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)

A rat liver nuclear envelope fraction isolated essentially by the technique of Monneron et al. (J. Cell Biol. 55, 104-125 (1972) is characterized by high levels of glucose-6-phosphatase and 5'-nucleotidase. A broadly specific nucleoside triphosphatase activity is present. Cytochromes b5 and P-450 as well as NADPH- and NADH-cytochrome c reductase activities are present but at lower levels than found in microsomes. Cytochrome c oxidase activity is low. RNA polymerase activity is absent from the nuclear envelope fraction. Cytochemistry shows that glucose-6-phosphatase activity is strong and restricted to the nuclear envelope of nuclei. 5'-Nucleotidase shows weak reaction deposit in whole nuclei but in contrast gives clear reaction deposit in isolated nuclear envelopes. Cytochemical reaction deposit due to nucleoside triphosphatase activity is not restricted to the nuclear envelope but is found to a larger extent within the nucleus.
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PMID:An enzymic analysis of a nuclear envelope fraction. 18 34

The mouse compared with the rat, is more resistent to the acute toxic action of aflatoxin B1 and is refractory to its hepatocarcinogenic properties. Aflatoxin B1 inhibits DNA synthesis more strongly than RNA synthesis in the rat, and both nucleic acid syntheses more strongly in rat than in the mouse. Mouse hepatic microsomes, like those of the rat, are capable of metabolizing aflatoxin B1 in vitro in the presence of NADPH, to an active form which binds to DNA both in solution and in intact nuclei and also inhibits nuclear RNA synthesis. Non NADPH-dependent binding of aflatoxin B1 to nuclei is not effective in inhibiting RNA polymerase and is largely removed by washing with lipid solvents. Mouse nuclear RNA polymerases particularly Mn 2+ (NH4)2SO4 primed acitivity are more resistant to inhibition in vitro by activated aflatoxin B1 than are the corresponding enzyme activities in rat liver nuclei. This would appear to be due to the bound aflatoxin B1 being less efficient in the case of the mouse nucleus, in inhibiting RNA synthesis. Mouse liver slices exhibit a much lesser degree of inhibition of RNA synthesis by aflatoxin B1 than do rat liver slices. Accompanying this is a lower level of binding of aflatoxin B1 to subcellular particulate fractions in the mouse liver slice compared to the rat, this disparity being most marked in the case of the nuclear fraction. The suggestion is made that the resistance of RNA synthesis in the mouse liver, to aflatoxin B1, and perhaps also resistance to its toxicity, is dependent, not on a lower capacity to activate the toxin, but (a) on a less efficient inhibition of RNA synthesis by nuclear bound toxin, and (b) a detoxifying mechanism at least partially situated in the cytosol fraction.
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PMID:Some studies of the effects of aflatoxin B1 in vivo and in vitro on nucleic acid synthesis in rat and mouse. 126 46

Large quantities of a catalytically active protein have been produced in a cell free system. More than 10(9) copies of protein were produced from each DNA plasmid containing DNAfol, the bacterial gene encoding dihydrofolate reductase (DHFR). The strategy employed, denoted gene amplification with transcription/translation (GATT), involves sequential coupling of (i) DNA amplification by the polymerase chain reaction (PCR) and (ii) in vitro RNA transcription by T7 RNA polymerase, followed by (iii) translation of the run-off transcripts in a rabbit reticulocyte system. The protein product had the expected size (18 kDa) and catalyzed the NADPH-dependent reduction of 7,8-dihydrofolic acid to 5,6,7,8-tetrahydrofolic acid as efficiently as authentic DHFR. Potential applications of the strategy include large scale production of enzymes containing synthetic amino acids and facilitation of the characterization of the function of genes encountered in genomic mapping studies.
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PMID:Amplification of protein expression in a cell free system. 128 16

Oxygen enhanced the bactericidal activity of rifamycin SV to Escherichia coli K12. Anaerobically grown cells, which had a low level of superoxide dismutase, were more susceptible to the bactericidal activity than aerobically grown cells, which contained a high level of superoxide dismutase. Oxygen also enhanced the inhibition of RNA polymerase activity of rifamycin SV, when Mn2+ was used as a cofactor. Rifamycin S was reduced to rifamycin SV by NADPH catalyzed by cell-free extracts of Escherichia coli K12. These results indicate that the inhibition of bacterial growth by rifamycin SV is due to the production of active species of oxygen resulting from the oxidation-reduction cycle of rifamycin SV in the cells. The aerobic oxidation of rifamycin SV to rifamycin S was induced by metal ions, such as Mn2+, Cu2+, and Co2+. The most effective metal ion was Mn2+. In the presence of Mn2+, accompanying the consumption of 1 mol of oxygen and the oxidation of 1 mol of rifamycin SV, 1 mol of hydrogen peroxide and 1 mol of rifamycin S were formed. Superoxide was generated during the autoxidation of rifamycin SV. Superoxide dismutase inhibited the formation of rifamycin S, but scavengers for hydrogen peroxide and the hydroxyl radical did not affect the oxidation. A mechanism of Mn2+-catalyzed oxidation of rifamycin SV is proposed and its relation to bactericidal activity is discussed.
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PMID:Oxygen Enhancement of bactericidal activity of rifamycin SV on Escherichia coli and aerobic oxidation of rifamycin SV to rifamycin S catalyzed by manganous ions: the role of superoxide. 627 85

Monodehydroascorbate radical (MDA) reductase, an FAD-enzyme, is the first enzyme to be identified whose substrate is an organic radical and catalyzes the reduction of MDA to ascorbate by NAD(P)H. Its cDNA has been cloned from cucumber seedlings (Sano, S., and Asada, K. (1994) Plant Cell Physiol. 35, 425-437), and a plasmid was constructed in the present study that allowed a high level expression in Escherichia coli of the cDNA-encoding MDA reductase using the T7 RNA polymerase expression system. The recombinant MDA reductase was purified to a crystalline state, with a yield of over 20 mg/liter of culture, and it exhibited spectroscopic properties of the FAD similar to those of the enzyme purified from cucumber fruits during redox reactions with NADH and MDA. The red semiquinone of the FAD of MDA reductase was generated by photoreduction. p-Chloromercuribenzoate inhibited the reduction of the enzyme-FAD by NADH, and dicumarol suppressed electron transfer from the reduced enzyme to MDA. The specificity of electron acceptors of the recombinant enzyme appeared to be similar to that of MDA reductase, even though the amino acid sequence encoded by the cDNA was somewhat different from that of the enzyme purified from cucumber fruits. The Km values for NADH and NADPH of the recombinant enzyme indicated a high affinity of the enzyme for NADH. The reaction catalyzed by the enzyme did not exhibit saturation kinetics with MDA up to 3 microM. A second order rate constant for the reduction of the enzyme-FAD with NADH was 1.25 x 10(8) M-1 s-1, as determined by a stopped-flow method, and its value decreased with increases in ionic strength, an indication of the enhanced electrostatic guidance of NADH to the enzyme-FAD.
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PMID:Molecular characterization of monodehydroascorbate radical reductase from cucumber highly expressed in Escherichia coli. 754 69

We demonstrate here that stilbene estrogen (diethylstilbestrol) is converted to nuclear protein binding metabolite(s) both in vitro and in vivo. In vitro reaction of DES with nuclei from hamster liver or kidney in the presence of cumene hydroperoxide or NADPH revealed binding of [3H]DES in nuclear proteins (histones; nonhistones precipitable by 2% TCA, NH2; nonhistones soluble in 2% TCA, NH30). The binding was significantly inhibited by cytochromes P450 inhibitors. In an in vitro system [3H]DES quinone, one of the metabolites of DES, was able to bind to pure nonhistone proteins RNA polymerase and DNA polymerase. The binding of [3H]DES quinone to nonhistones RNA polymerase and DNA polymerase was inhibited by low molecular weight thiols, i.e. glutathione and cysteine, or thiol modifiers, such as n-ethylmaleimide, dithionitrobenzoic acid and hydroxymercuric benzoate. DES and DES metabolites inhibited transcriptional activity. In vivo [3H]DES was able to bind to nuclear proteins of hamster liver, kidneys and testes. The level of in vivo [3H]DES binding to all three types of nuclear proteins (histones, NH2, NH30) in the kidney (target organ) was two or more fold higher than that observed in the liver or testis (nontarget organs). Four nuclear NH30 proteins (mol wts.: 56, 37, 33 and 28 kDa) were irreversibly bound to [3H]DES in vivo. The in vivo binding of [3H]DES to transcriptionally active chromatin NH30 proteins also was observed. The data reported here establish that DES was able to bind to liver or kidney nuclear proteins in vitro, which was catalyzed by nuclear enzymes when fortified with an appropriate cofactor. DES quinone may be one of the protein binding metabolites. DES and DES metabolites inhibited transcriptional activity. The level of in vivo binding of [3H] DES to nuclear proteins of kidney (target organ) was double in comparison with that observed in liver or testis (nontarget organs). In vivo modifications in the chromatin proteins may be a factor in the development of DES-induced renal carcinogenesis is not clear.
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PMID:In vivo binding of diethylstilbestrol to nuclear proteins of kidneys of Syrian hamsters. 773 58

Introduction of the cadmium chloride water solution to experimental animals induces changes in biochemical parameters which characterize structural and functional activity of transcriptionally active and repressed chromatin fractions. In the intoxicated chromatin-active fraction the DNA/protein ratio increases and DNA-polymerase alpha-activity decreases while in repressed chromatin activity of RNA polymerase I decreases as compared with controls. Change in intensity of lipoperoxidation reactions may underlie the cadmium chloride genotoxicity. This thesis is proved by an augmented level of NADPH-induced lipoperoxidation in active chromatin fraction.
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PMID:[Effect of cadmium chloride on DNA-, RNA-polymerase activity and lipid peroxidation of chromatin fraction in rat liver]. 816 Feb 91

The phagocyte cytochrome b558, a heterodimer comprised of gp91phox and p22phox, is a flavocytochrome that mediates the transfer of electrons from NADPH to molecular oxygen in the respiratory burst oxidase. The human gene encoding the glycosylated gp91phox subunit is the site of mutations in X-linked chronic granulomatous disease (CGD). Reverse transcriptase-polymerase chain reaction was used to obtain a full-length clone for the murine gp91phox cDNA, which was 87% identical to the human gp91phox cDNA. The encoded murine protein had 39 amino acids out of 570 that differed from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in binding to NADPH, flavin, and the cytosolic oxidase subunit p47phox. Some substitutions altered potential N-glycosylation sites, which is likely to explain why the glycosylated murine protein migrates with an apparent molecular mass of 58 kD instead of 91 kD as seen for the human protein. Expression of murine gp91phox in a human myeloid cell line with a null gp91phox allele using a mammalian expression plasmid or a retroviral vector rescued stable expression of the p22phox subunit and fully reconstituted respiratory burst activity. This suggests that the murine gp91phox subunit forms a functional cytochrome b558 heterodimer with human oxidase subunits, consistent with the high degree of identity between the mouse and human proteins in domains implicated in cytochrome function.
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PMID:Cloning of murine gp91phox cDNA and functional expression in a human X-linked chronic granulomatous disease cell line. 863 51

It has been hypothesized that plants contain respiratory burst oxidase which, upon activation, oxidize NADPH and generate extracellular superoxide, O2.-. These proteins are proposed to play a central role in defence against pathogens. However, plant DNA sequences that encode proteins with similarity to components of respiratory burst oxidase have not previously been reported. This paper describes the complete cDNA and genomic DNA sequence of the rice rbohA (for respiratory burst oxidase homologue) gene. The predicted RbohA product is most similar to the main catalytic subunit, gp91phox, of the respiratory burst oxidase of neutrophils. Reverse transcriptase PCR detects rbohA transcripts in both roots and shoots of healthy rice plants.
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PMID:rbohA, a rice homologue of the mammalian gp91phox respiratory burst oxidase gene. 881 65

The principal goal of the present study was to test the hypothesis that cytokines modulate glucose transport in skeletal muscle by increasing nitric oxide production. Cultured L6 skeletal muscle cells were incubated in the presence of tumour necrosis factor-alpha, interferon-gamma or lipopolysaccharide (LPS) alone or in combination for 24 h. Neither cytokines nor LPS alone induced NO production, as measured by nitrite concentrations in the medium. However, when used in combination, the two cytokines significantly stimulated NO production, and this effect was synergistically enhanced by the presence of LPS. Reverse transcriptase-PCR (RT-PCR) analysis revealed that NO release was associated with the induction of inducible (macrophage-type) NO synthase (iNOS). The increase in iNOS expression was confirmed at the protein level by Western-blot analysis and NADPH/diaphorase histochemical staining. Cytokines and LPS markedly increased basal glucose transport in L6 myocytes. Insulin also stimulated basal glucose transport, but significantly less in cells chronically exposed to cytokines/LPS. The sensitivity of L6 muscle cells to insulin-stimulated glucose transport was also significantly decreased by cytokines/LPS treatment. The NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) inhibited nitrite production in cytokine/LPS-treated cells, and this prevented the increase in basal glucose transport and restored muscle cell responsiveness to insulin. Cytokines/LPS exposure significantly increased GLUT1 transporter protein levels but decreased GLUT4 expression in L6 cells. l-NAME treatment prevented the increase in GLUT1 protein content but failed to restore GLUT4 transporter levels. These results demonstrate that cytokines and LPS affect glucose transport and insulin action by inducing iNOS expression and NO production in skeletal muscle cells. The data further indicate that cytokines and LPS increase the expression of the GLUT1 transporter protein by an NO-dependent mechanism.
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PMID:Cytokines modulate glucose transport in skeletal muscle by inducing the expression of inducible nitric oxide synthase. 923 Jan 32


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