Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent evidence suggests that a number of non-phagocytic cell types may contain a superoxide generating NADPH oxidase. Studies to data on cultured human fibroblasts have primarily concerned the identification of cytochrome b558, whilst expression of other NADPH oxidase components have not been addressed. In this study we have investigated the expression of NADPH oxidase with particular reference to the cytosolic factors p47-phox and p67-phox. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that human fibroblasts express mRNA for p47-phox, p67-phox and p22-phox. Expression of the gp91-phox transcript was not detected, indicating that human fibroblasts may possess an NADPH oxidase isoenzyme. Western blot analysis of human fibroblast cytosol, using an anti-p47-phox antibody (JW-1), identified a 47 kDa protein. Cell-free reconstitution assays showed that fibroblast cytosol could initiate superoxide generation when mixed with either human fibroblast membranes (0.16 nmol superoxide/min/microgram membrane protein), or resting human neutrophil membranes (0.20 nmol superoxide/min/microgram membrane protein). These data indicate that the expression of p47-phox and p67-phox by human fibroblasts may contribute to the cells' generation of superoxide.
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PMID:The functional expression of p47-phox and p67-phox may contribute to the generation of superoxide by an NADPH oxidase-like system in human fibroblasts. 798 96

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

Low-level generation of reactive oxygen species (ROS) by endothelial cells in response to a variety of stimuli has been observed; however, the enzyme system responsible is unknown. Using a variety of techniques, we examined for components of the phagocyte superoxide-generating NADPH oxidase to elucidate whether this enzyme could be a source of endothelial-derived ROS. Superoxide generation on addition of 100 microM NAD(P)H to human umbilical vein endothelial cell (HUVEC) sonicates (using lucigenin-enhanced chemiluminescence) was partially inhibited on addition of the flavoenzyme inhibitor diphenyliodonium (IDP). Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated expression of gp91phox, p22phox, p67phox, and p47phox in four independent HUVEC isolates. Expression of p22phox was also confirmed by Northern blotting. RT-PCR for tumor necrosis factor-alpha was negative, indicating an absence of mononuclear cell contamination (a potential source of NADPH oxidase). Immunoperoxidase staining, using anti-p47phox (JW-1)- and anti-p67phox (JW-2)-specific antibodies, showed protein expression of these cytosolic components. However, heme spectroscopy failed to indicate the presence of the low-potential cytochrome b558. These data indicate that cultured human endothelial cells express both mRNA and protein for cytosolic components of the phagocyte superoxide-generating NADPH oxidase. However, because the cytochrome b558 heme could not be conclusively demonstrated, a contribution of the phagocyte NADPH oxidase to endothelial oxidant generation may be unlikely.
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PMID:Expression of phagocyte NADPH oxidase components in human endothelial cells. 889 60

The immortalized human chondrocyte cell line C-20/A4 has the ability to produce superoxide constitutively at low levels of 5.4 x 10(-2) nmol/min/10(6) cells (S.E.M. = +/-0.5, n = 30) and at raised levels upon stimulation with ionomycin and phorbol 12-myristate 13-acetate. Priming and anti-priming effects of interleukin (IL)-1 beta and IL-4, respectively, are also demonstrated. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification using oligonucleotide primers to components of the NADPH oxidase enzyme complex showed mRNA expression of p22-phox, p40-phox and p47-phox. Western blot analysis using polyclonal antisera indicated the presence of the p47-phox p67-phox polypeptide components. These results show that the C-20/A4 cells contain an NADPH oxidase-like complex, similar to that found in other cell types, which produces superoxide anions.
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PMID:Detection of protein and mRNA of various components of the NADPH oxidase complex in an immortalized human chondrocyte line. 918 52

Porcine articular chondrocytes have the capacity to release superoxide in response to the addition of the calcium ionophore ionomycin in a concentration-dependent manner. This activity was not stimulated by the addition of fMetLeuPhe or the kinase activator phorbol myristate acetate (PMA). However, this release of superoxide was inhibited by iodonium diphenyl (IDP), suggesting the involvement of NADPH oxidase. Reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotides designed against the known sequences for the human phagocyte NADPH oxidase showed the expression of p22-phox, p40-phox, and p47-phox mRNA, while Western blot analysis of chondrocyte extracts using polyclonal antisera raised against the human phagocyte NADPH oxidase suggested the presence of the p67-phox polypeptide. These results suggest that porcine articular chondrocytes can release reactive oxygen species using a NADPH oxidase-like complex.
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PMID:Detection of superoxide and NADPH oxidase in porcine articular chondrocytes. 929 50

Cultured human endothelial cells (EC) exposed to atherogenic low-density lipoprotein levels have increased reactive oxygen species (ROS) generation. The enzyme responsible for this ROS production elevation is unknown. We have examined for the presence of a functional leukocyte-type NADPH oxidase in EC to elucidate whether this enzyme could be the ROS source. The plasma membrane fraction of disrupted EC showed a reduced-minus-oxidized difference spectra with absorption peaks identical to those observed in the spectra of the leukocyte NADPH oxidase component, cytochrome b558. Western-blot analysis, using anti-gp91 -phox. anti -p22-phox. anti -p47-phox. and anti -p67-phox antibodies, demonstrated the protein expression of NADPH oxidase subunits in EC. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed the mRNA expression of gp91-phox, p22-phox, p47-phox, and p67-phox in EC. Sonicates from unstimulated EC produced no measurable superoxide; whereas, exogenously applied arachidonic acid activated superoxide generation in a manner that was dependent upon the presence of NADPH and both membrane and cytosolic fractions combined. Apocynin, a specific leukocyte NADPH oxidase inhibitor, was shown by Western-blot analysis of membrane and cytoplasmic fractions to inhibit the translocation of p47-phox to the membrane of stimulated EC. These findings support the presence of a functionally active leukocyte-type NADPH oxidase in EC. NADPH oxidase could be the major cellular ROS source in EC perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.
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PMID:Identification of a functional leukocyte-type NADPH oxidase in human endothelial cells :a potential atherogenic source of reactive oxygen species. 1059 57

We previously reported increased aortic reactive oxygen species (ROS) production in mineralocorticoid (deoxycorticosterone acetate [DOCA]-salt) hypertensive rats. In the present study, we tested the hypothesis that NADH/NADPH oxidase is responsible for increased ROS production, namely superoxide (O(2-)), in aorta from the DOCA-salt rat. Treatment of aortic rings from DOCA-salt rats with the NO synthase inhibitor N-nitro-L-arginine and the xanthine oxidase inhibitor allopurinol did not significantly change O(2-) production. Furthermore, de-endothelialization of aorta from DOCA-salt rats did not affect O(2-) production compared with that of sham-operated rats. Thus, xanthine oxidase and uncoupled endothelial NO synthase were not responsible for increased O(2-) production in the DOCA-salt rats. In contrast, treatment with the NADPH oxidase inhibitor apocynin significantly decreased O(2-) production in aortic rings from DOCA-salt rats compared with sham-operated rats. Moreover, long-term administration of apocynin (in drinking water, 1.5 mmol/L, 28 days) to DOCA-salt rats significantly decreased systolic blood pressure compared with that of rats treated with DOCA-salt alone. Furthermore, O(2-) production in aortic rings from DOCA-salt rats treated with apocynin for 28 days was reduced compared with that of untreated DOCA-salt rats. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that DOCA-salt rats have significantly greater mRNA levels of the NADPH oxidase subunit p22phox than do sham-operated rats. These findings suggest that NADPH oxidase is increased and is responsible for increased O(2-) production and possibly contributes to increased blood pressure in the DOCA-salt hypertensive rat.
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PMID:NADH/NADPH oxidase and enhanced superoxide production in the mineralocorticoid hypertensive rat. 1171 6

The development of nitrate tolerance has been found to be associated with vascular production of superoxide anion (O2-*), generated mainly by the eNOS and NADPH oxidase pathways. The aim of our study was to investigate whether long-term angiotensin-converting enzyme inhibition by ramipril is able to protect against nitrate tolerance in the aortas of eNOS-deficient (eNOS-/-) mice and to assess the implication of the NADPH oxidase pathway. Therefore, 3 types of treatment were given to wild-type (WT) and eNOS-/- mice: group 1 received ramipril for 5 weeks and a co-treatment with ramirpil plus nitroglycerine (NTG) during the last 4 days, group 2 received only NTG, and group 3 served as control. Relaxations to NTG (0.1 nmol/L to 0.1 mmol/L) were determined on U44619, a thromboxane analogue, precontracted rings, and O2-* production were assessed on aorta homogenates with the lucigenin-enhanced chemiluminescence technique. Cyclic guanosine monophosphate and reverse-transcriptase-polymerase chain reaction analyses were performed on whole mouse aortas. In WT group 2, the concentration-effect curves to NTG were significantly shifted to the right: the pD2 was 6.16 +/- 0.17 (n = 6) vs 6.81 +/- 0.10 (n = 6) in WT group 3 (not exposed to NTG; P < 0.05) and O2-* production was enhanced from 100% +/- 11% (n = 9) to 191% +/- 21% (n = 6; P < 0.01). In contrast, in WT group 1, the rightward shift was abolished: the pD2 value was 6.73 +/- 0.13 (n = 6; NS vs group 3 WT) and O2-* production was 117% +/- 6% (n = 7; NS vs group 3 WT). In eNOS groups 1 and 3, similar data were observed: the pD2 values were 7.58 +/- 0.08 and 7.38 +/- 0.11 (NS) vs 6.89 +/- 0.20 in eNOS group 2 (n = 6; P < 0.01). In the WT mice aortas, ramipril treatment significantly increased the cyclic guanosine monophosphate levels (reflecting nitric oxide availability), which returned to control values after in vivo co-treatment with a bradykinin BK2 antagonist (Icatibant). In both strains, candesartan, an AT1 blocker, was also able to protect against the development of nitrate tolerance. Moreover, before NTG exposure, ramipril treatment decreased p22phox and gp91phox (essential NADPH oxidase subunits) mRNA expression in aortas from both mice strains. In conclusion, long-term ramipril treatment in mice protects against the development of nitrate tolerance by counteracting NTG-induced increase in O2 production, which involves a direct interaction with the NADPH oxidase pathway and seems to be completely independent of the eNOS pathway.
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PMID:Ramipril treatment protects against nitrate-induced oxidative stress in eNOS-/- mice: An implication of the NADPH oxidase pathway. 1689 13

Experiments were performed to elucidate the mechanism of action of a 7-day oral administration of the sulfur-containing angiotensin-converting enzyme (ACE) inhibitor 3-thienylalanine-ornithyl-proline (TOP; 10 mg/kg/d) on endothelial dysfunction and oxidative stress compared with that of captopril (control; 40 mg/kg/d) in spontaneously hypertensive rats. The differential expression of messenger RNA by real-time reverse-transcriptase-polymerase chain reaction and protein by Western blot analysis was assessed for the markers nicotinamide adenine dinucleotide phosphate oxidase, p22phox, endothelial nitric oxide (NO) synthase, and AT1 receptor. Furthermore, TOP-induced vascular relaxation was also investigated using rat aortic rings in an organ bath. TOP significantly downregulated both messenger RNA and protein expressions of p22phox and AT1 receptor; the latter facilitates vasoconstriction through angiotensin II. In addition, TOP upregulated endothelial NO synthase, thus enhancing the production of NO. Vascular studies revealed that TOP caused endothelium-dependent vasorelaxation. In conclusion, unlike the free sulfur in captopril, the thiophene ring in TOP may act as a better scavenger of free radicals. Therefore, TOP exerted more significant antihypertensive effects than captopril, not only through angiotensin-converting enzyme inhibition but also through more effective antioxidation, because the inherent thiophene moiety resulted in the enhanced production of NO.
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PMID:Sulfur-containing angiotensin-converting enzyme inhibitor 3-thienylalanine-ornithyl-proline activates endothelial function and expression of genes involved in Renin-Angiotensin system. 2323 42