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)

A full-length inducible nitric oxide synthase (iNOS) gene has been sequenced for the first time outside the mammals, and the gene organization compared with that already determined for human iNOS. While there are some differences from the human gene, overall the exons show remarkable conservation in sequence and organization. As in human, the trout iNOS gene has 27 exons, with 18 of the trout exons being identical in size with the equivalent human exons. The cofactor-binding domains are found in the same exons and in some cases are absolutely conserved. Differences include the start of the ORF in exon 3 instead of exon 2, resulting in a deletion at the 5' end of the trout iNOS protein. Exon 27 also shows a large difference in size and although the trout exon is larger this is due to the length of the 3'-UTR. Several non-mammalian features are notable, and include a conserved potential glycosylation site in chicken and fish, and an insertion at the boundary of exons 20 and 21 in fish. The intron sizes in trout were generally much smaller than in human iNOS, making the trout iNOS gene approximately half the size of the human gene. Analysis of RNA secondary structure revealed two regions with complementarity, which could interfere with reverse transcription. Using a trout fibroblast cell line (RTG-2 cells), it was shown by reverse transcriptase (RT)-PCR that virus infection was a good inducer of iNOS expression. However, when using a combination of Superscripttrade mark II for reverse transcription and primers at the 5' end of the gene only very weak products were amplified, in contrast with the situation when primers at the 3' end of the gene were used, or ThermoScripttrade mark-derived cDNA was used. The impact of such results on RT-PCR analysis of iNOS expression in trout is discussed.
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PMID:Molecular cloning, gene organization and expression of rainbow trout (Oncorhynchus mykiss) inducible nitric oxide synthase (iNOS) gene. 1153 35

Coxsackievirus B3 (CVB3)-induced myocarditis in NMRI mice represents a model for studying the pathogenesis of this chronic heart disease. Previously, we reported on specific cytokine patterns during the acute stage of myocarditis since cytokines are thought to play the important role in this cardiomyopathy. In this study, the expression of various cytokine mRNAs and CVB3-RNA kinetics was examined with particular emphasis on the late phase of myocarditis, by using reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization (ISH) and immunohistochemistry (IHC). In addition, replicating and persisting CVB3-RNAs were semiquantified by PCR-ELISA. Distinct histopathological changes responsible for ongoing heart disease were found and characterized by increased fibrosis, persistent cellular infiltration and degenerated necrotic myocytes. One of the most important findings of this study was that the mRNA-expression of TNF- alpha, IL-1 alpha, interferon- gamma, IL-10, IL-18, macrophage inflammatory protein-1 alpha (MIP-1 alpha), transforming growth factor- beta (TGF- beta) and inducible nitric oxide synthase (iNOS) persisted as long as 98 days after the virus infection. The induction of IL-10 as well as IFN- gamma mRNAs was also verified by ISH and IHC at days 28 and 98 p.i. The clearly apparent persistence of the viral genomes in the myocardium of infected mice was confirmed by seminested PCR, ISH, and PCR-enzyme linked immunoabsorbent assay (ELISA), showing the highest amount of viral RNA in myocardial cells at day 7 after infection. These data indicate that the persistence of viral RNA is associated with persistently high levels of cytokine mRNAs which, when translated, could severely contribute to pathological changes and injury of connective tissue in the chronic stage of myocarditis.
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PMID:Persistent expression of cytokines in the chronic stage of CVB3-induced myocarditis in NMRI mice. 1154 41

Cytokine-induced beta-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by interleukin-1beta (IL-1beta), and its activity promotes the expression of several beta-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1beta + gamma-interferon [IFN-gamma])-induced expression of NF-kappaB in beta-cell apoptosis, rat beta-cells were infected with the recombinant adenovirus AdIkappaB((SA)2), which contained a nondegradable mutant form of inhibitory kappaB (IkappaB((SA)2), with S32A and S36A) that locks NF-kappaB in a cytosolic protein complex, preventing its nuclear action. Expression of IkappaB((SA)2) inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-kappaB. Cytokine-induced gene expression of several NF-kappaB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIkappaB((SA)2), as established by reverse transcriptase-polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, beta-cell survival after IL-1beta + IFN-gamma treatment was significantly improved by IkappaB((SA)2) expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-kappaB activation, under in vitro conditions, has primarily a pro-apoptotic function in beta-cells.
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PMID:Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis. 1157 1

Streptococcus agalactiae (group B streptococcus [GBS]) is a leading cause of neonatal pneumonia, sepsis, and meningitis. Early-onset GBS pneumonia is characterized by marked pulmonary epithelial and endothelial cell injury. Innate proinflammatory responses to GBS infection that may contribute to the respiratory pathology include the synthesis and release of cytokines, prostaglandins, and nitric oxide (NO). The hypothesis that NO is directly induced in lung epithelial cells by invading GBS or indirectly induced by cytokines released by GBS-infected mononuclear cells was tested. A549 transformed human respiratory epithelial cells were directly cultured with GBS, cocultured with GBS-infected human mononuclear cells or purified macrophages, or exposed to conditioned culture medium from human mononuclear cells infected by GBS. The culture medium of A549 cultures was assayed for NO secretion, and the cell lysates were tested for presence of inducible nitric oxide synthase (iNOS) mRNA by reverse transcriptase PCR (RT-PCR). GBS-treated A549 cells neither secreted detectable NO nor expressed iNOS mRNA. GBS interaction with human mononuclear cells, however, stimulated release of soluble factors that readily induced iNOS mRNA expression and NO secretion by A549 cells. Inflammatory mediator-induced nitric oxide (NO) production by alveolar epithelium may exceed that of other lung cell types such as macrophages, and induction during GBS infection may play a significant role in pulmonary defense or free-radical-mediated lung injury.
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PMID:Cytokine responses to group B streptococci induce nitric oxide production in respiratory epithelial cells. 1174 62

Chronic exercise upregulates endothelial nitric oxide synthase (eNOS) gene expression. Whether the expression of inducible nitric oxide synthase (iNOS) is affected by exercise is unknown. We therefore investigated the effects of chronic exercise on iNOS and eNOS expression in isolated rat aortic endothelial and smooth muscle cells separately. Five-week-old male Wistar rats were randomly divided into control and exercise groups. After 10 weeks of running training, animals were sacrificed under ether anesthesia. The standard curve quantitative competitive reverse transcriptase-polymerase chain reaction method was used to quantify NOS mRNA expression in isolated endothelial/smooth muscle cells. To evaluate the functional role of iNOS, we examined phenylephrine-induced vascular responses with or without pretreatment with aminoguanidine. We found that (1) expression of iNOS and eNOS mRNA in endothelial cells was increased by chronic exercise and (2) chronic exercise blunted phenylephrine-induced vascular responses, probably by increasing NO release via iNOS. Our results show that chronic exercise increases both iNOS and eNOS gene expression in endothelium. These alterations may be partially responsible for the change in vascular response after exercise.
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PMID:Chronic exercise increases both inducible and endothelial nitric oxide synthase gene expression in endothelial cells of rat aorta. 1191 82

In the present study, the mediator role of p38 kinase, a member of the mitogen-activated protein kinase (MAPK) family, was studied in lipopolysaccharide-induced inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in J774 mouse macrophages and T-84 human colon epithelial cells. Two pyridinyl imidazole inhibitors of p38 MAPK, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole (SB203580) and 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-imidazole (SB202190), stimulated NO production at low drug concentrations, maximal stimulation occurring at 1 microM drug concentration. In contrast, higher concentrations inhibited NO production, which was>90% at 30 microM drug concentration. The bi-directional effect was found in both cell types tested. Negative control compound SB202474, which is structurally related but does not inhibit p38, did not stimulate NO production but inhibited it at 30 microM concentration. A chemically different p38 inhibitor 2-methyl-4-phenyl-5-(4-pyridyl)oxazole (SC68376) had only a stimulatory action on NO production. Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analysis of iNOS showed that both stimulatory and inhibitory effects of SB203580 occurred at the level of iNOS expression. SB203580 did not alter lipopolysaccharide-induced NF-kappaB activation as detected by electrophoretic mobility shift assay (EMSA). The data show that pyridinyl imidazoles SB203580 and SB202190 have a bi-directional effect on NO production through iNOS pathway depending on the drug concentration used. The inhibitory effect was unrelated to inhibition of p38 MAPK, whereas the stimulatory effect is most likely mediated by p38 MAPK dependent mechanism, suggesting a novel mechanism for regulation of iNOS expression, which is common for murine and human cells.
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PMID:P38 mitogen-activated protein kinase inhibitor SB203580 has a bi-directional effect on iNOS expression and NO production. 1242 38

Ovine lentivirus (OvLV) also known as maedi-visna virus, infects and replicates primarily in macrophages. This investigation examined the role of nitric oxide in the replication of OvLV in cultured macrophages. Peripheral blood mononuclear cells were collected from OvLV-free sheep and cultured in Teflon coated flasks at a high concentration of lamb serum. The cells were subsequently infected with OvLV strain 85/34. OvLV replication was assessed under different experimental treatments by comparison of reverse transcriptase (RT) activity in culture supernatant. Cultures that were treated with exogenous nitric oxide via S-nitroso-acetylpenicillamine did not have altered levels of RT activity compared to cultures treated with the inactive control compound, acetylpenicillamine. However, blockage of nitric oxide production by treatment with aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), led to a significant rise in RT activity. This rise in RT activity was partially reversed in aminoguanidine treated cultures by L-arginine, the normal substrate for iNOS. Finally, the number of viral antigen producing cells was also quantified after aminoguanidine treatment and found to be significantly higher than untreated cultures. Collectively, these results indicate that nitric oxide is a negative regulator of OvLV replication in macrophages.
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PMID:Inhibition of nitric oxide enhances ovine lentivirus replication in monocyte-derived macrophages. 1245 65

The expression of IL-1beta and inducible nitric oxide synthase (iNOS) from iNOS hypo (GB2, B(6)B(6)) and hyper (K-strain, B(15)B(15)) responder chickens was examined. Compared to GB2, macrophages from K-strain expressed higher iNOS mRNA as quantitated by reverse transcriptase polymerase (RT-PCR) chain reaction after stimulation with 1 microgram/ml of Escherichia coli (E. coli) lipopolysaccharide (LPS). On the contrary, IL-1beta mRNA expression was comparable between K and GB2 macrophages at 3h post-LPS stimulation but persisted up to 9h only in GB2 macrophages. The LPS-inducible interleukin-1 (IL-1) surface receptor expression, measured by flow cytometry, was higher in GB2 than on K-strain macrophages. Blocking of IL-1 receptor by the anti-IL-1 receptor antibody reduced the LPS-mediated iNOS expression by 50% as quantified by competitive RT-PCR. Furthermore, iNOS activity (nitrite) was also reduced to 50%. However, this magnitude of inhibition was similar in both K and GB2 macrophages. While these observations suggest that IL-1beta is involved in mediating LPS-induced iNOS expression and activity, the differential response of GB1 and K-strain macrophages in terms of LPS-induced iNOS expression and activity is unlikely to be modulated by IL-1beta.
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PMID:Interleukin-1beta does not contribute to genetic strain-based differences in iNOS expression and activity in chicken macrophages. 1254 27

It has been previously shown that the inducible nitric oxide (NO) synthase (iNOS; NOS-2) is elevated after hemorrhage, and that iNOS-derived NO participates in the upregulation of inflammation as well as lung and liver injury postresuscitation from shock. The purpose of this study was to elucidate the time course of iNOS mRNA expression, as well as the cellular and subcellular localization of iNOS protein in the liver posthemorrhage in rats subjected to varying durations of hemorrhagic shock (HS; mean arterial blood pressure [MAP] = 40 mmHg) with or without resuscitation. Expression of iNOS mRNA in rat liver by real-time reverse transcriptase (RT)-PCR demonstrated iNOS upregulation in shocked animals as compared with their sham counterparts as early as 60 min after the initiation of hemorrhage. By 1 h of HS, iNOS protein was detectable in rat liver by immunofluorescence, and this expression increased with time. Immunofluorescence localized iNOS primarily to the hepatocytes, and in particular to hepatocytes in the centrilobular regions. This analysis, confirmed by immunoelectron microscopy, revealed that iNOS colocalizes with catalase, a peroxisomal marker. Furthermore, we determined that iNOS mRNA is detectable by RT-PCR in liver biopsies from human subjects with HS (MAP < 90 mmHg) associated with trauma (n = 18). In contrast, none of the seven nontrauma surgical patients studied had detectable iNOS mRNA in their livers. Collectively, these results suggest that hepatic iNOS expression, associated with peroxisomal localization, is an early molecular response to HS in experimental animals and possibly in human patients with trauma with HS.
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PMID:Characterization of the expression of inducible nitric oxide synthase in rat and human liver during hemorrhagic shock. 1257 18

Nitric oxide (NO) is a biological mediator that is synthesized from L-arginine by the nitric oxide synthase (NOS) family. We investigated the expression of iNOS in bone marrow (BM) mononuclear cells (MNCs) using a reverse transcriptase polymerase chain reaction (RT-PCR) assay and the concentration of NO from BM serum by measuring the metabolite NO(2)(-) in 13 patients with aplastic anemia (AA) compared with 10 normal controls who were donors for allogeneic bone marrow transplantation (BMT). All samples of BM MNCs in patients with AA expressed iNOS mRNA, but iNOS was not expressed in patients who were treated successfully with allogeneic BMT. Normal control samples and samples from leukemia patients who had bone marrow aplasia after chemotherapy did not show significant iNOS expression. When we measured the density of bands for both iNOS and beta(2)-microglobin expressed as the iNOS/beta(2)-microglobin density ratio, there was a significant difference in the ratio between AA and normal controls (0.88+/-0.15 vs 0.26+/-0.05, P<0.001). The BM serum NO(2)(-) concentration in the patients with AA was significantly higher than that of normal controls (88.1+/-32.8 microM vs 48.8+/-8.6 microM, P=0.002). In addition, there was a significant correlation between the NO(2)(-) concentration and the calculated iNOS/beta(2)-microglobin density ratio (r=0.567, P=0.01). These findings suggest that upregulation of iNOS expression for local NO production may contribute in part to the pathogenesis of AA.
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PMID:Increased inducible nitric oxide synthase expression and nitric oxide concentration in patients with aplastic anemia. 1260 89


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