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)

Although the neurohypophyseal hormone oxytocin (OT) is best know for its role in reproduction, OT also stimulates natriuresis at physiological plasma levels. This effect is mediated via specific renal OT receptors (OTRs). In the present study, we have characterized rat renal OTR gene transcripts and assessed their regulation during gestation and in response to gonadal steroid treatment. Using a specific rat OTR probe, two major OTR messenger RNA (mRNA) bands [6.7 and 4.8 kilobases (kb)] were detected in renal extracts, corresponding to two of the three bands present in rat uterus. In contrast to the dramatic rise of OTR mRNA levels at term in the uterus and pituitary, renal OTR mRNA levels underwent a strong more than 3-fold decrease at term. Binding studies using a iodinated specific OT antagonist revealed a concomitant decrease in renal OT-binding sites. On the other hand, estrogen (E2) treatment led to an increase in renal OTR mRNA levels, as is also the case in the uterus and pituitary. However, the predominant E2-induced mRNA species were shorter (3.6 and 3.2 kb) than those present in control rat kidneys (6.7 and 4.8 kb). Analysis by reverse transcriptase-PCR and 5'- and 3'-directed complementary DNA probes indicated that the E2-induced OTR mRNA transcripts possessed the same coding region, but contained a shortened 3'-untranslated region. Binding studies showed that E2 treatment also led to an increase in renal OT-binding sites, suggesting that the shortened OTR transcripts encoded a functional receptor. The present study indicates that the uterine-type OTR gene is expressed in rat kidneys, but that the mechanisms controlling the expression of this gene in the two tissues are markedly different. The differential tissue-specific regulation of OTR gene expression may represent a mechanism by which circulating OT can assume a multifunctional role in both reproduction and sodium homeostasis.
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PMID:Renal oxytocin receptor messenger ribonucleic acid: characterization and regulation during pregnancy and in response to ovarian steroid treatment. 877 Aug 90

We report a method that allows accurate, absolute quantification of gene expression in a single reverse transcriptase (RT)-PCR reaction. This method makes use of novel high-performance liquid chromatography (HPLC) technology to resolve and quantify the products of competitive, mutant RNA PCRs. The HPLC technique allows rapid, high resolution of reaction products. On-line UV detection eliminates the need for radiolabel or other tracers. The HPLC technique also demonstrates that these competition reactions readily generate heteroduplex products. The ability of HPLC to resolve and quantify heteroduplex products is fundamental to the accuracy of the technique. Accurate measurements of gene expression have been obtained over four orders of magnitude and experiments employing predetermined quantities of specific native RNA input have demonstrated the ability of the system to provide absolute estimates of gene expression. Large size differences between native and mutant RNA inputs affected reverse transcriptase (RT) efficiency, but not PCR amplification efficiency. However, the magnitude of the RT efficiency effect can be estimated, is reproducible, and can therefore be adjusted by a calculated correction factor. The RT efficiency difference can been eliminated by reduction in the magnitude of the sequence difference between native and mutant RNA so that no correction factor is required. The application of the technique to quantification of expression of the alpha 1 subunit of sodium, potassium-ATPase in microdissected nephron segments is demonstrated.
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PMID:Accurate and absolute quantitative measurement of gene expression by single-tube RT-PCR and HPLC. 880 71

The effects of dietary sodium intake on the gene expression of the renin-angiotensin system (RAS) were investigated in rat central and peripheral tissues in a single set of experiment. Northern and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques were used to detect mRNA expression in rats fed a low- or a high-sodium diet (5 or 500 mmol Na+/kg diet) for 20 days. Plasma and renal renin levels were elevated in rats maintained on the low-sodium diet. Sodium deprivation enhanced the expression of angiotensinogen, renin, AT1A and AT1B receptor subtypes in the hypothalamus, but suppressed them in the brainstem. Kidney and adrenal levels of those mRNAs were also enhanced in the sodium-restricted rats. Both AT1A and AT1B mRNAs changed in a similar magnitude in each tissue examined upon dietary sodium intake. AT1A was the predominant receptor subtype of AT1 in all the tissues examined in the present study except the adrenal gland. The present study demonstrated that dietary sodium modulated the gene expression of the RAS components in the central and peripheral tissues. It also showed that the RAS components in the brainstem and hypothalamus were differentially expressed upon sodium deprivation. This suggests different roles of the RAS in these tissues in maintaining body fluid homeostasis in response to different sodium intakes.
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PMID:Gene expression of central and peripheral renin-angiotensin system components upon dietary sodium intake in rats. 895 82

The Na+/Ca2+ exchanger is an important plasma-membrane Ca2+ transport protein. Functional studies of the effects of removal of external Na+ on airway smooth muscle tone, and studies of Na+-dependent Ca2+ fluxes in membrane vesicles have provided indirect evidence of an important role for Na+/Ca2+ exchange in regulating airway smooth muscle tone. Recent molecular studies have identified seven isoforms of the Na+/Ca2+ exchanger (NCX1) gene in other tissues, but direct molecular characterization of the airway smooth muscle Na+/Ca2+ exchanger has not previously been performed. We have therefore used a reverse transcriptase-polymerase chain (RT-PCR) reaction method and DNA sequencing to study the Na+/Ca2+ exchanger isoform present in human airway smooth muscle. The dominant isoform was found to be virtually identical to the NACA3 isoform originally described in rabbit kidney cortex. The DNA sequence of the Na+/Ca2+ exchanger exon B that we obtained is previously unpublished for any human tissue. This study provides the first definitive molecular evidence of the existence of an Na+/Ca2+ exchanger in airway smooth muscle. The molecular characterization of the human airway smooth muscle Na+/Ca2+ exchanger will facilitate future electrophysiologic studies of its function.
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PMID:Molecular characterization of the human airway smooth muscle Na+/Ca2+ exchanger. 896 66

Mutations in a Cl- channel (cystic fibrosis transmembrane conductance regulator or CFTR) are responsible for the cystic fibrosis (CF) phenotype. Increased Na+ transport rates are observed in CF airway epithelium, and recent studies suggest that this is due to an increase in Na+ channel open probability (Po). The Xenopus renal epithelial cell line, A6, expresses both cAMP-activated 8-picosiemen (pS) Cl- channels and amiloride-sensitive 4-pS Na+ channels, and provides a model system for examining the interactions of CFTR and epithelial Na+ channels. A6 cells express CFTR mRNA, as demonstrated by reverse transcriptase-polymerase chain reaction and partial sequence analysis. A phosphorothioate antisense oligonucleotide, complementary to the 5' end of the open reading frame of Xenopus CFTR, was used to inhibit functional expression of CFTR in A6 cells. Parallel studies utilized the corresponding sense oligonucleotide as a control. CFTR protein expression was markedly reduced in cells incubated with the antisense oligonucleotide. Incubation of A6 cells with the antisense oligonucleotide led to inhibition of forskolin-activated amiloride-insensitive short circuit current (Isc). After a 30-min exposure to 10 microM forskolin, 8-pS Cl- channel activity was detected in only 1 of 31 (3%) cell-attached patches on cells treated with antisense oligonucleotide, compared to 5 of 19 (26%) patches from control cells. A shift in the single-channel current-voltage relationship derived from antisense-treated cells was also consistent with a reduction in Cl- reabsorption. Both amiloride-sensitive Isc and Na+ channel Po were significantly increased in antisense-treated, forskolin-stimulated A6 cells, when compared with forskolin-stimulated controls. These data suggest that the regulation of Na+ channels by CFTR is not limited to respiratory epithelia and to epithelial cells in culture overexpressing CFTR and epithelial Na+ channels.
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PMID:Expression of the cystic fibrosis phenotype in a renal amphibian epithelial cell line. 899 2

In a murine model of rickettsial disease in which, as in human rickettsioses, endothelial cells are the major target of infection, depletion of IFN-gamma or TNF-alpha converts a sublethal infection into a uniformly fatal disease with overwhelming rickettsial growth and decreased nitric oxide (NO) synthesis. The kinetics of NO production and rickettsial survival and growth were examined on Days 1, 2, and 3 after inoculation of endothelial cells with Rickettsia conorii under four different experimental conditions: (a) no cytokine treatment, (b) treatment with IFN-gamma and TNF-alpha, (c) treatment with cytokines and NG monomethyl-L-arginine, a competitive inhibitor of NO synthesis, and (d) treatment with sodium nitroprusside, a source of NO. Endothelial cells were examined for the presence of inducible nitric oxide synthase mRNA by specific reverse transcriptase-PCR after stimulation with IFN-gamma and TNF-alpha. Cytokine-stimulated and unstimulated rickettsiae-infected endothelial cells were examined by electron microscopy to observe the cellular and rickettsial events. Transformed and diploid mouse endothelial cells stimulated by the combination of recombinant murine IFN-gamma and TNF-alpha killed intracellular Rickettsia conorii by a mechanism that required the synthesis of NO. The antirickettsial effect and NO synthesis were inhibited by treatment of endothelial cells with NG monomethyl-L-arginine. Addition of nitroprusside, which released NO, also exerted a strong antirickettsial effect in the absence of IFN-gamma and TNF-alpha. Endothelial inducible nitric oxide synthase mRNA was detected 4 hours after cytokine stimulation, increased substantially at 8 hours, and decreased to low levels by 72 hours. Ultrastructural evaluation revealed that endothelial cells effected rickettsial killing in association with autophagy. Double membranes of endothelial cell granular endoplasmic reticulum surrounded rickettsiae, which were also observed being destroyed within phagolysosomes. This study demonstrated for the first time that endothelial cells are capable of killing rickettsiae. When stimulated by the combination of IFN-gamma and TNF-alpha, mouse endothelial cells kill Rickettsia conorii by an NO-dependent mechanism. Within the endothelium, NO exerts a rickettsicidal effect.
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PMID:Cytokine-induced, nitric oxide-dependent, intracellular antirickettsial activity of mouse endothelial cells. 901 Apr 56

The efficacy of angiotensin converting enzyme (ACE) inhibitors is well known to prevent the formation of angiotensin II (Ang II) by these agents. The objective of the present study was to evaluate the hemodynamic, biochemical, and morphological responses to Ang II receptor blockade with E-4177, 3-[(2'-carboxybiphenyl-4-yl) methyl]-2-cyclopropyl-7-methyl 3H-imidazol[4,5-b] pyridine, in rats with a healing myocardial infarction that had been induced by the surgical occlusion of the left main coronary artery. The left ventricular weight increased 8 and 12 weeks after infarction in comparison to that in sham-operated rats. Among the rats with experimental infarction, treatment with E-4177 significantly decreased the left ventricular weight. Although the infarct size was not affected by E-4177, its administration ameliorated the elevated end-diastolic pressure and reduced the systolic pressure. The effects of this agent on the levels of Ang II type 1 (AT1) receptor mRNA and ACe mRNA were evaluated in the non-infarcted myocardium by reverse transcriptase polymerase chain reaction and binding assays. Treatment with E-4177 reduced both the elevated AT1 mRNA and the number of Ang II receptors, but not the ACE mRNA or ACE activity. While the receptor affinity remained unchanged with this agent, the collagen concentration was decreased. On the other hand, the depressed Na+/Ca2+ exchange activity was restored in the non-infarcted myocardium at 8 and 12 weeks after injury to the level seen in the sham-operated rats. These findings suggest that the AT1 receptor antagonist, E-4177, has a beneficial effect on the hemodynamics in spite of the lack of any improvement in the infarct size. These observations may be partly attributed to the prevention of angiotensin II formation during the period of post-infarction healing.
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PMID:Regression of hypertrophy after myocardial infarction is produced by the chronic blockade of angiotensin type 1 receptor in rats. 901 34

Nucleoside transport may be involved in the regulation of extracellular levels of adenosine, an inhibitory neuromodulator in the central nervous system. Previous reports have provided functional evidence for Na+-dependent nucleoside transport in rat brain. We isolated total RNA from various regions of rat brain and tested for the presence of mRNA for two recently cloned Na+/nucleoside cotransporters using reverse transcriptase PCR (RT-PCR). Messenger RNA for a pyrimidine-selective Na+/nucleoside cotransporter mRNA (rCNT1) was detected in samples from each brain region tested by RT-PCR amplification of a 309-bp DNA product. Southern blot and sequence analysis confirmed that this product was derived from rCNT1 mRNA. A purine-selective Na+/nucleoside cotransporter mRNA (rCNT2, also termed SPNT) was detected throughout brain by amplifying a 235-bp DNA product, the sequence of which was identical to that published. These experiments demonstrate the presence of both rCNT1 and rCNT2 mRNA in rat brain.
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PMID:Demonstration of the existence of mRNAs encoding N1/cif and N2/cit sodium/nucleoside cotransporters in rat brain. 901 95

Somatostatin modulates important physiologic functions of the kidney, including mesangial cell contraction, glomerular prostaglandin synthesis, and phosphate, water and sodium excretion. In diabetic nephropathy, somatostatin inhibits renal hypertrophy. High affinity somatostatin receptors are expressed in the kidney. Circulating somatostatin concentrations, however, are generally well below the affinity constants of known somatostatin receptors. Thus, we hypothesized that somatostatin is produced in the kidney and released locally to act in an autocrine/paracrine manner. Using reverse transcriptase and polymerase chain reaction (RT-PCR) analysis, we found that fresh human renal cortex and cultured human mesangial cells express somatostatin mRNA. Restriction enzyme and Southern blot analysis confirmed that RT-PCR cDNA products were derived from somatostatin mRNA. Radioimmunoassay of mesangial cell culture supernatants demonstrated SS-immunoreactive peptide (87 +/- 30 pg/ml compared to 19 +/- 9 pg/ml in medium not exposed to cells; P < 0.05). In contrast, renal cells did not transcribe detectable levels of vasoactive intestinal peptide (VIP) or neuropeptide Y (NPY) mRNA, nor did they synthesize measurable peptide. Our results demonstrate that renal cells produce somatostatin and suggest that kidney-derived somatostatin may regulate renal function in an autocrine/paracrine manner. Characterization of this pathway may lead to novel methods to alter the course of diabetic nephropathy and other renal diseases.
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PMID:Somatostatin expression in human renal cortex and mesangial cells. 909 50

Deletions and other genome rearrangements are associated with carcinogenesis and inheritable diseases. The pink-eyed unstable (pun) mutation in the mouse is caused by duplication of a 70-kb internal fragment of the p gene. Spontaneous reversion events in homozygous pun/pun mice occur through deletion of a duplicated sequence. Reversion events in premelanocytes in the mouse embryo detected as black spots on the gray fur of the offspring were inducible by the carcinogen x-rays, ethyl methanesulfonate, methyl methanesulfonate, ethyl nitrosourea, benzo[a]pyrene, trichloroethylene, benzene, and sodium arsenate. The latter three carcinogens are not detectable with several in vitro or in vivo mutagenesis assays. We studied the molecular mechanism of the carcinogen-induced reversion events by cDNA analysis using reverse transcriptase-PCR method and identified the induced reversion events as deletions. DNA deletion assays may be sensitive indicators for carcinogen exposure.
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PMID:Carcinogens induce reversion of the mouse pink-eyed unstable mutation. 911 32


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