease (DD) and Hailey-Hailey disease (HHD) have eluded clinicians and scientists for more than 60 years. DD is characterized by loss of adhesion between suprabasal epidermal cells associated with abnormal keratinization, while loss of epidermal cell-to-cell adhesion is predominant in HHD. The genes for both conditions have recently been identified using candidate positional cloning approaches. The gene for DD (ATP2A2) encodes a calcium transport ATPase of the sarco (endo)plasmic reticulum (SERCA2) Verboomen et al. [1992: Biochem J 286(Pt 2):591-595], while the gene for HHD (ATP2C1) codes for a secretory pathway for calcium and manganese transport ATPase of the Golgi apparatus (SPCA1) Hu et al. [2000: Nat Genet 24:61-65]. These results have provided completely new insights into the role of calcium and/or manganese in maintaining skin integrity. Although the precise disease mechanisms remain to be understood, these discoveries open a new field in research for the understanding and the treatment of these distressing disorders. © 2004 Wiley-Liss, Inc.

TITLE:Protein kinase C Ī“ and Īµ mediate positive inotropy in adult ventricular myocytes 

[2-s2.0-17844366317] Refers [2-s2.0-0038519661] LEVEL: 3
To examine cardiac contractile regulation and protein kinase C (PKC) translocation in parallel, the ? and ? isoforms of PKC were fused to green fluorescent protein (GFP) and expressed in adult rat ventricular myocytes maintained in short term culture. PKC-?-GFP and PKC-?-GFP were predominantly cytosolic until phorbol dibutyrate (PDBu) was introduced. PKC-?-GFP redistributed preferentially to perinuclear structures that co-localized with a Golgi marker, whereas PKC-?-GFP redistributed preferentially to the surface sarcolemma. Myocyte contractile function was assessed by monitoring twitch shortening with field stimulation at 0.5:Hz, 22:°C. In myocytes expressing PKC-?-GFP, PDBu caused a transient negative inotropic response followed by a robust and sustained positive inotropic response that paralleled perinuclear PKC-? accumulation. In PKC-?-GFP myocytes, PDBu caused a sustained negative inotropic response that paralleled accumulation at the surface sarcolemma, but this response did not differ from myocytes expressing GFP alone. At higher expression levels, PKC-?-GFP myocytes responded more like PKC-?-GFP myocytes including perinuclear accumulation and a sustained positive inotropic response. Positive inotropic responses were markedly attenuated if PKC translocation was biased toward the surface sarcolemma by use of a more hydrophobic PKC activator, and were completely and selectively blocked by the PKC antagonist bis-indoylmaleimide. In contrast, transient and sustained negative inotropic responses were selectively blocked by the Ca2+-dependent PKC isoform antagonist Go6976. The data indicate that the novel PKC isoforms ? and ? have little effect on contractility when accumulating at the cell surface, but produce a strong positive inotropic response upon accumulation at the Golgi or other intracellular sites. © 2005 Elsevier Ltd. All rights reserved.

TITLE:Calcium pumps and keratinocytes: Lessons from Darier's disease and Hailey-Hailey disease 

[2-s2.0-3042823935] Refers [2-s2.0-0038519661] LEVEL: 3
Darier's disease and Hailey-Hailey disease are autosomal dominantly inherited skin disorders in which desmosomal adhesion between keratinocytes is abnormal. ATP2A2 and ATP2C1 have been identified as the causative genes for Darier's disease and Hailey-Hailey disease, respectively. ATP2A2 encodes the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) pump, while ATP2C1 encodes a secretory pathway Ca2+/Mn2+-ATPase (SPCA1) found in the Golgi apparatus. We review recent work into the function of these pumps in human keratinocytes and discuss how mutations in these genes might cause these diseases by altering the formation or stability of desmosomes.

TITLE:Calcium signalling in human spermatozoa: A specialized 'toolkit' of channels, transporters and stores 

[2-s2.0-33645837651] Refers [2-s2.0-0038519661] LEVEL: 3
Ca2+ is a ubiquitous intracellular messenger which encodes information by temporal and spatial patterns of concentration. In spermatozoa, several key functions, including acrosome reaction and motility, are regulated by cytoplasmic Ca2+ concentration. Despite the very small size and apparent structural simplicity of spermatozoa, evidence is accumulating that they possess sophisticated mechanisms for regulation of cytoplasmic Ca2+ concentration and generation of complex Ca2+ signals. In this review, we consider the various components of the Ca2+-signalling 'toolkit' that have been characterized in somatic cells and summarize the evidence for their presence and activity in spermatozoa. In particular, data accumulated over the last few years show that spermatozoa possess one (and probably two) Ca2+ stores as well as a range of plasma membrane pumps and channels. Selective regulation of the various components of the 'toolkit' by agonists probably allows spermatozoa to generate localized Ca2+ signals despite their very small cytoplasmic volume, permitting the discrete and selective activation of cell functions. © 2006 Oxford University Press.

TITLE:Packing interactions between transmembrane helices alter ion selectivity of the yeast Golgi Ca2+/Mn2+-ATPase PMR1 

[2-s2.0-0042818078] Refers [2-s2.0-0038519661] LEVEL: 3
PMR1 is the yeast secretory pathway pump responsible for high affinity transport of Mn2+ and Ca2+ into the Golgi, where these ions are sequestered and effectively removed from the cytoplasm. Phenotypic growth assays allow for convenient screening of side chains important for Ca2+ and Mn2+ transport. Earlier we demonstrated that mutant Q783A at the cytoplasmic interface of M6 could transport Ca2+ but not Mn2+. Scanning mutagenesis of side chains proximal to residue Gln-783 in membrane helices M2, M4, M5, and M6 revealed additional residues near the cytoplasmic interface, notably Leu-341 (M5), Phe-738 (M5), and Leu-785 (M6) that are sensitive to substitution. Importantly, we obtained evidence for a packing interaction between Val-335 in M4 and Gln-783 in M6 that is critical for Mn2+ transport. Thus, mutant V335G mimics the Mn 2+ transport defect of Q783A and mutant V335I can effectively suppress the Mn2+-defective phenotype of Q783A. These changes in ion selectivity were confirmed by cation-dependent ATP hydrolysis using purified enzyme. Other substitutions at these sites are tolerated individually, but not in combination. Exchange of side chains at 335 and 783 also results in ion selectivity defects, suggesting that the packing interaction may be conformation-sensitive. Homology models of M4, M5, and M6 of PMR1 have been generated, based on the structures of the sarcoplasmic reticulum Ca 2+-ATPase. The models are supported by data from mutagenesis and reveal that Gln-783 and Val335 show conformation-sensitive packing at the cytoplasmic interface. We suggest that this region may constitute a gate for access of Mn2+ ions.

TITLE:Hailey-Hailey disease as an orthodisease of PMR1 deficiency in Saccharomyces cerevisiae 

[2-s2.0-16344374273] Refers [2-s2.0-0038519661] LEVEL: 3
The term orthodisease has recently been introduced to define human disorders in which the pathogenic gene has orthologs in model organism genomes. Here, we describe Hailey-Hailey disease (HHD), a blistering skin disorder caused by haploinsufficiency of ATP2C1 as an orthodisease from a Saccharomyces cerevisiae perspective. ATP2C1 encodes the human secretory pathway Ca 2+/Mn2+ ATPase hSPCA1 and is orthologous to the PMR1 gene in S. cerevisiae. hSPCA1 fully complements PMR1 deficiency in yeast and pmr1?S. cerevisiae has proved to be a valuable tool to screen ATP2C1 mutations and address potential pathogenic/pharmacologic mechanisms in HHD. Consequently, this human skin disorder is an ideal example of an orthodisease. © 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

TITLE:Characteristics of prolonged Ca2+ release events associated with the nuclei in adult cardiac myocytes 

[2-s2.0-11844269174] Refers [2-s2.0-0038519661] LEVEL: 3
Confocal microscopy was used to study the properties of nuclear Ca 2+ regulation in adult ventricular myocytes. Prolonged nuclear Ca2+ release (PNCR) events were identified in both intact and permeabilized rat myocytes. PNCR occurred spontaneously and was restricted to localized regions at the ends of the elongated nuclei. Typically, PNCR took the form of a rapid rise in [Ca2+] followed by a maintained plateau. The mean duration of PNCR (1.78±0.19 seconds) was markedly greater than the half decay time for cytosolic Ca2+ sparks (31.2±0.56 ms) obtained under the same conditions. The PNCR width at half maximum amplitude (5.0±0.2 ?m) was also significantly greater than that of cytosolic Ca2+ sparks (2.6±0.05 ?m) obtained under the same conditions. Experiments involving the use of syto-11 to accurately locate the nuclei demonstrated that PNCR originates from the nuclear envelope or a closely associated structure. The spatial spread of PNCR was asymmetrical, with greater diffusion of Ca2+ toward the center of the nucleus than the cytosol. Both PNCR and Ca2+ sparks were abolished by interventions that deplete SR Ca2+ stores or inhibit RYR activation. Experiments on intact, electrically stimulated cells revealed that diffusion of Ca2+ from the ends of the nucleus toward the center is a prominent feature of the nucleoplasmic Ca2+ transient. The possibility that recruitment of Ca2+ release sites involved in PNCR might influence the temporal and spatial characteristics of the nucleoplasmic [Ca2+] transient is considered.

TITLE:Calcium transport and signaling in the mammary gland: Targets for breast cancer 

[2-s2.0-33646121976] Refers [2-s2.0-0038519661] LEVEL: 3
The mammary gland is subjected to extensive calcium loads during lactation to support the requirements of milk calcium enrichment. Despite the indispensable nature of calcium homeostasis and signaling in regulating numerous biological functions, the mechanisms by which systemic calcium is transported into milk by the mammary gland are far from completely understood. Furthermore, the implications of calcium signaling in terms of regulating proliferation, differentiation and apoptosis in the breast are currently uncertain. Deregulation of calcium homeostasis and signaling is associated with mammary gland pathophysiology and as such, calcium transporters, channels and binding proteins represent potential drug targets for the treatment of breast cancer. © 2005 Elsevier B.V. All rights reserved.

TITLE:Calcium secretion into milk 

[2-s2.0-22744458789] Refers [2-s2.0-0038519661] LEVEL: 3
Ionized calcium ([Ca2+]) is present in milk at concentrations around 3 mM, a concentration that drives the formation of complexes with citrate, phosphate, and casein, thereby generating compounds that carry the major portion of calcium in milk. In humans and cows, where it has been studied, changes in milk calcium appear to be regulated by the amount of citrate and casein in milk rather than changes in [Ca2+]. Most or all of the calcium in milk is likely derived through exocytosis of secretory vesicles derived from the Golgi compartment where a calcium ATPase mediates transport from the cytoplasm. The identity of the transporters is not yet certain but gene expression for the plasma membrane calcium ATPase, PMCA2bw, and the secretory pathway calcium ATPase, SPCA, is highly upregulated during lactation. Currently nothing appears to be known about the mechanisms that mediate transport of calcium across the basolateral membrane of the alveolar cell. © Springer Science + Business Media, Inc. 2005.

TITLE:Identification of new Golgi complex specific proteins by direct organelle proteomic analysis 

[2-s2.0-33745711870] Refers [2-s2.0-0038519661] LEVEL: 3
The Golgi complex is in the crossroad of the endocytic and secretory pathways. Its function is to post-translationally modify and sort proteins and lipids, and regulate the membrane balance in the cell. To understand the structure-function relationship of the Golgi complex the Golgi proteome has to be identified first. We have used a direct organelle proteomic analysis to identify new Golgi complex proteins. Enriched stacked Golgi membrane fractions from rat livers were isolated, and the proteins from these membranes were subsequently digested into peptides. The peptides were fractionated by cation-exchange chromatography followed by protein identification by automated capillary-LC/ESI-MS/MS analysis and database searches. Two different search programs, ProID and MASCOT were used. This resulted in a total of 1125 protein identifications in two experiments. In addition to the known Golgi resident proteins, a significant number of unknown proteins were identified. Some of these were further characterized in silica using different programs to provide insight into their structure, intracellular localization and biological functions. The Golgi localization of two of these newly identified proteins was also confirmed by indirect immunofluorescence. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

TITLE:Cell death suppressor arabidopsis bax inhibitor-1 is associated with calmodulin binding and ion homeostasis 

[2-s2.0-34247245285] Refers [2-s2.0-0038519661] LEVEL: 3
Cell death suppressor Bax inhibitor-1 (BI-1), an endoplasmic reticulum membrane protein, exists in a wide range of organisms. The split-ubiquitin system, overlay assay, and bimolecular fluorescence complementation analysis demonstrated that Arabidopsis (Arabidopsis thaliana) BI-1 (AtBI-1) interacted with calmodulin in yeast (Saccharomyces cerevisiae) and in plant cells. Furthermore, AtBI-1 failed to rescue yeast mutants lacking Ca2+ ATPase (Pmr1 or Spf1) from Bax-induced cell death. Pmr1 and Spf1, p-type ATPases localized at the inner membrane, are believed to be involved in transmembrane movement of calcium ions in yeast. Thus, the presence of intact Ca2+ ATPases was essential for AtBI-1-mediated cell death suppression in yeast. To investigate the effect of AtBI-1 on calcium homeostasis, we evaluated sensitivity against cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase in AtBI-1-overexpressing or knock-down transgenic Arabidopsis plants. These plants demonstrated altered CPA or ion stress sensitivity. Furthermore, AtBI-1-overexpressing cells demonstrated an attenuated rise in cytosolic calcium following CPA or H2O2 treatment, suggesting that AtBI-1 affects ion homeostasis in plant cell death regulation. © 2006 American Society of Plant Biologists.

TITLE:Autosomal-dominant calcium ATPase disorders 

[2-s2.0-33750062919] Refers [2-s2.0-0038519661] LEVEL: 3
Darier disease (DD) and Hailey-Hailey disease (HHD) are the only known autosomal-dominant Ca2+ ATPase disorders. Epidermal symptoms selectively occur in the affected individuals, the precise reason for which is still not fully understood. Here, we review the clinical, epidermal, and molecular features of the two genodermatoses. It is concluded that epidermal Ca2+ regulation disturbances and epigenetic factors may play an even more prominent role in the pathogenesis of DD and HHD than earlier appreciated. © 2006 The Society for Investigative Dermatology.

TITLE:Novel subcellular locations and functions for secretory pathway Ca 2+/Mn2+-ATPases 

[2-s2.0-33745675350] Refers [2-s2.0-0038519661] LEVEL: 3
Secretory pathway Ca2+/Mn2+-ATPases (SPCAs) are important for maintenance of cellular Ca2+ and Mn2+ homeostasis, and, to date, all SPCAs have been found to localize to the Golgi apparatus. The single Drosophila SPCA gene (SPoCk) was identified by an in silico screen for novel Ca2+-ATPases. It encoded three SPoCk isoforms with novel, distinct subcellular specificities in the endoplasmic reticulum (ER) and peroxisomes in addition to the Golgi. Furthermore, expression of the peroxisome-associated SPoCk isoform was sexually dimorphic. Overexpression of organelle-specific SPoCk isoforms impacted on cytosolic Ca2+ handling in both cultured Drosophila cells and a transporting epithelium, the Drosophila Malpighian (renal) tubule. Specifically, the ER isoform impacted on inositol (1,4,5)-trisphosphate-mediated Ca2+ signaling and the Golgi isoform impacted on diuresis, whereas the peroxisome isoform colocalized with Ca 2+ "spherites" and impacted on calcium storage and transport. Interfering RNA directed against the common exons of the three SPoCk isoforms resulted in aberrant Ca2+ signaling and abolished neuropeptide-stimulated diuresis by the tubule. SPoCk thus contributed to both of the contrasting requirements for Ca2+ in transporting epithelia: to transport or store Ca2+ in bulk without compromising its use as a signal. Copyright © 2006 the American Physiological Society.

TITLE:Calcium signalling and reactive oxygen species in non-excitable cells 

[2-s2.0-33646579530] Refers [2-s2.0-0038519661] LEVEL: 3
Reactive oxygen species can induce several biological processes by stimulating signal transduction components such as cytosolic free calcium concentration. The physiological significance of the role of biological oxidants in the regulation of calcium signalling pathway as well as the mechanisms of the oxidant-stimulation of signal transduction are discussed in this review. © 2006 Bentham Science Publishers Ltd.

TITLE:The MLCK-mediated Ī±1-adrenergic inotropic effect in atrial myocardium is negatively modulated by PKCĪµ signaling 

[2-s2.0-33749353241] Refers [2-s2.0-0038519661] LEVEL: 3
The present study examined the role of myosin light chain kinase (MLCK), PKC isozymes, and inositol 1,4,5-trisphosphate (IP3) receptor in the positive inotropic effect of ?1-adrenergic stimulation in atrial myocardium. We measured inotropic effects of phenylephrine (0.3-300 ?M) in isolated left atrial preparations (1 Hz, 37°C, 1.8 mM Ca 2+, 0.3 ?M nadolol) from male 8-week FVB mice (n = 200). Phenylephrine concentration-dependently increased force of contraction from 1.5 ± 0.1 to 2.8 ± 0.1 mN (mean ± s.e.m., n = 42), which was associated with increased MLC-2a phosphorylation at serine 21 and 22 by 67% and translocation of PKC? but not PKC? to membrane (+30%) and myofilament (+50%) fractions. MLCK inhibition using ML-7 or wortmannin right-shifted the concentration-response curve of phenylephrine, reducing its inotropic effect at 10 ?M by 73% and 81%, respectively. The compound KIE1-1 (500 nM), an intracellularly acting PKC? translocation inhibitor peptide, prevented PKC? translocation and augmented the maximal inotropic effect of phenylephrine by 40%. In contrast, inhibition of Ca2+-dependent PKC translocation (KIC1-1, 500 nM) had no effect. Chelerythrine, a PKC inhibitor, decreased basal force without changing the inotropic effect of phenylephrine. The IP3 receptor blocker 2-APB (2 and 20 ?M) concentration- dependently decreased basal force, but did not affect the concentration-response curve of phenylephrine. These results indicate that activation of MLCK is required for the positive inotropic effect of ?1-adrenergic stimulation, that the Ca2+-independent PKC? negatively modulates this effect, and that PKC? and IP3 receptor activation is not involved. © 2006 Nature Publishing Group. All rights reserved.

TITLE:A PMR1-like calcium ATPase of Aspergillus fumigatus: Cloning, identification and functional expression in S. cerevisiae 

[2-s2.0-23944489892] Refers [2-s2.0-0038519661] LEVEL: 3
The understanding of the controlling factors of calcium homeostasis in Aspergillus fumigatus is very poor, although this ion is involved in several important events of these particular cells. We have cloned, identified and expressed for functional complementation a PMR1-like Ca2+-ATPase gene from A. fumigatus. The Afpmr1 gene encodes a protein of 1061 deduced amino acids, containing all the conserved subdomains found in other P-type ATPases: the phosphatase region, phosphorylation site, FITC labelling site, ATP binding domain; E386, N871, D875 amino acid residues for calcium ion interaction and Q880, a residue that alters ion selectivity in PMR1. The expressed AfPMR1 in S. cerevisiae K616 strain functionally complemented the deficient growth in EGTA (5-20 mM)- and MnCl2 (4 mM)-containing medium. These results demonstrate the first evidence of a Ca2+-ATPase in A. fumigatus and strongly suggest a role for this enzyme in calcium and manganese homeostasis. Copyright © 2005 John Wiley & Sons, Ltd.

TITLE:PfluĢˆgers Archiv and the advent of modern electrophysiology: From the first action potential to patch clamp 

[2-s2.0-0442292064] Refers [2-s2.0-0038519661] LEVEL: 3
This short review recollects the many essential milestones in electrophysiology that were published in Pflu?gers Archiv. These involve the first measurement of an action potential by J. Bernstein, the requirement of Na+ for the generation of excitation, the prediction of a lipoid membrane surrounding cells by E. Overton, the physical explanation of the resting membrane potential by J. Bernstein, the first detailed description of the conductance properties of excitable tissues by L. Hermann, and more recently the publication of the patch-clamp method by E. Neher and B. Sakmann.

TITLE:The calcium-sensing receptor regulates plasma membrane calcium adenosine triphosphatase isoform 2 activity in mammary epithelial cells: A mechanism for calcium-regulated calcium transport into milk 

[2-s2.0-36348944695] Refers [2-s2.0-0038519661] LEVEL: 3
The calcium-sensing receptor (CaR) regulates transepithelial calcium transport into milk by mammary epithelial cells. Using a genome-wide screening strategy, we identified the plasma membrane calcium ATPase isoform 2 (PMCA2) as a potential downstream target of the CaR. We show that PMCA2 expression in the mouse mammary gland increases during lactation and that PMCA2 is localized solely to the apical plasma membrane of mammary epithelial cells. In milk from deafwaddler mice, which have mutations in the gene encoding PMCA2, calcium concentrations were reduced, confirming its importance in calcium transport into milk. Furthermore, in cultured primary and EpH4 mouse mammary epithelial cells, CaR stimulation up-regulated calcium-dependent ATPase activity in plasma membrane preparations. By small interfering RNA-mediated gene knockdown of PMCA2, we show that PMCA2 accounts for the preponderance of calcium-ATPase activity. We also show that reduction of CaR expression with small interfering RNA eliminates the ability of extracellular calcium to elicit an increase in calcium-dependent ATPase activity in EpH4 cell membranes. These results demonstrate that activation of the CaR increases PMCA2 activity in mouse mammary epithelial cells, providing a mechanism for the regulation of transepithelial calcium transport by calcium in the lactating mouse mammary gland. Copyright © 2007 by The Endocrine Society.

TITLE:Calcium gradients and the Golgi 

[2-s2.0-33750499961] Refers [2-s2.0-0038519661] LEVEL: 3
Changes in intracellular free calcium regulate many intracellular processes. With respect to the secretory pathway and the Golgi apparatus, changes in calcium concentration occurring either in the adjacent cytosol or within the lumen of the Golgi act to regulate Golgi function. Conversely, the Golgi sequesters calcium to shape cytosolic calcium signals as well as initiate them by releasing calcium via inositol-1,4,5-triphosphate (IP3) receptors, located on Golgi membranes. Local calcium transients juxtaposed to the Golgi (arising from release by the Golgi or other organelles) can activate calcium dependent signalling molecules located on or around the Golgi. This review focuses on the reciprocal relationship between the cell biology of the Golgi apparatus and intracellular calcium homeostasis. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Importance of calcium 

[2-s2.0-24344439773] Refers [2-s2.0-0038519661] LEVEL: 3
Calcium is the most abundant mineral in the body. Calcium regulates many cellular processes and has important structural roles in living organisms. Skeletal muscle structure and function, polymerisation of fibrin and the conduction of impulses in the nervous system are regulated by calcium. Calcium is an important intracellular messenger in protozoa, plants, and animals. Calcium-transporting systems which are located in the plasma membrane and in the organelles, regulate the ionic concentration of calcium in various compartments according to the different demands of the physiological cycle and these systems upregulate calcium entry by the action of several hormones and calcium binding proteins. Opening of calcium influx channels increases the cytosolic calcium concentrations but high calcium concentrations are toxic to the cell. Because of this toxicity; calcium is rapidly removed from the cytosol by calcium pumps and exchangers. Changes in cytosolic calcium concentrations cause a wide range of cellular responses. Cellular calcium is known to play an important role in apoptosis and the accumulation of calcium can induce various apoptotic pathways in the cell. Maintenance of the cellular calcium homeostasis has various benefits for human health and the deficiency of calcium causes many pathological conditions. © Tu?bi?tak.

TITLE:Molecular Chaperones and Protein Quality Control 

[2-s2.0-33646127577] Refers [2-s2.0-23744457478] LEVEL: 3
In living cells, both newly made and preexisting polypeptide chains are at constant risk for misfolding and aggregation. In accordance with the wide diversity of misfolded forms, elaborate quality-control strategies have evolved to counter these inevitable mishaps. Recent reports describe the removal of aggregates from the cytosol; reveal mechanisms for protein quality control in the endoplasmic reticulum; and provide new insight into two classes of molecular chaperones, the Hsp70 system and the AAA+ (Hsp100) unfoldases. © 2006 Elsevier Inc. All rights reserved.

TITLE:Cotranslocational Degradation Protects the Stressed Endoplasmic Reticulum from Protein Overload 

[2-s2.0-33747175431] Refers [2-s2.0-23744457478] LEVEL: 3
The ER's capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58IPK/DNAJC3, a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58IPK recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58IPK-/- mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58IPK is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells. © 2006 Elsevier Inc. All rights reserved.

TITLE:Protein disulfide isomerase: the structure of oxidative folding 

[2-s2.0-33746298040] Refers [2-s2.0-23744457478] LEVEL: 3
Cellular functions hinge on the ability of proteins to adopt their correct folds, and misfolded proteins can lead to disease. Here, we focus on the proteins that catalyze disulfide bond formation, a step in the oxidative folding pathway that takes place in specialized cellular compartments. In the endoplasmic reticulum of eukaryotes, disulfide formation is catalyzed by protein disulfide isomerase (PDI); by contrast, prokaryotes produce a family of disulfide bond (Dsb) proteins, which together achieve an equivalent outcome in the bacterial periplasm. The recent crystal structure of yeast PDI has increased our understanding of the function and mechanism of PDI. Comparison of the structure of yeast PDI with those of bacterial DsbC and DsbG reveals some similarities but also striking differences that suggest directions for future research aimed at unraveling the catalytic mechanism of disulfide bond formation in the cell. © 2006 Elsevier Ltd. All rights reserved.

TITLE:Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response 

[2-s2.0-33845480131] Refers [2-s2.0-23744457478] LEVEL: 3
The protein folding capacity of the endoplasmic reticulum (ER) is regulated by the unfolded protein response (UPR). The UPR senses unfolded proteins in the ER lumen and transmits that information to the cell nucleus, where it drives a transcriptional program that is tailored to re-establish homeostasis. Using thin section electron microscopy, we found that yeast cells expand their ER volume at least 5-fold under UPR-inducing conditions. Surprisingly, we discovered that ER proliferation is accompanied by the formation of autophagosome-like structures that are densely and selectively packed with membrane stacks derived from the UPR-expanded ER. In analogy to pexophagy and mitophagy, which are autophagic processes that selectively sequester and degrade peroxisomes and mitochondria, the ER-specific autophagic process described utilizes several autophagy genes: they are induced by the UPR and are essential for the survival of cells subjected to severe ER stress. Intriguingly, cell survival does not require vacuolar proteases, indicating that ER sequestration into autophagosome-like structures, rather than their degradation, is the important step. Selective ER sequestration may help cells to maintain a new steady-state level of ER abundance even in the face of continuously accumulating unfolded proteins. © 2006 Bernales et al.

TITLE:The cytoplasmic Hsp70 chaperone machinery subjects misfolded and endoplasmic reticulum import-incompetent proteins to degradation via the ubiquitin-proteasome system 

[2-s2.0-33846107847] Refers [2-s2.0-23744457478] LEVEL: 3
The mechanism of protein quality control and elimination of misfolded proteins in the cytoplasm is poorly understood. We studied the involvement of cytoplasmic factors required for degradation of two endoplasmic reticulum (ER)-import-defective mutated derivatives of carboxypeptidase yscY (?ssCPY* and ?ssCPY*-GFP) and also examined the requirements for degradation of the corresponding wild-type enzyme made ER-import incompetent by removal of its signal sequence (?ssCPY). All these protein species are rapidly degraded via the ubiquitin-proteasome system. Degradation requires the ubiquitin-conjugating enzymes Ubc4p and Ubc5p, the cytoplasmic Hsp70 Ssa chaperone machinery, and the Hsp70 cochaperone Ydj1p. Neither the Hsp90 chaperones nor Hsp104 or the small heat-shock proteins Hsp26 and Hsp42 are involved in the degradation process. Elimination of a GFP fusion (GFP-cODC), containing the C-terminal 37 amino acids of ornithine decarboxylase (cODC) directing this enzyme to the proteasome, is independent of Ssalp function. Fusion of ?ssCPY* to GFP-cODC to form ?ssCPY*- GFP-cODC reimposes a dependency on the Ssa1p chaperone for degradation. Evidently, the misfolded protein domain dictates the route of protein elimination. These data and our further results give evidence that the Ssa1p-Ydj1p machinery recognizes misfolded protein domains, keeps misfolded proteins soluble, solubilizes precipitated protein material, and escorts and delivers misfolded proteins in the ubiquitinated state to the proteasome for degradation. © 2006 by The American Society for Cell Biology.

TITLE:Generating disulfides in multicellular organisms: Emerging roles for a new flavoprotein family 

[2-s2.0-34347204504] Refers [2-s2.0-23744457478] LEVEL: 3
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TITLE:Coordinate regulation of phospholipid biosynthesis and secretory pathway gene expression in XBP-1(S)-induced endoplasmic reticulum biogenesis 

[2-s2.0-34147172715] Refers [2-s2.0-23744457478] LEVEL: 3
Development of the expansive endoplasmic reticulum (ER) present in specialized secretory cell types requires X-box-binding protein-1 (Xbp-1). Enforced expression of XBP-1(S), a transcriptional activator generated by unfolded protein response-mediated splicing of Xbp-1 mRNA, is sufficient to induce proliferation of rough ER. We previously showed that XBP-1(S)-induced ER biogenesis in fibroblasts correlates with increased production of phosphatidylcholine (PtdCho), the primary phospholipid of the ER membrane, and enhanced activities of the choline cytidylyltransferase (CCT) and cholinephosphotransferase enzymes in the cytidine diphosphocholine (CDP-choline) pathway of PtdCho biosynthesis. Here, we report that the level and synthesis of CCT, the rate-limiting enzyme in the CDP-choline pathway, is elevated in fibroblasts overexpressing XBP-1(S). Furthermore, overexpression experiments demonstrated that raising the activity of CCT, but not cholinephosphotransferase, is sufficient to augment PtdCho biosynthesis in fibroblasts, indicating that XBP-1(S) increases the output of the CDP-choline pathway primarily via its effects on CCT. Finally, fibroblasts overexpressing CCT up-regulated PtdCho synthesis to a level similar to that in XBP-1(S)-transduced cells but exhibited only a small increase in rough ER and no induction of secretory pathway genes. The more robust XBP-1(S)-induced ER expansion was accompanied by induction of a wide array of genes encoding proteins that function either in the ER or at other steps in the secretory pathway. We propose that XBP-1(S) regulates ER abundance by coordinately increasing the supply of membrane phospholipids and ER proteins, the key ingredients for ER biogenesis. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:The protective and destructive roles played by molecular chaperones during ERAD (endoplasmic-reticulum-associated degradation) 

[2-s2.0-34250745700] Refers [2-s2.0-23744457478] LEVEL: 3
Over one-third of all newly synthesized polypeptides in eukaryotes interact with or insert into the membrane or the lumenal space of the ER (endoplasmic reticulum), an event that is essential for the subsequent folding, post-translational modification, assembly and targeting of these proteins. Consequently, the ER houses a large number of factors that catalyse protein maturation, but, in the event that maturation is aborted or inefficient, the resulting aberrant proteins may be selected for ERAD (ER-associated degradation). Many of the factors that augment protein biogenesis in the ER and that mediate ERAD substrate selection are molecular chaperones, some of which are heat- and/or stress-inducible and are thus known as Hsps (heat-shock proteins). But, regardless of whether they are constitutively expressed or are inducible, it has been assumed that all molecular chaperones function identically. As presented in this review, this assumption may be false. Instead, a growing body of evidence suggests that a chaperone might be involved in either folding or degrading a given substrate that transits through the ER. A deeper appreciation of this fact is critical because (i) the destruction of some ERAD substrates results in specific diseases, and (ii) altered ERAD efficiency might predispose individuals to metabolic disorders. Moreover, a growing number of chaperone-modulating drugs are being developed to treat maladies that arise from the synthesis of a unique mutant protein; therefore it is critical to understand how altering the activity of a single chaperone will affect the quality control of other nascent proteins that enter the ER. © 2007 Biochemical Society.

TITLE:Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells 

[2-s2.0-33646238977] Refers [2-s2.0-23744457478] LEVEL: 3
The folding environment in the endoplasmic reticulum (ER) depends on multiple abundant chaperones that function together to accommodate a range of substrates. The ways in which substrate engagement shapes either specific chaperone dynamics or general ER attributes in vivo remain unknown. In this study, we have evaluated how changes in substrate flux through the ER influence the diffusion of both the lectin chaperone calreticulin and an inert reporter of ER crowdedness. During acute changes in substrate load, the inert probe revealed no changes in ER organization, despite significant changes in calreticulin dynamics. By contrast, inhibition of the lectin chaperone system caused rapid changes in the ER environment that could be reversed over time by easing new substrate burden. Our findings provide insight into the normal organization and dynamics of an ER chaperone and characterize the capacity of the ER to maintain homeostasis during acute changes in chaperone activity and availability. © 2006 by The National Academy of Sciences of the USA.

TITLE:The unfolded protein response of B-lymphocytes: PERK-independent development of antibody-secreting cells 

[2-s2.0-35548935675] Refers [2-s2.0-23744457478] LEVEL: 3
When B-lymphocytes differentiate into plasma cells, immunoglobulin (Ig) heavy and light chain synthesis escalates and the entire secretory apparatus expands to support high-rate antibody secretion. These same events occur when murine B-cells are stimulated with lipopolysaccharide (LPS), providing an in vitro model in which to investigate the differentiation process. The unfolded protein response (UPR), a multi-pathway signaling response emanating from the endoplasmic reticulum (ER) membrane, allows cells to adapt to increasing demands on the protein folding capacity of the ER. As such, the UPR plays a pivotal role in the differentiation of antibody-secreting cells. Three specific stress sensors, IRE1, PERK/PEK and ATF6, are central to the recognition of ER stress and induction of the UPR. IRE1 triggers splicing of Xbp-1 mRNA, yielding a transcriptional activator of the UPR termed XBP-1(S), and activation of the IRE1/XBP-1 pathway has been reported to be required for expansion of the ER and antibody secretion. Here, we provide evidence that PERK is not activated in LPS-stimulated splenic B-cells, whereas XBP-1(S) and the UPR transcriptional activator ATF6 are both induced. We further demonstrate that Perk-/- B-cells develop and are fully competent for induction of Ig synthesis and antibody secretion when stimulated with LPS. These data provide clear evidence for differential activation and utilization of distinct UPR components as activated B-lymphocytes increase Ig synthesis and differentiate into specialized secretory cells. © 2007 Elsevier Ltd. All rights reserved.

TITLE:Disease-associated mutations affect GPR56 protein trafficking and cell surface expression 

[2-s2.0-34548390149] Refers [2-s2.0-23744457478] LEVEL: 3
Bilateral frontoparietal polymicrogyria (BFPP) is a congenital brain malformation resulting in irregularities on the surface of the cortex, where normally convoluted gyri are replaced by numerous (poly) and noticeably smaller (micro) gyri. Individuals with BFPP suffer from epilepsy, mental retardation, language impairment and motor developmental delay. Mutations in the gene-encoding G protein-coupled receptor 56 (GPR56) cause BFPP; however, it remains unclear how these mutations affect GPR56 function. Here, we examine the biochemical properties and protein trafficking of wild-type and mutant GPR56. We demonstrate that GPR56 protein undergoes two major modifications, GPS domain-mediated protein cleavage and N-glycosylation, and that the N-terminal fragment can be released from the cell surface. In contrast to the wild-type protein, disease-associated GPR56 missense mutations in the tip of the N-terminal domain (R38Q, R38W, Y88C and C91S) produce proteins with reduced intracellular trafficking and poor cell surface expression, whereas the two mutations in the GPS domain (C346S and W349S) produce proteins with dramatically impaired cleavage that fail to traffic beyond the endoplasmic reticulum. Cell-trafficking impairments are abrogated in part by pharmacological chaperones that can partially rescue mutant GPR56 cell surface expression. These data demonstrate that some BFPP-associated mutations in GPR56 impair trafficking of the mutant protein to the plasma membrane, thus providing insights into how BFPP-associated mutations affect GPR56 function. © The Author 2007. Published by Oxford University Press. All rights reserved.

TITLE:Oxidoreductase interactions include a role for ERp72 engagement with mutant thyroglobulin from the rdw/rdw rat dwarf 

[2-s2.0-34250372166] Refers [2-s2.0-23744457478] LEVEL: 3
Newly synthesized thyroglobulin (Tg), the secretory glycoprotein that serves as precursor in thyroid hormone synthesis, normally forms transient covalent protein complexes with oxidoreductases of the endoplasmic reticulum (ER). The Tg-G2320R mutation is responsible for congenital hypothyroidism in rdw/rdw rats, in which a lack of secondary thyroid enlargement (goiter) implicates death of thyrocytes as part of disease pathogenesis. We found that mutant Tg-G2320R was retained within the ER with no detectable synthesis of thyroxine, had persistent exposure of free cysteine thiols, and was associated with activated ER stress response but incomplete ER-associated degradation (ERAD). Tg-G2320R associated with multiple ER resident proteins, most notably ERp72, including covalent Tg-ERp72 interactions. In PC Cl3 thyrocytes, inducible overexpression of ERp72 increased the ability of cells to maintain Tg cysteines in a reduced state. Noncovalent interactions of several ER chaperones with newly synthesized Tg-G2320R diminished over time in parallel with ERAD of the mutant protein, yet a small ERAD-resistant Tg fraction remained engaged in covalent association with ERp72 even 2 days post-synthesis. Such covalent protein aggregates may set the stage for apoptotic thyrocyte cell death, preventing thyroid goiter formation in rdw/rdw rats. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:Translation attenuation by PERK balances ER glycoprotein synthesis with lipid-linked oligosaccharide flux 

[2-s2.0-33847339711] Refers [2-s2.0-23744457478] LEVEL: 3
Endoplasmic reticulum (ER) homeostasis requires transfer and subsequent processing of the glycan Glc3Man9GlcNAc2 (G3M9Gn2) from the lipid-linked oligosaccharide (LLO) glucose3mannose9N-acetylglucosamine 2-P-P-dolichol (G3M9Gn2-P-P-Dol) to asparaginyl residues of nascent glycoprotein precursor polypeptides. However, it is unclear how the ER is protected against dysfunction from abnormal accumulation of LLO intermediates and aberrant N-glycosylation, as occurs in certain metabolic diseases. In metazoans phosphorylation of eukaryotic initiation factor 2? (eIF2?) on Ser51 by PERK (PKR-like ER kinase), which is activated by ER stress, attenuates translation initiation. We use brief glucose deprivation to simulate LLO biosynthesis disorders, and show that attenuation of polypeptide synthesis by PERK promotes extension of LLO intermediates to G3M9Gn2-P-P-Dol under these substrate-limiting conditions, as well as counteract abnormal N-glycosylation. This simple mechanism requires eIF2? Ser51 phosphorylation by PERK, and is mimicked by agents that stimulate cytoplasmic stress-responsive Ser51 kinase activity. Thus, by sensing ER stress from defective glycosylation, PERK can restore ER homeostasis by balancing polypeptide synthesis with flux through the LLO pathway. © The Rockefeller University Press.

TITLE:Substrate-Specific Requirements for UGT1-Dependent Release from Calnexin 

[2-s2.0-34447339935] Refers [2-s2.0-23744457478] LEVEL: 3
Newly synthesized glycoproteins displaying monoglucosylated N-glycans bind to the endoplasmic reticulum (ER) chaperone calnexin, and their maturation is catalyzed by the calnexin-associated oxidoreductase ERp57. Folding substrates are eventually released from calnexin, and terminal glucoses are removed from N-glycans. The UDP-glucose:glycoprotein glucosyltransferase (UGT1, UGGT, GT) monitors the folding state of polypeptides released from calnexin and adds back a glucose residue on N-glycans of nonnative polypeptides, thereby prolonging retention in the calnexin chaperone system for additional folding attempts. Here we show that for certain newly synthesized glycoproteins UGT1 deletion has no effect on binding to calnexin. These proteins must normally complete their folding program in one binding event. Other proteins normally undergo multiple binding events, and UGT1 deletion results in their premature release from calnexin. For other proteins, UGT1 deletion substantially delays release from calnexin, unexpectedly showing that UGT1 activity might be required for a structural maturation needed for substrate dissociation from calnexin and export from the ER. © 2007 Elsevier Inc. All rights reserved.

TITLE:Conserved structural and functional properties of D-domain containing redox-active and -inactive protein disulfide isomerase-related protein chaperones 

[2-s2.0-34249681821] Refers [2-s2.0-23744457478] LEVEL: 3
The structure and mode of binding of the endoplasmic reticulum protein disulfide isomerase-related proteins to their substrates is currently a focus of intensive research. We have recently determined the crystal structure of the Drosophila melanogaster protein disulfide isomerase-related protein Wind and have described two essential substrate binding sites within the protein, one within the thioredoxin b-domain and another within the C-terminal D-domain. Although a mammalian ortholog of Wind (ERp29/28) is known, conflicting interpretations of its structure and putative function have been postulated. Here, we have provided evidence indicating that ERp29 is indeed similar in both structure and function to its Drosophila ortholog. Using a site-directed mutagenesis approach, we have demonstrated that homodimerization of the b-domains is significantly reduced in vitro upon replacement of key residues at the predicted dimerization interface. Investigation of Wind-ERp29 fusion constructs showed that mutants of the D-domain of ERp29 prevent transport of a substrate protein (Pipe) in a manner consistent with the presence of a discrete, conserved peptide binding site in the D-domain. Finally, we have high-lighted the general applicability of these findings by showing that the D-domain of a redox-active disulfide isomerase, from the slime mold Dictyostelium discoideum, can also functionally replace the Wind D-domain in vivo. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

TITLE:Structural design of cage and coat scaffolds that direct membrane traffic 

[2-s2.0-34147129193] Refers [2-s2.0-23744457478] LEVEL: 3
Trafficking within the exocytic and endocytic pathways of eukaryotic cells involves the generation of caged transport carriers that mediate communication between compartments through vesicle budding and fusion. Structural studies of vesicle cage structures using X-ray crystallography and cryo-electron microscopy approaches reveal new insight into cargo-dependent coat assembly mechanisms. Clathrin and coat protein complex II (COPII) use conserved primary element ?-solenoid and WD40 structural motifs found in self-assembling cage scaffolds to generate unique geometries that sort cargo and produce vesicles. These studies emphasize molecular and structural principles that reflect the properties of self-assembling nanomachines to regulate cargo capacity in trafficking pathways. © 2007 Elsevier Ltd. All rights reserved.

TITLE:Does S-methyl methanethiosulfonate trap the thiol-disulfide state of proteins? 

[2-s2.0-33847219039] Refers [2-s2.0-23744457478] LEVEL: 3
S-Methyl methanethiosulfonate (MMTS) is a reagent used to trap the natural thiol-disulfide state of the proteins. The efficiency of trapping mixed disulfides in vivo has been found to be higher for MMTS than for the more commonly used N-ethylmaleimide. MMTS has also been used for studying protein S-nitrosylation and the role of catalytic and structural cysteines on enzyme activities. However, the treatment of a protein with MMTS can potentially generate additional protein disulfide bonds. These results indicate that in vitro MMTS is able to form both intramolecular and intermolecular protein disulfide bonds in addition to dithiomethane adducts. © Mary Ann Liebert, Inc.

TITLE:Polarized membrane distribution of potassium-dependent ion pumps in epithelial cells: Different roles of the N-glycans of their Ī² subunits 

[2-s2.0-34548331715] Refers [2-s2.0-23744457478] LEVEL: 3
The Na,K-ATPases and the H,K-ATPases are two potassium-dependent homologous heterodimeric P2-type pumps that catalyze active transport of Na+ in exchange for K+ (Na,K-ATPase) or H+ in exchange for K+ (H,K-ATPase). The ubiquitous Na,K-ATPase maintains intracellular ion balance and membrane potential. The gastric H,K-ATPase is responsible for acid secretion by the parietal cell of the stomach. Both pumps consist of a catalytic ?-subunit and a glycosylated ?-subunit that is obligatory for normal pump maturation and trafficking. Individual N-glycans linked to the ?-subunits of the Na,KATPase and H,K-ATPase are important for stable membrane integration of their respective ? subunits, folding, stability, subunit assembly, and enzymatic activity of the pumps. They are also essential for the quality control of unassembled ?-subunits that results in either the exit of the subunits from the ER or their ER retention and subsequent degradation. Overall, the importance of N-glycans for the maturation and quality control of the H,K-ATPase is greater than that of the Na,K-ATPase. The roles of individual N-glycans of the ?-subunits in the post-ER trafficking, membrane targeting and plasma membrane retention of the Na,K-ATPase and H,K-ATPase are different. The Na,K-ATPase ?1-subunit is the major ?-subunit isoform in cells with lateral location of the pump. All three N-glycans of the Na,K-ATPase ?1-subunit are important for the lateral membrane retention of the pump due to glycan-mediated interaction between the ?1-subunits of the two neighboring cells in the cell monolayer and cytosolic linkage of the ?-subunit to the cytoskeleton. This intercellular ?1-?1 interaction is also important for formation of cell-cell contacts. In contrast, the N-glycans unique to the Na,K-ATPase ?2-subunit,which has up to eight N-glycosylation sites, contain apical sorting information. This is consistent with the apical location of the Na,K-ATPase in normal and malignant epithelial cells with high abundance of the ?2-subunit. Similarly, all seven N-glycans of the gastric H,K-ATPase ?-subunit determine apical sorting of this subunit. © Humana Press Inc. 2007.

TITLE:SCFCdc4-mediated degradation of the Hac1p transcription factor regulates the unfolded protein response in Saccharomyces cerevisiae 

[2-s2.0-33846794446] Refers [2-s2.0-23744457478] LEVEL: 3
The Saccharomyces cerevisiae basic leucine zipper transcription factor Hac1p is synthesized in response to the accumulation of unfolded polypeptides in the lumen of the endoplasmic reticulum (ER), and it is responsible for up-regulation of ?5% of all yeast genes, including ER-resident chaperones and protein-folding catalysts. Hac1p is one of the most short-lived yeast proteins, having a half-life of ?1.5 min. Here, we have shown that Hac1p harbors a functional PEST degron and that degradation of Hac1p by the proteasome involves the E2 ubiquitin-conjugating enzyme Ubc3/Cdc34p and the SCF Cdc4 E3 complex. Consistent with the known nuclear localization of Cdc4p, rapid degradation of Hac1p requires the presence of a functional nuclear localization sequence, which we demonstrated to involve basic residues in the sequence 29RKRAKTK35. Two-hybrid analysis demonstrated that the PEST-dependent interaction of Hac1p with Cdc4p requires Ser146 and Ser149. Turnover of Hac1p may be dependent on transcription because it is inhibited in cell mutants lacking Srb10 kinase, a component of the SRB/mediator module of the RNA polymerase II holoenzyme. Stabilization of Hac1p by point mutation or deletion, or as the consequence of defects in components of the degradation pathway, results in increased unfolded protein response element-dependent transcription and improved cell viability under ER stress conditions. © 2007 by The American Society for Cell Biology.

TITLE:Blocking translocation of cell surface molecules from the ER to the cell surface by intracellular antibodies targeted to the ER 

[2-s2.0-33947315300] Refers [2-s2.0-23744457478] LEVEL: 3
Intracellular antibodies (intrabodies) constitute a potent tool to neutralize the function of target proteins inside specific cell compartments (cytosol, nucleus, mitochondria and ER). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals and complements or replaces knockdown techniques such as anti-sense-RNA, RNAi and RNA aptamers. This article focuses on intrabodies targeted to the ER. Intracellular antibodies expressed and retained inside the ER (ER intrabodies) are shown to be highly efficient in blocking the translocation of secreted and cell surface molecules from the ER to the cell surface. The advantage of ER intrabodies over cytoplasmic intrabodies is that they are correctly folded and easier to select. A particular advantage of the intrabody technology over existing ones is the possibility of inhibiting selectively post-translational modifications of proteins. The main applications of ER intrabodies so far have been (i) inactivation of oncogenic receptors and (ii) functional inhibition of virus envelope proteins and virus-receptor molecules on the surface of host cells. In cancer research, the number of in vivo mouse models for evaluation of the therapeutic potential of intrabodies is increasing. In the future, endosomal loc