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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The transcription factor NNR from Paracoccus denitrificans was expressed in a strain of Escherichia coli carrying a plasmid-borne fusion of the melR promoter to lacZ, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO(+) donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the E. coli reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a "positive control" phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.
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PMID:Heterologous NNR-mediated nitric oxide signaling in Escherichia coli. 1105 88

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

Leptin was shown to exhibit similar to cholecystokinin (CCK) cytoprotective activity against acute gastric lesions, but its role in ulcer healing has not been examined. The aims of this study were: (1) to compare the effects of exogenous leptin to those of CCK on the course of healing of chronic gastric ulcers; (2) to study the gene and protein expression of leptin at the ulcer margin during ulcer healing; and (3) to assess the effects of leptin administration on the mucosal gene expression of main growth factor such as transforming growth factor alpha (TGFalpha). Gastric ulcers were produced in rats by the acetic acid method. Rats with ulcers were divided in following treatment groups: (1) vehicle; (2) leptin (10 microg/kg i.p.); (3) CCK (10 microg/kg s.c.); and (4) leptin or CCK with or without tyrphostin A46 (200 microg/kg i.p.), an inhibitor of epidermal growth factor (EGF)-receptor tyrosine kinase or NG-nitro-L-arginine (20 mg/kg i.g.), a blocker of nitric oxide synthase. Animals were euthanized 9 days after ulcer induction. The area of gastric ulcers and the gastric blood flow at the ulcer area were determined. In addition, mucosal biopsy samples were taken from the ulcer area for histological evaluation as well as for the determination of mRNA and protein expression for leptin and constitutive nitric oxide synthase (cNOS) and inducibile nitric oxide synthase (iNOS) by reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot, respectively. In addition, the gene expression for TGFalpha was analyzed by RT-PCR. Both leptin and CCK reduced significantly the ulcer area as compared to vehicle-treated group by approximately 50%. The treatment with tyrphostin or N(G)-nitro-L-arginine reversed in part the acceleration of ulcer healing by leptin and CCK. The expression of leptin mRNA and protein was significantly increased at the ulcer edge. The leptin-induced acceleration of ulcer healing was associated with increased expression of transcripts for TGFalpha as well as increased mRNA and protein expression for cNOS and iNOS at the ulcer margin. We conclude that leptin accelerates ulcer healing by mechanisms involving the up-regulation of TGFalpha and increased production of nitric oxide due to up-regulation of cNOS and iNOS in the ulcer area.
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PMID:Role of leptin in ulcer healing. 1123 Sep 99

We determined that the highly pathogenic avian reovirus strain 176 (ARV-176) possesses an enhanced ability to establish productive infections in HD-11 avian macrophages compared to avian fibroblasts. Conversely, the weakly pathogenic strain ARV-138 shows no such macrophagotropic tendency. The macrophage infection capability of the two viruses did not reflect differences in the ability to either induce or inhibit nitric oxide production. Moderate increases in the ARV-138 multiplicity of infection resulted in a concomitant increase in macrophage infection, and under such conditions the kinetics and extent of the ARV-138 replication cycle were equivalent to those of the highly infectious ARV-176 strain. These results indicated that both viruses are apparently equally capable of replicating in an infected macrophage, but they differ in the ability to establish productive infections in these cells. Using a genetic reassortant approach, we determined that the macrophagotropic property of ARV-176 reflects a post-receptor-binding step in the virus replication cycle and that the ARV-176 M2 genome segment is required for efficient infection of HD-11 cells. The M2 genome segment encodes the major mu-class outer capsid protein (muB) of the virus, which is involved in virus entry and transcriptase activation, suggesting that a host-specific influence on ARV entry and/or uncoating may affect the likelihood of the virus establishing a productive infection in a macrophage cell.
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PMID:Avian reovirus major mu-class outer capsid protein influences efficiency of productive macrophage infection in a virus strain-specific manner. 1133 82

In the present study, we examined the effects of the aqueous extract of Rhodiola sachalinensis root (RSE) on the expression of inducible nitric oxide (NO) synthase (iNOS) gene in RAW264.7 macrophages. RSE synergistically increased NO synthesis in interferon-gamma-primed macrophages. Reverse transcriptase polymerase chain reaction and Northern blotting analysis revealed that RSE may provide a second triggering signal for the synergistic induction of iNOS mRNA expression. Thus, iNOS-mediated NO synthesis in response to RSE may be one mechanism whereby this herbal medicine elicits its therapeutic effects.
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PMID:The aqueous extract of Rhodiola sachalinensis root enhances the expression of inducible nitric oxide synthase gene in RAW264.7 macrophages. 1137 93

Inhaled nitric oxide gas (iNO) vasodilates the pulmonary circulation. The effective "dose" of iNO for chronic treatment of pulmonary hypertension is unknown. Increased abundance of pulmonary mRNA for preproendothelin-1 (ppET-1) with its associated increase in endothelin-1 (ET-1) could contribute to the development of both clinical and experimental pulmonary hypertension. The benefit of iNO therapy may be from inhibition of ET-1 production. The present study was designed to compare the effects of two therapeutic concentrations of NO gas, 10 parts per million (p.p.m.) and 100 p.p.m. on the steady-state level of mRNA for ppET-1 and nitric oxide synthase (NOS III), in cultured bovine pulmonary artery endothelial cells. Uptake of NO gas was assessed by measurement of nitrite anions in the medium. The mRNA for ppET-1 and NOS III was determined by semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR). After 4 h exposure to 100 p.p.m. NO in air, nitrite anions levels were 1.6 microM in the endothelial cell media as opposed to 0.23 microM with 10 p.p.m. NO. The levels were 0.02 microM in control cells exposed to air alone. Exposure to 100 p.p.m. NO reduced the steady state levels of mRNA for ppET-1, but not NOSIII mRNA levels. By comparison 10 p.p.m. NO did not affect levels of either mRNA.
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PMID:Nitric oxide gas decreases endothelin-1 mRNA in cultured pulmonary artery endothelial cells. 1189 Jul 39


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