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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Drug metabolizing enzymes, particularly those involved in the metabolism of carcinogenic chemicals, were characterized in cultured human keratinocytes. Using immunoblotting experiments, we analysed the expression of phase I enzymes, cytochrome P4501A1 (CYP1A1) and NADPH reductase, and phase II enzymes, phenol UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) isoform pi, in the presence of either classical inducers (i.e. 3-methylcholanthrene, dimethylbenz[a]anthracene, phenobarbital, and clofibrate) or all-trans retinoic acid (RA). This study has shown that the expression of CYP1A1 and UGT is concomitantly induced by 3-methylcholanthrene, dimethylbenz[a]anthracene, and RA, and that of NADPH reductase is only enhanced by phenobarbital and RA. In contrast, the expression of GST pi was not affected by the inducers. Using the reverse transcriptase-polymerase chain reaction, we have demonstrated that the effects of 3-methylcholanthrene, dimethylbenz[a]anthracene and RA on CYP1A1 expression correlate with an increase of CYP1A1 mRNA level. Our results indicate that, with the exception of clofibrate, xenobiotics and RA differentially modulate the expression of drug metabolizing enzymes.
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PMID:Constitutive and inducible expression of drug metabolizing enzymes in cultured human keratinocytes. 775 27

11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) catalyzes the conversion of active cortisol to inactive cortisone, and regulates the access of cortisol to both the mineralocorticoid and glucocorticoid receptors. Two isoforms of 11 beta-HSD have been described, the cloned "type 1" NADP(H)-dependent dehydrogenase/oxo-reductase and a high affinity NAD-dependent dehydrogenase (type 2). In the fetus, 11 beta-HSD activity may serve to protect developing tissues from cortisol excess or may modulate the permissive actions of glucocorticoids. We have studied 11 beta-HSD activity and mRNA levels in human mid-gestational fetal tissues. Tissue homogenates were incubated with either 0.1 mumol/L cortisol and 400 mumol/L NAD, 2.5 mumol/L cortisol and 400 mumol/L NADP, or 0.1 mumol/L cortisone wither either 400 mumol/L NADPH or NADH. No activity (< 2.5% conversion) was observed in fetal tissues using either cortisone or 2.5 mumol/L cortisol as a substrate. 11-oxo-reductase activity was observed in maternally-derived decidua. In keeping with these activity studies, northern blot analysis of fetal tissue RNA and PCR-reverse transcriptase of type 1 11 beta-HSD mRNA indicated 11 beta-HSD mRNA in decidua, but failed to detect any type 1 11 beta-HSD mRNA transcripts in fetal tissues. In contrast when 0.1 mumol/L cortisol was used as a substrate in the presence of NAD, 11 beta-HSD activity was ubiquitous with highest levels seen in the kidney (131 +/- 16 (SE) pmoles cortisone formed/h/mg.protein) > lung > gonad > liver > colon. 11 beta-HSD activity in fetal tissues is mediated by the type 2, high affinity, isoform. The widespread distribution of this novel isoform suggests that it may play an important role in fetal development. Type 1 11 beta-HSD mRNA and activity are absent in mid-gestational fetal tissues, but present in maternally-derived decidua, suggesting that its ontogeny is a late-gestational of post-natal event.
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PMID:Type 2 11 beta-hydroxysteroid dehydrogenase in human fetal tissues. 820 Sep 59

In plants tetrapyrrole synthesis is initially light regulated on the level of 5-aminolevulinate (ALA) synthesis. ALA is formed from glutamate in three enzymatic steps. Glutamyl tRNA reductase (GluTR) catalyses the NADPH-dependent reduction of glutamyl tRNA to glutamate 1-semialdehyde. GluTR is encoded by a low-copy gene family consisting of three to four genes. Three different cDNA clones are presented. Full-length clones BHA1 and 87 differ in the length of the 3' untranslated region and code for a 58.5 kDa protein. The sequence of the partial clone, BHA13, contains at least 87 base mismatches in the coding region for the mature GluTR resulting in 11 amino acid substitutions. Synthesis of a recombinant mature and a truncated GluTR lacking the first 19 amino terminal amino acids in Escherichia coli lead only in the latter case to complementation of an E. coli hemA mutant. Steady-state level of BHA1- and BHA13-specific mRNA encoding GluTR were analysed by Northern blot hybridization using cDNA-specific oligo nucleotides and quantitative reverse transcriptase-polymerase chain reaction. Accumulation of the two RNA species is light induced in greening barley and controlled during cellular development. In contrast to BHA13, BHA1 transcripts are present in roots and are elevated after cytokinin treatment of dark-grown seedlings. Furthermore, BHA1 mRNA shows oscillation under circadian growth conditions. GluTR transcript levels correlate with the capacity for ALA synthesis indicating that the rate-limiting substrate flux through the ALA synthesizing pathway can be at least partially attributed to GluTR expression. Consequences of the initial control of the chlorophyll metabolic pathway on the level of ALA formation are discussed.
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PMID:Members of a low-copy number gene family encoding glutamyl-tRNA reductase are differentially expressed in barley. 869 65

Human bronchial epithelial cells (BEC), a primary defense against inhaled materials, are the progenitor cells for bronchogenic carcinomas and have important metabolic capabilities. We used reverse transcriptase-polymerase chain reaction (RT-PCR) to identify xenobiotic metabolism enzymes expressed in primary BEC and alveolar macrophages (AM) of non-smoking volunteers. Cytochromes P450 (CYP) 1A1, 1B1, 2B7, 2E1, and 4B1 and microsomal epoxide hydrolase (mEH) were expressed in BEC but not AM. CYP2F1 was expressed in BEC, but it was expressed at barely detectable levels or not at all in AM. NADPH oxidoreductase (NADPH OR), microsomal glutathione transferase (GST 12), glutathione transferase mu, phenol sulfotransferase (PST), thermolabile phenol sulfotransferase (TL PST), and the clara cell-specific gene, CC10 were expressed in both BEC and AM. CYP3A4 and glucuronosyl transferases-1 and 2 were not expressed in either BEC or AM. In contrast to primary BEC, of the genes evaluated, the immortalized human bronchial epithelial cell line BEP2D constitutively expressed only CYP1A1, CYP2E1, NADPH OR, glucuronosyl transferase 1, GST 12, GST mu, PST, TL PST, and CC10. The loss of xenobiotic metabolism enzyme gene expression in the BEP2D cell line may result from either reduced exposure to inducing agents, or loss of differentiative characteristics in culture. It is clear from the data comparing BEC and AM that there are important intertissue differences in expression of xenobiotic metabolism enzymes.
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PMID:Xenobiotic metabolism enzyme gene expression in human bronchial epithelial and alveolar macrophage cells. 884 77

We have isolated a 1276-base pair cDNA from a rat heart cDNA library that encodes a novel thioredoxin (Trx2) of 166 amino acid residues with a calculated molecular mass of 18.2 kDa. Trx2 possesses the conserved thioredoxin-active site, Trp-Cys-Gly-Pro-Cys, but lacks structural cysteines present in all mammalian thioredoxins. Trx2 also differs from the previously described rat thioredoxin (Trx1) by the presence of a 60-amino acid extension at the N terminus. This extension has properties characteristic for a mitochondrial translocation signal, and the cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein of 12.2 kDa. Western blot analysis from cytosolic, peroxisomal, and mitochondrial rat liver cell fractions confirmed mitochondrial localization of Trx2. Northern blot and reverse transcriptase-polymerase chain reaction analyses revealed that Trx2 hybridized to a 1.3-kilobase message, and it was expressed in several tissues with the highest expression levels in heart, muscle, kidney, and adrenal gland. N-terminally truncated recombinant protein was expressed in bacteria and characterized biochemically. Trx2 possessed a dithiol-reducing enzymatic activity and, with mammalian thioredoxin reductase and NADPH, was able to reduce the interchain disulfide bridges of insulin. Furthermore, Trx2 was more resistant to oxidation than Trx1.
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PMID:Cloning and expression of a novel mammalian thioredoxin. 900 39

The lack of sequence information and clones of porcine pulmonary artery endothelial cell (PAEC) constitutive nitric oxide synthase (ecNOS) cDNA limits comparative analysis between porcine and human PAEC. Therefore, we cloned, characterized and expressed the ecNOS cDNA from porcine PAEC. Two oligonucleotide primers were designed based on the published human ecNOS cDNA sequence and used to clone porcine PAEC ecNOS using 5' and 3' rapid amplification of cDNA ends reverse transcriptase polymerase chain reaction technique. A full-length ecNOS cDNA was cloned and sequenced, representing a protein of 1205 amino acids with a molecular mass of 134 kDa. A mammalian expression vector (pcDNA3) containing this cDNA was transfected into COS-7 cells, and ecNOS activity was detected by monitoring the formation of [3H]-citrulline from [3H]-L-arginine. Expression of ecNOS activity was predominantly associated (> 90%) with the total membrane fraction of these transfected cells. The deduced amino acid sequence of porcine ecNOS cDNA, containing binding sites for NADPH, flavin adenine dinucleotide and bound flavin mononucleotide, shows 94% identity to human ecNOS. The molecular weight of porcine ecNOS mRNA was estimated to be 4.7 kb by Northern blot analysis, similar to human ecNOS mRNA. This suggests that porcine ecNOS is similar to human ecNOS in deduced amino acid sequence and structure.
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PMID:Molecular cloning, characterization and expression of a nitric oxide synthase from porcine pulmonary artery endothelial cells. 914 2

Complementary DNA clones of mRNAs for sheep and goat NADPH-ferredoxin reductases (ferredoxin NADP+ oxidoreductase, EC 1.18.1.2) were isolated by the reverse transcriptase polymerase chain reaction method, and the complete nucleotide sequences of the coding and 3'-flanking regions of these cDNA clones were determined. Comparative analysis using the deduced amino acid sequences of NADPH-ferredoxin reductases clarified the interspecific conservation of the ferredoxin-binding and flavin adenine dinucleotide (FAD)-binding regions, confirming the results reported previously. During this study, we happened to identify an alternatively spliced mRNA that completely lacks exon 3, just adjacent to the FAD-binding region of the sheep NADPH-ferredoxin reductase cDNA clone. In the screening of other alternatively spliced mRNAs of NADPH-ferredoxin reductases derived from several steroidogenic organs, such as adrenocortices, testes, and ovaries of sheep and goats, only one kind of alternatively spliced mRNA as described above was detected in sheep adrenocortices. Then, we constructed Escherichia coli expression systems for these two forms of mRNA and analyzed their enzymatic properties. We found that the ability of the alternatively spliced NADPH-ferredoxin reductase protein to transfer electrons to ferredoxin is completely abolished because FAD binding is inhibited.
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PMID:Molecular cloning of sheep and goat ferredoxin reductase messenger ribonucleic acids, and identification of an alternatively spliced form of sheep ferredoxin reductase. 916 Jul 36

Bronchial epithelial cells (BEC) are the progenitors of bronchogenic carcinomas and are exposed to polycyclic aromatic hydrocarbon (PAH) procarcinogens through inhalation of combustion products. PAH are converted to carcinogenic molecules through a combination of monoxygenation by cytochrome p450 (CYP) enzymes in the presence of NADPH oxidoreductase (OR) and hydrolysis by microsomal epoxide hydrolase (mEH). In artificial systems, the relative expression of these genes determines whether carcinogenic or noncarcinogenic species are generated during metabolism. This relationship was explored in humans by using quantitative competitive reverse transcriptase polymerase chain reaction amplification to determine the range of expression of CYP1A1, CYP1B1, mEH, and NADPH OR in BEC recovered from 10 nonsmokers and 9 smokers. CYP2B7 expression was evaluated because, although little is known of its substrate specificity, it is expressed at high levels in human lung tissue. CYP1A1 and CYP1B1 were expressed in BEC at significantly different levels (P < 0.05) in the 9 smokers at 1.4 +/- 2.3 x 10(4) and 2.4 +/- 3.2 x 10(3) molecules/10(6) beta-actin molecules (mean +/- STD), respectively, but each was measurable in only one of the 10 nonsmokers. There was significant inter-individual variation (P < 0.05) in both CYP1A1 and CYP1B1 expression among the subjects for whom sufficient data were obtained. The inducibility of human BEC CYP1A1 gene by PAH exposure was confirmed in vitro by incubating cultured immortalized human BEC with beta-naphthoflavone and observing a > 6-fold induction of CYP1A1 after 24 h. In contrast to BEC, alveolar macrophages expressed CYP1A1 at low (30-70 molecules/10(6) beta-actin molecules) to unmeasurable levels in both smokers and nonsmokers. There was no significant difference in expression of mEH, CYP2B7, or NADPH OR in smokers compared with nonsmokers. The inter-individual variation in absolute and relative expression of PAH metabolism enzymes in BEC reported here supports the hypothesis that inter-individual variation in ability to activate/inactivate inhaled PAH carcinogens accounts for at least some of the inter-individual variation in risk for bronchogenic carcinoma.
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PMID:Quantitative RT-PCR measurement of cytochromes p450 1A1, 1B1, and 2B7, microsomal epoxide hydrolase, and NADPH oxidoreductase expression in lung cells of smokers and nonsmokers. 922 17

Superoxide radical (O2-) is ubiquitously critical to the bioactivity of endothelial nitric oxide. In angiotensin-dependent hypertension, vascular O2- levels rise and impede endothelium/nitric oxide-dependent vascular relaxation. We have reported that the major O2- source in the rabbit aorta is adventitial fibroblast phagocyte-like NADPH oxidase and shown that angiotensin (Ang) II treatment of adventitial fibroblasts causes a concentration-dependent increase in particulate NADPH-dependent O2-. From cultured rabbit aortic adventitial fibroblasts treated or not treated with Ang II, we prepared particulate fractions and measured lucigenin-enhanced chemiluminescence. Because [Sar1,Thr8]-Ang II, a generalized antagonist of Ang II and plausible inhibitor of the conversion of Ang II, reversed Ang II (10 nmol/L)-induced NADH- and NADPH-dependent O2- to basal levels, we tested the effect of the inhibitor of aminopeptidase N, amastatin (10 micromol/L), and found no effect on Ang II-stimulated O2-. Ang(1-7), Ang III, and Ang IV also were not effective in stimulating O2- levels at concentrations similar to those of Ang II. Kinetic analysis showed a rise in NADPH oxidase O2- production in response to Ang II, which peaks at 3 hours and returns to basal levels by 16 hours. p67phox, a cytosolic factor, appears to be affected at both the level of transcription and protein synthesis because actinomycin and cycloheximide individually inhibited the observed effect. A partial sequence of p67phox was recovered by reverse transcriptase from mRNA harvested from cultured rabbit aortic adventitial fibroblasts. Furthermore, the p67phox mRNA transcript in aortic fibroblasts is induced by Ang II before the peak of NADPH oxidase by Northern analysis and ribonuclease protection assays. These data suggest that Ang II stimulates NAD(P)H oxidase O2- generation in fibroblasts of aortic adventitia via transcriptional activation of p67phox. These data also provide preliminary evidence for the regulation of factors of the NADPH oxidase and potentially provide a novel means by which to abrogate the development of O2(-)-dependent hypertension.
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PMID:Angiotensin II induces p67phox mRNA expression and NADPH oxidase superoxide generation in rabbit aortic adventitial fibroblasts. 971 63

1. S-1153, a non-nucleoside agent that is under development in the USA as a new anti-HIV agent, has potent antiviral activity based on the inhibition of reverse transcriptase. 2. S-1153 was incubated with rat liver microsomes and NADPH, and seven metabolites were formed. The main metabolites were identified as the S-oxide, N-oxide and sulphone of S-1153. 3. Two other minor metabolites were assumed to be S-1153 hydroxylated on the isopropyl moiety. 4. Our findings confirmed the existence of at least three oxidative metabolic pathways of S-1153.
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PMID:Structural determination of metabolites of S-1153, a new, potent, non-nucleoside, anti-HIV agent in rat liver microsomes. 976 29


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