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Cytoplasmic Protein (cytoplasmic + protein)
Selected Abstracts,-synuclein has a dynamic intracellular localizationCYTOSKELETON, Issue 8 2006Irina Surgucheva Abstract ,-Synuclein is a member of the synuclein family consisting of three proteins. Within the last several years increasing attention has focused on these proteins because of their role in human diseases. ,-Synuclein relevance to Parkinson's disease is based on mutations found in familial cases of the disease and its presence in filaments and inclusion bodies in sporadic cases. ,-Synuclein is implicated in some forms of cancer and ocular diseases, while ,-synuclein may antagonize their pathological functions. In this paper we present data on the localization and properties of ,-synuclein in several neuronal and nonneuronal cell cultures. We show that contrary to the current opinion, ,-synuclein is not an exclusively cytoplasmic protein, but has a dynamic localization and can associate with subcellular structures. It is present in the perinuclear area and may be associated to centrosomes. On late steps of mitosis ,-synuclein is not found in the centrosomes, and redistributes to the midbody in telophase. Under stress conditions a translocation of ,-synuclein from the perinuclear area to the nucleus occurs exhibiting nucleocytoplasmic shuttling. ,-Synuclein overexpression reduces neurite outgrowth in a greater extent then ,-synuclein overexpression. These data support the view that ,-synuclein may change its intracellular localization and associate with subcellular structures in response to intracellular signaling or stress. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] The ,II isotype of tubulin is present in the cell nuclei of a variety of cancersCYTOSKELETON, Issue 2 2004I-Tien Yeh Abstract Tubulin, the subunit protein of microtubules, has generally been thought to be exclusively a cytoplasmic protein in higher eukaryotes. We have previously shown that cultured rat kidney mesangial cells contain the ,II isotype of tubulin in their nuclei in the form of an ,,II dimer [Walss et al., 1999: Cell Motil. Cytoskeleton 42:274,284, 1999]. More recently, we examined a variety of cancerous and non-cancerous cell lines and found ,II in the nuclei of all of the former and only a few of the latter (Walss-Bass et al., 2002: Cell Tissue Res. 308:215,223]. In order to determine if ,II -tubulin occurs in the nuclei of actual cancers as well as in cancer cell lines, we used the immunoperoxidase method to look for nuclear ,II in a variety of tumors excised from 201 patients. We found that 75% of these tumors contain ,II in their nuclei. Distribution of nuclear ,II was highly dependent on the type of cancer, with 100% of the colon and prostate cancers, but only 19% of the skin tumors, having nuclear ,II. Nuclear ,II was particularly marked in tumors of epithelial origin, of which 83% showed nuclear ,II, in contrast to 54% in tumors of non-epithelial origin. In many cases, ,II staining occurred very strongly in the nuclei and not in the cytoplasm; in other cases, ,II was present in both. In many cases, particularly metastases, otherwise normal cells adjacent to the tumor also showed nuclear ,II, suggesting that cancer cells may influence nearby cells to synthesize ,II and localize it to their nuclei. Our results have implications for the diagnosis, biology, and chemotherapy of cancer. Cell Motil. Cytoskeleton 57:96,106, 2004. © 2004 Wiley-Liss, Inc. [source] Differential targeting of components of the dystrophin complex to the postsynaptic membraneEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2001Sophie Marchand Abstract Accumulating evidence points to the participation of dystroglycan in the clustering of nicotinic acetylcholine receptors at the neuromuscular junction [Côtéet al.. (1999) Nature Genet., 3, 338,342]. Dystroglycan is part of a multimolecular complex, either associated with dystrophin (the dystrophin-associated protein complex) at the sarcolemma or with utrophin (the utrophin-associated protein complex) at the neuromuscular junction. Understanding the assembly of this complex at the developing synapse led us to investigate, in Torpedo electrocyte, the intracellular routing and the targeting of several of its components, including dystroglycan, syntrophin, dystrophin and dystrobrevin. We previously demonstrated that acetylcholine receptors and rapsyn, the 43-kDa receptor-associated protein at the synapse, are cotargeted to the postsynaptic membrane via the exocytic pathway [Marchand et al.. (2000) J. Neurosci., 20, 521,528]. Using cell fractionation, immunopurification and immuno-electron microscope techniques, we show that ,-dystroglycan, an integral glycoprotein that constitutes the core of the dystrophin-associated protein complex localized at the innervated membrane, is transported together with acetylcholine receptor and rapsyn in post-Golgi vesicles en route to the postsynaptic membrane. Syntrophin, a peripheral cytoplasmic protein of the complex, associates initially with these exocytic vesicles. Conversely, dystrophin and dystrobrevin were absent from these post-Golgi vesicles and associate directly with the postsynaptic membrane. This study provides the first evidence for a separate targeting of the various components of the dystrophin-associated protein complex and a step-by-step assembly at the postsynaptic membrane. [source] Annexin A1 subcellular expression in laryngeal squamous cell carcinomaHISTOPATHOLOGY, Issue 6 2008V A F Alves Aims:, Annexin A1 (ANXA1) is a soluble cytoplasmic protein, moving to membranes when calcium levels are elevated. ANXA1 has also been shown to move to the nucleus or outside the cells, depending on tyrosine-kinase signalling, thus interfering in cytoskeletal organization and cell differentiation, mostly in inflammatory and neoplastic processes. The aim was to investigate subcellular patterns of immunohistochemical expression of ANXA1 in neoplastic and non-neoplastic samples from patients with laryngeal squamous cell carcinomas (LSCC), to elucidate the role of ANXA1 in laryngeal carcinogenesis. Methods and results:, Serial analysis of gene expression experiments detected reduced expression of ANXA1 gene in LSCC compared with the corresponding non-neoplastic margins. Quantitative polymerase chain reaction confirmed ANXA1 low expression in 15 LSCC and eight matched normal samples. Thus, we investigated subcellular patterns of immunohistochemical expression of ANXA1 in 241 paraffin-embedded samples from 95 patients with LSCC. The results showed ANXA1 down-regulation in dysplastic, tumourous and metastatic lesions and provided evidence for the progressive migration of ANXA1 from the nucleus towards the membrane during laryngeal tumorigenesis. Conclusions:, ANXA1 dysregulation was observed early in laryngeal carcinogenesis, in intra-epithelial neoplasms; it was not found related to prognostic parameters, such as nodal metastases. [source] The cytoplasmic tail of the ,3 integrin subunit promotes neurite outgrowth in PC12 cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2005Nadja Mechai Abstract Binding of integrins to proteins of the extracellular matrix (ECM) provides structural and signaling information for biological processes such as cell proliferation, migration, neurite outgrowth, and differentiation. Integrins represent a family of heterodimeric transmembrane cell surface receptors. Besides connecting the ECM with the cytoskeleton, integrins also induce various signaling pathways in response to ligand binding. Integrin ligation leads to cytoplasmic protein,protein interactions requiring both integrin cytoplasmic tails. These sequences are initiation points for focal adhesion formation and subsequent signal transduction cascades. In this study, we addressed the question of whether the short cytoplasmic tail of the ,3 integrin subunit of ,3,1 integrin is required for ,3,1 integrin-dependent processes. For this purpose, cDNA representing the extracellular and transmembrane domain of the interleukin 2 receptor (IL2R) , subunit and the cytoplasmic sequence of the ,3 integrin subunit was transfected into PC12 cells. Autonomous expression of the cytoplasmic ,3 tail does not affect attachment but leads to inhibition of neuronal differentiation on laminin 5. This indicates that the cytoplasmic ,3 sequence is not required for cell attachment but is necessary for long-term adhesion and for the reorganization of the cytoskeleton that precedes neuronal differentiation. Inhibition of neurite outgrowth by chimeric IL2R-,3 can be rescued by treatment of transfected cells with the pharmacological inhibitor Y27632, which inhibits the RhoA downstream effector Rho kinase ,. © 2005 Wiley-Liss, Inc. [source] Complementary DNA cloning of rat spetex-1, a spermatid-expressing gene-1, encoding a 63 kDa cytoplasmic protein of elongate spermatidsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Hiroshi Iida Abstract We used differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene designated as spetex-1, which had an open reading frame of 1,668-length nucleotides encoding a protein of 556 amino acids. Spetex-1 mRNA was highly expressed in testis, and weekly expressed in lung, intestine, and spleen. Spetex-1 expression in the rat testes was detected first at 3 weeks in postnatal development and continued to be detected up to adulthood. A search in the databases showed that the amino acid sequence of spetex-1 was 82% identical to that of its mouse homologue found in the databases. Both rat spetex-1 and the mouse homologue contained Ser-X (X,=,His, Arg, or Asn) repeats in the middle portion of the proteins. In situ hybridization revealed that spetex-1 mRNA was expressed in haploid spermatids of step 7,18 within the seminiferous epithelium. Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spetex-1 protein was not expressed in spermatogonia, spermatocytes, and round spermatids in adult rat testis, but was specifically detected in the residual cytoplasm of elongate spermatids of step 15,18 as well as in residual bodies engulfed by Sertoli cells. We interpreted these data as a potential role of spetex-1 in spermatogenesis, especially in cell differentiation from late elongate spermatids to mature spermatozoa. Mol. Reprod. Dev. 68: 385,393, 2004. © 2004 Wiley-Liss, Inc. [source] Characterisation of the CipC-like protein AFUA_5G09330 of the opportunistic human pathogenic mould Aspergillus fumigatusMYCOSES, Issue 4 2010Bettina Bauer Summary,Aspergillus fumigatus is currently the major airborne fungal pathogen that menaces immunocompromised individuals. Germination of inhaled conidia is a hallmark of the early infection process, but little is known about the underlying mechanisms. The intention of our ongoing studies is the identification of A. fumigatus proteins that are differentially expressed during germination and may provide insights in the germination process. Using a proteomic approach, we identified AFUA_5G09330 as a major hyphal-specific protein. This result was confirmed using monoclonal antibodies generated in this study. AFUA_5G09330 belongs to a fungal-specific protein family. The eponymous CipC protein of A. nidulans has been shown to be induced by concanamycin A, and transcriptional data from Cryptococcus neoformans demonstrate a strong up-regulation of the expression of a homologous gene during infection. Our data provide evidence that AFUA_5G09330 is a monomeric, cytoplasmic protein. We found no evidence for an overexpression of AFUA_5G09330 induced by concanamycin A or other stress conditions. AFUA_5G09330 is exclusively found in the hyphal morphotype that enables an invasive growth of A. fumigatus during infection. Further studies are required to define the biological function of this hyphae-specific protein and its potential relevance for the pathogenicity of A. fumigatus. [source] Immunohistochemical comparison of ,-catenin expression by human normal epidermis and epidermal tumorsTHE JOURNAL OF DERMATOLOGY, Issue 11 2007Keiko FUKUMARU ABSTRACT ,-Catenin, a cytoplasmic protein that binds directly to the intracellular domain of cadherin, controls various functions such as cell adhesion. In many human carcinomas, E-cadherin-mediated cell,cell adhesion is lost or disturbed and related to metastasis. The purpose of this study was to compare the expression of ,-catenin in the normal epidermal keratinocytes and samples from cutaneous benign and malignant epidermal tumors in 140 patients. Our study population consisted of 140 patients with benign or malignant epidermal tumors. Using immunohistochemical methods, we compared the expression of ,-catenin in their normal epidermal keratinocytes, and in samples from 61 benign (seborrheic keratosis, n = 33; verruca vulgaris, n = 14; keratoacanthoma, n = 14), and 79 malignant (Bowen's disease, n = 18; basal cell carcinoma, n = 33; squamous cell carcinoma, n = 28) epidermal tumors. ,-Catenin was found to be expressed in the cell membrane of normal keratinocytes. Compared to other cell components of the normal epidermis, basal cells showed the strongest ,-catenin expression in all 140 patients. While absent in three of 61 benign tumors, compared to normal basal cells, the expression of ,-catenin in the other 58 tumors was not significantly different; it was reduced in 71 of 79 malignant tumors (P < 0.0001). In Bowen's disease, the expression of ,-catenin on the tumor cell membrane was reduced, however, strong expression was seen in the nuclei and cytoplasm. Our results suggest that ,-catenin expression on the membrane of keratinocytes is associated with the differentiation of normal keratinocytes but not with their stage of differentiation, nor with the proliferation ability of epidermal tumor cells. [source] DNA vaccination against foot-and-mouth disease via electroporation: study of molecular approaches for enhancing VP1 antigenicityTHE JOURNAL OF GENE MEDICINE, Issue 9 2006Suk-Am Kim Abstract Background Foot-and-mouth disease virus (FMDV) affects susceptible livestock animals and causes disastrous economic impact. Immunization with plasmid expressing VP1 that contains the major antigenic epitope(s) of FMDV as cytoplasmic protein (cVP1) failed to elicit full protection against FMDV challenge. Materials and methods In this study, mice were immunized via electroporation with four cDNA expression vectors that were constructed to express VP1 of FMDV, as cytoplasmic (cVP1), secreted (sVP1), membrane-anchored (mVP1) or capsid precursor protein (P1), respectively, to evaluate whether expression of VP1 in specific subcellular compartment(s) would result in better immune responses. Results Electroporation enhanced immune responses to vectors expressing cVP1 or P1 and expedited the immune responses to vectors expressing sVP1 or mVP1. Immunization of mice via electroporation with mVP1 cDNA was better than sVP1 or cVP1 cDNA in eliciting neutralizing antibodies and viral clearance protection. Vaccination with P1 cDNA, nonetheless, yielded the best immune responses and protection among all four cDNAs that we tested. Conclusions These results suggest that the antigenicity of a VP1 DNA vaccine can be significantly enhanced by altering the cellular localization of the VP1 antigen. Electroporation is a useful tool for enhancing the immune responses of vectors expressing VP1 or P1. By mimicking FMDV more closely than that of transgenic VP1 and eliciting immune responses favorably toward Th2, transgenic P1 may induce more neutralizing antibodies and better protection against FMDV challenge. Copyright © 2006 John Wiley & Sons, Ltd. [source] All-trans retinoic acid affects subcellular localization of a novel BmNIF3l protein: functional deduce and tissue distribution of NIF3l gene from silkworm (Bombyx mori),ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2010Jianqing Chen Abstract A novel cDNA sequence encoding a predicted protein of 271 amino acids containing a conserved NIF3 domain was found from a pupal cDNA library of silkworm. The corresponding gene was named BmNIF3l (Bombyx mori NGG1p interacting factor 3-like). It was found by bioinformatics that BmNIF3l gene consisted of five exons and four introns and BmNIF3l had a high degree of homology to other NIF3-like proteins, especially in the N-terminal and C-terminal regions. A His-tagged BmNIF3l fusion protein with a molecular weight of approximately 33.6,kDa was expressed and purified to homogeneity. We have used the purified fusion protein to produce polyclonal antibodies against BmNIF3l for histochemical analysis. Subcellular localization revealed that BmNIF3l is a cytoplasmic protein that responds to all-trans retinoic acid (ATRA). Western blotting and real-time reverse transcription polymerase chain reaction showed that the expression level of BmNIF3l is higher in tissues undergoing differentiation. Taken together, the results suggest that BmNIF3l functions in transcription. © 2010 Wiley Periodicals, Inc. [source] Notching up another pathwayBIOESSAYS, Issue 5 2002Keith Brennan The Notch proteins play a vital role in cell fate decisions in both invertebrate and vertebrate development. Careful analysis of this role has led to a model of signalling downstream of these receptors, via the CSL (CBF1, Suppressor of Hairless, Lag-1) family of transcription factors. There have been suggestions, however, that Notch can signal through other pathways. In the current paper, Ramain et al.1 provide compelling evidence for Notch signalling through a CSL-independent pathway and they demonstrate that the cytoplasmic protein, Deltex, is required for this signal. In addition, they show that Wnt signalling may regulate this Deltex-dependent signal. BioEssays 24:405,410, 2002. © 2002 Wiley Periodicals, Inc. [source] Attenuation of Flightless I, an actin-remodelling protein, improves burn injury repair via modulation of transforming growth factor (TGF)-,1 and TGF-,3BRITISH JOURNAL OF DERMATOLOGY, Issue 2 2009D.H. Adams Summary Background, The pathophysiological mechanisms involved in burn injury repair are still not fully understood but include processes involving cellular proliferation, migration and adhesion. The actin cytoskeleton is intricately involved in these key wound repair processes. Flightless I (Flii), an actin-remodelling protein and transcriptional regulator, is an important regulator of wound healing. Objectives, To investigate the function of Flii gene expression in burn injury repair. Methods, Partial-thickness scald wounds were created on Flii heterozygous (Flii+/,), wild-type (WT) and Flii transgenic (FliiTg/+) mice. Burns were assessed using histology and immunohistochemistry, real-time quantitative polymerase chain reaction and biochemical analysis. Results, Flii expression, while upregulated in burn injuries, was significantly lower in the wounds of Flii+/, vs. WT vs. FliiTg/+ mice and healing was improved in Flii+/, mice with their burns healing faster than WT and FliiTg/+. Pro-scarring transforming growth factor (TGF)-,1 protein and gene expression were reduced in Flii+/, burns while antiscarring TGF-,3 was significantly elevated. Anti-,-smooth muscle actin (,-SMA) was decreased in Flii+/, burns suggesting a decrease in contractile myofibroblasts in the developing scars. Although Flii is primarily a nuclear and cytoplasmic protein it is also released by wounded cells. Intradermal injection of Flii-neutralizing antibodies (FliAbs) to WT burn wounds significantly improved their healing, indicating a potential novel approach for treating burns. Decreased TGF-,1 and elevated TGF-,3 expression were observed in FliAb-treated burns, which may contribute to their observed improvement in healing. Conclusions, Strategies aimed at reducing Flii expression, for example using neutralizing antibodies, may lead to improved burn outcomes. [source] Protein tandem repeats , the more perfect, the less structuredFEBS JOURNAL, Issue 12 2010Julien Jorda We analysed the structural properties of protein regions containing arrays of perfect and nearly perfect tandem repeats. Naturally occurring proteins with perfect repeats are practically absent among the proteins with known 3D structures. The great majority of such regions in the Protein Data Bank are found in the proteins designed de novo. The abundance of natural structured proteins with tandem repeats is inversely correlated with the repeat perfection: the chance of finding natural structured proteins in the Protein Data Bank increases with a decrease in the level of repeat perfection. Prediction of intrinsic disorder within the tandem repeats in the SwissProt proteins supports the conclusion that the level of repeat perfection correlates with their tendency to be unstructured. This correlation is valid across the various species and subcellular localizations, although the level of disordered tandem repeats varies significantly between these datasets. On average, in prokaryotes, tandem repeats of cytoplasmic proteins were predicted to be the most structured, whereas in eukaryotes, the most structured portion of the repeats was found in the membrane proteins. Our study supports the hypothesis that, in general, the repeat perfection is a sign of recent evolutionary events rather than of exceptional structural and (or) functional importance of the repeat residues. [source] The surface-associated elongation factor Tu is concealed for antibody binding on viable pneumococci and meningococciFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2008Jan Kolberg Abstract Proteome analyses revealed that elongation factor-Tu (EF-Tu) is associated with cytoplasmic membranes of Gram-positive bacteria and outer membranes of Gram-negative bacteria. It is still debatable whether EF-Tu is located on the external side or the internal side of the membranes. Here, we have generated two new monoclonal antibodies (mAbs) and polyclonal rabbit antibodies against pneumococcal EF-Tu. These antibodies were used to investigate the amount of surface-exposed EF-Tu on viable bacteria using a flow cytometric analysis. The control antibodies recognizing the pneumococcal surface protein A and phosphorylcholine showed a significant binding to viable pneumococci. In contrast, anti-EF-Tu antibodies did not recognize pneumococcal EF-Tu. However, heat killing of pneumococci lacking capsular polysaccharides resulted in specific antibody binding to EF-Tu and, moreover, increased the exposure of recognized phosphorylcholine epitopes. Similarly, our EF-Tu-specific antibodies did not recognize EF-Tu of viable Neisseria meningitidis. However, pretreatment of meningococci with ethanol resulted in specific antibody binding to EF-Tu on outer membranes. Importantly, these treatments did not destroy the membrane integrity as analysed with control mAbs directed against cytoplasmic proteins. In conclusion, our flow cytrometric assays emphasize the importance of using viable bacteria and not heat-killed or ethanol-treated bacteria for surface-localization experiments of proteins, because these treatments modulate the cytoplasmic and outer membranes of bacteria and the binding results may not reflect the situation under physiological conditions. [source] Atg17 recruits Atg9 to organize the pre-autophagosomal structureGENES TO CELLS, Issue 5 2009Takayuki Sekito Autophagy is a degradation system of cytoplasmic proteins and organelles via formation of double-membrane vesicles called autophagosomes. In the yeast Saccharomyces cerevisiae, autophagosomes are formed via the pre-autophagosomal structure (PAS) in a manner dependent on Atg proteins. Under nutrient-rich condition, Atg9 is recruited to the PAS by binding to Atg11 for the Cvt pathway. However, because Atg9 is recruited to the PAS in atg11, cells in starved condition and autophagy is induced, autophagy-specific mechanism for the Atg9 recruitment to the PAS has been assumed. Here, we demonstrate that, in autophagy-inducing condition, Atg9 is recruited to the PAS in a manner dependent on Atg17. Atg9 physically interacts with Atg17 in the presence of rapamycin. This interaction requires Atg1, a protein kinase essential for autophagy. Consistently, the Atg17-dependent PAS localization of Atg9 requires Atg1. However, its kinase activity is dispensable for this process. It rather regulates the equilibrium of assembly and disassembly of Atg9 at the PAS. [source] Modulation of O-GlcNAc glycosylation during Xenopus oocyte maturation,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2004Tony Lefebvre Abstract O-linked N -acetylglucosamine (O-GlcNAc) glycosylation is a post-translational modification, which is believed antagonises phosphorylation. We have studied the O-GlcNAc level during Xenopus oocyte meiotic resumption, taking advantage of the high synchrony of this model which is dependent upon a burst of phosphorylation. Stimulation of immature stage VI oocytes using progesterone was followed by a 4.51,±,0.32 fold increase in the GlcNAc content, concomitantly to an increase in phosphorylation, notably on two cytoplasmic proteins of 66 and 97 kDa. The increase of O-GlcNAc for the 97 kDa protein, which we identified as ,-catenin was partly related to its accumulation during maturation, as was demonstrated by the use of the protein synthesis inhibitor,cycloheximide. Microinjection of free GlcNAc, which inhibits O-glycosylated proteins,lectins interactions, delayed the progesterone-induced maturation without affecting the O-GlcNAc content. Our results suggest that O-GlcNAc glycosylation could regulate protein,protein interactions required for the cell cycle kinetic. © 2004 Wiley-Liss, Inc. [source] Expression of GFAT1 and OGT in podocytes: Transport of glucosamine and the implications for glucose uptake into these cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2010Dorota Rogacka Glutamine:fructose-6-phosphate amidotransferase (GFAT) and N -acetylglucosaminyltransferase (OGT) participate in glucosamine (GlcN) production and its utilization in O -glycosylation, one of key post-translational modifications of nuclear and cytoplasmic proteins. For this purpose, cells require a high rate of intracellular production of GlcN and/or significant GlcN delivery. We studied the expression of GFAT1 and OGT and measured uptake of glucose and GlcN in cultured rat podocytes, the main cellular component of glomerular filtration barrier. RT-PCR revealed the presence of both GFAT1 and OGT mRNA. Immunofluorescence of GFAT1 has shown staining signal diffused within the cytoplasm of the cell body and processes. However, OGT was distinctly visible around the nucleus and, in diffuse form, within the cytoplasm of cell bodies and processes. Glucose was transported (1.3,±,0.2,nmol/min/mg protein) mainly by facilitative transporter systems whilst GlcN uptake (1.1,±,0.2,nmol/min/mg protein) in a significant part, involved a sodium-dependent transporter. There was interplay between glucose and GlcN uptake. In the presence of GlcN (50,µM), the rate of glucose uptake decreased by about 50%. The rate of GlcN uptake decreased by 28% in the presence of 5.6,mM glucose. Our results suggest that cultured podocytes possess limited ability to synthesize GlcN internally and therefore may need to receive GlcN from the extracellular environment. J. Cell. Physiol. 225: 577,584, 2010. © 2010 Wiley-Liss, Inc. [source] O-linked ,-N-acetylglucosaminylation in mouse embryonic neural precursor cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 16 2009Makoto Yanagisawa Abstract In neural stem cells (NSCs), glycoconjugates and carbohydrate antigens are known not only to serve as excellent cell surface biomarkers for cellular differentiation and development but also to play important functional roles in determining cell fate. O-linked ,-N-acetylglucosamine (O-GlcNAc), which modifies nuclear and cytoplasmic proteins on the serine and threonine residues, is also expected to play an important regulatory role. It is not known, however, whether O-GlcNAc is expressed in NSCs or what the function of this expression is. In this study, we evaluated the patterns and possible functions of O-GlcNAcylation in mouse embryonic neuroepithelial cells (NECs), which are known to be rich in NSCs. We confirmed the expression of O-GlcNAc transferase, O-GlcNAcase, and several O-GlcNAcylated proteins in NECs. Treatment of NECs with O-GlcNAcase inhibitors, PUGNAc and streptozotocin, induced robust accumulation of O-GlcNAc in NECs and reduction of number of NECs. In O-GlcNAcase inhibitor-treated NECs, the Ras-mitogen-activated protein kinase pathway and the phosphatidylinositol 3-kinase-Akt pathway, important for proliferation and survival, respectively, were intact, but caspase-3, an executioner for cell death, was activated. These results suggest the possibility that O-GlcNAc is involved in cell death signaling in NECs. Furthermore, in NECs, we identified an O-GlcNAc-modified protein, Sp1 transcription factor. Our study is the first to evaluate expression and functions of O-GlcNAc in NECs. © 2009 Wiley-Liss, Inc. [source] Leucine-rich repeat proteins of synapsesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 13 2007Jaewon Ko Abstract Leucine-rich repeats (LRRs) are 20,29-aa motifs that mediate protein,protein interactions and are present in a variety of membrane and cytoplasmic proteins. Many LRR proteins with neuronal functions have been reported. Here, we summarize an emerging group of synaptic LRR proteins, which includes densin-180, Erbin, NGL, SALM, and LGI1. These proteins have been implicated in the formation, differentiation, maintenance, and plasticity of neuronal synapses. © 2007 Wiley-Liss, Inc. [source] Methionine sulphoxide reductase is an important antioxidant enzyme in the gastric pathogen Helicobacter pyloriMOLECULAR MICROBIOLOGY, Issue 5 2004Praveen Alamuri Summary The ability of Helicobacter pylori to colonize the stomach requires that it combat oxidative stress responses imposed by the host. The role of methionine sulfoxide reductase (Msr), a methionine repair enzyme, in H. pylori stress resistance was evaluated by a mutant analysis approach. An msr mutant strain lacked immunologically detectable sulphoxide reductase protein and also showed no enzyme activity when provided with oxidized methionines as substrates. The mutant strain showed diminished growth compared to the parent strain in the presence of chemical oxidants, and showed rapid viability loss when exposed to oxidizing conditions. The stress resistance and enzyme activity could be recovered by complementing the mutant with a functional copy of the msr gene. Upon fractionation of parent strain and the complemented mutant cells into membranes and cytoplasmic proteins, most of the immunologically detectable Msr was localized to the membrane, and this fraction contained all of the Msr activity. Qualitative detection of the whole cell protein pattern using 2,4-dinitro phenyl hydrazine (DNPH) showed a far greater number of oxidized protein species in the mutant than in the parent strain when the cells were subjected to oxygen, peroxide or s-nitrosoglutathione (GSNO) induced stress. Importantly, no oxidized proteins were discerned in either strain upon incubation in anaerobic conditions. A mutant strain that synthesized a truncated Msr (corresponding to the MsrA domain) was slightly more resistant to oxidative stress than the msr strain. Mouse colonization studies showed Msr is an important colonization factor, especially for effective longer-term (14 and 21 days) colonization. Complementation of the mutant msr strain by chromosomal insertion of a functional gene restored mouse colonization ability. [source] Membrane localization itself but not binding to IICBGlc is directly responsible for the inactivation of the global repressor Mlc in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 3 2004Yuya Tanaka Summary Mlc is a global transcriptional repressor involved in the regulation of genes linked to glucose metabolism. The activity of Mlc is modulated through the interaction with a major glucose transporter, IICBGlc, in response to external glucose. To understand how IICBGlc,Mlc interaction controls the repressor activity of Mlc, we attempted to isolate Mlc mutants that retain the ability to repress target genes even in the presence of glucose. The Mlc mutants were tested for their ability to interact with IICBGlc. Mutants in which a single amino acid substitution occurs in the N-terminal portion were no longer able to bind to IICBGlc, suggesting that the N-terminal region of Mlc is primarily responsible for the interaction with IICBGlc. To examine whether the Mlc,IICBGlc interaction and/or the membrane localization of Mlc per se are essential for the inactivation of Mlc, the properties of several hybrid proteins in which either IIBGlc or Mlc is fused to membrane proteins were analysed. The cytoplasmic IIBGlc domain failed to inhibit the Mlc action although it retains the ability to bind Mlc in cells. However, it gained the ability to inhibit the Mlc activity when it was fused to a membrane protein LacY. In addition, we showed that Mlc is inactivated when fused to membrane proteins but not when fused to cytoplasmic proteins. We conclude that the IICBGlc,Mlc interaction is dispensable for the inactivation of Mlc, and that membrane localization is directly responsible for the inactivation of Mlc. [source] Plasticity of Cadherin,Catenin Expression in the Melanocyte LineagePIGMENT CELL & MELANOMA RESEARCH, Issue 4 2000ALICE JOUNEAU Cadherins are calcium-dependent cell adhesion receptors with strong morphoregulatory functions. To mediate functional adhesion, cadherins must interact with actin cytoskeleton. Catenins are cytoplasmic proteins that mediate the interactions between cadherins and the cytoskeleton. In addition to their role in cell,cell adhesion, catenins also participate in signaling pathways that regulate cell growth and differentiation. Cadherins and catenins appear to be involved in melanocyte development and transformation. Here, we investigated the function of cadherin,catenin complexes in the normal development and transformation of melanocytes by studying the patterns of expression of the cell,cell adhesion molecules, E-, N- and P-cadherin, and the expression of their cytoplasmic partners, ,-, ,- and ,-catenin, during murine development. Similar analyses were performed in vitro using murine melanoblast, melanocyte, and melanoma cell lines in the presence and absence of keratinocytes, the cells with which melanocytes interact in vivo. Overall, the results suggest that the expression of cadherins and catenins is very plastic and depends on their environment as well as the transformation status of the cells. This plasticity is important in fundamental cellular mechanisms associated with normal and pathological ontogenesis, as well as with tumorigenesis. [source] MscL of Bacillus subtilis prevents selective release of cytoplasmic proteins in a hypotonic environmentPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2009Thijs R. H. M. Kouwen Abstract Bacillus subtilis serves as an excellent model to study protein secretion at a proteomic scale. Most of the extracellular proteins are exported from the cytoplasm via the secretory (Sec) pathway. Despite extensive studies, the secretion mechanisms of about 25% of the extracellular proteins are unknown. This suggests that B. subtilis makes use of alternative mechanisms to release proteins into its environment. In search for novel pathways, which contribute to biogenesis of the B. subtilis exoproteome, we investigated a possible role of the large conductance mechanosensitive channel protein MscL. We compared protein secretion by MscL deficient and proficient B. subtilis cells. MscL did not contribute to secretion under standard growth conditions. Unexpectedly, we discovered that under hypo-osmotic shock conditions specific, normally cytoplasmic proteins were released by mscL mutant cells. This protein release was selective since not all cytoplasmic proteins were equally well released. We established that this protein release by mscL mutant cells cannot be attributed to cell death or lysis. The presence of MscL, therefore, seems to prevent the specific release of cytoplasmic proteins by B. subtilis during hypo-osmotic shock. Our unprecedented findings imply that an unidentified system for selective release of cytoplasmic proteins is active in B. subtilis. [source] | |||||||||||||||||||||||||||||||