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 devised a micro-suspension-test to evaluate disinfectants against human immunodeficiency virus type-1 (HIV-1) and confirmed its reliability. Suspensions of persistently HIV-1-infected Molt-4 cells were used as targets of disinfectants and residual infectivity was measured by an infectivity assay: after cocultivation with uninfected Molt-4 cells reverse transcriptase activity (RTA) in the supernatant and giant cell formation (GCF) were monitored. Our new infectivity assay consists of a short-term assay, that is RTA and GCF monitoring on the second day of co-culture, and a long-term assay, that is RTA monitoring up to the 28th day of co-culture. The sensitivity of the short-term assay was 1 x 10(3) infected cells and that of the long-term assay 1 x 10(1) infected cells. All the chemical disinfectants examined in this study showed dose- and time-dependent inactivation of HIV-1. By 5-minute contact with ethanol, glutaraldehyde, formalin, sodium hypochlorite and povidone-iodine, HIV-1 was effectively inactivated at concentrations of 20, 0.01, 5, 0.05 and 0.1%, respectively. Since the micro-suspension-test is easy and sensitive, we recommend it as a method for evaluating disinfectants against HIV-1.
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PMID:A micro-suspension-test for evaluation of disinfectants against human immunodeficiency virus. 760 86

Anti-HIV-active polysaccharides and polyphenols were isolated from the brown seaweed Fucus vesiculosus by hot H2O extraction of both the intact and the homogenized algae. This was followed by XAD2 chromatography and by sequential precipitation of the non-adsorbed compounds with glacial HOAc and thereafter with EtOH. The precipitate was solubilized, dialyzed against distilled H2O, and chromatographed on SP-Sephadex C25 and on QAE-Sephadex A25. This was followed by gel filtration on Sephadex G50 and Sephadex G100 and finally by hplc on a Shodex Ionpak S-804 column. For comparison, the commercial product fucoidan, a sulfated algal polysaccharide, was also further purified by the chromatographic techniques mentioned above. The isolated freeze-dried fractions obtained by these procedures were tested for inhibition of both HIV-induced syncytium formation and HIV reverse transcriptase enzyme activity. Some of these fractions inhibited both of these activities at concentrations that were not cytotoxic.
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PMID:A new procedure for the isolation of anti-HIV compounds (polysaccharides and polyphenols) from the marine alga Fucus vesiculosus. 768 38

We and others have described methods to label specific nucleic acid sequences in fixed cells by reverse in situ transcription (IST). They are simple alternatives to the tedious steps of in situ hybridization with labeled probes. We have favored use of thermostable DNA polymerases after heat denaturation of template secondary structure, accompanied by synthesis of cDNA from an annealed primer, but the approach has been limited by the low reverse transcriptase (RT) activity of Taq polymerase and delayed detection methods. We have improved the technique by the use of recombinant Thermus thermophilus (rTth) DNA polymerase and fluorescein-12-dUTP (FIST). Jurkat T lymphocytes were stimulated with ionomycin + phorbol myristate acetate to produce interleukin-2 (IL-2) mRNA in vitro overnight. They were cytospun onto slides and fixed in 70% ethanol + 30% DEPC-treated water, acetone, and air-dried. The slides were placed on a temperature-controlled heating block, and the cell spot was covered with a plastic coverslip. The temperature was raised to 95 degrees C, and 5-10 microliters of modified Perkin-Elmer/Cetus rTth RT reaction mix was injected under the edge of the coverslip. Each 10 microliters of mix in DEPC-water contained 10 mM Tris-HCl, pH 8.3, 90 mM KCl, 1 mM MnCl2, 1 mM dithiothreitol, 10 U placental ribonuclease inhibitor, 0.125 mM dA,C,GTPs, 0.1 mM fluorescein-12-dUTP, 2 U rTth DNA polymerase, and 4 pM 22-mer oligonucleotide primer, which spanned the second intron of IL-2. After 3 min at 95 degrees C, 1 min at 50 degrees C and 10 min at 72 degrees C, the slides were washed in 0.5 x phosphate-buffered saline, pH 7.0, at 42 degrees C, in 70% ethanol, 100% ethanol, and air-dried. The cells were mounted in antifade solution (2% n-propyl gallate in 70% glycerol), and could be viewed immediately by fluorescence microscopy. Image analysis showed that stimulated Jurkat cells were brighter than uninduced controls or those treated with RNase or without polymerase or primer. FIST appears to be useful for the detection of specific mRNAs in single cells.
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PMID:In situ transcription with Tth DNA polymerase and fluorescent nucleotides. 798 81

The reverse transcriptase (RT)/polymerase chain reaction (PCR) methodology was used with a set of primers that corresponds to two conserved regions of the cytochrome P450s of subfamily 2C, in order to identify the members of this group of enzymes that are expressed at the RNA level in rat brain. Seven RT/PCR clones were sequenced from female brain, and four were found to code for P450 2C7 while three for P450 2C12. Using the same methodology nine RT/PCR clones were sequenced from olfactory lobes of ethanol treated male rats and three were found to code for P450 2C6, one for P450 2C11, three for P450 2C12 and two for P450 2C23. Neither ethanol administration nor hypophysectomy or growth hormone treatment had significant effects on the expression levels of these cytochromes in the brain.
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PMID:Identification of the major cytochrome P450s of subfamily 2C that are expressed in brain of female rats and in olfactory lobes of ethanol treated male rats. 832 27

Vasopressin mRNA content was studied by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in the hypothalami of rats chronically treated with ethanol (EtOH). Quantitative RT-PCR allows for the accurate measurement of peptide mRNA levels in discrete regions of the brain of individual animals. EtOH markedly reduced the level of vasopressin mRNA. Furthermore, salt loading was ineffective in inducing a significant increase in vasopressin mRNA level in EtOH-treated rats, unlike in controls. The present results suggest that EtOH not only decreases vasopressin mRNA content in the rat hypothalamus, but also impairs its capacity to respond to salt loading.
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PMID:Chronic ethanol intake decreases vasopressin mRNA content in the rat hypothalamus: a PCR study. 841 69

A high prevalence of antibodies to the hepatitis C virus (anti-HCV) has been demonstrated among patients with alcoholic liver disease, whereas the prevalence of HCV viremia in these patients remains uncertain. The aims of this study were to determine the prevalence of anti-HCV in alcoholic patients both with and without clinically apparent liver disease and to determine the presence of HCV RNA in those patients who tested positive for anti-HCV by RIBA II (Chiron Corporation, Emeryville, CA). One hundred male patients consecutively admitted to an alcoholic rehabilitation program were included. Group 1 was comprised of 40 patients with clinically apparent liver disease. Group 2 was comprised of 60 patients without clinically apparent liver disease. Anti-HCV was performed by a second-generation ELISA assay and confirmed by RIBA II. HCV RNA was performed by Quantiplex assay (Chiron Corporation) and a nested reverse transcriptase-polymerase chain reaction. No significant differences were found between the two groups with regards to age, quantity and duration of alcohol intake, or accepted risk factors for HCV. The overall prevalence of anti-HCV in our patients was 23%, with 43% of these in group 1 and 10% in group 2. HCV RNA tested positive in 94% of the anti-HCV-positive patients in group 1 and in 67% of the anti-HCV-positive patients in group 2. These data suggest that HCV infection is an important cofactor in the pathogenesis of liver disease among alcoholic patients.
Alcohol Clin Exp Res 1995 Oct
PMID:Hepatitis C virus in alcoholic patients with and without clinically apparent liver disease. 856 Dec 87

The mechanisms responsible for ethanol-mediated teratogenesis have not been resolved. However, possible etiologies include the local formation of the teratogen acetaldehyde or oxygen radicals by fetal ethanol-oxidizing enzymes. As alcohol dehydrogenases are expressed at very low concentrations in human embryonic tissues, the ethanol-inducible P450 enzyme, CYP2E1, could be the sole catalyst of fetal ethanol oxidation. With this in mind, we examined the expression of this P450 in liver samples from fetuses ranging in gestational age from 16 to 24 weeks. Immunoblot analysis of fetal liver microsomes revealed the presence of a protein immunoreactive with CYP2E1 antibodies that exhibited a slightly lower molecular weight than that found in adult liver samples. Embryonic CYP2E1 expression was further confirmed by the reverse transcriptase reaction with RNA from a 19-week gestational fetal liver used as template. Catalytic capabilities of human fetal microsomes were assessed by measurement of the rate of ethanol oxidation to acetaldehyde, which were 12-27% of those exhibited by adult liver microsomes. Immunoinhibition studies with CYP2E1 antibodies revealed that the corresponding antigen was the major catalyst of this reaction in both fetal and adult tissues. We then assessed whether embryonic CYP2E1 was, like the adult enzyme, inducible by xenobiotics. Treatment of primary fetal hepatocyte cultures with either ethanol or clofibrate demonstrated a 2-fold increase in CYP2E1 levels compared with untreated cells. Collectively, our results indicate that CYP2E1 is present in human fetal liver, that the enzyme is functionally similar to CYP2E1 from adults, and that fetal hepatocyte CYP2E1 is inducible in culture by xenobiotics, including ethanol. Because fetal CYP2E1 mediates ethanol metabolism, the enzyme may play a pivotal role in the local production of acetaldehyde and free radicals, both of which have potential deleterious effects on the developing fetus.
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PMID:Expression, induction, and catalytic activity of the ethanol-inducible cytochrome P450 (CYP2E1) in human fetal liver and hepatocytes. 863 58

A simple and efficient procedure was described for the isolation of total RNA from the fission yeast Schizosaccharomyces pombe. The present study demonstrated that the quality and the quantity of S. pombe RNA were increased by substituting phenol/chloroform mixture for phenol as a deproteinizing agent in the first vortexing step and using an ice bath instead of a dry ice-ethanol bath in the freezing step. Additionally, this protocol had the advantage of extracting total RNA without any degradation of S. pombe cells. Furthermore, the high amounts and quality of RNA extracted by this modified procedure enabled us to perform some experiments such as Northern blot, S1 mapping, primer extension, and reverse transcriptase reaction-polymerase chain reaction (RT-PCR) without further RNA purification. We suggest that this procedure is very useful to analyse primary structures and steady-state levels of RNA from S. pombe.
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PMID:A simple and efficient method for the isolation of total RNA from the fission yeast Schizosaccharomyces pombe. 867 17

We recently showed that alcohol significantly suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) production by whole blood and total mononuclear cells from healthy subjects as measured by bioassay. In the current study, we further examined the effect of alcohol on LPS-induced TNF-alpha gene expression by semiquantitative solution PCR and in situ reverse transcriptase PCR (RT-PCR) hybridization methods. Peripheral blood mononuclear cells were cultured with LPS (10 micrograms/ml) for 4 to 8 h with or without different concentrations of ethanol (0.1, 0.2, and 0.3% [vol/vol]). Total RNA from treated and untreated cultures was extracted and used for solution PCR analysis. Treated and untreated cells were subjected to both conventional in situ hybridization and RT-PCR in situ hybridization. In solution RT-PCR in vitro analysis, alcohol significantly suppressed TNF-specific message. In conventional in situ hybridization, the effect of alcohol on TNF-alpha gene expression was poorly detected. However, when cells were subjected to RT-PCR prior to in situ hybridization, cells treated with alcohol significantly suppressed expression of the message for TNF-alpha. These studies confirm our earlier finding that alcohol suppressed the production of TNF-alpha by LPS-induced whole blood cells and peripheral blood mononuclear cells. Furthermore, these studies also demonstrate that the RT-PCR in situ technique is a powerful tool for detecting and amplifying specific genes in whole cells when limited numbers of cells are available for RNA extraction.
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PMID:Alcohol inhibits lipopolysaccharide-induced tumor necrosis factor alpha gene expression by peripheral blood mononuclear cells as measured by reverse transcriptase PCR in situ hybridization. 880 2

We describe an adapted version of the Chomczynski and Sacchi [Anal Biochem (1987) 162:156-159] RNA isolation procedure that can be performed in less than 1 h on small (<15 mg) tissue samples, using commonly available reagents. Our modifications included: (1) one rather than two precipitation steps in the aqueous phase with 99% ethanol and (2) elimination of the 1-h incubation step at -20 degrees C. Our adaptations resulted in RNA yield (microg/mg of tissue) and purity (260/280 nm ratios) comparable to those of the original procedure. Furthermore, the isolated RNA was successfully utilized in reverse transcriptase-polymerase chain reaction assays, suggesting that it was essentially free of carry-over contaminants that could inhibit enzymatic reactions. When tested on tissue sample sizes of 7-12 mg, our adapted procedure allowed the recovery of enough total RNA for use in techniques such as Northern blot analysis. Our modified technique is therefore an inexpensive alternative to commercially available kits when isolating good-quality RNA from very small tissue samples, such as those obtained from needle biopsies.
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PMID:Rapid RNA isolation without the use of commercial kits: application to small tissue samples. 904 53


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