Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Localization of tenascin-C in vivo and cell culture experiments in vitro have provided evidence for stromal production of tenascin-C in malignant tumors of a variety of organs. Here we raised the question of whether the mesenchymal stroma in the case of endometrial adenocarcinoma is the unique source of tenascin-C. Therefore, the expression of tenascin-C mRNA by human endometrial adenocarcinoma cells and endometrial stroma cells was investigated. Several preparations of endometrial stroma cells produced tenascin-C mRNA. Using a serum-free defined cell culture medium, production of tenascin-C mRNA could be increased by adding either serum or 20 ng TGF-beta/mL to the cell culture medium. Reverse transcriptase polymerase chain reaction analysis revealed that five out of six endometrial adenocarcinoma cell lines produced tenascin-C mRNA. Northern blot experiments and ribonuclease protection assays provided evidence that the number of copies of tenascin-C mRNA was small. Analysis of expressed splice variants by reverse transcriptase polymerase chain reaction analysis revealed the abundance of one major splice variant that lacked all potential alternatively spliced fibronectin type-III-like repeats. Regarding larger splice variants, all fragment sizes that could theoretically originate from seven alternatively spliced fibronectin type-III-like repeats were observed. Evaluating relative signal intensities, the splice variants containing a single fibronectin type-III-like repeat and the variant possessing all but one alternatively spliced repeats were most frequent. In summary, evidence is provided that tenascin-C can originate from both tissue compartments of the human endometrium stroma and (tumor) epithelium. Splice variant analysis revealed a high number of splice variants and a relative high proportion of variants that have so far been regarded as minor constituents of expressed tenascin-C.
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PMID:Expression of tenascin-C by human endometrial adenocarcinoma and stroma cells: heterogeneity of splice variants and induction by TGF-beta. 959 65

Two catalytic functions were required, minimally, for the appearance of DNA in evolution: a ribonucleotide reductase (RNR) and a reverse transcriptase (RT). If one accepts the explanatory strength of the RNA world model, it is clear that DNA molecules arose in the RNA world at some stage during the early evolution of cells. I suggest that competition for limited and valuable resources such as nucleotides, amino acids, and sugars made an early appearance among RNA cells, RNA viruses, viroids, and RNA plasmids. Structural and functional similarities between the different types of polymerases favor the simple hypothesis that the first RTs were RNA polymerase mutants that preferentially joined together preexisting deoxyribonucleotide triphosphates (dNTPs) using RNA templates. What was the role of dNTPs inside cells before DNA was synthesized and tested by natural selection? The oxygen atom that is removed by the reductase is of crucial importance to many ribozyme functions, since the 2'-OH is a strong nucleophile that forms transitional states during catalysis. Consequently, a RNR may have been used by cellular parasites to inhibit ribozyme action. Thus, DNA may have been, initially, an inert by-product of retrotranscription in lineages that acquired RTs and could synthesize DNA molecules using cellular RNA templates to detoxify the intracellular environment. DNA was useless as template until a transcriptase (DNA-dependent RNA polymerase) evolved that could copy (-)DNA to reconstitute the (+)RNA genome, indeed a successful way of confronting ribonuclease threats in the RNA world.
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PMID:Inhibition of ribozymes by deoxyribonucleotides and the origin of DNA. 969 60

Cytokine-driven activation of hepatic stellate cells (HSC) in tissue injury and inflammation is a key pathogenetic event in liver fibrogenesis leading to an expanded pool of matrix producing myofibroblasts (MFB) which represent the transformed counterpart of HSC. We hypothesize that expansion of the pool of MFB might also be accomplished by modulation of apoptosis, which plays an opposite and complementary role to mitosis in the cellular homeostasis. We characterized the susceptibility of HSC in primary culture and of MFB in secondary culture to apoptosis induced by the soluble Fas ligand (sFasL) and related the effects to the expression levels of Fas (APO-1/CD95) and some major proapoptotic and contra-apoptotic protooncogenes. MFB showed a dose-dependent apoptotic reaction upon exposure to sFasL as evidenced by a strong increase of nucleosomal DNA fragments, loss of cellular DNA, positive TUNEL reaction, and annexin staining. The effect was found only if protein synthesis (cycloheximide) or RNA synthesis (actinomycin D) were arrested. HSC maintained for various times in primary culture were completely resistant to sFasL in combination with cycloheximide, but in late primary cultures (day 7 onward) an increasing susceptibility to sFasL-mediated apoptosis was developed. By semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and alkaline phosphatase-anti-alkaline phosphatase staining Fas receptor was identified both in HSC and MFB at comparable expression levels. The expression of the contra-apoptotic protooncogenes bcl-2 and bcl-xl was found to be much stronger in early HSC than in late HSC and MFB as shown by ribonuclease protection assay. The expression of bcl-2 was additionally confirmed by semiquantitative RT-PCR and immunoblotting. Proapoptotic bax was found in comparable quantities at the RNA level in HSC and MFB but at the protein level MFB showed increased bax expression. It is concluded that transformation of HSC to MFB is paralleled by an increasing sensitivity to sFasL-mediated apoptosis, which might be related to a strong decrease of bcl-2 and bcl-xl expression, leading to a preponderance of proapoptotic gene expression in MFB. Modulation of apoptotic susceptibility of transforming HSC could be an important complementary pathway in the pathogenesis of fibrosis.
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PMID:Transformation-dependent susceptibility of rat hepatic stellate cells to apoptosis induced by soluble Fas ligand. 969 16

The M1 protein of influenza virus inhibits the in vitro transcriptase activity of ribonucleoprotein cores from virions. This inhibitory activity is thought to be relevant in vivo because accumulation of M1 at the late stages of viral replication may be the cue to halt viral mRNA production. A model influenza reporter genome was used to explore the effect of M1 on the activity of the influenza virus transcriptase complex within cultured cells. Expression of M1 in cells bearing the model influenza virus reporter genome was accompanied by a reduction of CAT gene expression to 12% of control levels. Quantification of RNA by ribonuclease protection assay revealed that the influenza reporter genome mRNA levels in M1-expressing cells were reduced by approximately 74% compared with those of cells expressing a control protein. These findings are consistent with the proposed model in which M1 is responsible for limiting viral transcription during late stages of infection. By expressing truncated forms of M1, the inhibitory activity was found to reside within the amino-terminal half of the M1 protein. Two independent inhibitory domains were identified in this region: one between amino acid residues 1-90 and the other spanning residues 91-127.
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PMID:The matrix 1 protein of influenza A virus inhibits the transcriptase activity of a model influenza reporter genome in vivo. 974 Jul 76

Ternary complexes of RNA polymerase containing the DNA template and nascent RNA are the intermediates in transcript elongation in all cells. We have footprinted the RNA transcript with single-strand-specific ribonucleases in ternary complexes of Escherichia coli RNA polymerase. When complexes are treated with elevated levels of ribonucleases A and T1, the nascent transcript can be cleaved to within 3-4 nucleotides of the 3'-terminus. Ternary complexes containing ribonuclease-cleaved transcripts as short as 3 nucleotides remain stable and active, ensuring that the cleavage occurred within an active ternary complex. However, cleavage by ribonuclease I is restricted, and gives a limited digest product of about 16 nt. At lower concentrations of ribonuclease T1, two regions of partial protection are seen. The first region extends through the first 15-16 nucleotides from the 3'-OH terminus; the second region extends from position 30 out to position 45. We interpret these regions of partial protection as defining two RNA product binding sites on the RNA polymerase that bind the product to the enzyme during elongation. Our results rule out the existence of a stable RNA-DNA hybrid in these ternary complexes of greater than 3 base pairs in length.
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PMID:Structural analysis of ternary complexes of Escherichia coli RNA polymerase: ribonuclease footprinting of the nascent RNA in complexes. 989 Sep 1

Bone marrow-culture-derived macrophages activated with interferon-gamma and lipopolysaccharide produced less nitric oxide (NO) when cultured with vesicular stomatitis virus (VSV)-infected BALB/c3T3 (3T3-VSV) than macrophages activated in an identical manner and cultured alone, with uninfected BALB/c3T3 (3T3), or with P815. However, all four groups of macrophages produced nearly the same amount of interleukin-6 (IL-6). Addition of VSV to activated macrophages did not change the amount of NO produced. The amount of NO generated by two non-macrophage sources of NO was not affected by the presence of either P815 or 3T3-VSV. Reverse transcriptase-polymerase chain reaction showed a decrease in the amount of inducible nitric oxide synthase (iNOS) but not IL-6 mRNA from macrophages cocultured with 3T3-VSV compared with macrophages cocultured with P815. The reduction in iNOS mRNA was confirmed by ribonuclease protection assay. When RAW 264.7 transfected with an iNOS regulatory construct were activated and incubated with 3T3-VSV there was a decrease in the expression of the reporter luciferase gene and NO production but not IL-6 production compared with cells incubated with either medium alone or with P815.
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PMID:Interaction with vesicular stomatitis virus-infected BALB/c3T3 cells inhibits the synthesis of nitric oxide in activated murine bone marrow culture-derived macrophages. 1033 88

X-linked Charcot-Marie-Tooth disease (CMTX) is an inherited demyelinating neuropathy caused by mutations in the gene encoding the gap junction protein connexin32 (Cx32). Despite the identification of over 160 different mutations in the Cx32 coding sequence, it is not known whether the mutations cause the disease manifestations through a loss of Cx32 function or through toxic effects on peripheral nerve. We created transgenic mice with a frameshift mutation at codon 175 (175fs), identified in a large CMTX pedigree. Light microscopic examination of the peripheral nerves from adult transgenic animals showed no pathological features. Western blotting did not show transgenic Cx32 protein in any of the 26 lines, although expression of transgenic messenger RNA was detected by reverse-transcriptase polymerase chain reaction and by ribonuclease protection assay. Our findings indicate that the 175fs mutation results in a loss of Cx32 function, without additional toxic effects.
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PMID:Studies in transgenic mice indicate a loss of connexin32 function in X-linked Charcot-Marie-Tooth disease. 1041 40

Reverse transcriptase-polymerase chain reaction (PCR) was used to clone two esterase cDNAs from a diazinon-resistant field population of horn flies that expresses qualitative and quantitative differences in esterases compared with a susceptible population. The open reading frame from one of the esterase cDNAs, HialphaE7, exhibits substantial amino-acid identity to an esterase associated with diazinon resistance in Lucilia cuprina. RNA Northern blots showed that HialphaE7 mRNA was more abundant in the diazinon-resistant population than the susceptible population. DNA copy number analysis did not reveal major differences in HialphaE7 gene copy number between the two populations. The full-length cDNA to HialphaE7 was cloned and sequenced, and found to contain all of the highly conserved sequence elements associated with carboxyl/cholinesterases. The HialphaE7 homologs in diazinon-resistant strains of L. cuprina and Musca domestica have been shown to possess an amino-acid substitution conferring diazinon hydrolytic activity to the esterase enzyme. This amino-acid substitution was not found in diazinon-resistant horn flies examined by allele-specific PCR. Individual flies from the resistant field population were phenotyped as diazinon-resistant or diazinon-susceptible by topical diazinon application bioassays and total RNA isolated and hybridized to HialphaE7 probe in ribonuclease protection assays. HialphaE7 transcript was expressed at a five-fold higher level in resistant female individual flies than in susceptible female individuals.
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PMID:Cloning of a horn fly cDNA, HialphaE7, encoding an esterase whose transcript concentration is elevated in diazinon-resistant flies. 1098 98

Although 3':5' cyclic adenosine monophosphate (cAMP) is known to modulate cytokine production in a number of cell types, little information exists regarding cAMP-mediated effects on this synthetic function of human airway smooth-muscle (HASM) cells. We examined the effect of increasing intracellular cAMP concentration ([cAMP](i)) on tumor necrosis factor (TNF)-alpha-induced regulated on activation, normal T cells expressed and secreted (RANTES) and interleukin (IL)-6 secretion from cultured HASM cells. Pretreatment of HASM with prostaglandin (PG) E(2), forskolin, or dibutyryl cAMP inhibited TNF-alpha-induced RANTES secretion but increased TNF-alpha-induced IL-6 secretion. Moreover, stimulation with PGE(2), forskolin, or dibutyryl cAMP alone increased basal IL-6 secretion in a concentration-dependent manner. SB 207499, a specific phosphodiesterase type 4 inhibitor, augmented the inhibitory effects of PGE(2) and forskolin on TNF-alpha-induced RANTES. Collectively, these data demonstrate that increasing [cAMP](i) in HASM effectively increases IL-6 secretion but reduces RANTES secretion promoted by TNF-alpha. Reverse transcriptase/polymerase chain reaction and ribonuclease protection assays suggested that these opposite effects of increased [cAMP](i) on TNF-alpha- induced IL-6 and RANTES secretion may occur at the transcriptional level. Accordingly, we examined the effects of TNF- alpha and cAMP on the regulation of nuclear factor (NF)-kappaB, a transcription factor known to modulate cytokine synthesis in numerous cell types. Stimulation of HASM cells with TNF-alpha increased NF-kappaB DNA-binding activity. However, increased [cAMP](i) in HASM neither activated NF-kappaB nor altered TNF-alpha- induced NF-kappaB DNA-binding activity. These results were confirmed using a NF-kappaB-luciferase reporter assay. Together, our data suggest that TNF-alpha-induced IL-6 and RANTES secretion may be associated with NF-kappaB activation, and that inhibition of TNF-alpha-stimulated RANTES secretion and augmentation of IL-6 secretion by increased [cAMP](i) in HASM cells occurs via an NF-kappaB-independent mechanism.
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PMID:Tumor necrosis factor-alpha-induced secretion of RANTES and interleukin-6 from human airway smooth-muscle cells. Modulation by cyclic adenosine monophosphate. 1110 33

We have identified a new cyclic-nucleotide phosphodiesterase isoform, PDE3A, and cloned its cDNA from cultured aortic myocytes. The nucleotide sequence of its coding region is similar to that of the previously cloned myocardial isoform except for the absence of the initial 300-400 nt that are present in the latter, as confirmed by reverse-transcriptase-mediated PCR, 5' rapid amplification of cDNA ends and a ribonuclease protection assay. Expression in Spodoptera frugiperda (Sf9) cells yields a protein with catalytic activity and inhibitor sensitivity typical of the PDE3 family. The recombinant protein's molecular mass of approx. 131 kDa is compatible with translation from an ATG sequence corresponding to nt 436-438 of the myocardial PDE3A coding region. Antibodies against residues 424-460 (nt 1270-1380) and 1125-1141 (nt 3373-3423) of the myocardial isoform react with an approx. 118 kDa band in Western blots of homogenates of human aortic myocytes, whereas antibodies against residues 29-42 (nt 85-126) do not react with any bands in these homogenates. Our results suggest that a vascular smooth-muscle isoform ('PDE3A2') is a product of the same gene as the longer myocardial ('PDE3A1') and the shorter placental ('PDE3A3') isoforms and is generated pre-translationally in a manner that results in the absence of the 145 N-terminal amino acids of PDE3A1.
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PMID:Identification of a novel isoform of the cyclic-nucleotide phosphodiesterase PDE3A expressed in vascular smooth-muscle myocytes. 1111 97


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