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)

We developed a simplified protocol for sensitive quantitation of mRNA using polymerase chain reaction (PCR) amplification of cDNA made by reverse transcriptase (RT), as resolved with capillary electrophoresis (CE) and detected with laser-induced fluorescence (LIF). The conditions required for adequate accuracy of the simplified version of the RT/PCR quantitation, in which a single concentration of external standard and amplification to within or near the plateau phase are used, were established for assay of mRNAs expressed at high, moderate, and low abundance. The mRNAs for the cytosolic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH) and the growth-associated protein GAP-43 in cultured SN49 neuroblastoma cells were used as target genes for high and moderate levels of expression, respectively. Using cultured mouse microglial cells (BV-2), we demonstrated the utility of this RT/PCR/CE/LIF protocol to quantitate a low-abundance mRNA, encoding a form of nitric oxide synthase (i-NOS) induced by treatment with endotoxin. The appearance of i-NOS mRNA after endotoxin treatment of BV-2 cells was confirmed by Northern blot analysis and in situ hybridization histochemistry, and functional enzyme activity was followed by release of nitric oxide (as nitrite) into the medium. The many advantages of the 'single-point' RT/PCR/CE/LIF protocol for quantitating mRNAs of interest include: simplified protocol, elimination of the use of radiotracers, high sensitivity and precision, and semi-automation of the quantitation phase of analysis.
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PMID:Simplified RT/PCR quantitation of gene transcripts in cultured neuroblastoma (SN49) and microglial (BV-2) cells using capillary electrophoresis and laser-induced fluorescence. 881 12

Rat aortic endothelial cells were found to contain both constitutive and lipopolysaccharide (LPS)-inducible arginase activity. Studies were performed to determine whether induction of nitric oxide synthase (NOS) by LPS and cytokines is accompanied by sufficient arginase induction to render arginine concentrations rate limiting for high-output NO production. Unactivated cells contained abundant arginase activity accompanied by continuous urea formation. LPS induced the formation of both inducible NOS (iNOS) and arginase, and this was accompanied by increased production of NO, citrulline, and urea. Immunoprecipitation experiments revealed the constitutive presence of arginase-I in both unactivated and LPS-activated cells and arginase-II induction by LPS. Arginase-I and iNOS were verified by reverse transcriptase-polymerase chain reaction. Induction of large amounts of iNOS by LPS plus several cytokines resulted in large quantities of NO, citrulline, and NG-hydroxy-L-arginine (NOHA), but urea production was markedly diminished. Decreased urea production was attributed to increased formation of NOHA, the precursor to NO and citrulline and a potent inhibitor of arginase-I activity with an inhibitory constant of 10-12 microM. Inhibition of iNOS activity by NG-methyl-L-arginine decreased NO and NOHA production and increased urea production. This study reveals for the first time that substantial arginase activity is present constitutively in rat aortic endothelial cells, a different isoform of arginase is induced by LPS, and intracellular arginase activity can be markedly inhibited during cytokine induction of iNOS because of NOHA formation. The inhibition of arginase activity that occurs by NOHA during marked iNOS induction may be a mechanism to ensure sufficient arginine availability for high-output production of NO.
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PMID:Arginase activity in endothelial cells: inhibition by NG-hydroxy-L-arginine during high-output NO production. 894 18

Messenger RNA encoding inducible NO synthase (iNOS) was measured by competitive reverse transcriptase polymerase chain reaction (cRT-PCR) and ribonuclease protection assays in spinal cords from mice at varying stages of experimental allergic encephalomyelitis (EAE) and from control mice. iNOS mRNA was increased in spinal cords from mice with acute EAE. cRT-PCR assays revealed a 10-20-fold increase in iNOS mRNA in spinal cords during acute EAE compared with the level observed in normal mouse spinal cords. Functional iNOS activity, as assessed by assay of calcium-independent citrulline production, was also significantly increased in spinal cords from mice with acute EAE in comparison to normal controls. The correlation of functional iNOS expression with active disease in EAE in consistent with a pathogenic role for excess NO in this model of cell-mediated central nervous system autoimmunity.
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PMID:Inducible nitric oxide synthase gene expression and enzyme activity correlate with disease activity in murine experimental autoimmune encephalomyelitis. 898 14

In the present study we examined the effects of hypobaric hypoxia on neuronal (n) and endothelial (e) nitric oxide synthase (NOS) gene expression in the central and peripheral nervous system. Adult rats were exposed either to normoxia (room air) on to hypobaric hypoxia (0.4 atm) for 4, 12 or 24 h and cerebellum and nodose ganglion representing the central and peripheral neurons, respectively, were removed. Messenger RNAs encoding n- and eNOS as well as beta-actin were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) technique. Hypoxia increased nNOS mRNA expression with maximal changes occurring after 12 h wherein mRNA levels were increased by 10.4 +/- 1.3 and 2 +/- 0.4 fold in nodose ganglion and cerebellum, respectively. Hypoxia, on the other hand, had no significant effect on eNOS and beta-actin mRNA levels. Analysis of nNOS protein and enzyme activity showed near doubling of these variables in both tissues after 24 h of hypoxia, indicating that nNOS protein levels are increased and that the protein is functionally active. These observations demonstrate that 12-24 h of hypobaric hypoxia selectively activates nNOS gene expression, which is reflected in an increase in nNOS protein in central and peripheral neurons. It is suggested that up-regulation of nNOS leads to increased generation of nitric oxide, which in turn may contribute to the readjustments of cardio-respiratory systems during the early stages of chronic hypoxia.
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PMID:Activation of nitric oxide synthase gene expression by hypoxia in central and peripheral neurons. 903 52

We have previously proposed that pro-inflammatory cytokines and nitric oxide (NO) contributed to reversible myocardial depression in patients with sepsis and congestive heart failure. Sepsis and heart failure are also associated with refractoriness to beta-adrenoceptor agonists. Therefore, the chronotropic effects of cytokines and the NO synthase inhibitor, NG-methyl-L-arginine (NMA), on beta-adrenoceptor stimulation of neonatal cardiac myocytes were studied. Tumor necrosis factor alpha, interleukin-1 beta and interleukin-6 but not interleukin-4 or interleukin-5 significantly enhanced spontaneous beating rates compared to untreated myocytes in serum-free media for 48 h (P < 0.01; n = 12 for each). NMA also significantly enhanced spontaneous beating rates (P < 0.01; n = 12 for each). Only interleukin-1 beta treatment resulted in significant nitrite production, immunohistochemical staining for inducible nitric oxide synthase and detection of inducible NO synthase messenger RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). However, tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, and NMA each completely blocked the positive chronotropic effects of the beta-adrenoceptor agonist, isoproterenol (P < 0.01; n = 12 for each). These findings are most consistent with an inducible NO synthase-independent effect of cytokines and NMA on the chronotropic responses of neonatal cardiac myocytes to beta-adrenoceptor stimulation. This effect of cytokines and NMA on adrenergic signaling may involve a myocardial constitutive NO synthase or an NO-independent mechanism.
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PMID:Cytokines and nitric oxide synthase inhibitor as mediators of adrenergic refractoriness in cardiac myocytes. 905 50

Although nitric oxide (NO) is a well documented effector molecule in rodent macrophages, its significance in human mononuclear phagocytic cells has been controversial. The foreign body inflammatory reaction around loosened joint replacement implants leads to formation of an osteolytic granulomatous pseudo-synovial membrane rich in activated macrophages. We studied 13 specimens of interface membrane tissue collected from revision surgery of aseptically loosened hip and knee prostheses for the presence of inducible NO synthase (iNOS). The presence of iNOS was demonstrated immunohistochemically in 10 of these specimens. Within the tissue this enzyme was confined to macrophages and vascular endothelial cells. iNOS activity was demonstrated biochemically by measuring the calcium-independent generation of citrulline from L-arginine, and the presence of iNOS mRNA was demonstrated using reverse transcriptase polymerase chain reaction. NO synthesis in the interface tissue may be an important factor in the maintenance of the inflammatory and osteolytic processes.
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PMID:Nitric oxide synthase is expressed in human macrophages during foreign body inflammation. 906 Aug 26

Erectile dysfunction is mainly due to the inability of the cavernosal smooth muscle of the penis to undergo complete relaxation. In the aging rat model, erectile dysfunction is accompanied by a reduction of penile smooth muscle compliance and, in very old animals, by a decrease in penile nitric oxide synthase (NOS), which is responsible for the synthesis of the mediator of penile erection, nitric oxide (NO). We have investigated whether the stimulation of penile NOS expression by local induction or gene therapy can mitigate erectile dysfunction in the aged rat. A mix of iNOS (inducible NOS) inducers was continuously delivered to the penises of 5- ("adult"), 20- ("old"), and 30- ("very old") mo-old rats for 3-6 days, and the erectile response to electrical field stimulation of the cavernosal nerve was measured. The erectile dysfunction observed in old and very old rats as compared to adult animals was ameliorated by treatment with iNOS inducers. Penile iNOS was detectable in the penis of these rats by Western blot, NADPH diaphorase, and NOS activity assays. Inducible NOS was inducible in vitro in both rat and human corpora cavernosal tissue and in rat penile smooth muscle cells (RPSMC), as shown by Western blots. However, NO synthesis in cavernosal tissue upon iNOS protein induction remained low, indicating that the increased NOS levels were under physiological control. The iNOS cDNA was cloned from induced RPSMC mRNA and generated by reverse transcriptase polymerase chain reaction (RT-PCR) from induced human penile smooth muscle cells and corporal tissue. The coding regions from both the rat (RPiNOS) and human (HPiNOS) penile iNOS showed several amino acid differences from their analogous isoform in nonpenile tissues. RPiNOS cDNA injected into the penis mitigated the aging-associated erectile dysfunction. The iNOS construct was detected in cavernosal tissue by PCR, and its expression by RT-PCR and Western blots. These results open the way for the possible use of NOS isoforms in the management of erectile dysfunction.
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PMID:Cloning of rat and human inducible penile nitric oxide synthase. Application for gene therapy of erectile dysfunction. 909 78

Nitric oxide (NO) is a cellular mediator and regulator of multiple biologic functions. NO released by alveolar macrophages (AM) is suggested to play a role in mediating pulmonary injury. In murine and rat macrophages, the expression of inducible NO synthase (iNOS) and the release of NO are well established. However, the existence of such a pathway in other species remains controversial. In this study, we examined NO production and iNOS expression by AM from rats and hamsters, two laboratory animal species that are characterized by their disparate pulmonary responses to various inhaled irritants/toxicants. AM were treated with lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), or tumor necrosis factor-alpha (TNF-alpha) in vitro, and nitrite, the stable oxidation product of NO, was assayed by the Griess reaction. Rat AM produced NO in a dose- and time-dependent manner upon stimulation with LPS and/or IFN-gamma, but not with TNF-alpha. Surprisingly, hamster AM did not release detectable levels of NO after the same treatment. Although iNOS expression was demonstrated in rat AM by immunocytochemical and Western blot analyses, no induction of iNOS expression could be found in hamster AM. Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we found that rat and hamster AM could be induced to express iNOS mRNA after treatment with LPS and IFN-gamma. The results presented here indicate that hamster AM, in contrast to rat AM, lack the ability to express iNOS protein and to generate NO in response to LPS, IFN-gamma, or TNF-alpha in vitro. In conclusion, our data suggest striking differences in iNOS regulation and NO production by AM from rats and hamsters, two rodent species that are commonly used in biomedical research and well-known for their disparate responses to pulmonary irritants/toxicants.
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PMID:Species differences in NO formation by rat and hamster alveolar macrophages in vitro. 911 52

We have previously reported an interaction of nitric oxide (NO) and cyclic 3',5'-guanosine monophosphate (cGMP) in erythropoietin (Epo) production. Further studies have been carried out to clarify the role of NO in the hypoxic regulation of Epo production in Epo producing human hepatocellular carcinoma (Hep3B) cells, which produce Epo in response to physiological stimuli. Our reverse transcriptase-polymerase chain reaction (RT-PCR) technique revealed the expression of iNOS mRNA in Hep3B cells after incubation under hypoxic (1% O2) conditions for 6 hr. Hypoxia also significantly increased medium levels of nitrite in Hep3B cells. In order to investigate the role of NO in Epo production in Hep3B cells under normoxic (20% O2) conditions, we have studied the effects of interferon-gamma (IFN-gamma) on Epo production. IFN-gamma is known to induce iNOS and enhance the production of NO. IFN-gamma produced significant increases in medium levels of Epo and nitrite. IFN-gamma also significantly increased cGMP levels in Hep3B cells. Furthermore, NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, significantly decreased IFN-gamma induced elevations in medium levels of Epo and nitrite as well as cGMP levels in Hep3B cells. These results provide further support for an important role of the NO/cGMP system in hypoxic regulation of Epo production in Hep3B cells.
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PMID:Inducible nitric oxide synthase expression and erythropoietin production in human hepatocellular carcinoma cells. 912 39

1. We have investigated the mechanism by which L-arginine stimulates membrane depolarization, an increase of intracellular calcium ([Ca2+]i) and insulin secretion in pancreatic beta-cells. 2. L-Arginine failed to affect beta-cell metabolism, as monitored by NAD(P)H autofluorescence. 3. L-Arginine produced a dose-dependent increase in [Ca2+]i, which was dependent on membrane depolarization and extracellular calcium. 4. The cationic amino acids L-ornithine, L-lysine, L-homoarginine (which is not metabolized) and NG-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor) produced [Ca2+]i responses similar to that produced by L-arginine. The neutral nitric oxide synthase inhibitors NG-nitro-L-arginine (L-NNA) and N omega-monomethyl-L-arginine (L-NAME) also increased [Ca2+]i. D-Arginine was ineffective. 5. L-Arginine did not affect whole-cell Ca2+ currents or ATP-sensitive K+ currents, but produced an inward current that was carried by the amino acid. 6. The reverse transcriptase-polymerase chain reaction demonstrated the presence of messenger RNA for the murine cationic amino acid transporters mCAT2A and mCAT2B within the beta-cell. 7. L-Arginine did not affect beta-cell exocytosis as assayed by changes in cell capacitance. 8. Our data suggest that L-arginine elevates [Ca2+]i and stimulates insulin secretion as a consequence of its electrogenic transport into the beta-cell. This uptake is mediated by the mCAT2A transporter.
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PMID:Electrogenic arginine transport mediates stimulus-secretion coupling in mouse pancreatic beta-cells. 913 Jan 59


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