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 surveyed the occurrence of unique restriction sites on the cDNAs of viroids, virusoids, and plant viral satellite RNAs that have a circular RNA as an intermediate of replication and found that four such sites would linearize their circular cDNAs. A rapid and simple method was then developed for cloning a naturally occurring viroid from Nematanthus wettsteinii plants. First-strand cDNA was synthesized using random hexanucleotide DNA primers and M-MuLV reverse transcriptase (Superscript RT). Second-strand DNA was synthesized by employing the replacement synthesis method using Escherichia coli RNase H, E. coli DNA polymerase I, E. coli DNA ligase, and beta-NAD+. The circular double-stranded DNA was analyzed for the presence of commonly available, unique restriction sites and subsequently linearized with a selected restriction enzyme. The linear cDNA was ligated to dephosphorylated plasmid vector pGEM 3Z f(+) and cloned in E. coli strain DH5 alpha. This cDNA cloning procedure is suitable for cloning sequence variants of well-characterized viroids, virusoids, certain plant viral satellite RNAs, and new such pathogens of unknown sequence.
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PMID:A rapid and versatile method for cloning viroids or other circular plant pathogenic RNAs. 138 86

We have described a strain of Escherichia coli that expresses high levels of enzymatically active, soluble, HIV-1 reverse transcriptase (A. Hizi, C. McGill, and S. H. Hughes, Proc. Natl. Acad. Sci. USA, 85, 1218-1222, 1988). The clone can be used as a source of the enzyme and to generate and characterize mutations in the reverse transcriptase. We have made a series of small in-frame insertions in the region that encodes the reverse transcriptase. When the mutant plasmids are reintroduced into E. coli, they induce the synthesis of mutant forms of the enzyme. With one interesting exception, the reduction in RNA-dependent DNA polymerizing activity seen in the mutants correlates well with the degree of sequence conservation among the various reverse transcriptases. Insertions into regions that are evolutionarily conserved have a more profound effect on RNA-dependent DNA polymerase activity than do insertions into regions that are less conserved. The exception to this simple correlation is that a small insertion into the region encoding RNase H gives rise to a protein with essentially no RNA-dependent DNA polymerase activity. We suggest that this mutation may affect the ability of the reverse transcriptase to fold properly, which might explain our previous observation that small carboxyl terminal deletions profoundly affect RNA-dependent NAD polymerase activity.
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PMID:Effects of small insertions on the RNA-dependent DNA polymerase activity of HIV-1 reverse transcriptase. 247 Jan 95

Mineralocorticoid receptors (MRs) are nonselective in vitro, binding corticosterone, cortisol, and aldosterone with similar affinity. In the distal nephron in vivo, MRs are selectively activated by aldosterone despite much higher glucocorticoid levels. This has been suggested to reflect the action of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which catalyzes rapid inactivation of corticosterone to 11-dehydrocorticosterone (cortisol to cortisone). However, cellular models of this effect have not been reported, and a recent study suggested that properties intrinsic to MR contribute to aldosterone selectivity. We have screened clonal mammalian cell lines for 11 beta-HSD activity. Pig kidney epithelial LLC-PK1 cells expressed by far the greatest 11 beta-HSD activity. In cell homogenates, this was NAD-dependent, with Km for corticosterone of 34.4 nM and cortisol of 89.7 nM. Intact LLC-PK1 cells showed similar apparent Km for corticosterone (13.9 nM) and cortisol (79.4 nM); only 11 beta-dehydrogenation was detected. These biochemical data indicate the expression of the type 2 isoform, 11 beta-HSD2. Using primers to conserved regions of 11 beta-HSD2, a reverse transcriptase-polymerase chain reaction product was obtained from LLC-PK1 cell RNA. Sequence analysis revealed close homology to previously cloned 11 beta-HSD2 cDNAs from several species. LLC-PK1 cell 11 beta-HSD activity was inhibited by carbenoxolone (IC50 approximately 10(-8) M) and high concentrations of estradiol or progesterone (10(-7) and 10(-6) M), but was induced at lower estradiol concentrations (10(-8) and 10(-9) M). To examine whether the 11 beta-HSD2 activity in LLC-PK1 cells regulates corticosterone access to MR, cells were transfected with the corticosteroid-inducible mouse mammary tumor virus long terminal repeat-luciferase reporter construct. Cell transfection by a lipofection method did not alter 11 beta-HSD activity in LLC-PK1 cells. LLC-PK1 cells expressed low levels of MR (13.9 fmol/mg protein, dissociation constant (Kd) 0.3 x 10(-9) M for aldosterone) and glucocorticoid receptors (GR; 18.5 fmol/mg protein, Kd 0.3 x 10(-9) M for dexamethasone). Transfection with mouse mammary tumor virus long terminal repeat-luciferase reporter construct alone suggested that the endogenous levels of MR and GR were insufficient to affect transcription. However, cotransfection of LLC-PK1 cells with pRShMR, an MR expression plasmid, allowed at least 50-fold induction of luciferase with 10(-8) M aldosterone; the ED50 0.3 x 10(-9) M closely reflects the in vitro affinity of MR for aldosterone. Corticosterone only weakly induced luciferase (maximum of 6-fold induction).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:LLC-PK1 cells model 11 beta-hydroxysteroid dehydrogenase type 2 regulation of glucocorticoid access to renal mineralocorticoid receptors. 758 9

The inactivation of physiological glucocorticoids by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) confers mineralocorticoid specificity to certain aldosterone target tissues. However, 11 beta-HSD activity in a human mineralocorticoid-responsive tissue has never been characterized. The present studies describe the features of 11 beta-HSD in the cultured human colonic epithelial cell line, T84. The 11 beta-HSD activity of T84 cells resided in the microsomal fraction and showed a marked preference for NAD rather than NADP as cofactor. NAD or NADP (200 microM) increased the conversion of corticosterone to 11-dehydrocorticosterone by 24.1 +/- 2.1 and 0.5 +/- 0.7 pmol.mg protein-1.20 min-1, respectively, indicating a > 40-fold preference for NAD vs. NADP. The Michaelis constant values for corticosterone and cortisol were 11.3 +/- 1.5 and 79.8 +/- 10 nM, respectively. The T84 11 beta-HSD was inhibited by 11-dehydrocorticosterone in a noncompetitive fashion [inhibition constant (Ki) = 180 +/- 9.6 nM] and by carbenoxolone in a competitive fashion (Ki = 17.4 +/- 1.3 nM). The expression of mineralocorticoid receptors in these cells was demonstrated by reverse transcriptase-polymerase chain reaction of mRNA isolated from T84 cells and by [3H]aldosterone binding studies. The coexpression of this NAD-dependent isoform of 11 beta-HSD and mineralocorticoid receptors is consistent with the view that the NAD-dependent isoform is responsible for the specificity of mineralocorticoid responses.
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PMID:NAD-dependent 11 beta-hydroxysteroid dehydrogenase in cultured human colonic epithelial cells. 761 67

11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) catalyzes the conversion of active cortisol to inactive cortisone, and regulates the access of cortisol to both the mineralocorticoid and glucocorticoid receptors. Two isoforms of 11 beta-HSD have been described, the cloned "type 1" NADP(H)-dependent dehydrogenase/oxo-reductase and a high affinity NAD-dependent dehydrogenase (type 2). In the fetus, 11 beta-HSD activity may serve to protect developing tissues from cortisol excess or may modulate the permissive actions of glucocorticoids. We have studied 11 beta-HSD activity and mRNA levels in human mid-gestational fetal tissues. Tissue homogenates were incubated with either 0.1 mumol/L cortisol and 400 mumol/L NAD, 2.5 mumol/L cortisol and 400 mumol/L NADP, or 0.1 mumol/L cortisone wither either 400 mumol/L NADPH or NADH. No activity (< 2.5% conversion) was observed in fetal tissues using either cortisone or 2.5 mumol/L cortisol as a substrate. 11-oxo-reductase activity was observed in maternally-derived decidua. In keeping with these activity studies, northern blot analysis of fetal tissue RNA and PCR-reverse transcriptase of type 1 11 beta-HSD mRNA indicated 11 beta-HSD mRNA in decidua, but failed to detect any type 1 11 beta-HSD mRNA transcripts in fetal tissues. In contrast when 0.1 mumol/L cortisol was used as a substrate in the presence of NAD, 11 beta-HSD activity was ubiquitous with highest levels seen in the kidney (131 +/- 16 (SE) pmoles cortisone formed/h/mg.protein) > lung > gonad > liver > colon. 11 beta-HSD activity in fetal tissues is mediated by the type 2, high affinity, isoform. The widespread distribution of this novel isoform suggests that it may play an important role in fetal development. Type 1 11 beta-HSD mRNA and activity are absent in mid-gestational fetal tissues, but present in maternally-derived decidua, suggesting that its ontogeny is a late-gestational of post-natal event.
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PMID:Type 2 11 beta-hydroxysteroid dehydrogenase in human fetal tissues. 820 Sep 59

Intrauterine growth retardation (IUGR) resulting from placental insufficiency is a common complication of pregnancy. Bilateral uterine artery ligation of the pregnant rat is a model which mimics intrauterine growth retardation in the human. IUGR rat fetuses have altered hepatic energy and redox states, with reduced fetal hepatic ATP/ADP ratio, increased cytosolic NAD+/NADH ratio, and decreased mitochondrial NAD+/NADH ratio. These critical changes in energy metabolism contribute to IUGR. The effects of these changes at the molecular level are largely unknown. To address these effects we compared hepatic mRNA populations of IUGR and normal fetuses and neonates using mRNA differential display, a polymerase chain reaction-based method for assaying transcriptional differences under various conditions. We isolated and sequenced 18 cDNA products whose mRNA levels were elevated in IUGR compared with normal fetal and neonatal liver. These analyses demonstrated that NADH-ubiquinone oxireductase subunit 4L mRNA (ND-4L) was significantly increased in liver of IUGR fetuses and neonates. This suggested that IUGR may be associated with altered expression of genes involved in the generation of ATP and NADH. Therefore, we measured mRNA levels of adenine-nucleotide translocator-2 (ANT-2), glucose-6-phosphate dehydrogenase (G6PD), mitochondrial malate dehydrogenase (MMD), ornithine transcarbamylase (OTC), and phosphofructokinase-2 (PFK-2) using a semiquantitative reverse transcriptase-polymerase chain reaction-based technique. In the IUGR fetus, ND-4L, ANT-2, G6PD, and MMD mRNA levels were significantly elevated; PFK-2 mRNA levels were unchanged, and OTC levels were decreased. In the IUGR newborn rat, mRNA levels of all 6 enzymes were increased suggesting that the metabolic state of the growth retarded newborn remains abnormal after birth. Uteroplacental insufficiency affects the immediate and long-term metabolic milieu of the growth retarded animal, and forces specific adjustments, including the expression of mRNA encoding enzymes involved with hepatic energy production.
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PMID:Altered hepatic gene expression of enzymes involved in energy metabolism in the growth-retarded fetal rat. 892 56

We searched for the presence of calretinin (CR) in 12 colonic cancer cell lines using immunohistochemistry, Western blot analysis and the reverse transcriptase-polymerase chain reaction (RT-PCR). We were able to demonstrate that calretinin is expressed in the cell lines HT-29, WiDr, LoVo, LS180, CO112, CO115. SW480, SW620, COLO205 and SK-CO-1, while no detectable amounts were found in the cell lines SW1116 and Caco-2. In general, rapidly proliferating cell lines expressed calretinin, whereas the protein was absent from cell lines with a low multiplication rate. A possible role for calretinin in maintaining the proliferative cycle of tumor cells is discussed.
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PMID:Heterogeneity of expression of the calcium-binding protein calretinin in human colonic cancer cell lines. 904 11

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

To date, two isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD) have been characterized: a low affinity, NADP+-dependent isoform (11betaHSD1) and a high affinity, NAD+-dependent isoform which metabolizes dexamethasone and is inhibited by cortisone (11betaHSD2). Having previously reported a relationship between ovarian 11betaHSD activities and conception in women undergoing in vitro fertilization (IVF-ET), the objective of the present study was to identify which isoforms of 11betaHSD metabolize glucocorticoids in cultures of human granulosa-lutein cells. In both intact cells and cell homogenates, two distinct 11betaHSD activities were identified with differing affinities for cortisol (Km = 490 nM and 2.6 microM). Even at low concentrations, cortisol oxidation was preferentially supported by NADP+ and was independent of NAD+. Although inhibited by the hemisuccinate ester of glycyrrhetinic acid, carbenoxolone, the predominant 11betaHSD activity in intact cells was resistant to end-product inhibition. Intact cells were also able to reduce [3H]cortisone (Km = 190 nM) but did not metabolize [3H]dexamethasone. 11BetaHSD1 mRNA was expressed in 23 of 28 cell cultures whereas 11betaHSD2 mRNA was not expressed in any of the 22 independent cultures studied by reverse transcriptase-polymerase chain reaction (RT-PCR). We conclude that human granulosa-lutein cells express both type 11betaHSD and a novel isoform of this enzyme. While the low affinity 11beta-dehydrogenase and 11-ketosteroid reductase activities exhibit properties consistent with 11betaHSD1, the high affinity 11beta-dehydrogenase differs from 11betaHSD2 in that it is NADP+-dependent, does not metabolize dexamethasone and is resistant to end-product inhibition.
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PMID:Isoforms of 11beta-hydroxysteroid dehydrogenase in human granulosa-lutein cells. 932 45

1. Presynaptic injection of cyclic ADP-ribose (cADPR), a modulator of the ryanodine receptor, increased the postsynaptic response evoked by a presynaptic spike at an identified cholinergic synapse in the buccal ganglion of Aplysia californica. 2. The statistical analysis of long duration postsynaptic responses evoked by square depolarizations of the voltage-clamped presynaptic neurone showed that the number of evoked acetylcholine (ACh) quanta released was increased following cADPR injection. 3. Overloading the presynaptic neurone with cADPR led to a transient increase of ACh release followed by a depression. 4. cADPR injections did not modify the presynaptic Ca2+ current triggering ACh release. 5. Ca2+ imaging with the fluorescent dye rhod-2 showed that cADPR injection rapidly increased the free intracellular Ca2+ concentration indicating that the effects of cADPR on ACh release might be related to Ca2+ release from intracellular stores. 6. Ryanodine and 8-amino-cADPR, a specific antagonist of cADPR, decreased ACh release. 7. ADP-ribosyl cyclase, which cyclizes NAD+ into cADPR, was present in the presynaptic neurone as shown by reverse transcriptase-polymerase chain reaction experiments. 8. Application of NAD+, the substrate of ADP-ribosyl cyclase, increased ACh release and this effect was prevented by both ryanodine and 8-amino-cADPR. 9. These results support the view that Ca(2+)-induced Ca2+ release might be involved in the build-up of the Ca2+ concentration which triggers ACh release, and thus that cADPR might have a role in transmitter release modulation.
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PMID:Cyclic ADP-ribose and calcium-induced calcium release regulate neurotransmitter release at a cholinergic synapse of Aplysia. 951 1


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