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Alpha Subunit (alpha + subunit)
Selected AbstractsInterleukin-5 does not influence differential transcription of transmembrane and soluble isoforms of IL-5R, in vivoEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 3 2006Jonas Byström Abstract:, Interleukin-5 (IL-5) promotes signal transduction and expansion of eosinophil colonies in bone marrow via interactions with its heterodimeric receptor (IL-5R). Two variants encoding soluble forms of the alpha subunit (sIL-5R,) have been described, although the signals promoting and/or limiting differential transcription remain to be clarified. Objectives:,Our intent was to explore the role of IL-5 in regulating differential transcription of these splice variants in vivo. Methods:,We have designed a quantitative reverse transcriptase-polymerase chain reaction assay to detect transcripts encoding the transmembrane, soluble 1 and 2 forms of IL-5R, in two strains of wild-type (BALB/c and C57BL/6) and corresponding IL-5 gene-deleted mice. Wild-type mice respond to S. mansoni infection with a gradual increase in serum IL-5 and eosinophilia, which is not observed in IL-5 gene-deleted mice. Results and conclusions:,We find that IL-5 is not necessary for differential splicing to occur in vivo, as all three forms of the IL-5R, are detected in both strains of IL-5 gene-deleted mice, with ratios of transcript expression (transmembrane : soluble 1 : soluble 2) that were indistinguishable from their wild-type counterparts. Differential splicing does vary markedly between strains, potentially because of local effects of strain-specific polymorphisms. [source] Pigment epithelium-derived factor binds to cell-surface F1 -ATP synthaseFEBS JOURNAL, Issue 9 2010Luigi Notari Pigment epithelium-derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothelial cells. Given that protein fingerprinting suggested a match of a , 60 kDa PEDF-binding protein in bovine retina with Bos taurus F1 -ATP synthase ,-subunit, and that F1Fo -ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding of PEDF to F1. Size-exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F1. Real-time binding as determined by surface plasmon resonance demonstrated that yeast F1 interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces. PEDF blocked interactions between F1 and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF-binding and angiostatin-binding sites on F1. Surfaces of endothelial cells exhibited affinity for PEDF-binding proteins of , 60 kDa. Antibodies to F1,-subunit specifically captured PEDF-binding components in endothelial plasma membranes. The extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell-surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell-surface F1, these results show that PEDF is a ligand for endothelial cell-surface F1Fo -ATP synthase. They suggest that PEDF-mediated inhibition of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity. Structured digital abstract ,,MINT-7711286: angiostatin (uniprotkb:P00747) physically interacts (MI:0915) with F-ATPase alpha subunit (uniprotkb:P07251), F-ATPase beta subunit (uniprotkb:P00830), F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase delta subunit (uniprotkb:Q12165) and F-ATPase epsilon subunit (uniprotkb:P21306) by competition binding (MI:0405) ,,MINT-7711113: angiostatin (uniprotkb:P00747) physically interacts (MI:0915) with F-ATPase epsilon subunit (uniprotkb:P21306), F-ATPase delta subunit (uniprotkb:Q12165), F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase beta subunit(uniprotkb:P00830) and F-ATPase alpha subunit (uniprotkb:P07251) by surface plasmon resonance (MI:0107) ,,MINT-7711060: F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase beta subunit (uniprotkb:P00830), F-ATPase alpha subunit (uniprotkb:P07251) and PEDF (uniprotkb:P36955) physically interact (MI:0915) by molecular sieving (MI:0071) ,,MINT-7711313: F-ATPase epsilon subunit (uniprotkb:P21306), F-ATPase delta subunit (uniprotkb:Q12165), PEDF (uniprotkb:P36955), F-ATPase alpha subunit (uniprotkb:P07251), F-ATPase beta subunit (uniprotkb:P00830) and F-ATPase gamma subunit(uniprotkb:P38077) physically interact (MI:0915) by molecular sieving (MI:0071) ,,MINT-7711083: PEDF (uniprotkb:P36955) physically interacts (MI:0915) with F-ATPase epsilon subunit (uniprotkb:P21306), F-ATPase delta subunit (uniprotkb:Q12165), F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase beta subunit (uniprotkb:P00830) and F-ATPase alpha subunit (uniprotkb:P07251) by surface plasmon resonance (MI:0107) [source] FLR-2, the glycoprotein hormone alpha subunit, is involved in the neural control of intestinal functions in Caenorhabditis elegansGENES TO CELLS, Issue 10 2009Akane Oishi The intestine plays an essential role in organism-wide regulatory networks in both vertebrates and invertebrates. In Caenorhabditis elegans, class 1 flr genes (flr-1, flr-3 and flr-4) act in the intestine and control growth rates and defecation cycle periods, while class 2 flr genes (flr-2, flr-5, flr-6 and flr-7) are characterized by mutations that suppress the slow growth of class 1 flr mutants. This study revealed that flr-2 gene controls antibacterial defense and intestinal color, confirming that flr-2 regulates intestinal functions. flr-2 encoded the only glycoprotein hormone alpha subunit in C. elegans and was expressed in certain neurons. Furthermore, FLR-2 bound to another secretory protein GHI-1, which belongs to a family of lipid- and lipopolysaccharide-binding proteins. A ghi-1 deletion mutation partially suppressed the short defecation cycle periods of class 1 flr mutants, and this effect was enhanced by flr-2 mutations. Thus, FLR-2 acts as a signaling molecule for the neural control of intestinal functions, which is achieved in a functional network involving class 1 and class 2 flr genes as well as ghi-1. These results are informative to studies of glycoprotein hormone signaling in higher animals. [source] Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neuronsGLIA, Issue 10 2010Nader D. Halim Abstract Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH,). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorlyated PDH,. Dephosphorylation of astrocytic PDH, restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. © 2010 Wiley-Liss, Inc. [source] Single nucleotide polymorphisms in the hypoxia-inducible factor-1 gene and colorectal cancer riskMOLECULAR CARCINOGENESIS, Issue 9 2010Gudrun Knechtel Abstract With an incidence of about 300,000 new cases colorectal cancer (CRC) is the second leading cause of cancer-related death in Europe and the United States. Environmental and genetic factors influence CRC risk. Hypoxia-inducible factor-1 (HIF-1), a heterodimeric protein composed of two subunits, HIF-1 alpha and HIF-1 beta, plays a critical role in oxygen homeostasis and is involved in angiogenesis and cell proliferation. The gene for the HIF-1 alpha subunit (HIF1A) carries two common missense mutations,P582S (rs11549465) and A588T (rs11549467),which both have been related to increased trans-activation capacity of HIF1A. In our case,control study we investigated the association between these polymorphisms and CRC risk. We investigated 381 patients with histologically confirmed CRC and 2156 control subjects. HIF1A genotypes were determined by exonuclease (TaqMan) assays. For determination of microvessel density (MVD) tumor sections were stained using a mouse monoclonal antibody recognizing the pan-endothelial marker CD31. In a multivariate logistic regression analysis including age and sex neither the HIF1A 582S allele (Odds ratio: 1.204; 95% confidence interval 0.911,1.592; P,=,0.193) nor the 588T allele was significantly associated with CRC (Odds ratio: 0.851; 95% confidence interval 0.444,1.631; P,=,0.626). However, in an exploratory analysis, the HIF1A 588T allele was associated with tumor localization (P,=,0.016) and tumor size (P,=,0.003). MVD was similar in tumors of patients carrying HIF1A 588T allele and patients without this rare allele. We conclude that functional polymorphisms in the HIF1A gene do not modify CRC risk but maybe associated with clinic-pathological features of the disease. © 2010 Wiley-Liss, Inc. [source] Widespread distribution of a lexA -regulated DNA damage-inducible multiple gene cassette in the Proteobacteria phylumMOLECULAR MICROBIOLOGY, Issue 1 2004Marc Abella Summary The SOS response comprises a set of cellular functions aimed at preserving bacterial cell viability in front of DNA injuries. The SOS network, negatively regulated by the LexA protein, is found in many bacterial species that have not suffered major reductions in their gene contents, but presents distinctly divergent LexA-binding sites across the Bacteria domain. In this article, we report the identification and characterization of an imported multiple gene cassette in the Gamma Proteobacterium Pseudomonas putida that encodes a LexA protein, an inhibitor of cell division (SulA), an error-prone polymerase (DinP) and the alpha subunit of DNA polymerase III (DnaE). We also demonstrate that these genes constitute a DNA damage-inducible operon that is regulated by its own encoded LexA protein, and we establish that the latter is a direct derivative of the Gram-positive LexA protein. In addition, in silico analyses reveal that this multiple gene cassette is also present in many Proteobacteria families, and that both its gene content and LexA-binding sequence have evolved over time, ultimately giving rise to the lexA lineage of extant Gamma Proteobacteria. [source] UPs and downs in bacterial transcription initiation: the role of the alpha subunit of RNA polymerase in promoter recognitionMOLECULAR MICROBIOLOGY, Issue 4 2000Richard L. Gourse In recent years, it has become clear that promoter recognition by bacterial RNA polymerase involves interactions not only between core promoter elements and the , subunit, but also between a DNA element upstream of the core promoter and the , subunit. DNA binding by , can increase transcription dramatically. Here we review the current state of our understanding of the , interaction with DNA during basal transcription initiation (i.e. in the absence of proteins other than RNA polymerase) and activated transcription initiation (i.e. when stimulated by transcription factors). [source] The pathogenesis of cell death in Parkinson's disease , 2007MOVEMENT DISORDERS, Issue S17 2007C. Warren Olanow MD Abstract A number of factors have been implicated in the pathogenesis of cell death in Parkinson's disease (PD). These include oxidative stress, mitochondrial dysfunction, inflammation, excitotoxicity, and apoptosis. While the precise pathogenic mechanism leading to neurodegeneration in PD is not known, there is considerable evidence suggesting that cell death occurs by way of a signal-mediated apoptotic process. PD is also characterized by intracellular proteinaceous inclusions or Lewy bodies. Proteolytic stress arises as a consequence of the excessive production of misfolded proteins, which exceed the capacity of the ubiquitin-proteasome system to degrade them. Recent genetic and laboratory studies support the possible relevance of proteolytic stress to both familial and sporadic forms of PD. Postmortem studies have shown that in the SNc of sporadic PD patients there are reduced levels of the alpha subunit of the 20S proteasome and reduced proteolytic enzyme activities. A determination as to the precise cause of cell death in PD, and the identification of specific targets for the development of drugs that might modify disease progression is one of the most critical goals in PD research. It is anticipated that over the next few years there will be a flurry of scientific activity examining the mechanism of cell death and putative neuroprotective interventions. © 2007 Movement Disorder Society [source] Abnormal Vesicular Trafficking in Mouse Models of Hermansky,Pudlak SyndromePIGMENT CELL & MELANOMA RESEARCH, Issue 2000RICHARD T. SWANK Hermansky,Pudlak Syndrome (HPS) is a group of related multigenic recessively inherited disorders which causes abnormalities in the biosynthesis and/or function of three related organelles; melanosomes, platelet-dense granules and lysosomes. These lead, in turn, to hypopigmentation, prolonged bleeding and ceroid deposition. Positional cloning strategies have identified five mouse HPS genes. Two orthologous human diseases (HPS1 and HPS2) have likewise been identified. At least four of the five mouse genes encode proteins involved in the regulation of intracellular vesicle trafficking. The pearl (HPS2) and mocha genes encode the beta3A and delta subunits, respectively, of the AP-3 adaptor complex, which captures organelle membrane proteins at the trans-Golgi apparatus. The protein products of the pallid and gunmetal genes are also important components of the vesicular trafficking machinery. The former interacts with a t-SNARE, syntaxin13, and the latter is the alpha subunit of Rab geranylgeranyltransferase, which renders Rab proteins sufficiently lipophilic to function at their target membranes. The pale ear (HPS1) gene encodes a ubiquitously expressed protein of unknown function. Recent physiological studies have shown that mouse HPS mutants, like their human HPS counterparts, have variably reduced lifespans and may have lung abnormalities. [source] Proteome analysis of human androgen-independent prostate cancer cell lines: Variable metastatic potentials correlated with vimentin expressionPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 12 2007Mingfu Wu Abstract To better understand the molecular mechanisms of prostate cancer (PCA) dissemination and to develop new anti-metastasis therapies, key regulatory molecules involved in PCA metastasis were identified in two human androgen-independent PCA cell lines, highly metastatic 1E8-H and lowly metastatic 2B4-L cells. Through 2-DE and MS analyses, 12 proteins with different expression levels in the two cell lines were identified. The following proteins were found to be significantly up-regulated in 1E8-H cells compared with 2B4-L cells: gp96 precursor, calreticulin precursor, vimentin (VIM), Hsp90,, peroxiredoxin 2, HNRPH1, ezrin, T-complex protein 1, alpha subunit, and hypothetical protein mln2339. In contrast, heart L -lactate dehydrogenase H chain, annexin I, and protein disulfide isomerase were notably down-regulated in 1E8-H cells compared with 2B4-L cells. To our knowledge, this study is the first to demonstrate that up-regulation of VIM expression positively correlates with the invasion and metastasis of androgen-independent PCA. [source] Distribution of the protein IMPACT, an inhibitor of GCN2, in the mouse, rat, and marmoset brainTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 5 2008Simone Bittencourt Abstract IMPACT is an inhibitor of GCN2, a kinase that phosphorylates the alpha subunit of the translation initiation factor 2 (eIF2,). GCN2 has been implicated in regulating feeding behavior and learning and memory in mice. IMPACT is highly abundant in the brain, suggesting its relevance in the control of GCN2 activation in the central nervous system. We describe here the distribution of IMPACT in the brain of rodents (mice and rats) and of a primate (marmoset) using highly specific antibodies raised against the mouse IMPACT protein. Neurons expressing high levels of IMPACT were found in most areas of the brain. In the hippocampal formation the lack of IMPACT in the dentate gyrus granule cells was striking. The hypothalamus is exceptionally rich in neurons expressing high levels of IMPACT, particularly in the suprachiasmatic nucleus. The only exception to this pattern was the ventromedial nucleus. The thalamic neurons are mostly devoid of IMPACT, with the exception of the paraventricular, reuniens and reticular nuclei, and intergeniculate leaf. The brainstem displayed high levels of IMPACT. For the marmoset, IMPACT expression in the brain is not as prominent when compared to other organs. In the marmoset brain the pattern of IMPACT expression was similar to rodents in most areas, except for the very strong labeling of the Purkinje cells, the lack of IMPACT-positive neurons in the nucleus reuniens, and weak labeling of interneurons in the hippocampus. GCN1, the activator of GCN2 to which IMPACT binds, is widely distributed in all neuronal populations, and all IMPACT-positive cells were also GCN1-positive. The data presented herein suggest that IMPACT may be involved in biochemical homeostatic mechanisms that would prevent GCN2 activation and therefore ATF4 (CREB-2) synthesis in neurons. J. Comp. Neurol. 507:1811,1830, 2008. © 2008 Wiley-Liss, Inc. [source] Simultaneous activation of JAK1 and JAK2 confers IL-3 independent growth on Ba/F3 pro-B cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2005Huei-Mei Huang Abstract JAK1 and JAK2 are tyrosine kinases involved in the regulation of cell proliferation, differentiation, and survival. These proteins may play a key role in mediating the effects of the cytokine IL-3 on hematopoietic cells. IL-3 induces tyrosine phosphorylation of both JAK1 and JAK2. However, it is not clear whether the activation of JAK1, JAK2, or both is sufficient to confer factor-independent growth in IL-3 dependent cells. To address this issue, fusion proteins CD16/CD7/JAK (CDJAK), comprised of a CD16 extracellular domain, a CD7 transmembrane domain, and a JAK cytoplasmic region (either a wild-type JAK or a dominant negative mutant of JAK) were constructed. We established several Ba/F3 derivatives that stably overexpress the conditionally active forms of either CDJAK1, CDJAK2, or both these fusion proteins. In this study, the autophosphorylation of CDJAK1 or CDJAK2 was induced by crosslinking with anti-CD16 antibody. We demonstrated that, like their wild-type counterparts, CDJAK1 and CDJAK2 were preassociated with the IL-3 receptor beta and alpha subunits, respectively. Furthermore, the simultaneous activation of both CDJAK1 and CDJAK2 fusion proteins, but not either one alone, led to the tyrosine phosphorylation of the IL-3 receptor beta subunit, the activation of downstream signaling molecules, including STAT5, Akt, and MAPK, and the conferring of factor-independent growth to IL-3-dependent Ba/F3 cells. Coexpression of dominant negative mutants CDJAK1KE or CDJAK2KE with wild type CDJAK2 or CDJAK1, respectively, inhibited these activation activities. These results suggest that JAK1 and JAK2 must work cooperatively and not independently and that their actions are dependent on having normal kinase activity to trigger downstream signals leading to IL-3 independent proliferation and survival of Ba/F3 cells. © 2005 Wiley-Liss, Inc. [source] Impaired inhibitory G-protein function contributes to increased calcium currents in rats with diabetic neuropathyJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 2 2002KE Hall There is a growing body of evidence that sensory neuropathy in diabetes is associated with abnormal calcium signaling in dorsal root ganglion (DRG) neurons. Enhanced influx of calcium via multiple high-threshold calcium currents is present in sensory neurons of several models of diabetes mellitus, including the spontaneously diabetic BioBred/Worchester (BB/W) rat and the chemical streptozotocin (STZ)-induced rat. We believe that abnormal calcium signaling in diabetes has pathologic significance as elevation of calcium influx and cytosolic calcium release has been implicated in other neurodegenerative conditions characterized by neuronal dysfunction and death. Using electrophysiologic and pharmacologic techniques, the present study provides evidence that significant impairment of G-protein-coupled modulation of calcium channel function may underlie the enhanced calcium entry in diabetes. N- and P-type voltage-activated, high-threshold calcium channels in DRGs are coupled to mu opiate receptors via inhibitory G(o)-type G proteins. The responsiveness of this receptor coupled model was tested in dorsal root ganglion (DRG) neurons from spontaneously-diabetic BB/W rats, and streptozotocin-induced (STZ) diabetic rats. Intracellular dialysis with GTPgammaS decreased calcium current amplitude in diabetic BB/W DRG neurons compared with those of age-matched, nondiabetic controls, suggesting that inhibitory G-protein activity was diminished in diabetes, resulting in larger calcium currents. Facilitation of calcium current density (I(DCa)) by large-amplitude depolarizing prepulses (proposed to transiently inactivate G proteins), was significantly less effective in neurons from BB/W and STZ-induced diabetic DRGs. Facilitation was enhanced by intracellular dialysis with GTPgammaS, decreased by pertussis toxin, and abolished by GDPbetaS within 5 min. Direct measurement of GTPase activity using opiate-mediated GTPgamma[(35)S] binding, confirmed that G-protein activity was significantly diminished in STZ-induced diabetic neurons compared with age-matched nondiabetic controls. Diabetes did not alter the level of expression of mu opiate receptors and G-protein alpha subunits. These studies indicate that impaired regulation of calcium channels by G proteins is an important mechanism contributing to enhanced calcium influx in diabetes. [source] G protein-independent neuromodulatory action of adenosine on metabotropic glutamate signalling in mouse cerebellar Purkinje cellsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2007Toshihide Tabata Adenosine receptors (ARs) are G protein-coupled receptors (GPCRs) mediating the neuromodulatory actions of adenosine that influence emotional, cognitive, motor, and other functions in the central nervous system (CNS). Previous studies show complex formation between ARs and metabotropic glutamate receptors (mGluRs) in heterologous systems and close colocalization of ARs and mGluRs in several central neurons. Here we explored the possibility of intimate functional interplay between Gi/o protein-coupled A1 -subtype AR (A1R) and type-1 mGluR (mGluR1) naturally occurring in cerebellar Purkinje cells. Using a perforated-patch voltage-clamp technique, we found that both synthetic and endogenous agonists for A1R induced continuous depression of a mGluR1-coupled inward current. A1R agonists also depressed mGluR1-coupled intracellular Ca2+ mobilization monitored by fluorometry. A1R indeed mediated this depression because genetic depletion of A1R abolished it. Surprisingly, A1R agonist-induced depression persisted after blockade of Gi/o protein. The depression appeared to involve neither the cAMP-protein kinase A cascade downstream of the alpha subunits of Gi/o and Gs proteins, nor cytoplasmic Ca2+ that is suggested to be regulated by the beta-gamma subunit complex of Gi/o protein. Moreover, A1R did not appear to affect Gq protein which mediates the mGluR1-coupled responses. These findings suggest that A1R modulates mGluR1 signalling without the aid of the major G proteins. In this respect, the A1R-mediated depression of mGluR1 signalling shown here is clearly distinguished from the A1R-mediated neuronal responses described so far. These findings demonstrate a novel neuromodulatory action of adenosine in central neurons. [source] | ||||||||||||||||