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)

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

Chronic granulomatous disease (CGD) is a genetic disorder of NADPH oxidase in which phagocytes are defective in generating reactive oxidants. CGD patients suffer from recurrent infections and exuberant and persistent tissue granuloma formation. We hypothesized that abnormal granulomata in CGD may result from aberrant T-cell-mediated cytokine responses. To assess Th-1-type cytokine responses and granulomata, we challenged p47(phox-/-) and wild-type mice with avirulent (SmD) or virulent (SmT) variants of Mycobacterium avium 2-151. To assess Th-2-type cytokine responses and granulomata, we used Schistosoma mansoni eggs (SME). Mononuclear cells were harvested, and cytokine responses were determined by enzyme-linked immunosorbent assay or reverse transcriptase PCR. Following SmD or SmT challenge, splenocytes from p47(phox-/-) and wild-type mice generated similar polar Th-1 responses (increased levels of gamma interferon and basal levels of interleukin 4 [IL-4] and IL-5). By 8 weeks after SmT challenge, exuberant splenic granulomata developed in p47(phox-/-) and wild-type mice. After SME challenge, thoracic lymph node mononuclear cells from p47(phox-/-) and wild-type mice generated similar mixed Th-1 and Th-2 cytokine responses to SME antigen and concanavalin A. Peak lung granuloma sizes and rates of regression were similar in p47(phox-/-) and wild-type mice. These results suggest that exuberant granulomatous inflammation in CGD is probably not the result of skewing of T-cell responses toward the Th-1 or Th-2 pole. Appropriate regression of established tissue granulomata in p47(phox-/-) mice challenged with SME suggests that abnormal granuloma formation in CGD is stimulus dependent and is not an invariant feature of the disease.
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PMID:The p47(phox-/-) mouse model of chronic granulomatous disease has normal granuloma formation and cytokine responses to Mycobacterium avium and Schistosoma mansoni eggs. 1008

Deficiencies in neutrophil NADPH oxidase proteins have been demonstrated in humans with chronic granulomatous disease. However, no spontaneous mutation in murine NADPH oxidase has been reported. In this study we report that neutrophils from the diabetic mouse strains, C57BL/6J-m heterozygous lean (lepr(db/+)) and homozygous obese (lepr(db/db)) mice produced no superoxide on stimulation. An absence of intact p47(phox) but not other oxidase proteins was observed in both mouse strains through the use of immunoblotting. Molecular analysis by reverse transcriptase-polymerase chain reaction identified three abnormal p47phox mRNA transcripts. Sequencing of genomic DNA of p47(phox) revealed a point mutation at the -2 position of exon 8, which is consistent with aberrant splicing of the p47(phox) transcript. These results indicate that the C57BL/6J-m db/db and db/+ mice are the first spontaneously derived murine model of NADPH oxidase deficiency involving a p47(phox) mutation.
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PMID:P47(phox)-deficient NADPH oxidase defect in neutrophils of diabetic mouse strains, C57BL/6J-m db/db and db/+. 1067 May 82

Superoxide anions (O(*-)(2)) induce oxidative stress and reduce endothelial NO availability by peroxynitrite formation. In human endothelial cells gp91(phox) was identified as the limiting subunit of the forming NAD(P)H oxidase. Because endothelin-1 (ET-1) is considered as a pro-atherosclerotic stimulus, we analyzed the effect of ET-1 on gp91(phox) expression and O(*-)(2) generation in primary cultures of human umbilical vein endothelial cells (HUVECs). The gp91(phox) mRNA expression was quantified by standard calibrated competitive reverse transcriptase-polymerase chain reaction. ET-1 induces gp91(phox) mRNA expression in HUVEC (max. after 1 h). The induction of gp91(phox) expression was dose-dependent, reaching its maximum at 10 nmol/L ET-1. The increased gp91(phox) expression is mediated by endothelial receptor type B (ET(B)). Furthermore, ET-1 augments O(*-)(2) generation in human endothelial cells as measured by coelenterazine chemiluminescence. These data support a new mechanism: how ET-1 increases oxidative stress in the vessel wall leading to endothelial dysfunction and enhanced susceptibility to atherosclerosis.
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PMID:Endothelin-1 induces NAD(P)H oxidase in human endothelial cells. 1072 Apr 82

Evidence is rapidly accumulating that low-activity-reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases homologous to that in phagocytic cells generate reactive oxygen species as signaling intermediates in both endothelium and vascular smooth muscle. We therefore explored the possibility of such an oxidase regulating growth of airway smooth muscle (AWSM). Proliferation of human AWSM cells in culture was inhibited by the antioxidants catalase and N-acetylcysteine, and by the flavoprotein inhibitor diphenylene iodonium (DPI). Membranes prepared from human AWSM cells generated superoxide anion (O) measured by superoxide dismutase-inhibitable lucigenin chemiluminescence, with a distinct preference for NADPH instead of reduced nicotinamide adenine dinucleotide as substrate. Chemiluminescence was also inhibited by DPI, suggesting the presence of a flavoprotein containing oxidase generating O as a signaling molecule for cell growth. Examination of human AWSM cells by reverse transcriptase-polymerase chain reaction consistently demonstrated transcripts with sequences identical to those reported for p22(phox). Transfection with p22(phox) antisense oligonucleotides reduced human AWSM proliferation. Inhibition of NADPH oxidase activity with DPI prevented serum-induced activation of nuclear factor-kappaB (NF-kappaB), and overexpression of a superrepressor form of the NF-kappaB inhibitor IkappaBalpha significantly reduced human AWSM growth. These findings suggest that an NADPH oxidase containing p22(phox) regulates growth-factor responsive human AWSM proliferation, and that the oxidase signals in part through activation of the prototypical redox-regulated transcription factor NF-kappaB.
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PMID:NADPH oxidase promotes NF-kappaB activation and proliferation in human airway smooth muscle. 1188 Mar 5

We report here two atypical cases of X-linked CGD patients (first cousins) in which cytochrome b(558) is present at a normal level but is not functional (X91+). The mutations were localized by single-strand conformational polymorphism of reverse transcriptase-polymerase chain reaction amplified fragments and then identified by sequence analysis. They consisted in two base substitutions (C919 to A and C923 to G), changing His303 to Asn and Pro304 to Arg in the cytosolic gp91phox C-terminal tail. Mismatched polymerase chain reaction and genomic DNA sequencing showed that mothers had both wild-type and mutated alleles, confirming that this case was transmitted in an X-linked fashion. A normal amount of FAD was found in neutrophil membranes, both in the X91+ patients and their parents. Epstein-Barr virus-transformed B lymphocytes from the X91+ patients acidified normally upon stimulation with arachidonic acid, indicating that the mutated gp91phox still functioned as a proton channel. A cell-free translocation assay demonstrated that the association of the cytosolic factors p47phox and p67phox with the membrane fraction was strongly disrupted. We concluded that residues 303 and 304 are crucial for the stable assembly of the NADPH oxidase complex and for electron transfer, but not for its proton channel activity.
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PMID:Molecular and functional characterization of a new X-linked chronic granulomatous disease variant (X91+) case with a double missense mutation in the cytosolic gp91phox C-terminal tail. 1199 83

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

During the host defense process, neutrophils migrate into infected tissues where they become activated, resulting in the assembly of a superoxide anion-generating complex known as the NADPH oxidase. Despite the importance of this system in animal host defense, almost nothing is known about the NADPH oxidase in neutrophils from wild ruminant species. In the present studies, we provide a molecular analysis of the bison leukocyte NADPH oxidase. Using reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends, we cloned and sequenced the full-length cDNAs for five bison NADPH oxidase components: p22(phox), p40(phox), p47(phox) and p67(phox), and gp91(phox). When compared to other species, the deduced amino acid sequences of the bison homologs were most similar to those of bovine. Interestingly, a bison p40(phox) alternative splice product was isolated, which was similar to that observed for human p40(phox) in that the cDNAs contained sequence from intron 8. Consistent with the high degree of similarity between bison and bovine amino acid sequences, immunoblot analysis showed that the bison homologs migrated similarly to their bovine counterparts. Overall, these studies show that the bison and bovine NADPH oxidase genes are highly conserved between these two species, despite their divergence from a common ancestor over 1 million years ago.
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PMID:Molecular analysis of the bison phagocyte NADPH oxidase: cloning and sequencing of five NADPH oxidase cDNAs. 1222 6

High glucose (HG) is the underlying factor contributing to long term complications of diabetes mellitus. The molecular mechanisms transforming the glomerular mesangial cell phenotype to cause nephropathy including diacylglycerol-sensitive protein kinase C (PKC) are still being defined. Reactive oxygen species (ROS) have been postulated as a unifying mechanism for HG-induced complications. We hypothesized that in HG an interaction between ROS generation, from NADPH oxidase, and PKC suppresses mesangial Ca2+ signaling in response to endothelin-1 (ET-1). In primary rat mesangial cells, growth-arrested (48 h) in 5.6 mM (NG) or 30 mm (HG) glucose, the total cell peak [Ca2+]i response to ET-1 (50 nM) was 630 +/- 102 nM in NG and was reduced to 159 +/- 15 nM in HG, measured by confocal imaging. Inhibition of PKC with phorbol ester down-regulation in HG normalized the ET-1-stimulated [Ca2+]i response to 541 +/- 74 nM. Conversely, an inhibitory peptide specific for PKC-zeta did not alter Ca2+ signaling in HG. Furthermore, overexpression of conventional PKC-beta or novel PKC-delta in NG diminished the [Ca2+]i response to ET-1, reflecting the condition observed in HG. Likewise, catalase or p47phox antisense oligonucleotide normalized the [Ca2+]i response to ET-1 in HG to 521 +/- 58 nM and 514 +/- 48 nM, respectively. Pretreatment with carbonyl cyanide m-chlorophenylhydrazone or rotenone did not restore Ca2+ signaling in HG. Detection of increased intracellular ROS in HG by dichlorofluorescein was inhibited by catalase, diphenyleneiodonium, or p47phox antisense oligonucleotide. HG increased p47phox mRNA by 1.7 +/- 0.1-fold as measured by reverse transcriptase-PCR. In NG, H2O2 increased membrane-enriched PKC-beta and -delta, suggesting activation of these isozymes. HG-enhanced immunoreactivity of PKC-delta visualized by confocal imaging was attenuated by diphenyleneiodium chloride. Thus, mesangial cell [Ca2+]i signaling in response to ET-1 in HG is attenuated through an interaction mechanism between NADPH oxidase ROS production and diacylglycerol-sensitive PKC.
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PMID:High glucose-suppressed endothelin-1 Ca2+ signaling via NADPH oxidase and diacylglycerol-sensitive protein kinase C isozymes in mesangial cells. 1282 78

Recent reports indicate that NAD(P)H oxidase 1 (Nox1) mRNA undergoes alternative splicing, producing a short transcript (NOH-1S) encoding a novel H+ channel. Although the H+ transport properties of NOH-1S-transfected cells resemble those of many cells, the production of a NOH-1S protein was never documented. We characterized Nox1 transcripts in colon-derived cells and present evidence that mRNA splicing does not produce NOH-1S; rather, NOH-1S appears to be an artifact of template switching during cDNA synthesis. The NOH-1S transcript was not observed by Northern blotting, despite claims of its abundance based on RNase protection assays. The shortened cDNA was generated by avian myeloblastosis virus reverse transcriptase, but not by thermally stable reverse transcriptase under conditions that produce full-length Nox1. Analysis of shortened cDNAs detected NOH-1S sequence and other variants that differ at the alleged splice junction site. Although no appropriate RNA splicing sites were found within Nox1 to account for NOH-1S formation, we found repetitive sequence elements bordering the deleted region, which could promote intramolecular template switching during cDNA synthesis. Template switching was confirmed in vitro, where the deleted cDNA was generated by avian myeloblastosis virus reverse transcriptase from a synthetic, full-length Nox1 RNA template. A survey of the expressed sequence tags database suggests that similar switching phenomena occur between repetitive elements in other Nox family transcripts, indicating such cloning artifacts are common. In contrast, genuine RNA splicing does account for another Nox1 transcript lacking the entire exon 11, which is abundant in colon cells but encodes a protein incapable of supporting superoxide production.
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PMID:Analysis of mRNA transcripts from the NAD(P)H oxidase 1 (Nox1) gene. Evidence against production of the NADPH oxidase homolog-1 short (NOH-1S) transcript variant. 1537 66


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