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)

Administration of delavirdine, an HIV-1 reverse transcriptase inhibitor, to rats or monkeys resulted in apparent loss of hepatic microsomal CYP3A and delavirdine desalkylation activity. Human CYP3A catalyzes the formation of desalkyl delavirdine and 6'-hydroxy delavirdine, an unstable metabolite, while CYP2D6 catalyzes only desalkyl delavirdine. CYP2D6 catalyzed desalkyl delavirdine formation was linear with time (up to 30 min) but when catalyzed by cDNA expressed CYP3A4 or human liver microsomes the reaction rate declined progressively with time. Coincubation with triazolam showed that delavirdine caused a time- and NADPH-dependent loss of CYP3A4 activity in human liver microsomes as measured by triazolam 1'-hydroxylation. The catalytic activity loss was saturable and was characterized by a Ki of 21.6 +/- 8.9 microM and a kinact of 0.59 +/- 0.08 min-1. An apparent partition ratio of 41 was determined with cDNA expressed CYP3A4, based on the substrate depletion method. Incubation of [14C]delavirdine with microsomes from several species resulted in irreversible association with an approximately 50 kDa protein, as demonstrated by SDS-PAGE/autoradiography. Binding to the protein was NADPH dependent, glutathione insensitive, proportional to the level of CYP3A expression and was inhibited by ketoconazole, a specific CYP3A inhibitor. NADPH-dependent irreversible binding to human and rat total microsomal protein was demonstrated following exhaustive extraction of microsomal protein. Binding was decreased in the presence of glutathione and appeared to be related to expression level of CYP3A. These results suggest that delavirdine can inactivate CYP3A and has the potential to slow the metabolism of coadministered CYP3A substrates.
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PMID:Microsomal metabolism of delavirdine: evidence for mechanism-based inactivation of human cytochrome P450 3A. 976 59

Pulmonary cytochrome P450 monooxygenases metabolize xenobiotic chemicals, including those found in environmental tobacco smoke (ETS). Exposure to ETS beginning at birth has been shown to induce the P450 CYP1A1 by seven days of life. The effects of perinatal exposure to ETS of the rat lung on the expression of CYP1A1, 1B1, 2B1, and NADPH cytochrome P450 reductase were measured using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Timed pregnant dams and their pups were exposed to aged and diluted sidestream cigarette smoke (ADSS) as a surrogate for ETS for four hours/ day from gestational day 5 through postnatal day 21. For all genes analyzed, mRNA could be detected in the fetal lung beginning at gestational day 17 but were not altered by ADSS. In contrast, intraperitoneal injection of dams with beta-naphthoflavone significantly elevated both CYP1A1 and 1B1 at gestational day 21, indicating that these genes are inducible. Continued exposure to ADSS significantly induced CYP1A1 but not other P450 genes as early as one day after birth.. We conclude that (1) ADSS induces pulmonary CYP1A1 in the first day of life; (2) fetal cytochrome P450 genes are not induced by maternal exposure to ADSS; and (3) in the fetal lung, CYP1A1 and 1B1 can be induced by beta-naphthoflavone.
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PMID:Effect of in utero and postnatal exposure to environmental tobacco smoke on the developmental expression of pulmonary cytochrome P450 monooxygenases. 1071 27

We studied 11beta-hydroxysteroid dehydrogenase activities in the renal cell line LLC-PK1 and the effects of different steroids on them. Cortisol was oxidized in the presence of NAD as well as NADP, reflecting the presence of two different 11beta-HSD forms. Enzyme kinetics for cortisol 11beta-oxidation were: Vmax = 5.9 pmol/(min x mg), Km = 0.2 microM with NAD, and Vmax = 4.5 pmol/(min x mg), Km = 1.0 microM with NADP. Interestingly, no reverse reaction was observed when using cortisone and NADPH as substrate and cosubstrate, respectively. Exposure of cells to a variety of steroids had different effects on cortisol 11beta-oxidation rates with NADP compared to those with NAD. Dexamethasone initially (3-60 min of exposure) decreased the NAD-dependent 11beta-HSD activity to about 60%, which was no longer evident after 2 h or longer. By contrast, the 11beta-oxidation of cortisol with NADP increased by dexamethasone treatment of the cells, after a lagtime of about 2 h, and this effect was still evident after 32 h. The increase of 11beta-HSD activity with NADP by dexamethasone was concentration dependent (estimated EC50:125 nM). The antiglucocorticoid RU486 did not antagonize dexamethasone induction. Exposure of cells for 19 h to 1 microM cortisol, cortisone, progesterone, and estradiol also increased NADP-dependent cortisol 11beta-oxidation, but had no effect on the NAD-dependent 11beta-HSD activity. Immunoblot and reverse transcriptase-polymerase chain reaction experiments failed to detect any 11beta-HSD 1 protein or mRNA in these cells. Our observations suggest that in LLC-PK1 cells, two forms of 11beta-HSD exist, which differ in cosubstrate dependency, kinetics for cortisol, and modulation by steroids. Whereas the NAD-dependent form seems identical to renal 11beta-HSD 2, the NADP-dependent 11beta-HSD possibly resembles an as yet unknown third isoform.
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PMID:Characterization of 11beta-hydroxysteroid dehydrogenase activities in the renal cell line LLC-PK1: evidence for a third isoform? 1078 27

Phenobarbital (PB) induces various gene encoding drug/steroid-metabolizing enzymes such as cytochromes P450 (P450s) and transferases. Although the nuclear orphan constitutive active receptor (CAR) has been identified as a key transcription factor that regulates the induction of CYP2B, the full scope of CAR-regulated genes still remains a major question. To this end, reverse transcriptase-polymerase chain reaction and cDNA microarray techniques were employed to examine gene expression in wild-type and CAR-null mice. The results show that a total of 138 genes were detected to be either induced or repressed in response to PB treatment, of which about half were under CAR regulation. Including CYP2B10, CYP3A11, and NADPH-CYP reductase, CAR regulated a group of the PB-induced drug/steroid-metabolizing enzymes. Enzymes such as amino levulinate synthase 1 and squalene epoxidase displayed CAR-independent induction by PB. Cyp4a10 and Cyp4a14 represented the group of genes induced by PB only in CAR-null mice, indicating that CAR may be a transcription blocker that prevents these genes from being induced by PB. Additionally, the group of genes encoding enzymes and proteins involved in basic biological processes such as energy metabolism underwent the CAR-dependent repression by PB. Thus, CAR seems to have diverse roles, both as a positive and negative regulator, in the regulation of hepatic genes in response to PB beyond drug/steroid metabolism.
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PMID:Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital. 1175 99

A major source of vascular smooth muscle cell (VSMC) superoxide is NAD(P)H oxidase. However, the molecular characteristics and regulation of this enzyme are unclear. We investigated whether VSMCs from human resistance arteries (HVSMCs) possess a functionally active, angiotensin II (Ang II)-regulated NAD(P)H oxidase that contains neutrophil oxidase subunits, including p22phox, gp91phox, p40phox, p47phox, and p67phox. mRNA expression of gp91phox homologues, nox1 and nox4, was also assessed in HVSMCs, human aortic smooth muscle cells, and rat VSMCs. HVSMCs were obtained from resistance arteries from gluteal biopsies of healthy subjects. gp91phox and nox4, but not nox1, were detected in HVSMCs. Nox1 and nox4, but not gp91phox, were expressed in human aortic smooth muscle cells and rat VSMCs. All NAD(P)H oxidase subunits were present in HVSMCs as detected by reverse transcriptase-polymerase chain reaction and immunoblotting. Ang II increased NAD(P)H oxidase subunit abundance. These effects were inhibited by cycloheximide. Acute Ang II stimulation (10 to 15 minutes) increased p47phox serine phosphorylation and induced p47phox and p67phox translocation. This was associated with NAD(P)H oxidase activation. In cells transfected with gp91phox antisense oligonucleotides, Ang II-mediated actions were abrogated. NADPH-induced superoxide generation was reduced by gp91ds-tat and apocynin, inhibitors of p47phox-gp91phox interactions. Our results suggest that HVSMCs possess a functionally active gp91phox-containing neutrophil-like NAD(P)H oxidase. Ang II regulates the enzyme by inducing phosphorylation of p47phox, translocation of cytosolic subunits, and de novo protein synthesis. These novel findings provide insight into the molecular regulation of NAD(P)H oxidase by Ang II in HVSMCs. Furthermore, we identify differences in gp91phox homologue expression in VSMCs from rats and human small and large arteries.
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PMID:Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries: regulation by angiotensin II. 1206 24

Transaldolase (TAL) is an enzyme of the non-oxidative part of the pentose phosphate pathway which produces reductive potential in the form of NADPH as well as ribose 5-phosphate for incorporation into nucleotides. Developmental analysis via reverse transcriptase-polymerase chain reaction, immunoblots, enzymatic activity, in situ hybridization and immunocytochemistry demonstrate that TAL is expressed during all developmental stages in Drosophila. The tal locus is unique and contains two small introns. The first two introns in the human gene are situated at the same locations in the coding sequence as in Drosophila. Analysis of TAL protein levels and expression patterns reveals that it is also expressed in all larval tissues examined with particularly high levels in the fat body. Interestingly, we describe specific TAL expression only in non-neuronal cells in the larval brain.
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PMID:Characterization and tissue-specific expression of the Drosophila transaldolase gene. 1245 74

Recently we reported that the occurrence of lung adenoma caused by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was completely prevented by pretreatment of female A/J mice with 8-methoxypsoralen, a potent inhibitor of cytochrome P450 (P450 or CYP) 2A [Takeuchi et al. (2003) Cancer Res., 63, 7581-7583]. Thus, the aim of this study was to confirm that 8-methoxypsoralen exhibits chemopreventive effects by inhibiting CYP2A in the mouse lung. The involvement of CYP2A in the metabolic activation of NNK in the lung was first evidenced by the fact that the mutagenic activation of NNK by mouse lung microsomes was inhibited by 8-methoxypsoralen, coumarin and antibodies to rat CYP2A1. Supporting this, the mutagenic activation of NNK was efficiently catalyzed by mouse CYP2A4 and CYP2A5 co-expressed with NADPH-P450 reductase in a genetically engineered Salmonella typhimurium YG7108. The expression of mRNA for CYP2A5, but not for CYP2A4 or CYP2A12, in the mouse lung was proven by reverse transcriptase-polymerase chain reaction, probably indicating that CYP2A5 present in the mouse lung was involved in the metabolic activation of NNK. In accordance with these in vitro data, treatment of gpt delta transgenic mice with 8-methoxypsoralen prior to NNK completely inhibited the mutation of the gpt delta gene. The in vivo chemopreventive effects of 8-methoxypsoralen towards NNK-induced adenoma was seen only when the agent was given to female A/J mice prior to, but not posterior to, NNK, lending support to the idea that NNK is activated by CYP2A5 in the mouse lung as an initial step to cause adenoma. The inhibition by 8-methoxypsoralen of NNK-induced adenoma was seen in a dose-dependent manner: the dose to show apparent 50% suppression was calculated to be 1.0 mg/kg. To our surprise, CYP2A protein(s) was expressed in the lesion of NNK-induced lung adenomas, probably suggesting that 8-methoxypsoralen could inhibit the possible occurrence of further mutation of the adenoma cells induced by NNK. Based on these lines of evidence, we propose that 8-methoxypsoralen inhibits the CYP2A5-mediated metabolic activation of NNK in the mouse lung, leading to the prevention of NNK-induced adenoma.
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PMID:Mechanisms of chemopreventive effects of 8-methoxypsoralen against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mouse lung adenomas. 1595 17

Efavirenz is a non-nucleoside human immunodeficiency virus (HIV)-1 reverse transcriptase inhibitor used in combination therapy to treat HIV-1. Efavirenz metabolism is catalyzed primarily by the polymorphic enzyme P450 2B6. Metabolism of efavirenz by P450 2B6 and the naturally occurring P450 2B6.4 mutant led to the formation of 8-hydroxyefavirenz. Efavirenz inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin activity of the wild-type P450 2B6 enzyme in a time-, concentration-, and NADPH-dependent manner. However, the P450 2B6.4 variant was not inactivated by efavirenz. The ability of efavirenz to inactivate both enzymes was investigated using cyclophosphamide and bupropion, two structurally unrelated substrates of P450 2B6, as probes. Preincubations with efavirenz decreased the ability of the wild-type enzyme to hydroxylate both substrates to similar extents but had no effect on the activities of the mutant enzyme. Interestingly, the inactivation of the wild-type enzyme was completely reversible after 24 h of dialysis as determined by heme, reduced CO spectra, and activity loss. In contrast, 8-hydroxyefavirenz, a metabolite of efavirenz, was able to inactivate both enzymes irreversibly. These data suggest that incubations of P450 2B6 and P450 2B6.4 with either the parent compound efavirenz or the metabolite 8-hydroxyefavirenz in the reconstituted system result in the formation of two different reactive intermediates that lead to losses in enzymatic activity by two different mechanisms, one reversible and one irreversible.
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PMID:Metabolism of efavirenz and 8-hydroxyefavirenz by P450 2B6 leads to inactivation by two distinct mechanisms. 1661 50

Piperonyl butoxide (PBO), alpha-[2-(2-butoxyethoxy)ethoxy]-4,5-methylene-dioxy-2-propyltoluene, is widely used as a synergist for pyrethrins. In order to clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by PBO, molecular pathological analyses consisting of low-density microarray analysis and real-time reverse transcriptase (RT)-PCR were performed in male ICR mice fed a basal powdered diet containing 6000 or 0 ppm PBO for 1, 4, or 8 weeks. The animals were sacrificed at weeks 1, 4, and 8, and the livers were histopathologically examined and analyzed for gene expression using the microarray at weeks 1 and 4 followed by real-time RT-PCR at each time point. Reactive oxygen species (ROS) products were also measured using liver microsomes. At each time point, the hepatocytes of PBO-treated mice showed centrilobular hypertrophy and increased lipofuscin deposition in Schmorl staining. The ROS products were significantly increased in the liver microsomes of PBO-treated mice. In the microarray analysis, the expression of oxidative and metabolic stress-related genes--cytochrome P450 (Cyp) 1A1, Cyp2A5 (week 1 only), Cyp2B9, Cyp2B10, and NADPH-cytochrome P450 oxidoreductase (Por) was over-expressed in mice given PBO at weeks 1 and 4. Fluctuations of these genes were confirmed by real-time RT-PCR in PBO-treated mice at each time point. In additional real-time RT-PCR, the expression of Cyclin D1 gene, key regulator of cell-cycle progression, and Xrcc5 gene, DNA damage repair-related gene, was significantly increased at each time point and at week 8, respectively. These results suggest the possibility that PBO has the potential to generate ROS via the metabolic pathway and to induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in mice.
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PMID:Molecular pathological analysis for determining the possible mechanism of piperonyl butoxide-induced hepatocarcinogenesis in mice. 1701 48

Glutathione reductase (GR) plays an essential role in cell defense against reactive oxygen metabolites by sustaining the reduced status of an important antioxidant, glutathione. To address the effect of oxidative stresses on the intertidal copepod Tigriopus japonicus, we exposed specimens to hydrogen peroxide, heavy metals and different salinity levels, cloned and sequenced the oxidative stress-related GR gene. T. japonicus GR gene (Tigriopus GR) cDNA contained 1526 bp including an open reading frame (ORF) encoding 458 amino acids with a theoretical pI of 6.58 and a calculated molecular weight of 49.6 kDa. Tigriopus GR showed a high similarity to frog Xenopus laevis GR (identity 57%) and the filarial parasite, Onchocerca volvulus GR (identity 57%). Specific motifs such as flavin adenine dinucleotide-binding site (LVLGGGSGGIASARRAAEF), pyridine nucleotide-disulphide oxidoreductases class-I active site (GGTCVNVGCVP), and NADPH binding motif (GxGYIAx18Rx5R) were highly conserved in the deduced amino acid sequence of Tigriopus GR. Interestingly, its expression and enzyme characteristics were different from GR homologue of filarial parasite O. volvulus. To investigate the biochemical and enzymatic characteristics of Tigriopus GR protein, we constructed the expression vector, pCRT7/TOPO NT containing Tigriopus GR. Tigriopus pCRT7/TOPO NT/GR was expressed in Escherichia coli, and the soluble protein was purified by 6x His-tag chromatography. The recombinant Tigriopus GR enzyme was found to make homodimer complexes of approximately 108 kDa on 12% native gel electrophoresis and showed enzymatic activity with NADPH and oxidized glutathione (GSSG) as substrates. To analyze the gene expression of Tigriopus GR against different environmental stresses (hydrogen peroxide, salinity, and heavy metals), we performed real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR). Slight down-regulation in the expression of Tigriopus GR at the initial stage was observed upon exposure to hydrogen peroxide. The expression recovered in 2h, while there were significant changes upon heavy metal (Cu and Mn) exposures in a time-dependent manner. Also, Tigriopus GR expression was significantly increased with moderately high salt stress (24 and 40 ppt). In the case of low salt stress (0 and 12 ppt) the expression was found to be down-regulated. These findings provide a better understanding of cellular protection mechanisms in the intertidal copepod T. japonicus against the environmental stressors caused by non-optimal salt levels.
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PMID:Environmental stressors (salinity, heavy metals, H2O2) modulate expression of glutathione reductase (GR) gene from the intertidal copepod Tigriopus japonicus. 1707 28


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