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)

The purpose of this study was to determine the efficiency of semi-nested PCR in detecting hepatitis A virus (HAV) RNA. During a 2-year period (1990-1991), HAV RNA was searched for in shellfish from the French Brittany coasts using cRNA and vRNA probes. In January 1992, at the time of a hepatitis A outbreak, 28 stool samples were collected from infected patients (18 adults, 10 children) with anti-HAV IgM. Four samples from subjects with negative HAV serology were used as negative controls. Nucleic acid amplification (reverse-transcription-semi-nested PCR) was performed to detect HAV in stool. HAV RNA was purified by phenol-chloroform extraction and converted to cDNA using reverse transcriptase (Mu-MLV). After amplification, PCR products were visualized on an ethidium-bromide-stained gel and confirmed by hybridization with a specific digoxigenin-labelled oligoprobe. Samples were also studied by molecular hybridization with cRNA and vRNA probes. After onset of the illness, HAV RNA was detected over a longer time period by semi-nested PCR (16/28) than by hybridization (0/28). Even though biological diagnosis of hepatitis A will continue to rely on the detection of anti-HAV IgM, PCR should be useful in certain clinical cases (diagnosis of relapse) and for epidemiological and environmental monitoring of viruses.
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PMID:Development of RT-semi-nested PCR for detection of hepatitis A virus in stool in epidemic conditions. 793 9

Quantification of mRNA is important for studies of gene expression and gene regulation. We investigated the utility of the reverse transcriptase polymerase chain reaction (RT-PCR) approach in the quantification of mRNA from small cell numbers. To take into account the complex kinetics of the PCR amplification process and the nonlinear signal development during detection of PCR products, calibration curves were established on the basis of different, known, starting concentrations of cDNA fragments, different PCR cycle numbers, and different signal intensities. Detection of digoxigenin-labeled PCR products via an enzymatically generated chemiluminescent signal was found to give a reproducible and wider range of signal intensities compared to simple ethidium bromide staining. We applied this methodology to the quantification of immunoglobulin M (IgM) mRNA levels in human B cells. Using an in vitro culture system in which B cells differentiate into plasma cells, the kinetics of IgM mRNA expression were established during a 10-day culture period and a 180-fold mRNA increase was found.
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PMID:Semiquantitative, nonradioactive RT-PCR detection of immunoglobulin mRNA in human B cells and plasma cells. 801 Nov 69

The beta 3-adrenergic receptor (beta 3AR) has been purported to play important roles in a number of metabolic functions, suggesting that beta 3AR agonists might be useful as antidiabetic and antiobesity therapeutic agents. However, these assertions are based entirely on extensive metabolic studies with such agonists in rodents. To clarify the role that the beta 3AR might have in humans, we sought to define the tissue distribution of the beta 3AR in adult human tissue by the use of a highly specific and sensitive approach. Northern blots of selected tissues failed to reveal any beta 3AR mRNA, suggesting little or no expression. To detect minute amounts of transcripts, we developed a reverse transcriptase-polymerase chain reaction (RT-PCR) method that uses primers to amplify a region of the beta 3AR that has little homology with the closely related beta 1- and beta 2-AR genes, and we verified the specificity of this approach using plasmids containing the cloned human beta 1-, beta 2-, and beta 3AR genes. RT-PCR performed on as little as 20 ng of total RNA from 3T3-F442A cells, which expressed beta 3AR at very low levels (approximately 20 fmol/mg of protein), provided an easily detectable signal by ethidium bromide staining and Southern blotting of electrophoresed products. RT-PCR was performed on RNA obtained from 23 different human tissues, using primers for the beta 3AR, the beta 2AR, and beta-actin, which acted as a control. Whereas beta-actin and the beta 2AR were detected in virtually all tissues, RT-PCR using beta 3AR primers gave products from 13 tissues, including skeletal muscle, lung, adipose tissue, kidney, small intestine, pancreas, spleen, and adrenal gland. An end-labeled 50-nucleotide probe identical to an internal region of the expected beta 3AR product hybridized under low stringency conditions to seven of these products. However, sequencing of these products, which were somewhat smaller in molecular size than expected, did not reveal beta 3AR DNA sequence. Given the specificity and sensitivity of our approach, we conclude that the beta 3AR is not expressed to any significant degree in the adult human tissues studied, including adipose tissue and other metabolic sites.
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PMID:Lack of beta 3-adrenergic receptor mRNA expression in adipose and other metabolic tissues in the adult human. 838 99

Multidrug resistance to anticancer drugs proved to be related to the MDR1 gene which encodes the P-glycoprotein, an energy-dependent drug-efflux pump for lipophilic drugs. We investigated the expression of the MDR1 gene in clinical samples by RT-PCR. The subjects were all resected cases of 14 colorectal cancers, five gastric cancers, two esophageal cancers, two gallbladder cancers and 20 lung cancers. Adrenal gland was used as a positive control. Total RNA was extracted from a fresh tissue sample. The cDNA was synthesized from 1 microgram of total RNA using reverse transcriptase. With the above cDNA as the template, amplification of the 157-bp fragment of the MDR1 gene was performed using PCR. The PCR product was polyacrylamide gel-electrophoresed and ethidium bromide-stained. In addition, a dot blot analysis was performed to quantify the amount of PCR product. Since PCR was performed simultaneously under the same conditions, the PCR product was quantified at four stages, from (3+) to (-), to indicate the degree of expression of MDR1 mRNA. Adrenal gland showed (3+)-(2+) and colorectal cancer exhibited mostly (2+)-(1+). Both cancerous and non-cancerous areas evidenced a similar degree of expression in the cases of colorectal cancer. The MDR1 gene was expressed at low levels in other digestive tract cancers and in lung cancer. The levels of MDR1 expression revealed no correlation to either histological type or clinical stage. The present method may contribute to designing anti-cancer protocols.
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PMID:[Analysis of MDR1 (multidrug resistance) gene expression by RT-PCR]. 838 54

DNA in-vitro amplification when a PCR (polymerase chain reaction) method is used (Saiki et al., 1985) provides for a simple technique of marked amplification of a selected DNA fragment. The length of a DNA amplified fragment is determined by two synthetic primers which spontaneously (at an appropriate temperature) hybridize with the opposite ends of antiparallelly oriented strains of denatured DNA. The enzyme Taq polymerase completes the synthetisation of new DNA strains from the primers. Repetition of these cycles (denaturing, primer bonds, DNA synthesis) enhances the DNA amplification of a defined strain length to such an extent that is possible to prove this process by e.g. electrophoretic analysis. For the purposes of a proof of BVD-MD genome in cattle the fragment 315 bp was chosen from the virus-coding gene gp 80. The primers P1-5'-GTAGGTAGAGTGAAACCCGG-3' and P2-5'-CGGGACCTGGACTTCATAGC-3' (Hertig et al., 1991) determined the length of the amplified fragment. Virus RNA was isolated from the infectious BVD-MD-containing medium (Ph strain) using a phenol-chloroform (1:1) mixture, and before amplification it was transcribed to cDNA in the P2 presence by the effect of AMV reverse transcriptase. cDNA without isolation from the transcription reaction mixture was directly used for PCR. DNA was denatured at 94 degrees C for 10 minutes before the outset of amplification. These reaction conditions are suitable for the PCR method: P1 and P2 primer concentrations per 100 microliters reaction solution-1 microM, dNTP-100 microM, 2-4 U Taq polymerase, 25-35 amplification cycles with the temperature regime: 94 degrees C/l min, 56 degrees C/l min, 73 degrees C/l min, and prolonged incubation 73 degrees C/7 min in the last cycle. Proof of the amplified product 315 bp DNA-electrophoretic analysis of 1.5 to 2% agarose gel in TAE buffer and ethidium bromide staining of gel are suitable. The introduced PCR method gives opportunities for innovations of BVD-MD diagnosis in cattle on the basis of virus genome proof without any cell cultivation need.
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PMID:[In vitro amplification of genome fragments of the mucosal disease virus (BVD-MD) using the PCR method]. 839 38

A number of recent studies suggest that cells of the immune system, e.g. peripheral blood mononuclear cells (PBMC), can synthesize and process POMC and secrete POMC-derived peptides, such as ACTH and endorphins, upon immune and hormonal challenges. From this, it has been proposed that POMC-derived peptides originating from lymphoid cells can function as hormones, for instance in a lymphoid-adrenal axis. In view of the important physiological implications of this proposal, the present study was designed to investigate the expression of the POMC gene in human PBMC and the production by these cells of alpha-, beta-, and gamma-endorphins (alpha E, beta E, and gamma E) peptides that are established end products of the posttranslational processing of POMC. PBMC of individual donors were used uncultured (fresh cells) or cultured for 24 and 48 h in the presence and absence of Concanavalin-A (Con-A), bacterial lipopolysaccharide, phytohemagglutinin, or CRH, and vasopressin, conditions that reportedly stimulate POMC activity in those cells, to investigate the presence of POMC transcripts by analysis of total RNA with Northern blotting and the reverse transcriptase polymerase chain reaction (RT-PCR). Large scale preparations containing over 10(9) cells (fresh, cultured with and without Con-A) originating from several donors were examined for the presence of POMC transcripts by analysis of poly(A)+ RNA on Northern blots and for the presence of alpha E, beta E, and gamma E by gel filtration over Sephadex G-75 and reverse phase HPLC, followed by assay of the fractions in four endorphin RIA systems with different specificities. On the Northern blots of total RNA, no POMC transcripts were detectable. In poly(A)+ RNA preparations, no full-length POMC mRNA was found, and it was estimated that the concentration of POMC mRNA, if present, was below approximately 0.005 transcript/cell in Con-A-stimulated cells and still lower in unstimulated cells. In accord with literature data, an 800- to 900-nucleotide POMC transcript was detected in cultured PBMC, and the levels of this transcript were stimulated by Con-A. In all samples analyzed with RT-PCR, a transcript spanning most of exons 2 and 3 was detectable only on Southern blots of the RT-PCR product, but not on agarose gels stained with ethidium bromide. Chromatographic analysis of endorphin immunoreactivities in cell extracts revealed no qualitative differences between the immunoreactive profiles of fresh PBMC or PBMC cultured with or without Con-A.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Analysis of proopiomelanocortin (POMC) messenger ribonucleic acid and POMC-derived peptides in human peripheral blood mononuclear cells: no evidence for a lymphocyte-derived POMC system. 840 37

A synthetic, symmetry-based inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease, A77003, was evaluated for antiviral activity and cytotoxicity in vitro in human peripheral blood lymphocytes or cell lines H9, CEM, and U937. Toxicity and antiviral activity of the HIV-1 protease inhibitor were compared with those of the reverse transcriptase inhibitors zidovudine and 2',3'-dideoxy-2',3'-didehydrothymidine and human recombinant alpha and beta interferons. Production of infectious virus particles, cell-free p24 antigen, and cell-associated viral proteins was reduced 50% by the HIV-1 protease inhibitor at concentrations of 0.12 to 0.26 microM (50% effective concentration [EC50]) in acute infection and 0.2 to 1.7 microM (EC50) in persistent infection. Fluorescence-activated cell sorter analysis of U937 cells persistently infected with HIVIIIB using a monoclonal antibody to HIV also showed a reduction of cell-associated viral protein in A77003-treated cells. Furthermore, toxicity of A77003 assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay was not observed at greater than 100 times the EC50. A77003 was more effective in persistent HIV-1 infection than alpha and beta interferons (1,000 U/ml), while zidovudine and 2',3'-dideoxy-2',3'-didehydrothymidine were not active.
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PMID:Preclinical evaluation of antiviral activity and toxicity of Abbott A77003, an inhibitor of the human immunodeficiency virus type 1 protease. 843 Oct 7

To investigate whether the oral cavity is a potential reservoir and possible sanctuary for Helicobacter pylori, supragingival and subgingival plaques were analyzed by a Helicobacter genus-specific reverse transcriptase-polymerase chain reaction based on the sequence data of H. pylori 16S rRNA. The amplified 500-bp DNA fragment was identified by ethidium bromide staining after agarose gel electrophoresis and by Southern hybridization. Twenty-five dyspeptic patients were studied. Histologic examination of gastric biopsy specimens revealed that 18 had H. pylori gastritis and 7 did not. For seven of the 18 (38.8%) patients with proven H. pylori gastritis, H. pylori was also identified in their dental plaque. None of the patients without H. pylori gastritis had H. pylori in their dental plaque. The detection of H. pylori in dental plaque suggests that this H. pylori colonization is not restricted to the gastric mucosa and that this ecological niche may serve as a possible sanctuary which may be responsible for reinoculation of the stomach after topical anti-H. pylori therapies such as bismuth.
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PMID:Detection of Helicobacter pylori in dental plaque by reverse transcription-polymerase chain reaction. 846 87

We constructed a plasmid for the in vitro synthesis of a competitor RNA for use as an internal exogenous control during reverse transcriptase--PCR (RT-PCR) detection of epidermal growth factor receptor (EGFR) expression. The competitor RNA harbors a 32-base deletion compared with wild-type EGFR mRNA and generates a PCR product that is easily distinguished from the wild-type PCR product by agarose gel electrophoresis. We encountered the problem of heteroduplex formation during later stages of PCR, which could be solved by decreasing the PCR cycle number. This was accompanied by a significant loss of sensitivity. Sensitivity could be restored by using a novel and extremely sensitive DNA stain (SYBR Green I) instead of ethidium bromide.
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PMID:Quantitative detection of reverse transcriptase-PCR products by means of a novel and sensitive DNA stain. 857 92

Some intercalating and nonintercalating drugs have been tested as inhibitors on the DNA synthesis reaction catalyzed by avian myeloblastosis virus (AMV) reverse transcriptase, in the presence of polyriboadenylic acid (poly(rA)) and poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl)) as templates. In both cases, the inhibition was higher with the intercalating drug ethidium bromide than with the nonintercalating analog tetramethyl ethidium bromide. Ethidium bromide inhibited more efficiently the poly(rA)- than the poly(dAfl)-directed reverse transcriptase reaction; in the latter case, the inhibition was non-competitive in relation to TTP. On the other hand, the reaction catalyzed in the presence of the 2'-fluorinated polynucleotide as template was inhibited to a higher extent by other nonintercalating drugs, berenil, netropsin, and distamycin. The inhibitions of both reactions by dideoxy TTP, novobiocin and HPA-23 are also discussed.
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PMID:Inhibition of poly(2'-fluoro-2'-deoxyadenylic acid)-directed-reverse transcriptase activity. 858 54


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