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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A commercially available kit, Amplicor, was compared with a locally developed nested reverse-transcriptase (RT) PCR for qualitative detection of HCV-RNA. Sixty-one serum samples from sixty-one patients with liver disease, and 60 samples from 60 hemophiliacs without symptoms, but known to have been heavily exposed to hepatitis C virus, were investigated. There was a high degree of concordance between the two diagnostic tests (97%), the Amplicor kit being slightly more sensitive than the in-house PCR, when evaluated using serial dilutions of samples showing discrepant results. The relationship between viremia and abnormal ALT levels was studied in the two groups of patients. Among those with chronic liver disease, 8.3% of patients with viremia had normal ALT levels, whereas transaminases were normal in 20% of hemophiliacs with viremia. This points to ALT as being a poor marker of ongoing viral replication.
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PMID:Viremia in chronic hepatitis C patients evaluated by the Amplicor RT-PCR, a nested RT-PCR, and transaminase levels. 953 67

Previous studies have demonstrated that the c-kit encoded tyrosine kinase receptor and its ligand, steel factor (SLF), are critical for normal blood cell development. We have reported that transduction of the c-kit gene into single hematopoietic progenitor cells (HPC), CD34(+++) cells, from cord blood (CB) enhances erythroid colony formation via a SLF-dependent mechanism. We therefore decided to evaluate the impact on cell proliferation of co-transducing c-kit and SLF cDNAs into these cells. CD34(+++) cells were sorted as a population or as 1 cell/well for cells expressing the highest levels of CD34 and different levels of c-kit. Cells were then prestimulated with granulocyte macrophage (GM)-colony stimulating factor (CSF), interleukin (IL)-3, IL-6, erythropoietin (Epo) in the presence and absence of various concentrations of SLF. Cells were then transduced with SLF and/or c-kit cDNAs, and then assayed for colony formation with the same cytokine combination. At a single cell level, co-transduction with c-kit and SLF genes significantly enhanced colony formation compared with individual gene transduction, especially by erythroid and multipotential progenitors that responded to stimulation by added cytokines. Little or no growth was seen with the c-kit- and/or SLF-transduced cells without addition of cytokines. The degree of enhancement effected by co-transduction inversely correlated with the degree of expression of c-kit protein before transduction. Optimal enhancing effects were noted in CD34(+++) kit(Lo/-) cells co-transduced with both c-kit and SLF cDNAs. Reverse transcriptase-polymerase chain (RT-PCR) analysis of SLF mRNA expression in CD34(+++) cells and enzyme-linked immunoadsorbent assay (ELISA) measurement of secreted SLF protein demonstrated that the transduced SLF cDNA was expressed and soluble SLF was released in medium cultured with SLF gene transduced MACS-separated CD34(+) cells in the presence, but not in the absence, of IL-3, GM-CSF, IL-6, and Epo. These results demonstrate the enhancement of the proliferation of growth factor responsive HPC that express transduced c-kit and SLF genes.
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PMID:Co-transduction of cDNAs for c-kit and steel factor into single CD34+ cord blood cells further enhances the growth of erythroid and multipotential progenitors. 1117 93

Differences in methods of reverse-transcriptase (RT)-polymerase chain reaction (PCR)-based detection of tumour cells in the blood gives rise to conflicting results, and standardisation is urgently needed. This pilot study aimed to assess the variation of RT-PCR-based detection of tumour cells in blood between four different laboratories using a commercially available kit with a standardised protocol. This kit allows comparison of results from different laboratories and facilitates the investigation of the influence of pre-analytical parameters. All laboratories analysed identical sets of blood samples spiked with tumour cells in a concentration range of 1-100 tumour cells/ml. To study at which level variation was introduced, three kinds of sample sets were generated in which (i) tumour cell RNA was spiked in the RNA of mononuclear cells (MNC), (ii) tumour cells were spiked in isolated MNC, and (iii) tumour cells were spiked in blood. Real-time quantitative RT-PCR was used to detect and quantify cytokeratin 20 (CK20) expression, which is indicative for the presence of epithelial tumour cells. All laboratories were able to detect CK20 expression in all spiked-RNA samples with limited variation in expression levels between laboratories. There was a positive correlation between the amount of spiked tumour cell RNA and CK20 expression level. RT-PCR analysis of spiked-MNC samples resulted in more variation in the CK20 expression levels between laboratories, however again all spiked samples were reported to be positive by all of the laboratories. The evaluation of spiked-blood samples gave rise to considerable quantitative and qualitative variation between the laboratories. Our results underline the importance and need for standardisation and extended quality control studies in the field of pre-analytics.
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PMID:Reliability of quantitative reverse-transcriptase-PCR-based detection of tumour cells in the blood between different laboratories using a standardised protocol. 1256 93

The aim of this study was to evaluate efficacy of highly active antiretroviral therapy (HAART) in 64 HIV-1 positive treatment-naive and previously treated patients, with different viral load at baseline. The HAART regimen consisted of one protease inhibitor and two reverse-transcriptase inhibitors. Plasma RNA viral load was measured by RT-PCR (Roche Amplicor Monitor kit) at enrolment and at months 3, 6, 9, and 12 of follow-up. The viral load fell below the threshold of 200 copies/ml in 46.9% of patients at month 6 and this result lasted up to month 12 of follow-up in 42.2% of patients. In these patients the CD4+ cell count increased from a baseline with a median of 194 cells/mmc at month 12. Treatment failure occurred in 35.9% of patients and the proportion was higher among previously treated patients. 7 patients stopped therapy because of poor compliance and 5 because of adverse drug effects. Also in our cases the HAART regimen showed more efficacy in treatment-naive patients, whereas baseline viral load >1x105 was the cause of less efficacy of therapy.
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PMID:[Plasma HIV-1 RNA viral load up to one year of follow-up in 64 patients treated with HAART] 1275 86

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is a technique with the potential of improving the quantification of disseminated epithelial cells (DEC) in haematological tissues due to its exquisite sensitivity. This sensitivity may lead to false positivity. Immunocytochemistry (ICC) is regarded as the standard methodology to diagnose DEC. In this study, detection with ICC was compared with quantitative real-time RT-PCR for CK-19 and mammaglobin (hMAM) mRNA in bone marrow (BM) of patients with metastatic breast cancer (MBC). Bone marrow was aspirated from 14 control patients and from 29 patients with MBC. Mononuclear cells (MNC) were isolated. Immunostaining was carried out with the Epimet kit. Quantitative PCR was performed on the ABI Prism 7700. The CK-19 and hMAM mRNA quantities were normalised against beta-Actin and calculated relative to a calibrator sample (relative gene expression). All controls were negative by ICC and for hMAM expression measured by RT-PCR, whereas the median RGE value for CK-19 was 0.57. For the MBC patients, the median RGE for hMAM was 0 and 10 out of 25 (40%) tested positive. Median RGE for CK-19 was 2.9 and 20 out of 25 (80%) tested positive. With ICC, the median value was 1 stained cell per sample, and 15 out of 24 (62%) samples were positive. A correlation was observed between CK-19 and hMAM expression (r=0.7; P=0.0003), and between hMAM expression and ICC (r=0.6; P=0.003). CK-19 expression and ICC (r=0.9; P<0.0001) showed the strongest correlation. Reverse transcriptase-polymerase chain reaction for CK-19 resulted in a higher number of positive BM samples of patients with MBC than ICC. Since an excellent correlation is observed between ICC and RT-PCR, and RT-PCR is probably more sensitive with the advantage of being less observer dependent and thus also more easy to automate, we consider our quantitative real-time RT-PCR method as validated for the detection of DEC in the bone marrow of breast cancer patients.
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PMID:Real-time RT-PCR correlates with immunocytochemistry for the detection of disseminated epithelial cells in bone marrow aspirates of patients with breast cancer. 1550 29

The semiquantitative detection of Alicyclobacillus acidoterrrestris in orange juice by reverse-transcriptase polymerase chain reaction (RT-PCR) with a linear dynamic range of 2 x 10(5)-2 colony forming units (CFU)/mL in terms of cell count is described. Separation, detection, and quantification of the RT-PCR products were accomplished using the Agilent 2100 bioanalyzer in conjunction with the DNA 1000 LabChip kit. After 0 and 12 h of enrichment, it was possible to generate a linear standard curve between the amount of cells and amplicon concentration of RT-PCR and PCR products. Using this method, cell diminution was verified in samples of orange juice treated with a natural inhibitor (Sapindus saponaria), determining the persistence of viable cells. Semiquantitative RT-PCR using the Agilent 2100 bioanalyzer is a potentially useful approach for rapid in vitro determination of A. acidoterrestris and monitoring of inhibitor susceptibility for the orange juice-producing industry.
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PMID:Semiquantitative determination of Alicyclobacillus acidoterrestris in orange juice by reverse-transcriptase polymerase chain reaction and capillary electrophoresis--laser induced fluorescence using microchip technology. 1556 70

Genogroup I noroviruses from five genetic clusters and genogroup II noroviruses from eight genetic clusters were detected in stool extracts using degenerate primers and single-tube, real-time reverse transcription-PCR (RT-PCR) with SYBR Green detection. Two degenerate primer sets, designated MON 431-433 and MON 432-434, were designed from consensus sequences from the major clusters of norovirus based on the RNA-dependent RNA polymerase region of the norovirus genome. Viruses were extracted from stool samples within 20 min using a viral RNA extraction kit. Real-time RT-PCR for noroviruses generated semiquantitative results by means of the cycle threshold data and dilution endpoint standard curves. Presumptive product verification was achieved by evaluation of first-derivative melt graphs. Multiple clusters of noroviruses were identified simultaneously in a multiplex fashion by virtue of slight differences in melting temperature. The detection of 13 different genetic clusters suggests that the MON primers may serve as universal primers for most, if not all, of the noroviruses in a multiplex assay. Our technique provides a framework for broad application of real-time RT-PCR in clinical, environmental, and food testing laboratories for a wide range of noroviruses.
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PMID:Genogroup I and II noroviruses detected in stool samples by real-time reverse transcription-PCR using highly degenerate universal primers. 1557 15

Tetramethylpyrazine is the active ingredient of a Chinese herbal medicine. In this study, tetramethylpyrazine was tested for its activities in irradiated bone marrow stromal QXMSC1 cells. The proliferation of QXMSC1 cells was measured by MTS assay kit and flow cytometry. To identify proteins involved in the processes of cellular and molecular response of tetramethylpyrazine to irradiation damage, we comparatively analyzed the proteome of nonirradiated, irradiated and tetramethylpyrazine treated QXMSC1 cells. Reverse transcriptase polymerase chain reaction (RT-PCR) were used to validate the differentially expressed proteins. 20 Gy 60Co gamma irradition inhibited QMSC1 cells growth and tetramethylpyrazine could reverse of this action due to stimulating QXMSC1 cells from G1 to S progression. Proteomic analytical results showed that 18 spots were changed in irradiated QXMSC1 cells, and 15 spots matched with known proteins after database searching. The expression level of proteins such as translationally controlled tumor protein (TCTP), and galectin-3, were increased in irradiated QXMSC1 cells, while calmodulin, pyruvate kinase were decreased. Tetramethylpyrazine could prevent this change or reverse to some degree. The function of these proteins involves in hematopoiesis, cell cycle and signal transduction. The changes of these proteins were confirmed by RT-PCR at mRNA levels. This study suggested that stimulating proliferation via tetramethylpyrazine played an important role in the cure effect on irradiated QXMSC1 cells and was helpful to deeply understand the mechanism of tetramethylpyrazine at the molecular level.
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PMID:Proteomic analysis of the effects of tetramethylpyrazine on irradiated QXMSC1 cells. 1726 90

A one-step reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed and optimized for the detection of duck hepatitis virus type 1 (DHV-1) using the Viral Gene-spin viral DNA/RNA extraction kit. A pair of DHV-1-specific primers was designed against the gene encoding RNA-dependent RNA polymerase (3D gene). Using RNA prepared from duckling liver samples infected with two reference and seven Korean field isolates of DHV-1, one-step RT-PCR with DHV1-specific primers amplified a 467-bp fragment. Under the same conditions, no amplification was observed for 14 other avian pathogenic viruses and bacteria. Using RNA prepared from serial dilutions of the DHV-1 with the supernatant of the uninfected duckling liver homogenate (10% w/v), the one-step RT-PCR assay was found to be sensitive to 10 50% egg lethal dose (ELD50) 0.1 ml(-1) of DHV-1. Furthermore, this method detected DHV-1 from the livers and allantoic fluid of duck embryos dying before 3 days postinoculation (PI) and of chicken embryos that were chilled at 3 days PI. Therefore, this one-step RT-PCR method is rapid, sensitive, and reliable, and can be readily adapted for detection of DHV-1 from other clinical samples.
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PMID:Development of one-step reverse transcriptase-polymerase chain reaction to detect duck hepatitis virus type 1. 1762 80

Reverse transcriptase (RT) assay is commonly used to detect enzyme activity associated with retroviral-like particles. Previously, detection of RT activity in virus-infected cultures was done using a radioisotope-based assay system. However, assay systems, which detect the antigen directly(as opposed to antibody ELISA assays), have been developed. For diagnostic purposes, RT activity and p24 antigen capture assays are the two most commonly used methods for detection of retroviral infection. More recently, new non-radioactive assay systems have been developed. In this study, four non-radioactive reverse transcriptase kits were evaluated using samples obtained from a chimeric virus, simian/human immunodeficiency virus (SHIV) and SIV-infected cell cultures. The results showed that the magnesium kit was the most appropriate for detection of SIV and SHIV infection in cell culture supernatants.
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PMID:Evaluation of Non-radioactive ELISA assay Kits for Detection of Retroviral Reverse Transcriptase (RT) Activity Associated with Retroviral SIV and HIV. 1765 46


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