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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The aim of this study was to investigate the involvement of endothelins (ET) in brain injury. The effect of ET was studied in the isolated basilar artery (BA) taken from control, sham-operated, and cold-lesioned rats. Cold lesion was induced by application of a precooled (-78 degrees C) copper cylinder (outer diameter 5 mm) for 60 seconds to the intact dura over the parietal cortex. After precontraction with prostaglandin (PG) F2alpha, ET-3 (10(-10) to 10(-8) mol/L) dilated BA with a pD2 (negative log of the half-maximal concentration) of 9.06+/-0.031 (mean +/- SD) and a maximal effect (Emax) of 1.64+/-1.0 mN at 3 x 10(-9) mol/L in sham-operated animals. This dilation was reduced 24 and 48 hours after cold lesion by 33% and 73%, respectively, at 3 x 10(-9) mol/L. The effects of acetylcholine (10(-8) to 10(-4) mol/L) and sodium nitroprusside (10(-3) mol/L) were unaltered. Activation of the ETB receptor in thoracic aorta by the specific agonist IRL 1620 also resulted in a reduced dilation (51% by 48 hours after cold lesion). Reverse transcriptase-polymerase chain reaction of the BA showed unaltered expression of mRNA for the ETB receptor after cold lesion whereas ETB immunoreactivity in BA and in its intraparenchymal arteries was reduced at 24 and 48 hours. In contrast to the reduction of ET-3-induced dilation, the constrictor effects of ET-1 and ET-3 were retained after cold lesion. Endothelin-1 (10(-12) to 10(-6) mol/L) dose-dependently contracted segments of untreated control BA segments under resting conditions with a pD2 of 8.03+/-0.22 and an Emax of 6.35+/-0.70 mN. Further evidence that the constrictor ability of BA was not influenced by cold lesion is given by the unaltered response to 124 mmol/L K+ and 10(-6) mol/L serotonin. We conclude that the ETB receptor of BA after cold lesion is downregulated specifically, apparently at the posttranscriptional level. Because the ETB-mediated dilation in thoracic aorta was also reduced, downregulation of the ETB receptor apparently is not restricted to cerebral arteries. The nitric oxide-cyclic guanosine monophosphate system in BA is, however, intact.
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PMID:Delayed loss of ETB receptor-mediated vasorelaxation after cold lesion of the rat parietal cortex. 985 Jan 48

Bone marrow-culture-derived macrophages activated with interferon-gamma and lipopolysaccharide produced less nitric oxide (NO) when cultured with vesicular stomatitis virus (VSV)-infected BALB/c3T3 (3T3-VSV) than macrophages activated in an identical manner and cultured alone, with uninfected BALB/c3T3 (3T3), or with P815. However, all four groups of macrophages produced nearly the same amount of interleukin-6 (IL-6). Addition of VSV to activated macrophages did not change the amount of NO produced. The amount of NO generated by two non-macrophage sources of NO was not affected by the presence of either P815 or 3T3-VSV. Reverse transcriptase-polymerase chain reaction showed a decrease in the amount of inducible nitric oxide synthase (iNOS) but not IL-6 mRNA from macrophages cocultured with 3T3-VSV compared with macrophages cocultured with P815. The reduction in iNOS mRNA was confirmed by ribonuclease protection assay. When RAW 264.7 transfected with an iNOS regulatory construct were activated and incubated with 3T3-VSV there was a decrease in the expression of the reporter luciferase gene and NO production but not IL-6 production compared with cells incubated with either medium alone or with P815.
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PMID:Interaction with vesicular stomatitis virus-infected BALB/c3T3 cells inhibits the synthesis of nitric oxide in activated murine bone marrow culture-derived macrophages. 1033 88

Nitric oxide plays a significant but incompletely understood role in fibroblast function and cutaneous wound collagen synthesis; however, the participation of inducible nitric oxide synthase (iNOS) in gastrointestinal anastomotic healing has not been studied. Male Sprague-Dawley rats underwent single-layer left colonic anastomosis. Animals were killed at 24-hour intervals postoperatively and the anastomosis was excised. Parallel uninjured colon tissue samples were also analyzed. Reverse transcriptase-polymerase chain reaction confirmed the absence of iNOS messenger RNA in control colon and expression of the gene in anastomotic tissue on all study days. Northern hybridization demonstrated maximal iNOS messenger RNA transcription on day 1 with decreased levels on days 3 and 5. iNOS enzyme activity, measured biochemically by the conversion of [(3) H-arginine to [(3) H]-citrulline ex vivo, was also maximal on day 1 (7.35 +/- 1.34 pmol/mg protein/min [+/- standard error of the mean], n = 10) and decreased on days 3 (4.37 +/- 2.32 pmol/mg protein/min; n = 6) and 5 (2.80 +/- 0.92 pmol/mg protein/min; n = 6). Immunohistochemical staining demonstrated that (1) iNOS expression is confined to a discrete cell population in the region of the anastomosis containing inflammatory cells; (2) those cells assume a highly conserved position on the luminal edge of the proliferating scar; and (3) the iNOS-expressing cells are present throughout the fibroplastic phase of healing. To functionally assess the role of iNOS in colonic healing, rats were treated with a continuous intravenous infusion of S-methylisothiourea (a selective inhibitor of iNOS) at a dosage of 200 mg/kg/day for 5 days after anastomosis. There was a significantly reduced anastomotic bursting pressure in rats treated with the inhibitor as compared to rats treated with intravenous normal saline solution (108.4 +/- 13.2 mm Hg vs. 148.4 +/- 10.3 mm Hg; P <0.05). These results suggest that iNOS gene expression is induced during colonic anastomotic healing, that it is present through all phases of healing but is maximal through the inflammatory phase, and that iNOS activity is required for optimal anastomotic healing.
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PMID:Expression and function of inducible nitric oxide synthase during rat colon anastomotic healing. 1055 65

The muscles of the corpus cavernosum of the penis relax in response to stimulation of non-adrenergic, non-cholinergic nerves or nitric oxide (NO)-donating drugs to elicit erection. It is generally assumed that NO mediates this effect via activation of soluble guanylyl cyclase and a subsequent increase in cyclic guanosine 3', 5'-monophosphate concentration. However, there are no data on the expression of this enzyme in human corpus cavernosum. The purpose of the present study was the molecular characterization of NO-sensitive guanylyl cyclase in human corpus cavernosum. RNA was extracted from tissue samples obtained from seven patients undergoing penile prosthetic surgery or correction of penile deviation. Reverse transcriptase-polymerase chain reaction (RT-PCR) with specific primers for the subunits of NO-sensitive guanylyl cyclase was performed, and PCR products were subcloned and sequenced. Specific amplification products encoding the alpha(1), beta(1), alpha(2), and beta(2) subunits were detected. In addition, we isolated a transcript encoding a novel variant beta(2) subunit. To test whether this novel transcript arises by alternative splicing or whether it is encoded by a separate gene, a 4000-bp clone of the corresponding genomic DNA sequence was isolated. Sequence analysis suggests that the novel beta(2) variant arises by alternative splicing from the same gene as the beta(2) subunit on chromosome 13. In conclusion, our findings suggest the presence of different subunit mRNAs of NO-sensitive guanylyl cyclase in human corpus cavernosum.
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PMID:Expression of nitric oxide-sensitive guanylyl cyclase subunits in human corpus cavernosum. 1067 88

Although NO has been postulated to play important roles in host defences, it is potentially damaging for exposed cells, including for the macrophages producing the NO. Thus a network of radical acceptors and enzymes is thought to play an important redox-buffering role to protect cells against NO-mediated injury. We examined the properties of the redox systems superoxide dismutase (SOD)/catalase, glutathione (GSH) and thioredoxin (Trx), in regulating the viability of two human monocytic cell lines (THP1 and U937) exposed to the NO-generating compound diethylene triamine-nitric oxide (DETA-NO). We observed that NO-induced cytotoxic effects were time- and dose-dependent towards the two cell lines. After vitamin-induced differentiation in vitro with retinoic acid (RA) and 1,25-dihydroxy vitamin D(3) (VD), termed RA/VD, we observed that THP1 RA/VD cells became more resistant to NO-mediated cytotoxicity whereas the susceptibility of U937 cells was not modified. Using Western blotting and reverse-transcriptase PCR methods, we observed that gene transcription and protein expression of Trx and thioredoxin reductase were significantly increased upon RA/VD treatment and differentiation in THP1 cells. By contrast, SOD/catalase and GSH redox state remained unmodified. Finally, a stable transfectant THP1 line overexpressing Trx was found to be more resistant than THP1 control cells that were untransfected or transfected with an empty plasmid, when exposed to DETA-NO in vitro. In conclusion, we observed an inverse correlation between cell susceptibility to NO damaging effects and Trx expression, suggesting that the Trx system may have important preventative capacities towards NO-mediated cellular injury in monocytic macrophage cells.
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PMID:Protective effect of thioredoxin upon NO-mediated cell injury in THP1 monocytic human cells. 1069 4

The anterior pituitary gland produces neuronal nitric oxide synthase (nNOS) and nitric oxide regulates secretion of various anterior pituitary hormones. Estrogen has many functions in anterior pituitary cells including stimulation of prolactin (PRL) cell proliferation and secretion of various anterior pituitary hormones. However, the role of estradiol-17beta (E2) in regulating pituitary nNOS expression has not been previously examined. We studied the regulation of nNOS in normal pituitaries, and neoplastic GH3 pituitary tumors in order to analyze the effects of E2 on nNOS in pituitary cells. GH3 tumors expressed higher levels of nNOS proteins compared to normal pituitaries. Estrogen downregulated nNOS mRNA and protein in both estrogen-treated pituitaries with PRL cell hyperplasia and in GH3 tumors implanted into the flank of rats treated with E2 in silastic tubing. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated three alternatively spliced nNOS transcript isoforms--nNOSa, nNOSb, and nNOSc mRNAs--with distinct 5' untranslated first exons that arose from alternative splicing to a common second exon. All three spliced isoforms were found in the normal rat pituitary, whereas nNOSa and nNOSb, but not nNOSc, were expressed in GH3 tumors implanted into Wistar-Furth rats. E2 also downregulated the nNOSa alternative mRNA transcript isoform in vivo. These results indicate that the biological activity of nNOS in the normal rat anterior pituitary and in pituitary tumors is regulated by a complex pattern of alternative splicing and that some of these mRNA isoforms as well as nNOS protein are regulated by estrogen. Our results also indicate that the levels of nNOS and the alternatively spliced nNOS transcript between normal and GH3 pituitary tumors are different.
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PMID:Estrogen downregulates neuronal nitric oxide synthase in rat anterior pituitary cells and GH3 tumors. 1070 58

All-trans-retinoic acid (RA) is a cancer chemopreventive agent and a pluripotent morphogen. It belongs to the class of retinoids that, besides being inducers of differentiation and growth-inhibitos, exert immunomodulatory and anti-inflammatory functions by mechanisms that are not clearly understood. Macrophages play different roles in diverse physiological processes, including ones in orchestrating immune and inflammatory responses. Products of activated macrophages such as interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), interleukin-6, interleukin-8, and nitric oxide (NO) are important regulators of inflammatory reactions. In this study J774A. 1 cells, a murine macrophage cell line, was used to study the effects of RA on the production of NO, TNFalpha and IL-1beta. Cells were stimulated with lipopolysaccharide (LPS) with or without RA. RA depressed the levels of NO in a dose-dependent manner. NO production and subsequent nitrite accumulation in the media peaked at 24 h, plateaued at 48 h, and remained at the same level through 72 h. The presence of RA decreased TNFalpha levels, measured by both bioassay and enzyme-linked immunosorbent assay (ELISA), but these did not correlate with increased mRNA expression measured by reverse-transcriptase polymerase chain reaction at 6 h after LPS stimulation. IL-1beta protein production measured by both ELISA and bioassay decreased with RA treatment. IL-1beta mRNA expression was not affected by RA except at low doses. This study indicated that RA modulates cytokine production in J774A.1 macrophage cells. Inhibition of inflammatory cytokine production may play a role in the anti-inflammatory activity of RA. The results suggested that effects of RA are complex and are time and concentration dependent.
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PMID:Effect of all-trans-retinoic acid on cytokine production in a murine macrophage cell line. 1088 90

To gain insight into the glomerular capillary repair mechanisms in immunoglobulin A (IgA) nephropathy, we focused on vascular endothelial growth factor (VEGF-A) and nitric oxide (NO). Because abnormal glycosylation of serum IgA has been shown in IgA nephropathy, we examined whether VEGF-A and NO production by mesangial cells (MCs) could be modulated by aberrantly glycosylated (desialylated or degalactosylated) IgA. VEGF-A and NO synthase (NOS) gene expression were examined by reverse-transcriptase polymerase chain reaction (RT-PCR) or Northern blot analysis, and VEGF-A peptide, by capture enzyme-linked immunosorbent assay and NOS activity as production of tritium ([(3)H]) citrulline from [(3)H] arginine. Semiquantitative densitometric analysis of RT-PCR experiments showed a significant downregulation of VEGF-A messenger RNA (mRNA) in MCs incubated with aberrantly glycosylated IgA. This resulted in decreased release of VEGF-A in culture medium (P: < 0. 01). NOS activity and inducible NOS (iNOS) mRNA were enhanced by aberrantly glycosylated IgA (both P: < 0.01). No modulation of constitutive NOS mRNA was found. The depression of the VEGF-A production induced by aberrantly glycosylated IgA was mediated by NO because it was completely reversed by the NOS inhibitor, N:omega-nitro-L-arginine methyl ester. The NO donor, sodium nitroprusside, induced a bimodal modulation of VEGF; although low concentrations (0.0001 nmol/L) increased VEGF-A synthesis, greater concentrations (1,000 nmol/L) depressed it. In conclusion, we report negative control of VEGF-A synthesis in MCs by aberrantly glycosylated IgA, mediated by enhanced iNOS activity. We speculate that both increased iNOS activity and depressed VEGF-A synthesis might have a role in impairing vascular repair and favor sclerosis in IgA nephropathy.
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PMID:Aberrantly glycosylated IgA molecules downregulate the synthesis and secretion of vascular endothelial growth factor in human mesangial cells. 1109 49

Endothelial dysfunction anticipates the development of transplant coronary artery disease (TxCAD) observed more than 1 year after transplantation (HTx). We investigated whether in patients early after HTx myocardial inducible and constitutive nitric oxide synthases (iNOS; cNOS) are expressed and cardiac nitric oxide production occurs. Moreover, a possible relationship to alterations in endothelium dependent and independent vasomotor function was assessed. Forty-two transplant recipients were studied 37 +/- 5 days after HTx. Microvascular coronary flow velocity reserve (CFVR) was tested endothelium dependent (acetylcholine; 30 microg/min x 5 min/i.c.) and independent (adenosine; 160 microg/min x 5 min/i.c.) by Doppler flow wire. Flow velocity increase by a factor greater than 2 was considered normal. Quantitative coronary angiography was used to assess epicardial vasomotor function in response to the same stimuli. Myocardial iNOS and cNOS gene expression were detected by semiquantitative reversed transcriptase polymerase chain reaction. Plasma nitrite levels (microM) were measured by spectrophotometry. Cytokines (TNF-alpha, IL-6; pg/ml) were measured by enzyme linked immunosorbent assay. In 26.1% of patients (n = 11; group A) an impaired endothelium dependent CFVR (1.65 +/- 0.23 increase) was observed; in 73.9% (n = 31, group B) a normal endothelium dependent CFVR (3.0 +/- 0.7 increase; P = 0.003) was observed. Myocardial iNOS and cNOS gene expression did not differ between the two groups. Transcardiac cytokine production was noted in 58.8% of patients for IL-6 and in 53.3% for TNF-alpha. Coronary sinus (CS) levels of TNF-alpha, IL-6 and nitrite were higher in group A. A significant increase in nitrite production was found only in patients with impaired endothelium dependent CFVR (aorta: 43.9 +/- 3.7 vs CS: 52.8 +/- 5.6, P = 0.05), suggesting transcardiac nitric oxide production. In addition, CS nitrite levels correlated with CS TNF-alpha levels in patients with impaired CFVR (r = 0.44, P = 0.003). Microvascular endothelium dependent CFVR is impaired in 26% of patients early after HTx. Activation of cytokines and the NO pathway seem to be involved in this vasomotor dysfunction The association between cardiac nitric oxide production and TNF-alpha in this group indicates a chronic high immunologic process, which may represent an early and important target for therapy and prevention of TxCAD.
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PMID:An association between microvascular endothelial dysfunction, transcardiac nitric oxide production and pro-inflammatory cytokines after heart transplantation in humans. 1111 1

Nitric oxide produced through the action of inducible nitric oxide synthase (iNOS) is an important mediator in immune responses of the host. Various extracellular factors, including inflammatory stimuli, affect intracellular free Ca2+ levels ([Ca2+](i)), modulating cellular signalling and gene expression. In the present study we investigated the effects of increased ([Ca2+](i)) on NO production through the iNOS pathway in J774 macrophages. Thapsigargin (TG), a Ca2+-ATPase inhibitor, and the Ca2+ ionophore A23187 were used as tools to induce an increase in ([Ca2+](i)) in the cytosol. This increase was confirmed by the fura 2 method. The production of NO was measured as accumulated nitrite in the cell culture medium; iNOS protein and iNOS mRNA were detected by Western blotting and reverse-transcriptase-mediated PCR respectively. The activation of nuclear factor kappaB (NF-kappaB) was investigated by electrophoretic mobility-shift assay. TG (100 nM) induced a marked synthesis of iNOS mRNA, iNOS protein and NO in cells primed with a low concentration of endotoxin [lipopolysaccharide (LPS) 1 ng/ml], which on its own induced barely detectable NO synthesis. Stimulation by a high concentration of LPS (100 ng/ml) induced a marked expression of iNOS and NO production. Under these conditions, treatment with TG hindered the synthesis of iNOS protein and NO production by accelerating the degradation of iNOS mRNA. Treatment with TG (100 nM) did not affect the NF-kappaB activity induced by low (1 ng/ml) or high (100 ng/ml) concentrations of LPS. Viability of the cells was confirmed by the 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyaniline ("XTT") method; apoptosis was ruled out by propidium iodide staining and flow cytometry. A23187 (1 microM) also transiently increased ([Ca2+](i)) and had opposite effects on NO production depending on the LPS concentration. Our results show that increased ([Ca2+](i)) induced the stimulation or suppression of NO production through iNOS in macrophages depending on the state of cell activation. These findings suggest that the receptor-mediated increase in ([Ca2+](i)) might be an important factor in the control of the balance between the up-regulation and down-regulation of inflammatory genes, including that encoding iNOS, depending on the phase of the inflammatory response.
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PMID:Bi-directional effects of the elevation of intracellular calcium on the expression of inducible nitric oxide synthase in J774 macrophages exposed to low and to high concentrations of endotoxin. 1117 Nov 14


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