Home About us Contact
|
Home About us Contact | ||
|
|
||
Post-translational Regulation (post-translational + regulation)
Selected AbstractsPost-translational Regulation of Endothelial Nitric Oxide Synthase (eNOS) by Estrogens in the Rat VaginaTHE JOURNAL OF SEXUAL MEDICINE, Issue 5 2010Biljana Musicki PhD ABSTRACT Introduction., Estrogens control vaginal blood flow during female sexual arousal mostly through nitric oxide (NO). Although vascular effects of estrogens are attributed to an increase in endothelial NO production, the mechanisms of endothelial NO synthase (eNOS) regulation by estrogens in the vagina are largely unknown. Aims., Our hypothesis was that estrogens regulate eNOS post-translationally in the vagina, providing a mechanism to affect NO bioavailability without changes in eNOS protein expression. Methods., We measured eNOS phosphorylation and eNOS interaction with caveolin-1 and heat shock protein 90 (HSP90) in the distal and proximal vagina of female rats at diestrus, 7 days after ovariectomy and 2 days after replacement of ovariectomized rats with estradiol-17, (15 µg). Main Outcome Measures., Molecular mechanisms of eNOS regulation by estrogen in the rat vagina. Results., We localized phospho-eNOS (Ser-1177) immunohistochemically to the endothelium lining blood vessels and vaginal sinusoids. Estrogen withdrawal decreased phosphorylation of eNOS on its positive regulatory site (Ser-1177) and increased eNOS binding to its negative regulator caveolin-1 (without affecting eNOS/HSP90 interaction), and they were both normalized by estradiol replacement. Protein expressions of phosphorylated Akt (protein kinase B) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) were not affected by estrogen status, suggesting that the effect of estrogens on eNOS (Ser-1177) phosphorylation was not mediated by activated AKT or ERK1/2. eNOS phosphorylation on its negative regulatory site (Ser-114) was increased in the vagina by estrogen withdrawal and normalized by estradiol replacement, implying that the maintenance of low phosphorylation of eNOS on this site by estradiol may limit eNOS interaction with caveolin-1 and preserve the enzyme's activity. Total eNOS, inducible NOS, caveolin-1, and HSP90 protein expressions were not affected by ovariectomy or estradiol replacement in the distal or proximal vagina. Conclusions., These results define novel estrogen signaling mechanisms in the vagina which involve eNOS phosphorylation and eNOS,caveolin-1 interaction. Musicki B, Liu T, Strong TD, Lagoda GA, Bivalacqua TJ, and Burnett AL. Post-translational regulation of endothelial nitric oxide synthase (eNOS) by estrogens in the rat vagina. J Sex Med 2010;7:1768,1777. [source] Post-translational regulation of EAAT2 function by co-expressed ubiquitin ligase Nedd4-2 is impacted by SGK kinasesJOURNAL OF NEUROCHEMISTRY, Issue 4 2006Christoph Boehmer Abstract The human excitatory amino acid transporter (EAAT)2 is the major glutamate carrier in the mammalian CNS. Defective expression of the transporter results in neuroexcitotoxicity that may contribute to neuronal disorders such as amyotrophic lateral sclerosis (ALS). The serum and glucocorticoid inducible kinase (SGK) 1 is expressed in the brain and is known to interact with the ubiquitin ligase Nedd4-2 to modulate membrane transporters and ion channels. The present study aimed to investigate whether SGK isoforms and the related kinase, protein kinase B (PKB), regulate EAAT2. Expression studies in Xenopus oocytes demonstrated that glutamate-induced inward current (IGLU) was stimulated by co-expression of SGK1, SGK2, SGK3 or PKB. IGLU is virtually abolished by Nedd4-2, an effect abrogated by additional co-expression of either kinase. The kinases diminish the effect through Nedd4-2 phosphorylation without altering Nedd4-2 protein abundance. SGKs increase the transporter maximal velocity without significantly affecting substrate affinity. Similar to glutamate-induced currents, [3H] glutamate uptake and cell surface abundance of the transporter were increased by the SGK isoforms and down-regulated by the ubiquitin ligase Nedd4-2. In conclusion, all three SGK isoforms and PKB increase EAAT2 activity and plasma membrane expression and thus, may participate in the regulation of neuroexcitability. [source] Post-translational regulation of phosphatidylglycerolphosphate synthase in response to inositolMOLECULAR MICROBIOLOGY, Issue 4 2004Quan He Summary Phosphatidylglycerolphosphate synthase (Pgs1p) catalyses the committed step in the synthesis of cardiolipin (CL). This is the only step of CL synthesis that is regulated by inositol. We have shown previously that Pgs1p enzyme activity is decreased within minutes after supplementation with inositol, but PGS1 expression is unaltered. We utilized an epitope-tagged Pgs1p to determine if the rapid decrease in activity following inositol was because of degradation or inactivation of the protein. In this report, we show that, in response to inositol, the decrease in CL content and Pgs1p enzyme activity are associated with increased phosphorylation of Pgs1p, but not with degradation or mislocalization of the protein. This is the first evidence of phosphorylation of a phospholipid biosynthetic enzyme in response to inositol and identifies a new mechanism of inositol-mediated regulation. [source] Post-translational Regulation of Endothelial Nitric Oxide Synthase (eNOS) by Estrogens in the Rat VaginaTHE JOURNAL OF SEXUAL MEDICINE, Issue 5 2010Biljana Musicki PhD ABSTRACT Introduction., Estrogens control vaginal blood flow during female sexual arousal mostly through nitric oxide (NO). Although vascular effects of estrogens are attributed to an increase in endothelial NO production, the mechanisms of endothelial NO synthase (eNOS) regulation by estrogens in the vagina are largely unknown. Aims., Our hypothesis was that estrogens regulate eNOS post-translationally in the vagina, providing a mechanism to affect NO bioavailability without changes in eNOS protein expression. Methods., We measured eNOS phosphorylation and eNOS interaction with caveolin-1 and heat shock protein 90 (HSP90) in the distal and proximal vagina of female rats at diestrus, 7 days after ovariectomy and 2 days after replacement of ovariectomized rats with estradiol-17, (15 µg). Main Outcome Measures., Molecular mechanisms of eNOS regulation by estrogen in the rat vagina. Results., We localized phospho-eNOS (Ser-1177) immunohistochemically to the endothelium lining blood vessels and vaginal sinusoids. Estrogen withdrawal decreased phosphorylation of eNOS on its positive regulatory site (Ser-1177) and increased eNOS binding to its negative regulator caveolin-1 (without affecting eNOS/HSP90 interaction), and they were both normalized by estradiol replacement. Protein expressions of phosphorylated Akt (protein kinase B) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) were not affected by estrogen status, suggesting that the effect of estrogens on eNOS (Ser-1177) phosphorylation was not mediated by activated AKT or ERK1/2. eNOS phosphorylation on its negative regulatory site (Ser-114) was increased in the vagina by estrogen withdrawal and normalized by estradiol replacement, implying that the maintenance of low phosphorylation of eNOS on this site by estradiol may limit eNOS interaction with caveolin-1 and preserve the enzyme's activity. Total eNOS, inducible NOS, caveolin-1, and HSP90 protein expressions were not affected by ovariectomy or estradiol replacement in the distal or proximal vagina. Conclusions., These results define novel estrogen signaling mechanisms in the vagina which involve eNOS phosphorylation and eNOS,caveolin-1 interaction. Musicki B, Liu T, Strong TD, Lagoda GA, Bivalacqua TJ, and Burnett AL. Post-translational regulation of endothelial nitric oxide synthase (eNOS) by estrogens in the rat vagina. J Sex Med 2010;7:1768,1777. [source] Post-translational regulation of expression and conformation of an immunoglobulin domain in yeast surface displayBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Ranganath Parthasarathy Abstract Display of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding. CD47's extracellular domain is fused to the yeast mating protein Aga2p on the cell wall, and the resulting fusion protein binds several key antibodies, including a conformation-sensitive antibody. Site-by-site mutagenesis of CD47's five N-linked glycosylation sites progressively decreases expression levels on yeast, but folding appears stable. Cysteine mutations disrupt the expected core disulfide, and also decrease protein expression levels, though not to the extent seen with complete deglycosylation. However, with the core disulfide mutants, antibody binding proves to be lower than expected from expression levels and glycosylation is clearly reduced compared to wild-type. The results indicate that glycosylation regulates heterologous display on yeast more than core disulfides do and thus suggest bounds on directed evolution by post-translational processing. © 2005 Wiley Periodicals, Inc. [source] The taurine transporter: mechanisms of regulationACTA PHYSIOLOGICA, Issue 1-2 2006X. Han Abstract Taurine transport undergoes an adaptive response to changes in taurine availability. Unlike most amino acids, taurine is not metabolized or incorporated into protein but remains free in the intracellular water. Most amino acids are reabsorbed at rates of 98,99%, but reabsorption of taurine may range from 40% to 99.5%. Factors that influence taurine accumulation include ionic environment, electrochemical charge, and post-translational and transcriptional factors. Among these are protein kinase C (PKC) activation and transactivation or repression by proto-oncogenes such as WT1, c-Jun, c-Myb and p53. Renal adaptive regulation of the taurine transporter (TauT) was studied in vivo and in vitro. Site-directed mutagenesis and the oocyte expression system were used to study post-translational regulation of the TauT by PKC. Reporter genes and Northern and Western blots were used to study transcriptional regulation of the taurine transporter gene (TauT). We demonstrated that (i) the body pool of taurine is controlled through renal adaptive regulation of TauT in response to taurine availability; (ii) ionic environment, electrochemical charge, pH, and developmental ontogeny influence renal taurine accumulation; (iii) the fourth segment of TauT is involved in the gating of taurine across the cell membrane, which is controlled by PKC phosphorylation of serine 322 at the post-translational level; (iv) expression of TauT is repressed by the p53 tumour suppressor gene and is transactivated by proto-oncogenes such as WT1, c-Jun, and c-Myb; and (v) over-expression of TauT protects renal cells from cisplatin-induced nephrotoxicity. [source] Post-translational and cell type-specific regulation of CXCR4 expression by cytokinesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2003Hilke Brühl Abstract We have investigated the regulation and function of the chemokine receptor CXCR4 on neutrophils. CXCR4 is hardly detectable on neutrophils in the peripheral blood. However, overnight culture strongly up-regulates CXCR4 expression on the cell surface. The functional activity of CXCR4 on cultured neutrophils was confirmed by stromal cell-derived factor (SDF)-induced migration and up-regulation of the integrins CD11b and CD11c. CXCR4 surface expression on neutrophils but not on lymphocytes and monocytes is rapidly down-regulated after stimulation with TNF-, and IFN-,, resulting in significantly decreased SDF-induced functional responses of neutrophils. In contrast to surface expression, CXCR4 mRNA expression was several-fold increased in cytokine-stimulated neutrophils, suggesting a post-translational regulation. By confocal microscopy we demonstrate that CXCR4 is internalized after stimulation with TNF-, and IFN-,. The down-modulation of CXCR4 surface expression in response to TNF-, and IFN-, was fully reversible after cytokine removal. Further, CXCR4 down-modulation could be completely blocked by hypertonic sucrose and significantly reduced by chlorpromazine indicating the involvement of clathrin-coated pits. Internalization of CXCR4 by cytokines in a cell type-specific manner is a novel and functionally important mechanism of chemokine receptor regulation. [source] An integrated view of the regulation of NKG2D ligandsIMMUNOLOGY, Issue 1 2009Noam Stern-Ginossar Summary NKG2D is one of the best characterized activating receptors and is expressed on natural killer cells and on various T-cell subsets. This receptor recognizes several different ligands that are induced by cellular stresses. In this review, we described the mechanisms controlling the expression of NKG2D ligands, with the emphasis on post-transcriptional and post-translational regulation. [source] Antiproliferative activity of CCN3: Involvement of the C-terminal module and post-translational regulation,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2007A.M. Bleau Abstract Previous work had suggested that recombinant CCN3 was partially inhibiting cell proliferation. Here we show that native CCN3 protein secreted into the conditioned medium of glioma transfected cells indeed induces a reduction in cell proliferation. Large amounts of CCN3 are shown to accumulate both cytoplasmically and extracellularly as cells reach high density, therefore highlighting new aspects on how cell growth may be regulated by CCN proteins. Evidence is presented establishing that the amount of CCN3 secreted into cell culture medium is regulated by post-translational proteolysis. As a consequence, the production of CCN3 varies throughout the cell cycle and CCN3 accumulates at the G2/M transition of the cycle. We also show that CCN3-induced inhibition of cell growth can be partially reversed by specific antibodies raised against a C-terminal peptide of CCN3. The use of several clones expressing various portions of CCN3 established that the CT module of CCN3 is sufficient to induce cell growth inhibition. J. Cell. Biochem. 101: 1475,1491, 2007. © 2007 Wiley-Liss, Inc. [source] Down-regulation of protein l -isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulinJOURNAL OF NEUROCHEMISTRY, Issue 3 2002Julie Lanthier Abstract Protein l -isoaspartyl methyltransferase (PIMT) repairs the damaged proteins which have accumulated abnormal aspartyl residues during cell aging. Gene targeting has elucidated a physiological role for PIMT by showing that mice lacking PIMT died prematurely from fatal epileptic seizures. Here we investigated the role of PIMT in human mesial temporal lobe epilepsy. Using surgical specimens of hippocampus and neocortex from controls and epileptic patients, we showed that PIMT activity and expression were 50% lower in epileptic hippocampus than in controls but were unchanged in neocortex. Although the protein was down-regulated, PIMT mRNA expression was unchanged in epileptic hippocampus, suggesting post-translational regulation of the PIMT level. Moreover, several proteins with abnormal aspartyl residues accumulate in epileptic hippocampus. Microtubules component ,-tubulin, one of the major PIMT substrates, had an increased amount (two-fold) of l -isoaspartyl residues in the epileptic hippocampus. These results demonstrate that the down-regulation of PIMT in epileptic hippocampus leads to a significant accumulation of damaged tubulin that could contribute to neuron dysfunction in human mesial temporal lobe epilepsy. [source] Platelet nitric oxide synthase is activated by tyrosine dephosphorylation: possible role for SHP-1 phosphataseJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2006B. PATEL Summary.,Background:,Endothelial nitric oxide synthase (eNOS) activity in endothelial cells is regulated by post-translational phosphorylation of critical serine, threonine and tyrosine residues in response to a variety of stimuli. However, the post-translational regulation of eNOS in platelets is poorly defined. Objectives:,We investigated the role of tyrosine phosphorylation in the regulation of platelet eNOS activity. Methods:,Tyrosine phosphorylation of eNOS and interaction with the tyrosine phosphatase SHP-1 were investigated by coimmunoprecipitation and immunoblotting. An in vitro immunoassay was used to determine eNOS activity together with the contribution of protein tyrosine phosphorylation. Results:,We found platelet eNOS was tyrosine phosphorylated under basal conditions. Thrombin induced a dose- and time-dependent increase in eNOS activity without altering overall level of tyrosine phosphorylation, although we did observe evidence of minor tyrosine dephosphorylation. In vitro tyrosine dephosphorylation of platelet eNOS using a recombinant protein tyrosine phosphatase enhanced thrombin-induced activity compared to thrombin alone, but had no effect on endothelial eNOS activity either at basal or after stimulation with bradykinin. Having shown that dephosphorylation could modulate platelet eNOS activity we examined the role of potential protein phosphatases important for platelet eNOS activity. We found SHP-1 protein tyrosine phosphatase, co-associated with platelet eNOS in resting platelets, but does not associate with eNOS in endothelial cells. Stimulation of platelets with thrombin increased SHP-1 association with eNOS, while inhibition of SHP-1 abolished the ability of thrombin to induce elevated eNOS activity. Conclusions:,Our data suggest a novel role for tyrosine dephosphorylation in platelet eNOS activation, which may be mediated by SHP-1. [source] Primary and secondary metabolism, and post-translational protein modifications, as portrayed by proteomic analysis of Streptomyces coelicolorMOLECULAR MICROBIOLOGY, Issue 4 2002A. R. Hesketh Summary The newly sequenced genome of Streptomyces coelicolor is estimated to encode 7825 theoretical proteins. We have mapped approximately 10% of the theoretical proteome experimentally using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Products from 770 different genes were identified, and the types of proteins represented are discussed in terms of their anno-tated functional classes. An average of 1.2 proteins per gene was observed, indicating extensive post-translational regulation. Examples of modification by N-acetylation, adenylylation and proteolytic processing were characterized using mass spectrometry. Proteins from both primary and certain secondary metabolic pathways are strongly represented on the map, and a number of these enzymes were identified at more than one two-dimensional gel location. Post-translational modification mechanisms may therefore play a significant role in the regulation of these pathways. Unexpectedly, one of the enzymes for synthesis of the actinorhodin polyketide antibiotic appears to be located outside the cytoplasmic compartment, within the cell wall matrix. Of 20 gene clusters encoding enzymes characteristic of secondary metabolism, eight are represented on the proteome map, including three that specify the production of novel metabolites. This information will be valuable in the characterization of the new metabolites. [source] Evidence for post-translational regulation of NrtA, the Aspergillus nidulans high-affinity nitrate transporterNEW PHYTOLOGIST, Issue 4 2007Ye Wang Summary ,,Here, influx and efflux of , and net fluxes of and , were measured in Aspergillus nidulans mutants niaD171 and niiA5, devoid of nitrate reductase (NR) and nitrite reductase (NiR) activities, respectively. ,,Transcript and protein abundances of NrtA, the A. nidulans principal high-affinity transporter, were determined using semiquantitative reverse transcription-polymerase chain reaction and western blots, respectively. , influx in niaD171 was negligible relative to wild-type values, whereas efflux to influx ratios increased nine-fold. Nevertheless, NrtA mRNA and NrtA protein were expressed at levels more than two-fold and three-fold higher, respectively, in niaD171 than in the wild-type strain. ,,This is the first demonstration of diminished high-affinity influx associated with elevated transporter levels, providing evidence that, in addition to transcriptional regulation, control of NrtA expression operates at the post-translational level. This mechanism allows for rapid control of transport at the protein level, reduces the extent of futile cycling of that would otherwise represent a significant energy drain when influx exceeds the capacity for assimilation or storage, and may be responsible for the rapid switching between the on and off state that is associated with simultaneous provision of to mycelia absorbing . [source] A systems biology investigation of the MEP/terpenoid and shikimate/phenylpropanoid pathways points to multiple levels of metabolic control in sweet basil glandular trichomesTHE PLANT JOURNAL, Issue 3 2008Zhengzhi Xie Summary The glandular trichome is an excellent model system for investigating plant metabolic processes and their regulation within a single cell type. We utilized a proteomics-based approach with isolated trichomes of four different sweet basil (Ocimum basilicum L.) lines possessing very different metabolite profiles to clarify the regulation of metabolism in this single cell type. Significant differences in the distribution and accumulation of the 881 highly abundant and non-redundant protein entries demonstrated that although the proteomes of the glandular trichomes of the four basil lines shared many similarities they were also each quite distinct. Correspondence between proteomic, expressed sequence tag, and metabolic profiling data demonstrated that differential gene expression at major metabolic branch points appears to be responsible for controlling the overall production of phenylpropanoid versus terpenoid constituents in the glandular trichomes of the different basil lines. In contrast, post-transcriptional and post-translational regulation of some enzymes appears to contribute significantly to the chemical diversity observed within compound classes for the different basil lines. Differential phosphorylation of enzymes in the 2- C -methyl- d -erythritol 4-phosphate (MEP)/terpenoid and shikimate/phenylpropanoid pathways appears to play an important role in regulating metabolism in this single cell type. Additionally, precursors for different classes of terpenoids, including mono- and sesquiterpenoids, appear to be almost exclusively supplied by the MEP pathway, and not the mevalonate pathway, in basil glandular trichomes. [source] Repression of light signaling by Arabidopsis SPA1 involves post-translational regulation of HFR1 protein accumulationTHE PLANT JOURNAL, Issue 1 2005Jianping Yang Summary Arabidopsis uses two major classes of photoreceptors to mediate seedling de-etiolation. The cryptochromes (cry1 and cry2) absorb blue/ultraviolet-A light, whereas the phytochromes (phyA,phyE) predominantly regulate responses to red/far-red light. Arabidopsis COP1 represses light signaling by acting as an E3 ubiquitin ligase in the nucleus, and is responsible for targeted degradation of a number of photomorphogenesis-promoting factors, including HY5, LAF1, phyA, and HFR1. Distinct light signaling pathways initiated by multiple photoreceptors (including both phytochromes and cryptochromes) eventually converge on COP1, causing its inactivation and nuclear depletion. Arabidopsis SPA1, which encodes a protein structurally related to COP1, also represses light signaling under various light conditions. In this study, we present genetic evidence supporting that HFR1, which encodes a photomorphogenesis-promoting bHLH transcription factor, acts downstream of SPA1 and is required for different subsets of branch pathways of light signaling controlled by SPA1 under different light conditions. We show that SPA1 physically interacts with HFR1 in a yeast two-hybrid assay and an in vitro co-immunoprecipitation assay. We demonstrate that higher levels of HFR1 protein accumulate in the spa1 mutant background under various light conditions, including far-red, red, blue, and white light, whereas a marginal increase in HFR1 transcript level is only seen in dark- and far-red light-grown spa1-100 mutants. Together, our data suggest that repression of light signaling by Arabidopsis SPA1 likely involves post-translational regulation of HFR1 protein accumulation. [source] Expression analysis suggests novel roles for the plastidic phosphate transporter Pht2;1 in auto- and heterotrophic tissues in potato and ArabidopsisTHE PLANT JOURNAL, Issue 1 2004Christine Rausch Summary A cDNA encoding Pht2;1 from potato, a new member of the plant Pht2 gene family of low-affinity orthophosphate (Pi) transporters, was isolated. The expression pattern of the corresponding gene as well as its ortholog from Arabidopsis was analyzed and the encoded proteins were localized in the two plants. Pht2;1 expression is strongly upregulated by light in potato and Arabidopsis leaf tissue. RNA gel blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), promoter/GUS, and protein/green fluorescent protein (GFP) fusion studies, respectively, indicate that the gene is expressed in both auto- and heterotrophic tissues and its encoded protein is localized to the plastids. The similar patterns of Pht2;1 gene regulation in potato and Arabidopsis prompted us to screen publicly available gene expression data from 228 Arabidopsis oligonucleotide microarrays covering 83 different experimental conditions. Modulation of Pht2;1 transcript levels was overall moderate, except for a limited number of experimental conditions where Pht2;1 mRNA concentrations varied between 2- and 3.7-fold. Overall, these analyses suggest involvement of the Pht2;1 protein in cell wall metabolism in young, rapidly growing tissues, independent of other Pi transporters such as the high-affinity Solanum tuberosum Pi transporter 1 (StPT1). Cluster analysis allowed identification of colinear or antiparallel expression profiles of a small set of genes involved in post-translational regulation, and photosynthetic carbon metabolism. These data give clues about the possible biological function of Pht2;1 and shed light on the complex web of interactions in which Pht2;1 could play a role. [source] Inhibition of calcium-calmodulin kinase restores nitric oxide production and signaling in submandibular glands of a mouse model of salivary dysfunctionBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2004Florencia Rosignoli Nitric oxide is an intracellular and diffusible messenger of neurotransmitters involved in salivary secretion, as well as an inflammatory mediator in salivary gland diseases. It is synthesized by three different isoforms of nitric oxide synthase (NOS), each subject to a fine transcriptional, post-transcriptional and/or post-translational regulation. Our purpose was to study the possible mechanisms leading to NOS downregulation in submandibular glands of normal mice and in the nonobese diabetic (NOD) mouse model of salivary dysfunction with lower NOS activity. NOS activity and cGMP accumulation were determined by radioassays in submandibular glands of both mice in the presence of the protein kinase inhibitors KN-93 and bisindolylmaleimide. NOS I mRNA and protein expression and localization were assessed by RT,PCR, Western blot and immunohistochemistry. A downregulatory effect of calcium,calmodulin kinase II (CaMK II) on NOS activity in submandibular glands of both NOD and BALB/c mice was observed. Our results are consistent with a physiological regulation of NOS activity by this kinase but not by PKC in normal BALB/c mice. They are also supportive of a role for CaMK II in the lack of detectable NOS activity in submandibular glands of NOD mice. KN-93 also restored cGMP accumulation in NOD submandibular glands. The downregulation of NOS in NOD mice seems to be mainly mediated by this kinase rather than the result of a lower expression or different cellular localization of the enzyme. It was not related to different substrate or cofactors availability either. British Journal of Pharmacology (2004) 143, 1058,1065. doi:10.1038/sj.bjp.0705952 [source] A heat labile soluble factor from Bacteroides thetaiotaomicron VPI-5482 specifically increases the galactosylation pattern of HT29-MTX cellsCELLULAR MICROBIOLOGY, Issue 5 2001Miguel Freitas The aim of this work was to set up and validate an in vitro model to study a molecular response of an intestinal host cell line (HT29-MTX), to a non-pathogen microflora component. We found that Bacteroides thetaiotaomicron strain VPI-5482 had the capacity to change a specific glycosylation process in HT29-MTX cells via a mechanism that involved a soluble factor. Differentiated HT29-MTX cells were grown in the presence of 20% of spent culture supernatant from the B. thetaiotaomicron during 10 days. Glycosylation processes were followed using a large panel of lectins and analysed using confocal microscopy, western blotting and flow cytometry techniques. Our results show that a B. thetaiotaomicron soluble factor modified specifically the galactosylation pattern of HT29-MTX cells, whereas other glycosylation steps remained mainly unaffected. Further characterization of this soluble factor indicates that it is a heat labile, low molecular weight compound. Reverse transcript-PCR (RT-PCR) analysis was unable to show any significant change in mRNA expression level of the main galactosyltransferases expressed in HT29-MTX cells. By contrast, galactosyltransferase activities dramatically increased in HT29-MTX cells treated by the soluble extract of B. thetaiotaomicron, suggesting a post-translational regulation of these activities. Our in vitro model allowed us to study the cross-talk between a single bacteria and intestinal cells. The galactosylation process appears to be a target of this communication, thus uncovering a new window to study the functional consequences of co-operative symbiotic bacterial,host interactions. [source] | |||||||||||||