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)

It has been proposed that the biosynthesis of estrogens by the human endometrium may be of physiological significance during the menstrual cycle. Local estrogen production was also suggested to be important in the development of endometrial cancer; however, the presence or absence of aromatase enzyme activity in normal human endometrium is controversial. To address this issue, we used a sensitive technique capable of detecting mRNA transcripts present in only very low copy number. The polymerase chain reaction linked to reverse transcription (RT-PCR) was used to evaluate the presence or absence of aromatase cytochrome P450 (P450arom) transcripts in endometrial tissues (n = 7) and endometrial stromal cells (n = 9) under various culture conditions. RNA was isolated from four proliferative and three secretory tissue samples and from cultured endometrial stromal cells isolated from seven proliferative and two secretory endometria. Five sets of cultures were treated with medroxyprogesterone acetate (MPA), estradiol (E2), and forskolin. Additionally, RNA was isolated from decidualized endometrium obtained from a patient with tubal pregnancy. A single stranded cDNA was synthesized from total RNA using Moloney murine leukemia virus reverse transcriptase and a P450arom-specific oligonucleotide. The single stranded cDNA was used as a template for PCR and was amplified for 20-35 cycles using P450arom-specific primers. RNA from adipose tissue and placenta was amplified to provide positive controls, whereas myometrial RNA was used as a negative control. In two experiments involving two endometrial tissues and three sets of cells in culture, a rat P450arom cRNA was coamplified in each sample as an internal control to demonstrate that the remote possibility of RT-PCR failures in individual test samples cannot account for our negative results. By Southern or slot blot hybridization of the amplified fragments using human and rat P450arom-specific probes, we found no evidence for the presence of P450arom transcripts in normal endometrium, decidualized endometrium, or endometrial stromal cells in culture. In our hands, assay of aromatase activity using [3H]water release from [3H]androstenedione by endometrial stromal cells in culture treated with MPA and E2, did not reveal any detectable aromatase activity. The same cells responded to MPA plus E2 treatment by a significant increase in PRL secretion into the culture medium. Presently, RT-PCR is the most sensitive method available for the detection of specific mRNA species in low copy numbers. These findings are indicative of the absence of P450arom transcripts in normal human endometrium.
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PMID:Polymerase chain reaction amplification fails to detect aromatase cytochrome P450 transcripts in normal human endometrium or decidua. 768 41

We have used a simple method for detecting HIV-1 in the serum of infected individuals using the polymerase chain reaction (PCR). This is useful if only serum, or other specimens which would not be expected to harbour proviral DNA, is available for diagnostic testing. Viral RNA present in serum is bound to silica particles in the presence of a high concentration of guanidinium thiocyanate (GuSCN) which denatures any proteins present, specifically ribonucleases. After washing the RNA/silica pellet, the RNA is eluted in water and reverse transcribed using random primers and Moloney murine leukaemia virus reverse transcriptase in the presence of a modified PCR buffer. The resultant cDNA is amplified using nested PCR and the products of amplification are detected by gel electrophoresis and ethidium bromide staining. The identity of bands on the gel is confirmed using a digoxigenin-labelled oligomer probe. The method is a general one applicable to amplification of any RNA species.
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PMID:Detection of HIV-1 in serum, using reverse transcription and the polymerase chain reaction (RT-PCR). 768 79

During the early stages of human immunodeficiency virus (HIV) infection, although symptoms are absent and viral replication in peripheral blood mononuclear cells is low, substantial levels of HIV replication can be documented in lymphoid tissue [G. Pantaleo, C. Graziosi, J.F. Demarest, L. Butini, M. Montroni, C.H. Fox, J.M. Orenstein, D.P. Kotler, and A.S. Fauci, Nature (London) 362:355-358, 1993, and J. Embretsen, M. Zupancic, J.L. Ribas, A. Burke, P. Racz, K. Tenner-Tacz, and A.T. Haase, Nature (London) 362:359-362, 1993]. This observation suggests that earlier treatment of HIV infection may be indicated and that strategies for enhancing drug targeting to the lymphoid tissue reservoris of HIV infection may be beneficial. To address this issue, we synthesized dioleoylphosphatidyl-ddC (DOP-ddC) and dipalmitoylphosphatidyl-3'-azido-3'-deoxythymidine (DPP-AZT), phospholipid prodrugs which form lipid bilayers and which are readily incorporated into liposomes. The anti-HIV activity of DOP-ddC was similar to that of ddC in HIV type 1-infected HT4-6C cells, but DPP-AZT was considerably less active than AZT in HT4-6C cells. Liposomes containing DOP-[3H]ddC or DPP-[3H]AZT administered intraperitoneally to mice produced greater levels of total radioactivity over time in plasma, spleen, and lymphoid tissue relative to the results with [3H]ddC and [3H]AZT, respectively. DPP-AZT administered intraperitoneally in liposomes as a single daily dose to mice infected with Rauscher leukemia virus prevented increased spleen weight and reverse transcriptase levels in serum with a dose-response roughly comparable to that of AZT given continuously in the drinking water. DOP-ddC, DPP-AZT, and lipid conjugates of other antiretroviral nucleosides may provide higher levels of drug over time in plasma and in lymph nodes and spleen, important reservoirs of HIV infection, and may represent an interesting alternative approach to antiviral nucleoside treatment of AIDS.
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PMID:Phosphatidylazidothymidine and phosphatidyl-ddC: assessment of uptake in mouse lymphoid tissues and antiviral activities in human immunodeficiency virus-infected cells and in Rauscher leukemia virus-infected mice. 769 64

The backbone dynamics of Escherichia coli ribonuclease HI (RNase HI) in the picosecond to nanosecond time scale were characterized by a combination of measurements of 15N-NMR relaxation (T1, T2, and NOE), analyzed by a model-free approach, and molecular dynamics (MD) simulation in water. The MD simulations in water were carried out with long-range Coulomb interactions to avoid the artificial fluctuation caused by the cutoff approximation. The model-free analysis of the 15N-NMR relaxation indicated that RNase HI has a rotational correlation time of 10.9 ns at 27 degrees C. The generalized order parameter (S2) for the internal motions varied from 0.15 to 1.0, with an average value of 0.85, which is much larger than that of the RNase H domain of HIV-1 reverse transcriptase (0.78). Large internal motions (small order parameters) were observed in the N-terminal region (Leu2-Lys3), the loop between beta-strands A and B (Cys13-Gly15), the turn between alpha-helix I and beta-strand D (Glu61, His62), the loop between beta-strand D and alpha-helix II (Asp70-Tyr71), the loop between alpha-helices III and IV (Ala93-Lys96), the loop between beta-strand E and alpha-helix V (Gly123-His127), and the C-terminal region (Gln152-Val155). The effective correlation time observed in these regions varied from 0.45 ns (Glu61, Lys96) to 2.2 ns (Leu14). The order parameters calculated from the MD agreed well with those from the NMR experiment, with a few exceptions. The distributions of most of the backbone N-H vectors obtained by MD are approximately consistent with the diffusion-in-a-cone model. These distributions, however, were elliptic, with a long axis perpendicular to the plane defined by the N-H and N-C alpha vectors. Distributions supporting the axial fluctuation model or the jump-between-two-cones model were also observed in the MD simulation.
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PMID:Characterization of the internal motions of Escherichia coli ribonuclease HI by a combination of 15N-NMR relaxation analysis and molecular dynamics simulation: examination of dynamic models. 775 90

It is important to determine the stability of naked viral RNA in seawater, since false-positive results can occur when reverse transcriptase-PCR (RT-PCR) is used to detect viruses if the RT-PCR amplifies free RNA instead of RNA from intact viruses. An acid guanidinium thiocyanate-phenol-chloroform method was used to extract total RNA from a filtered poliovirus cell culture suspension. The sensitivity of detection in this viral RNA study was 600 fg when RT-PCR was used. The extracted total RNA was seeded into filtered and unfiltered seawater, and the resulting preparations were incubated at 4 degrees C and at room temperature (23 +/- 1 degrees C). Our results showed that the seeded RNA was more stable in filtered seawater than in unfiltered seawater at both temperatures. The viral RNA could not be detected by the RT-PCR after 2 days of incubation in unfiltered seawater and after 28 days of incubation in filter-sterilized seawater. Therefore, because of the relatively short life of viral RNA in natural water, the detection of virus in environmental samples by the RT-PCR was mainly due to the presence of well-protected viral particles and not due to the presence of naked viral RNA.
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PMID:Analysis of viral RNA persistence in seawater by reverse transcriptase-PCR. 788 16

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

A triplex reverse transcriptase PCR (RT-PCR) was developed to simultaneously detect poliovirus, hepatitis A virus (HAV), and rotavirus in sewage and ocean water. Sewage and ocean water samples seeded with the three different viruses were concentrated by ultrafiltration. The unseeded ocean water and sewage samples were concentrated by vortex flow filtration and/or ultrafiltration. Random hexamers and a rotavirus downstream primer were used to initiate reverse transcription. Three different sets of primers specific for poliovirus, HAV, and rotavirus cDNAs were mixed in the PCR mixture to amplify the target DNA. Three distinct amplified DNA products representing poliovirus, HAV, and rotavirus were identified by gel electrophoresis as 394-, 192-, and 278-bp sequences, respectively. Dot blot and Southern analyses were used to confirm the amplified products for each virus present in the environmental samples. Except for poliovirus, the sensitivity of triplex RT-PCR for the detection of rotavirus and HAV was found to be similar to that of monoplex RT-PCR, which uses only one set of primers to amplify a single type of virus. The triplex RT-PCR has greater advantages over monoplex RT-PCR for virus detection, namely, the rapid turnaround time and cost effectiveness.
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PMID:Detection of poliovirus, hepatitis A virus, and rotavirus from sewage and ocean water by triplex reverse transcriptase PCR. 807 20

Magnetic beads coated with antibodies against surface epitopes of the hepatitis A virus (HAV) captured efficiently viral particles from various types of samples. Contaminating substances, including particulate material, were removed by using a magnet to retain the beads during the washing procedure. A sensitive reverse transcriptase/nested polymerase chain reaction (RT-PCR) was developed to confirm the presence of viral particles on the beads. The above technique was shown to be useful for the detection of a laboratory strain of HAV added to polluted river water, sea water and fecal extracts.
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PMID:Detection of hepatitis A virus in clinical and environmental samples by immunomagnetic separation and PCR. 818 12

Hepatitis A virus (HAV) is a major cause of infectious hepatitis in humans. In this respect, bivalve mollusks pose a major health concern because they are filter feeders and can concentrate the virus up to 900-fold from contaminated water. Detection of HAV has been hampered because wild-type HAV grows poorly if at all in cell culture. Here we describe a technique for the detection of HAV in shellfish based on reverse transcription coupled with the polymerase chain reaction. RNA is isolated from hard-shell clam tissue and reverse transcribed with avian myeloblastosis virus reverse transcriptase. A portion of the cDNA pool is then amplified with primers specific for HAV. In experiments with an in vitro-synthesized HAV transcript, we were able to detect HAV sequence in the presence of a 200-million-fold excess of shellfish RNA. When intact virus was added to shellfish tissue before the isolation of RNA, the method was capable of detecting 10 viral RNA molecules in a reaction mixture.
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PMID:Detection of hepatitis A virus in Mercenaria mercenaria by coupled reverse transcription and polymerase chain reaction. 821 51

1. Renin mRNA is expressed in several extrarenal tissues, including brain. The aim of the present study was to quantify renin mRNA in the hypothalamus in response to a low NaCl diet and angiotensin converting enzyme inhibitor treatment, both of which are well-known stimuli of renin mRNA in kidney. 2. Groups of six Sprague-Dawley rats were given either a normal diet, low NaCl chow (0.04% NaCl), enalapril (0.25 mg/mL in drinking water), or low NaCl + enalapril, for 7 days. Renin mRNA in the hypothalamus was quantified by a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) technique. 3. Renin mRNA concentration in the hypothalamus of rats receiving a normal diet was 52 +/- 3 S.E. fg/microgram total RNA. Low NaCl had no effect (57 +/- 7), whereas values were significantly lower in rats treated with enalapril, either given alone (38 +/- 2; P = 0.002) or combined with a low NaCl diet (33 +/- 4; P = 0.003). 4. In conclusion, we have quantified renin mRNA in the rat hypothalamus and shown that it can be suppressed significantly by enalapril. This response is opposite to that seen in the kidney after enalapril.
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PMID:Renin mRNA concentration in rat hypothalamus is decreased by enalapril. 858 14


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