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)

Studies in normotensive rats showed that excessive fetal exposure to maternal glucocorticoids retards growth and programs hypertension in later life. This excessive exposure is proposed to occur due to a reduction of the placental barrier to maternal glucocorticoids that is provided by 11beta-hydroxysteroid dehydrogenase (11betaHSD). To assess the possible alterations of glucocorticoid placental barrier in two genetic models of hypertension - spontaneously hypertensive (SHR) and Dahl salt-sensitive rats (DS) and their normotensive counterparts Wistar-Kyoto (WKY) and Dahl salt-resistant rats (DR)-we performed real-time reverse transcriptase-polymerase chain reaction analysis and bioactivity measurements of placental 11betaHSD in the last third of gestation. Whereas 11betaHSD2 mRNA expression was not different among the investigated strains, 11betaHSD1 mRNA abundance was 2.4 times higher in WKY than in SHR and 9.6 times higher in DS than in DR placentae. The 11betaHSD2 activity studies performed in placental homogenates revealed activity that did not differ among the strains. Concomitant with 11betaHSD1 mRNA expression 11-oxoreductase activity was clearly evident in all strains and was higher in WKY and DS rats than in SHR and DR, respectively. Nevertheless, the net 11betaHSD activity of tissue fragments (11beta-dehydrogenase minus 11-oxoreductase) was tended toward dehydrogenase action, ie, toward corticosterone inactivation and was significantly lower in DS than in DR rats. The 11beta-dehydrogenase/11-oxoreductase ratio was less than 2:1 in SHR and WKY rats, whereas this ratio was 9:1 in DR and 4.5:1 in DS rats. These data suggest that the placental glucocorticoid barrier is not decreased in SHR rats in comparison with normotensive WKY but is lower in DS than in DR counterparts. It cannot be excluded, therefore, that the placental glucocorticoid barrier in Dahl rats influences the pathways that might lead to the sensitivity of blood pressure to high salt intake in later life.
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PMID:Placental 11beta-hydroxysteroid dehydrogenase in Dahl and spontaneously hypertensive rats. 1274 3

Microarrayed compound screening format (muARCS) is a novel high-throughput screening technology that uses agarose matrices to integrate various biochemical or biological reagents in the assay. To evaluate the feasibility of using the muARCS technology for nucleic acid polymerization assays, the authors developed HIV reverse transcription (RT) and E1-dependent human papillomavirus (HPV) replication assays in this format. HIV RT is an RNA-dependent DNA polymerase, whereas HPV E1 is a DNA helicase. To ensure the efficient capture of the nucleic acid polymerization reaction and to minimize the nonspecific binding, the authors used a SAM(2) biotin capture membrane in the assay. In both studies, the nucleic acid substrate was biotinylated on one end and was bound to the SAM(2) membrane. A low melting-point agarose gel containing the rest of the reaction components was first placed on a polystyrene sheet spotted with compounds to allow passive diffusion of the compounds into the gel. The gel was removed from the compound sheet and applied to the SAM(2) membrane with the immobilized nucleic acid template to initiate the polymerization. After the incubation, the membrane was washed with a high-salt buffer and exposed for imaging. Potential inhibitors can be seen as white spots on a dark background. The sensitivity for the known inhibitors appears to be comparable in muARCS as in a traditional 96-well plate assay. The methodology described in this paper further expands the applications of muARCS technology.
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PMID:Use of SAM2 biotin capture membrane in microarrayed compound screening (muARCS) format for nucleic acid polymerization assays. 1285 81

Splicing of the Saccharomyces cerevisiae mitochondrial DNA group II intron aI2 depends on the intron-encoded 62-kDa reverse transcriptase-maturase protein (p62). In wild-type strains, p62 remains associated with the excised intron lariat RNA in ribonucleoprotein (RNP) particles that are essential for intron homing. Studies of a bacterial group II intron showed that the DIVa substructure of intron domain IV is a high-affinity binding site for its maturase. Here we first present in vitro evidence extending that conclusion to aI2. Then, experiments with aI2 DIVa mutant strains show that the binding of p62 to DIVa is not essential for aI2 splicing in vivo but is essential for homing. Because aI2 splicing in the DIVa mutant strains remains maturase dependent, splicing must rely on other RNA-protein contacts. The p62 that accumulates in the mutant strains has reverse transcriptase activity, but fractionation experiments at high and low salt concentrations show that it associates more weakly than the wild-type protein with endogenous mitochondrial RNAs, and that phenotype probably explains the homing defect. Replacing the DIVa of aI2 with that of the closely related intron aI1 improves in vivo splicing but not homing, indicating that DIVa contributes to the specificity of the maturase-RNA interaction needed for homing.
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PMID:The DIVa maturase binding site in the yeast group II intron aI2 is essential for intron homing but not for in vivo splicing. 1461 20

Prostratin, a non-tumour promoting phorbol ester, exhibit a potent anti-HIV activity against human immunodeficiency virus type 1 (HIV-1). However, the antiviral mechanism of prostratin is not well defined. In the present study, we report that prostratin exhibits potent antiviral activity against different strains of HIV-1 (subtypes B and D), a clinical HIV isolate (L1), HIV-2 (ROD and EHO) and SIV (MAC251) with EC50-values ranging from 0.02-0.09 microg/ml. Prostratin was equally active against HIV strains resistant to the polyanionic binding inhibitor dextran sulphate, the fusion inhibitor T-20 (enfuvirtide), nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors (PIs). In contrast, prostratin lost 4.4- and 6.8-fold of its effect against the HIV strains resistant to AMD3100 and the quaternary ammonium salt QAS10+, respectively. As shown by time-of-addition experiments, prostratin needs to be present at the time of viral adsorption to exert its antiviral activity. We selected an HIV strain (NL4.3/PROS) resistant to prostratin in MT-4 cells. The sensitivity of NL4.3/PROS towards prostratin, dextran sulphate and QAS10+ was reduced by 3.2, 4.1 and >50-fold, respectively. However, NL4.3/ PROS was still sensitive to AMD3100, T-20, NRTIs (zidovudine and nevirapine) and a PI (ritonavir). Recombination of the gp160-gene of the NL4.3/PROS strain in a NL4.3 wild-type molecular clone fully rescued its phenotypic resistance. DNA sequencing of the NL4.3/PROS strain revealed mutations throughout the gp120 gene previously associated with resistance towards other HIV entry inhibitors. We concluded that prostratin inhibits the entry step of the replication cycle of HIV by interacting with a cellular target necessary for viral entry.
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PMID:Potent and selective inhibition of HIV and SIV by prostratin interacting with viral entry. 1496 38

This study was aimed to investigate the effects and the mechanism of mangiferin on chronic myeloid leukemia cell lines K562 cells in vitro. The antiproliferation effects of mangiferin on K562 leukemia cells were tested by tetrazolium salt (MTT) method; the apoptosis induced by mangiferin on K562 cell line was explored by means of cell morphology, DNA gel electrophoresis and flow cytometry. The changes in bcr/abl gene expression was detected by using reverse transcriptase (RT)-PCR. The results showed that five different concentrations of mangiferin (25 - 200 micromol/L) dose-dependently and time-dependently inhibited the proliferation of K562 cells, and induced apoptosis in K562 cell line. RT-PCR revealed that bcr/abl gene expression was down-regulated when K562 cells had been treated with different concentrations of mangiferin. In conclusion, mangiferin remarkably inhibits the proliferation of K562 leukemia cells in vitro, and induces apoptosis in K562 cell line probably through down-regulation of bcr/abl gene expression.
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PMID:[CML cell line K562 cell apoptosis induced by mangiferin]. 1549 16

Chloroplast metabolism is rapidly affected by salt stress. Photosynthesis is one of the first processes known to be affected by salinity. Here, we report that salinity inhibits chloroplast post-transcriptional RNA processing. A differentially expressed 680-bp cDNA, containing the 3' sequence of 16S rRNA, transcribed intergenic spacer, exon 1 and intron of tRNA(Ile), was isolated by differential display reverse transcriptase PCR from salt-grown jojoba (Simmondsia chinesis) shoot cultures. Northern blot analysis indicated that although most rRNA appears to be fully processed, partially processed chloroplast 16S rRNA accumulates in salt-grown cultures. Thus, salinity appears to decrease the processing of the rrn transcript. The possible effect of this decreased processing on physiological processes is, as yet, unknown.
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PMID:Salinity inhibits post transcriptional processing of chloroplast 16S rRNA in shoot cultures of jojoba (Simmondsia chinesis). 1564 9

Previous work demonstrated both acid and neutral, bile salt-independent retinyl ester hydrolase activities in rat liver homogenates. Here we present the purification, identification, and characterization of an acid retinyl ester hydrolase activity from solubilized rat liver microsomes. Purification to homogeneity was achieved by sequential chromatography using SP-Sepharose cation exchange, phenyl-Sepharose hydrophobic interaction, concanavalin A-Sepharose affinity and Superose 12 gel filtration chromatography. The isolated protein had a monomer molecular mass of approximately 62 kDa, as measured by mass spectrometry. Gel filtration chromatography of the purified protein revealed a native molecular mass of approximately 176 kDa, indicating that the protein exists as a homotrimeric complex in solution. The purified protein was identified as carboxylesterase ES-10 (EC 3.1.1.1) by N-terminal Edman sequencing and extensive LC-MS/MS sequence analysis and cross-reaction with an anti-ES-10 antibody. Glycosylation analysis revealed that only one of two potential N-linked glycosylation sites is occupied by a high mannose-type carbohydrate structure. Using retinyl palmitate in a micellar assay system the enzyme was active over a broad pH range and displayed Michaelis-Menten kinetics with a K(m) of 86 microm. Substrate specificity studies showed that ES-10 is also able to catalyze hydrolysis of triolein. Cholesteryl oleate was not a substrate for ES-10 under these assay conditions. Real time reverse transcriptase-PCR and Western blot analysis revealed that ES-10 is highly expressed in liver and lung. Lower levels of ES-10 mRNA were also found in kidney, testis, and heart. A comparison of mRNA expression levels in liver demonstrated that ES-10, ES-4, and ES-3 were expressed at significantly higher levels than ES-2, an enzyme previously thought to play a major role in retinyl ester metabolism in liver. Taken together these data indicate that carboxylesterase ES-10 plays a major role in the hydrolysis of newly-endocytosed, chylomicron retinyl esters in both neutral and acidic membrane compartments of liver cells.
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PMID:Isolation and characterization of a microsomal acid retinyl ester hydrolase. 1576 60

Rapamune, an inhibitor of the mammalian target of rapamycin, exhibits antiproliferative actions and is increasingly used as adjuvant therapy with calcineurin inhibitors. This study investigated the effect of Rapamune on functional and molecular markers in a rat model of calcineurin inhibitor-induced graft dysfunction. Prograf (6 mg), with or without addition of Rapamune (1 mg), was administered to salt-depleted male rats (n = 6/group). Urinary protein excretion and serum creatinine were measured. Rats were culled at 28 days, and messenger RNA expression of TGF-beta, MMP-2, MMP-9, TIMP-1, and collagen III was evaluated with reverse transcriptase polymerase chain reaction. Serum creatinine increased with Prograf (P = .01), but not Rapamune (P = .69) treatment, compared to controls at 28 days. The combination of Rapamune and Prograf produced a rise in serum creatinine at 7 (P = .007) and 14 (P = .01) days, but this was not observed at later time points. Urinary protein excretion was unaltered by any drug or combination. While confirming a synergistic effect of Rapamune and calcineurin inhibitors on renal function, these results suggest that sole therapy with Prograf produces inhibition of fibrotic gene expression. Rapamune alone has no deleterious effect on gene expression but addition of Rapamune cancels out the beneficial effects of Prograf.
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PMID:Prograf produces a molecular environment favoring antifibrosis, an effect reversed by the addition of rapamune. 1580 77

The combination of cyclosporine (CSA) and rapamycin (RAPA) is a potent and commonly used approach to immunosuppression following solid-organ transplantation. By applying varying doses of CSA and RAPA to the rat salt-depleted model, we aimed to find a dose combination that favored antiproliferation/antifibrosis rather than toxicity. Male Sprague-Dawley rats (350 to 500 g) were salt-depleted for 7 days prior to commencing CSA and RAPA treatment. Serum creatinine and urinary protein/creatinine ratios were measured. Fibrosis was estimated with Sirius red staining of extracellular collagen. mRNA expression of TGF-beta, MMP-2, MMP-9, TIMP-1, and collagen III was assessed with reverse transcriptase PCR. A rise in serum creatinine at 7 and 28 days was observed for CSA 15 mg/kg/d (P = .002) but not CSA 7.5 mg (P = .06) or RAPA 1 mg (P = .69) compared to controls. Twenty-four-hour urinary protein excretion was unchanged compared to controls for all drug doses and combinations. Of the dose combinations, CSA 7.5 mg/d + RAPA 0.5 mg/d produced the lowest serum creatinine for all time points, and inhibited profibrotic TIMP-1 (P = .017), while increasing antifibrotic MMP-2 (P = .009) mRNA expression, compared to CSA treatment alone. Expression of TGF-beta and collagen III was unaltered between groups. CSA treatment produced molecular and biochemical changes indicating renal damage. Addition of RAPA can attenuate this damage, but only with a dose reduction of both agents. The most favorable results were for the dose combination CSA 7.5 mg/kg/d plus RAPA 0.5 mg/kg/d.
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PMID:Cyclosporine and rapamycin act in a synergistic and dose-dependent manner in a model of immunosuppressant-induced kidney damage. 1584 49

A more sensitive version of ExaVir Load, a test that utilizes reverse transcriptase (RT) activity from virions in plasma to determine HIV-1 viral load, is described. The virions were immobilized on a gel that was washed, followed by lysis of the virions, elution of purified RT, and finally RT activity determination. The changes made to the original test were: (1) improved washing of the immobilized virions by addition of a non-lytic detergent to the wash buffer, (2) improved virion lysis procedure, including changes in salt, detergent and pH, (3) the use of larger sample volumes in the RT assay, and (4) prolonged RT reaction time. The alterations gave a tenfold increased sensitivity compared to the original version. The correlation between RT load by the current test and RNA PCR was the same as previously (r=0.90). Using colorimetric product detection, the average detection limit in a panel of 262 patient plasma from Stockholm was 0.5 fg RT/ml, corresponding to approximately 170 RNA copies/ml. None of 54 HIV-1 RNA negative samples exhibited RT. The amount of RT load positive samples were 19% for samples containing 50-400 RNA, 71% for samples with 400-1,500, and 100% among samples with >8,000 copies/ml (according to Roche Amplicor). The sensitivity could be increased further using fluorimetric detection. In conclusion, the modifications of the test described result in an important increase in sensitivity. It can now be regarded as a competitive alternative method for HIV viral load determinations.
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PMID:Improved HIV-1 viral load determination based on reverse transcriptase activity recovered from human plasma. 1590 97


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