Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purpose of this investigation was to examine the expression of three commonly used housekeeping genes -- glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta(2)-microglobulin (beta(2)M), and RNA polymerase 2a (polR2a) -- in elderly (E) compared to young (Y) subjects. Nine young subjects (22.7 +/- 3.4 yrs) and 11 elderly subjects (73.0 +/- 9.5 yrs) underwent a percutaneous skeletal muscle biopsy of the vastus lateralis. Equal concentrations of isolated mRNA from these samples were used to perform real-time polymerase chain reaction with primer/probe combinations specific to each gene of interest. The expression of GAPDH, beta(2)M, and polR2a was obtained as the value of cycle threshold (C(T)). An independent t-test with a level of significance at p < or = 0.05 was used to determine differences between groups. There was no difference in average C(T) of GAPDH between groups (p=0.869) (Y = 16.92 +/- 2.25 vs. E = 17.08 +/- 2.09) and polR2a (p = 0.089) (Y = 28.00 +/- 0.89 vs. E = 26.73 +/- 1.91). However, there was a significant difference (p < or = 0.05) in the average C(T) of beta(2)M (Y =21.79 +/- 0.44 vs. E = 21.05 +/- 0.51). The results indicate that special consideration needs to be made when selecting housekeeping genes for comparisons in real-time reverse-transcriptase polymerase chain reaction, depending upon the age of the populations of interest.
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PMID:Age-related changes in relative expression of real-time PCR housekeeping genes in human skeletal muscle. 1674 Dec 43

To examine the proteomes of 2 important causative agents of fish streptococcosis, Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408, we used 2-dimensional gel electrophoresis (2-DE) followed by mass spectrometry to generate 2-DE maps of these type strains. Silver-stained 2-DE gels of S. iniae ATCC29178 and L. garvieae KG9408 revealed approximately 320 and 300 spots, respectively, and immobilized pH gradient strips (13 cm, pH 4 to 7) revealed that the majority of the detected spots were concentrated in the pH range of 4.5 to 5.5. The spots were randomly selected from the 2-DE profiles and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry. The majority of the identified proteins were functionally related to energy and carbohydrate metabolism (e.g. enolase ATPase, glyceraldehyde-3-phosphate dehydrogenase) or translation and translocation (e.g. elongation factor G, elongation factor Tu, DNA-directed RNA polymerase alpha chain). These data, along with our partial 2-DE maps of S. iniae ATCC29178 and L. garvieae KG9408, may help suggest antigenic proteins for the development of effective diagnostic tools and vaccines against S. iniae and L. garvieae.
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PMID:Partial two-dimensional gel electrophoresis (2-DE) maps of Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408. 1687 93

Mechanical stimuli are known to have major influences on chondrocyte function. The molecular events that regulate chondrocyte responses to mechanical stimulation have been the subject of much study. Using an in vitro experimental system we have identified mechanotransduction pathways that control molecular and biochemical responses of human articular chondrocytes to cyclical mechanical stimulation, and how these responses differ in cells isolated from diseased cartilage. We have previously shown that mechanical stimulation of normal articular chondrocytes leads to a cell membrane hyperpolarisation. Within 1 hour following mechanical stimulation there is an increase in aggrecan mRNA levels. These responses are mediated via alpha5beta1 integrins, the neuropeptides substance P and NMDA, and the cytokine interleukin-4. In OA chondrocytes mechanical stimulation leads to cell membrane depolarisation, but no change in aggrecan mRNA at 1 hour. The depolarisation response is mediated via alpha5beta1 integrins, substance P and interleukin-4, but the cells show an altered response to NMDA. Having identified that the NMDA receptor is present in human articular cartilage and may play an important role in a chondroprotective mechanotransduction pathway, we were interested in whether other components associated with NMDA signalling may be involved in the chondrocyte mechanotransduction pathways. One such component is calcium/calmodulin-dependent protein kinase II (CaMKII). CaMKII mediates many cellular responses to elevated Ca2+ in a wide variety of cells and tissues. It is involved in the regulation of ion channels, cytoskeletal dynamics, gene transcription, neurotransmitter synthesis, insulin secretion, and cell division. CaMKII also shows a broad substrate specificity and is abundant in brain tissue, indicating that this kinase may play a number of roles in the functioning of the central nervous system. This kinase has been studied extensively in brain, but there is only a limited understanding of CaMKII in other tissues. CAMKII has four subunit isoforms (alpha,beta,gamma,delta). The alpha- and beta-isoforms have narrow distributions restricted mainly to neuronal tissues, but the gamma- and delta-isoforms are ubiquitously expressed within neuronal and non-neuronal tissues. The aim of this study was to investigate the expression of CaMKII in normal and OA cartilage and chondrocytes, and whether this enzyme is involved in the response of chondrocytes to cyclical mechanical stimuli. Reverse transcriptase-polymerase chain reaction (RT-PCR), using primers specific for the different CaMKII isoforms, was carried out to assess which isoforms are expressed in human articular chondrocytes. To assess whether CaMKII is expressed in human articular chondrocytes at the protein level, cultured chondrocytes were extracted and analysed by Western blotting using a pan-CaMKII antibody. Immunohistochemistry was carried out to investigate whether CaMKII is expressed by human articular chondrocytes in vivo. Frozen sections of normal, OA and ankle cartilage were incubated for one hour with CaMKII antibody and visualised using ABC and DAB. To assess the role of CaMKII in the mechanotransduction responses of normal and OA chondrocytes, human normal and OA articular chondrocytes were mechanically stimulated at 0.33 Hz, or by addition of recombinant IL-4 for 20 minutes. Cell responses to these stimuli, in the absence or presence of an inhibitor of CaMKII were assessed by measuring changes in cell membrane potential or changes in relative levels of aggrecan mRNA compared with the housekeeping gene GAPDH. Normal, OA, and ankle chondrocytes expressed the gamma and delta isoforms of CaMKII mRNA, but not the alpha and beta isoforms as demonstrated by RT-PCR. Western blotting showed a band at approximately 60 kDa consistent with the expression of CaMKII. Immunohistochemistry revealed the positive staining in the middle and deep zones, but not the superficial zone, of normal, OA, and ankle cartilage. The presence of a CaMKII inhibitor inhibits the membrane hyperpolarisation response and upregulation of aggrecan mRNA in normal chondrocytes following mechanical stimulation, but has no effect on the hyperpolarisation response to recombinant IL4. The depolarisation response of OA chondrocytes to mechanical stimulation is unaffected by the presence of the CaMKII inhibitor. The CaMKII isoforms gamma and delta are expressed in both normal and OA chondrocytes, both in vitro and in vivo, but are only involved in the response of normal chondrocytes to mechanical stimulation. This response is upstream of the effect of IL4. These findings are consistent with previous findings for the NMDA receptor, and suggest that dysregulation of NMDA-CaMKII signalling may be important in onset and progression of osteoarthritis.
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PMID:Calcium/calmodulin-dependent protein kinase II in human articular chondrocytes. 1691 96

Real-time PCR is frequently used for gene expression quantification due to its methodological sensitivity and reproducibility. The gene expression is quantified by normalization to one or more reference genes, usually beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPD) or to ribosomal RNA (18S). However, different environmental or pathological conditions might also influence the expression of normalizing genes, which could severely skew the interpretation of quantitative results. This study evaluates whether 16 genes frequently used as endogenous controls in expression studies, can serve as such for comparison of human brain tissues of chronic alcoholics and control subjects. The prefrontal and motor cortices that are affected differently by chronic alcohol consumption were analyzed. The reference genes that have no or small differences in expression in alcoholics and control subjects, were found to be specific for each region: beta-actin (ACTB) and ribosomal large P0 (RPLP0) for the prefrontal cortex while importin 8 (IPO8) and RNA polymerase II (POLR2A) for the motor cortex. Four out of sixteen analyzed genes demonstrated significant differences in expression between alcoholics and controls: phosphoglycerate kinase (PGK1), hypoxanthine phosphoribosyl transferase (HPRT1) and peptidylprolyl isomerase A (PPIA) in the motor cortex and beta-2-microglobulin (B2M) in the prefrontal cortex. Our study demonstrates the importance of validation of endogenous control genes prior to real-time PCR analysis of human brain tissues. Prescribed and non-prescribed drugs, pathological or environmental conditions along with alcohol abuse may differentially influence expression of reference genes.
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PMID:Validation of endogenous controls for quantitative gene expression analysis: application on brain cortices of human chronic alcoholics. 1718 56

Tumor necrosis factor-alpha (TNF-alpha) is a potent mediator of inflammation, inducing expression of a gene network mediated by NF-kappaB. Previously we found that TNF-alpha-induced reactive oxygen species (ROS) production is required for NF-kappaB action because antioxidants inhibited TNF-alpha-inducible IL-8 expression without affecting its nuclear translocation. Here, we further investigated this ROS pathway controlling NF-kappaB/RelA dependent gene expression. We observed that TNF-alpha enhanced ROS production approximately 2-fold 20 min after stimulation and significantly increased oxidative DNA damage (8-oxoguanine lesions) over controls. Treatment with chemically unrelated antioxidants specifically inhibited expression of TNF-inducible NF-kappaB-dependent genes without producing detectable cytotoxicity or affecting GAPDH expression. We found that TNF-alpha-induced NF-kappaB/RelA Ser(276) phosphorylation, a modification critical for its transcriptional activity, was inhibited by abrogation of the ROS signaling pathway, whereas NF-kappaB/RelA Ser(536) phosphorylation was not. Interestingly, antioxidant treatment selectively inhibited TNF-alpha-induced catalytic activity of cAMP dependent protein kinase A (PKAc) but not mitogen-stress related kinase-1 (MSK1), kinases known to phosphorylate RelA at Ser(276). Using PKAc inhibitors and siRNA mediated PKAc knockdown, TNF-alpha-induced Ser(276) phosphorylation and IL-8 expression were both significantly reduced, indicating PKAc is required for RelA Ser(276) phosphorylation. Consistently, a site mutation of Rel A (Ser(276) to Ala) in RelA-deficient embryonic fibroblasts failed to activate IL-8 Luciferase activity in response to TNF-alpha. Furthermore, TNF-alpha-inducible NF-kappaB/RelA interaction with the co-activator CBP/p300, essential for enhanceosome formation, was attenuated by antioxidant treatment. Using chromatin immunoprecipitation assay (ChIP), we observed that recruitment of p300 and RNA polymerase II (Pol II) to the IL-8 promoter was also abrogated by antioxidant. These results indicate that the ROS-mediated TNF-alpha-induced IL-8 transcription is regulated by NF-kappaB/RelA phosphorylation at the critical Ser(276) residue by PKAc, resulting in stable enhanceosome formation on target genes. These studies provide insight into a novel antioxidant-sensitive pathway that can be targeted to inhibit NF-kappaB-mediated inflammation.
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PMID:TNF-alpha-induced NF-kappaB/RelA Ser(276) phosphorylation and enhanceosome formation is mediated by an ROS-dependent PKAc pathway. 1731 4

The efficiency of protein synthesis for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was examined with several in vitro coupled transcription/translation protein synthesis systems based on Escherichia coli lysate, wheat germ, or reticulocyte lysate, and an in vitro translation system based on wheat germ extract. A significant amount of protein synthesis was observed only in systems based on E. coli using pET/G3PDH as the expression vector. A remarkable increase of protein synthesis was obtained in wheat germ using a pT(N)T expression vector which contains a 5'-globin leader sequence and a synthetic poly(A)(30) tail instead of pET. A significant difference of T7 RNA polymerase presence by Western blot analysis was not observed in the first four systems, and the difference of total RNA presence in each reaction mixture by Northern blot analysis seemed unrelated to protein synthesis. Although a small amount of protein was synthesized using RNA-encoding G3PDH transcribed in vitro with pET/G3PDH by an in vitro translation system, an extreme increase was observed using transcribed RNA with pEU/G3PDH, which contains T7 RNA promoter and a translation enhancer, Omega sequence. These results suggest that the presence of an enhancer sequence for translation is one of the critical steps for protein synthesis by a eukaryotic cell-free protein synthesis system.
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PMID:Efficiency of cell-free protein synthesis based on a crude cell extract from Escherichia coli, wheat germ, and rabbit reticulocytes. 1782 60

PAF, which is composed of Paf1, Cdc73, Ctr9, Leo1, and Rtf1, is a novel complex with multiple functions in transcription-related activities. The PAF complex interacts with histone-modifying enzymes and RNA polymerase II to regulate transcription. With general transcription regulatory potential in yeast, Hyrax/Cdc73 has been reported to associate with beta-catenin to control Wnt/Wg signal-specific transcription in Drosophila. Here, we present the first evidence of IL-6 signal-specific transcriptional regulation by SH2BP1/CTR9 in mammals. Upon LPS injection of mice, we observed transient induction of the mammalian PAF complex in the liver. Inhibition of CTR9 specifically abrogated expression of IL-6-responsive genes, but had no effect on genes constitutively expressed or induced by interferon-beta, TNFalpha, or IL-1beta. The PAF complex was found in the promoter regions of IL-6-responsive HP and FGGgamma, but not in the promoter region of constitutively active GAPDH. Transcriptional activation by STAT3 was inhibited when CTR9 siRNA was introduced, whereas transcriptional activation was enhanced by mCtr9 overexpression. IL-6-activated Stat3 was found to co-localize and interact with CTR9. In CTR9-depleted cells, decreased STAT3 association with the promoter regions, as well as impaired K4-trimethylation of histone H3 in the coding regions, of target genes was observed. These data suggest that CTR9 participates in the transcription of IL-6-responsive genes through the regulation of DNA association of STAT3 and modification of histone methylation.
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PMID:hCTR9, a component of Paf1 complex, participates in the transcription of interleukin 6-responsive genes through regulation of STAT3-DNA interactions. 1791 Nov 13

Gene silencing can be mediated by small interfering RNA (siRNA) and microRNA (miRNA). To investigate the potential application of using a precursor microRNA (pre-miRNA) backbone for gene silencing, we studied the inhibition efficiency of exogenous GFP and endogenous GAPDH by conventional shRNA- and pre-miRNA-designed hairpins, respectively. In this study, the conventional shRNA-, pre-miRNA-30-, and pre-miRNA-155-designed hairpins targeting either GFP or GAPDH were transfected into the HEK293 cells that were mediated by the pSilencer-4.1-neo vector, which carries a modified RNA polymerase II-type CMV promoter. Comparisons with conventional GFP shRNA showed that GFP levels were reduced markedly by pre-miRNA-30- and pre-miRNA-155-designed GFP shRNAs by fluorescence microscopy. The consistent results from semi-quantitative RT-PCR and Western blot analysis revealed that pre-miRNA-30- and pre-miRNA-155-designed GFP shRNAs could suppress GFP expression significantly. As for endogenous GAPDH, the results from semi-quantitative RT-PCR and Western blot analysis showed that pre-miRNA-30- and pre-miRNA-155-designed GAPDH shRNAs could suppress GAPDH expression even more efficiently than conventional GAPDH shRNA. Together, this study confirmed the efficiency of gene silencing mediated by pre-miRNA-30- and pre-miRNA-155-designed shRNAs, demonstrating that pre-miRNA-designed hairpins are a good strategy for gene silencing.
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PMID:An efficient method to enhance gene silencing by using precursor microRNA designed small hairpin RNAs. 1875 92

Lactococcus garvieae is an important etiological agent of lactococcosis in various fish species including olive flounder (Paralichthys olivaceus). In this study, proteomic and immunoproteomic analyses were employed to compare the antigenic profiles of strains KG9408, MS93003, and NSS9310 strains of L. garvieae. Proteomic analysis using two-dimensional gel electrophoresis (2-DE) revealed differences in five protein spots among the different L. garvieae strains. In immunoproteomic analysis, there was a significant difference in the 2-DE immunoblot profiles of the L. garvieae strains using sera collected from fish surviving infection with either L. garvieae strains KG9408 or NSS9310. These sera reacted with 8 and 7 unique antigenic protein spots, respectively. Heat shock protein (HSP) 70 and DNA-directed RNA polymerase were among the specific antigens recognized by the anti-NSS9310 serum. In addition, the anti-NSS9310 and anti-KG9408 olive flounder sera reacted with 25 common antigenic protein spots of all the L. garvieae strains, which included elongation factor (EF)-Tu, arginine deiminase (AD), inosine-5'-monophosphate dehydrogenase (IMPD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphomannomutase (PMM), L-lactate dehydrogenase (L-LDH), 6-phosphofructokinase and UDP-galactose 4-epimerase (UDP-galactose). Based on the present results, the 8 antigens recognized by the anti-KG9408 serum and the 25 common antigens recognized by both sera may serve as potential markers for developing an effective vaccine against this bacterium.
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PMID:Comparison of antigenic proteins from Lactococcus garvieae KG- and KG+ strains that are recognized by olive flounder (Paralichthys olivaceus) antibodies. 1955 79

We examined whether transcription elongation factors control constitutive transcription of the histone H1(0) and GAPDH genes. Chromatin immunoprecipitation demonstrated positive transcription elongation factor b (P-TEFb) and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) present together with RNA polymerase II (pol II) throughout the histone H1(0) gene, whereas negative elongation factor (NELF) was confined to the 5' region. Contrarily, DSIF, NELF and pol II were confined to the 5' region on the GAPDH. Inhibition of those factors affected the constitutive transcription of the histone H1(0) gene but not the GAPDH gene. Thus, NELF, DSIF and P-TEFb control constitutive transcription in a gene-specific manner.
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PMID:The transcription elongation factors NELF, DSIF and P-TEFb control constitutive transcription in a gene-specific manner. 1965 8


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