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Post-translational Mechanisms (post-translational + mechanism)
Selected AbstractsDepolarization promotes GAD 65-mediated GABA synthesis by a post-translational mechanism in neural stem cell-derived neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2008Nidhi Gakhar-Koppole Abstract Neuronal activity regulates neurogenesis and neuronal differentiation in the mammalian brain. The commencement of neurotransmitter expression establishes the neuronal phenotype and enables the formation of functional connectivity between neurons. In addition, release of neurotransmitters from differentiating neurons may modulate the behaviour of neural precursors. Here, we show that neuronal activity regulates ,-aminobutyric acid (GABA) expression in neurons generated from stem cells of the striatum and adult subventricular zone (SVZ). Differentiating neurons display spontaneous Ca2+ events, which are voltage-gated calcium channel (VGCC) dependent. Depolarization increases both the frequency of Ca2+ transients and the amount of Ca2+ influx in differentiating neurons. We show that depolarization-dependent GABA expression is regulated by the amplitude and not by the frequency of Ca2+ influx. Brief activation of VGCCs leads to Ca2+ influx that in turn promotes a rapid expression of GABA. Depolarization-dependent GABA expression does not require changes in gene expression. Instead, it involves cAMP-dependent protein kinase (PKA) and Ca2+ and phospholipid-dependent protein kinase (PKC) signalling. Activity increases the number of glutamic acid decarboxylase (GAD) 65-immunoreactive neurons in a PKA-dependent manner, without altering the expression of GAD 65, suggesting that depolarization promotes recruitment of GAD 65 by a post-translational mechanism. In line with this, depolarization does not permanently increase the expression of GABA in neurons derived from neural stem cells of the embryonic striatum, cortex and adult SVZ. Thus, neuronal activity does not merely accelerate neuronal differentiation but it may alter the mechanism of GABA synthesis in newly generated neurons. [source] Chronic Alcohol Consumption Disrupted Cholesterol Homeostasis in Rats: Down-Regulation of Low-Density Lipoprotein Receptor and Enhancement of Cholesterol Biosynthesis Pathway in the LiverALCOHOLISM, Issue 3 2010Zhigang Wang Background:, Chronic alcohol consumption causes alcoholic liver disease, which is associated, or initiated, with dysregulated lipid metabolism. Very recent evidence suggested that dysregulated cholesterol metabolism plays an important role in the pathogenesis of alcoholic fatty liver diseases, however, the effects of chronic alcohol exposure on cholesterol homeostasis have not been well studied and underlying mechanisms behind are still elusive. Methods:, Male Sprague,Dawley rats weighing 250 ± 5.5 g (mean ± SEM) divided into 2 groups (8 rats per group) and pair-fed with liquid diets containing (in percent of energy intake) 18% protein, 35% fat, 12% carbohydrate, and 35% either ethanol (ethanol diet) or an isocaloric maltose-dextrin mixture (control diet), according to Lieber and De Carli, for 4 weeks. Results:, Long-term excessive alcohol feeding to rats caused fatty liver and liver injury, which was associated with disrupted cholesterol homeostasis, characterized by increased hepatic cholesterol levels and hypercholesterolemia. Hepatic cholesterol increases were concomitant with constantly activated sterol regulatory element-binding protein-2 (SREBP-2) in the liver and increased expression of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, a rate-limiting enzyme for cholesterol de novo synthesis, indicating enhanced cholesterol biosynthesis. Alcohol-induced hypercholesterolemia was accompanied by decreased LDL receptor (LDLr) levels in the liver. Further investigations revealed that chronic alcohol exposure increased hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) contents to down-regulate LDLr via a post-translational mechanism. Moreover, alcohol feeding suppressed extracellular signal-regulated kinase (ERK) activation in the liver. In vitro studies showed that inhibition of ERK activation was associated with decreased LDLr expression in HepG2 cells. Conclusions:, Our study provides the first evidence that both increased PCSK9 expression and suppressed ERK activation in the liver contributes to alcohol-induced hypercholesterolemia in rats. [source] Wnt signaling stabilizes the DIXDC1 protein through decreased ubiquitin-dependent degradationCANCER SCIENCE, Issue 3 2010Lei Wang (Cancer Sci 2010; 101: 700,706) Wnt signaling plays key roles in development, cell growth, differentiation, polarity formation, neural development, and carcinogenesis. DIX Domain Containing 1 (DIXDC1), a novel component of the Wnt pathway, was recently cloned. DIXDC1 is the human homolog of Ccd1, a positive regulator of the Wnt signaling pathway during zebrafish neural patterning. Little has been known about DIXDC1 gene expression regulation. In the present study, we showed that the DIXDC1 protein was induced upon Wnt-3a stimulation, whereas the DIXDC1 mRNA level was not significantly increased after Wnt-3a treatment. Positive DIXDC1 staining was detected in colon cancer cells and was colocalized with ,-catenin staining. However, the DIXDC1 mRNA expression decreased in human colon cancer cells compared to the matched normal colon epithelial cells. Our further investigation showed that the DIXDC1 protein was degraded through the proteasome pathway, and the activation of canonical Wnt signaling decreased the ubiquitin-dependent degradation of both the ectopic and endogenous DIXDC1 protein. In order to explore the possible mechanism of the ubiquitination of DIXDC1, we found that the phosphorylation of DIXDC1 was inhibited by Wnt-3a. Collectively, these results indicate that canonical Wnt/,-catenin pathway activation might upregulate DIXDC1 through a post-translational mechanism by inhibiting the ubiquitin-mediated degradation of the DIXDC1 protein. [source] Co-ordination of osmotic stress responses through osmosensing and signal transduction events in fishesJOURNAL OF FISH BIOLOGY, Issue 8 2010T. G. Evans This review centres upon the molecular regulation of osmotic stress responses in fishes, focusing on how osmosensing and signal transduction events co-ordinate changes in the activity and abundance of effector proteins during osmotic stress and how these events integrate into osmotic stress responses of varying magnitude. The concluding sections discuss the relevance of osmosensory signal transduction to the evolution of euryhalinity and present experimental approaches that may best stimulate future research. Iterating the importance of osmosensing and signal transduction during fish osmoregulation may be pertinent amidst the increased use of genomic technologies that typically focus solely on changes in the abundances of gene products, and may limit insight into critical upstream events that occur mainly through post-translational mechanisms. [source] Activation of cyclin-dependent kinases CDC2 and CDK2 in hepatocellular carcinomaLIVER INTERNATIONAL, Issue 3 2002Kay K. W. Li Abstract: Background: The cyclin-dependent kinases (CDKs) CDC2 and CDK2 are key regulators of the cell cycle. The expression of the CDK alone does not necessary reflect their true activities because they are highly regulated by post-translational mechanisms. Human hepatocellular carcinoma (HCC) is one of the most common cancers in the world, but the kinase activities of CDKs in HCC have not been examined. Methods: Here we examined the protein expression and kinase activities associated with CDC2 and CDK2 in HCC and the corresponding non-tumorous liver tissues. Results: CDC2 and CDK2 are activated in HCC in over 70% and 80% of the cases, respectively, but have little correlation with clinical parameters and PCNA expression. Interestingly, PCNA was readily detectable in extracts from non-tumorous liver, but more than 60% of samples contain higher concentration of PCNA in HCC than the corresponding non-tumorous tissues. CDC2 and CDK2 are generally activated in the same HCC samples, but the extent of their activation varied significantly, suggesting that the pathways leading to the activation of CDC2 and CDK2 can be regulated independently. Both positive regulators of CDK activity like cyclins and CDKs, and negative regulators of CDK activity like p21CIP1/WAF1 and Thr14/Tyr15 phosphorylation were up-regulated in HCC. Conclusion: CDC2 and CDK2 are activated in HCC, and this may be due to a complex interplay between the level of the cyclin, CDK, CDK inhibitors, and inhibitory phosphorylation. [source] Copper-mediated reversal of defective laccase in a ,vph1 avirulent mutant of Cryptococcus neoformansMOLECULAR MICROBIOLOGY, Issue 4 2003Xudong Zhu Summary Previous studies have shown that a ,vph1 Cryptococcus neoformans mutant defective in vesicular acidification lacked several important virulence factors including a copper-containing laccase and was avirulent in a mouse model. In the present studies, we characterized laccase transcription and protein production to obtain insights into the mechanism of the vph1 mutation in this pathogen. Although transcription and protein expression were somewhat reduced, laccase protein was found to be successfully translated and correctly targeted to the cell wall in the ,vph1 mutant as shown by Western blot and immuno-electron microscopy, despite a complete lack of laccase activity. Laccase activity was substantially restored in metabolically active ,vph1 cells at 30°C by addition of 100 µM copper sulphate. This restoration by copper was found to occur through both transcriptional and post-translational mechanisms. Laccase transcriptional induction by copper was found to be dependent on enhancer region II within the 5,-untranslated region of CNLAC1. Copper was also found to restore partial activity to ,vph1 cells at 0°C, suggesting that cell wall laccase was expressed in the mutant as an apo-enzyme. Apo-laccase restoration by copper was found to be facilitated by an acidic environment, consistent with a role for the vacuolar (H+)-ATPase proton pump in copper assembly of laccase in C. neoformans. [source] Elevated carbon dioxide increases nitrate uptake and nitrate reductase activity when tobacco is growing on nitrate, but increases ammonium uptake and inhibits nitrate reductase activity when tobacco is growing on ammonium nitratePLANT CELL & ENVIRONMENT, Issue 11 2001P. Matt Abstract The influence of elevated [CO2] on the uptake and assimilation of nitrate and ammonium was investigated by growing tobacco plants in hydroponic culture with 2 mm nitrate or 1 mm ammonium nitrate and ambient or 800 p.p.m. [CO2]. Leaves and roots were harvested at several times during the diurnal cycle to investigate the levels of the transcripts for a high-affinity nitrate transporter (NRT2), nitrate reductase (NIA), cytosolic and plastidic glutamine synthetase (GLN1, GLN2), the activity of NIA and glutamine synthetase, the rate of 15N-nitrate and 15N-ammonium uptake, and the levels of nitrate, ammonium, amino acids, 2-oxoglutarate and carbohydrates. (i) In source leaves of plants growing on 2 mm nitrate in ambient [CO2], NIA transcript is high at the end of the night and NIA activity increases three-fold after illumination. The rate of nitrate reduction during the first part of the light period is two-fold higher than the rate of nitrate uptake and exceeds the rate of ammonium metabolism in the glutamate: oxoglutarate aminotransferase (GOGAT) pathway, resulting in a rapid decrease of nitrate and the accumulation of ammonium, glutamine and the photorespiratory intermediates glycine and serine. This imbalance is reversed later in the diurnal cycle. The level of the NIA transcript falls dramatically after illumination, and NIA activity and the rate of nitrate reduction decline during the second part of the light period and are low at night. NRT2 transcript increases during the day and remains high for the first part of the night and nitrate uptake remains high in the second part of the light period and decreases by only 30% at night. The nitrate absorbed at night is used to replenish the leaf nitrate pool. GLN2 transcript and glutamine synthetase activity rise to a maximum at the end of the day and decline only gradually after darkening, and ammonium and amino acids decrease during the night. (ii) In plants growing on ammonium nitrate, about 30% of the nitrogen is derived from ammonium. More ammonium accumulates in leaves during the day, and glutamine synthetase activity and glutamine levels remain high through the night. There is a corresponding 30% inhibition of nitrate uptake, a decrease of the absolute nitrate level, and a 15,30% decrease of NIA activity in the leaves and roots. The diurnal changes of leaf nitrate and the absolute level and diurnal changes of the NIA transcript are, however, similar to those in nitrate-grown plants. (iii) Plants growing on nitrate adjust to elevated [CO2] by a coordinate change in the diurnal regulation of NRT2 and NIA, which allows maximum rates of nitrate uptake and maximum NIA activity to be maintained for a larger part of the 24 h diurnal cycle. In contrast, tobacco growing on ammonium nitrate adjusts by selectively increasing the rate of ammonium uptake, and decreasing the expression of NRT2 and NIA and the rate of nitrate assimilation. In both conditions, the overall rate of inorganic nitrogen utilization is increased in elevated [CO2] due to higher rates of uptake and assimilation at the end of the day and during the night, and amino acids are maintained at levels that are comparable to or even higher than in ambient [CO2]. (iv) Comparison of the diurnal changes of transcripts, enzyme activities and metabolite pools across the four growth conditions reveals that these complex diurnal changes are due to transcriptional and post-transcriptional mechanisms, which act several steps and are triggered by various signals depending on the condition and organ. The results indicate that nitrate and ammonium uptake and root NIA activity may be regulated by the sugar supply, that ammonium uptake and assimilation inhibit nitrate uptake and root NIA activity, that the balance between the influx and utilization of nitrate plays a key role in the diurnal changes of the NIA transcript in leaves, that changes of glutamine do not play a key role in transcriptional regulation of NIA in leaves but instead inhibit NIA activity via uncharacterized post-transcriptional or post-translational mechanisms, and that high ammonium acts via uncharacterized post-transcriptional or post-translational mechanisms to stabilize glutamine synthetase activity during the night. [source] Molecular Mechanisms of Microtubular Organelle Assembly in TetrahymenaTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2000JACEK GAERTIG ABSTRACT. Thanks to recent technological advances, the ciliate Tetrahymena thermophila has emerged as an attractive model organism for studies on the assembly of microtubular organelles in a single cell. Tetrahymena assembles 17 types of distinct microtubules, which are localix.ed in cilia, cell cortex, nuclei, and the endoplasm. These diverse microtubules have distinct morphologies, stabilities, and associations with specific Microtubule-Assoeiated Proteins. For example, kinesin-111, a microtubular motor protein, is required for assembly of cilia and is preferentially targeted to microtubules of actively assembled, immature cilia. It is unlikely that the unique properties of individual microtubules are derived from the utilization of diverse tubulin genes, because Tetrahymena expresses only a single isotype of ,-and two isotypes of ,-tubulin. However, Tetrahymena tubulins are modified secondarily by a host of post-translational mechanisms. Each microtubule organelle type displays a unique set of secondary tubulin modifications. The results of systematic in vivo mutational analyses of modification sites indicate a divergence in significance among post-translational mechanisms affecting either ,-or ,-tubulin. Both acetylation and polyglycylation of ,-tubulin are not essential and their complete elimination does not change the cell's phenotype in an appreciable way. However, the multiple polyglycylation sites on ,-tubulin are essential for survival, and their partial elimination dramatically affects cell motility, growth and morphology. Thus, both high-precision targeting of molecular motors to individual organelles as well as organelle-specific tubulin modifications contribute to the creation of diverse microtubules in a single cytoplasm of Tetrahymena. [source] Loss of Nkx3.1 expression in the transgenic adenocarcinoma of mouse prostate modelTHE PROSTATE, Issue 16 2007Carlise R. Bethel Abstract BACKGROUND The transgenic adenocarcinoma of mouse prostate (TRAMP) model has been extensively characterized at the histological and molecular levels, and has been shown to mimic significant features of human prostate cancer. However, the status of Nkx3.1 expression in the TRAMP model has not been elucidated. METHODS Immunohistochemical analyses were performed using dorsal, lateral, and ventral prostate (VP) lobes from ages 6 to 30 weeks. Quantitative RT-PCR analyses were performed to determine relative mRNA expression. RESULTS Heterogeneous loss of Nkx3.1 was observed in hyperplastic lesions of the ventral, dorsal, and lateral lobes. At 6 weeks of age, the ventral lobe displayed profound loss of Nkx3.1. Diminished Nkx3.1 protein was observed in well- to moderately-differentiated cancer lesions of all lobes. Poorly differentiated (PD) tumors stained negatively for Nkx3.1. Quantitative RT-PCR analyses revealed the presence of Nkx3.1 mRNA in each lobe at all ages, albeit reduced to variable levels. CONCLUSIONS These data suggest that disease progression in the TRAMP model may be driven by loss of function of Nkx3.1, in addition to p53 and Rb. Lobe-specific disease progression in the TRAMP model correlates with the reduction of Nkx3.1 protein. Regulation of Nkx3.1 expression during tumorigenesis appears to occur by post-transcriptional and post-translational mechanisms. Prostate 67: 1740,1750, 2007. © 2007 Wiley-Liss, Inc. [source] Glucocorticoid resistance in a multiple myeloma cell line is regulated by a transcription elongation block in the glucocorticoid receptor gene (NR3C1)BRITISH JOURNAL OF HAEMATOLOGY, Issue 6 2009Beatriz Sánchez-Vega Summary Glucocorticoid (GC) effects are mediated by the glucocorticoid receptor (GR). Several studies have demonstrated that a lower number of receptors per cell were associated with poor GC response. The regulation of GR expression is complex; the levels of GR can be autologously regulated by its ligand and also by transcriptional, post-transcriptional and post-translational mechanisms. Using three human myeloma cell lines that parallel the development of GC resistance, this work describes the mechanism involved in the downregulation of GR expression. The decreased expression was neither due to mutations in the gene encoding GR, NR3C1, nor due to methylation of the promoters. A gradual decrease in NR3C1 transcripts was seen during the development of resistance, the level of expression of exon 1 to 2 RNA fragments remained the same in sensitive and resistant cell lines but a chromatin immunoprecipitation assay demonstrated that RNA polymerase II, detectable throughout exon 2 to 3 in the sensitive cells, was undetectable on exon 3 in the resistant variant, suggesting lower or no transcription at this site. These studies demonstrated that downregulation of NR3C1 mRNA in a resistant cell line involves a block to transcriptional elongation within intron B of NR3C1. This block may represent an important element in the regulation of GR expression. [source] | |||||||||||||