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)

We have introduced a reverse transcriptase polymerase chain reaction based method to measure mRNA levels of the melanogenesis enzymes tyrosinase, tyrosinase-related-protein 1 (TRP-1), and tyrosinase-related-protein 2 (TRP-2). Expression was determined by reverse transcriptase-competitive multiplex polymerase chain reaction of (i) melanogenesis enzyme transcripts and the "housekeeping" gene glyceraldehyde-3-phosphate dehydrogenase, and (ii) two internal standards consisting of mutated melanogenesis enzyme cDNA and mutated gene glyceraldehyde-3-phosphate dehydrogenase cDNA. This was investigated on in vitro cultured melanocytes in the presence of three different steroids; one glucocorticoid (betamethasone-17-valerate) and two sex steroids (diethylstilbestrol and estradiol). All three steroids lead to an increase of about 1.5-2.5-fold of tyrosinase transcripts. The amount of TRP-1 transcripts was likewise enhanced, but only moderately (approximately 1.5-fold). In contrast, TRP-2 transcripts were reduced by approximately 40% in number after betamethasone-17-valerate treatment, whereas the two sex steroids, diethylstilbestrol and estradiol, caused an upregulation of about 20-fold of the initial TRP-2 transcript level. We therefore suggest that hyperpigmentation during pregnancy or under contraceptive treatment is mediated by a direct induction of melanogenesis via sex steroids.
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PMID:Quantification of tyrosinase, TRP-1, and Trp-2 transcripts in human melanocytes by reverse transcriptase-competitive multiplex PCR--regulation by steroid hormones. 954 Sep 76

Detection and quantitation of circulating cancer cells in peripheral blood may improve cancer staging and monitoring. This study explored the feasibility of using circulating cancer cell detection in peripheral blood for the rapid assessment of chemotherapeutic response. Cytokeratin 19 mRNA was amplified by nested reverse transcriptase-PCR in the peripheral blood of 29 healthy volunteers, 33 pneumonia patients, and 86 lung cancer patients. Circulating cancer cells in the peripheral blood were semiquantitatively determined by taking the ratio of cytokeratin 19 band intensity from the second round of nested PCR to the glyceraldehyde-3-phosphate dehydrogenase band intensity from the first round of PCR amplification. The detection limit of the method was 1 cancer cell in 107 peripheral blood mononuclear cells. The positive detection rate was 40% for lung adenocarcinoma patients of all stages, 41% for squamous carcinoma patients of all stages, and 27% for small cell lung cancer patients. Only one control sample from a pneumonia patient showed a positive result (1.6%). The quantitative method reliably and sensitively estimated cancer cell numbers in the peripheral blood of lung cancer patients. Serial measurement of the relative number of circulating cancer cells correlated with the tumor burden and treatment response of patients. This method may help rapidly assess the efficacy of anticancer treatment, redefine cancer staging, and facilitate the design of better therapeutic strategies for the treatment of cancer patients.
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PMID:Detection and quantitation of circulating cancer cells in the peripheral blood of lung cancer patients. 966 88

To gain further information concerning the regulation by androgen of AR mRNA expression in cultured genital skin fibroblasts (GSF), we first developed a quantitative reverse transcription-competitive polymerase chain reaction (RT-PCR). This method used an ethidium bromide stain analysis of the PCR products for the accurate quantitation of low levels of human androgen receptor (hAR) mRNA in GSF. To control for variations due to sample preparation, and to minimize the disparity of the reverse transcriptase efficiency between samples after the RT procedure, we produced an initial PCR for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and then adjusted the amount of cDNA to that of this housekeeping gene. Competitive PCR for hAR was then immediately performed on normalized cDNA with a competitor DNA that exhibited a 13 bp deletion as compared to the 163 bp for the target fragment, and the PCR products were easily separated by 3.5% agarose gel electrophoresis. This quantitation procedure involved no additional steps, such as enzymatic cleavage of the PCR products, nor the use of radioactivity. In GSF from individuals, we found that the normal amount of AR mRNA was 5.6 attomoles/microg RNA, (+/-1.0, s.e.m.) with an intra- and an inter-assay of 8.4 and 14.7%, respectively. We observed a biphasic pattern of AR mRNA expression in normal human GSF in the presence of physiological concentration of androgen. Quantitative RT-PCR of AR mRNA may be useful for studying AR mRNA expression in experimental or clinical conditions.
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PMID:Quantitation of androgen receptor messenger RNA from genital skin fibroblasts by reverse transcription--competitive polymerase chain reaction. 971 9

Renal cell carcinoma (RCC) is a solid tumour of the kidney and is the most common renal neoplasm. Despite the presence of tumour infiltrating lymphocytes (TIL) in RCC, these tumours continue to progress in vivo suggesting a poor host immune response to the tumour, and the suppression of TIL effector function. Cytokines are key molecules that modulate the function of T cells. The possibility is investigated that the local production of cytokines in RCC contributes to immunosuppression of TIL. The expression of pro-inflammatory (IFN-gamma/IL-2) and immunosuppressive (IL-10/TGF-beta) cytokine mRNA transcripts was determined in RCC, normal kidney and peripheral blood of RCC patients using a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with cytokine-specific primers. Following Southern blot hybridization of the PCR products with internal radiolabelled oligonucleotide probes, cytokine transcript levels were measured by densitometry and expressed relative to the glyceraldehyde-3-phosphate dehydrogenase densitometry score. With the exception of IL-10, there were no differences in expression of cytokine mRNA transcripts between the peripheral blood of patients and normal healthy individuals. It was found that TGF-beta transcripts were well represented in normal kidney and RCC. In contrast, the expression of IFN-gamma transcripts, while low in the majority of samples, was significantly increased in RCC when compared to normal kidney (P=0.05). The IL-2 and IL-10 transcripts showed a more variable expression in normal kidney and RCC, with no significant differences in expression between the sample groups. The data demonstrating pro-inflammatory and immunosuppressive cytokine expression in RCC do not support a prominent immunosuppressive cytokine profile in these tumours.
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PMID:Expression of cytokine mRNA transcripts in renal cell carcinoma. 972 77

Renal cell carcinoma (RCC) is the most common renal neoplasm. Despite being infiltrated by tumour infiltrating lymphocytes (TIL), these TIL are unable to control tumour growth in vivo, suggesting that the cytotoxic capacity of TIL against RCC is impaired, or that the tumour cells are resistant to killing and therefore escape detection by the immune system. It is postulated that the expression of apoptotic regulatory molecules in RCC favours tumour cell survival. The present study has therefore determined the expression of Fas (APO-1/CD95), Fas ligand (Fas L) and bcl-2 in these tumours. The expression of Fas, Fas L and bcl-2 mRNA transcripts was determined in RCC, normal kidney and peripheral blood by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), following RNA extraction and cDNA synthesis from tissues and cell samples. Transcript levels were measured by densitometry after Southern blot hybridization of PCR products with internal radio-labelled oligonucleotide probes; a densitometry score was assigned to each hybridizing DNA band and expressed as a ratio of the glyceraldehyde-3-phosphate dehydrogenase content. In peripheral blood, the expression of Fas L and bcl-2 transcripts was similar between patients and normal healthy individuals; however, Fas transcript expression was significantly down-regulated in the patients' versus normal peripheral blood (P = 0.026). Most interestingly, significantly up-regulated Fas L expression was observed in RCC compared to normal kidney (P = 0.041). In contrast, bcl-2 transcripts were well represented in normal kidney but markedly decreased in RCC (P = 0.021). The expression of Fas transcripts in normal kidney and RCC was variable. These data demonstrate elevated expression of Fas L transcripts in RCC, but the functional relevance of this remains to be investigated.
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PMID:Expression of apoptotic regulatory molecules in renal cell carcinoma: elevated expression of Fas ligand. 1010 81

Recent studies have suggested that simvastatin may exert endothelial-protective and anti-ischemic effects via nitric oxide (NO) mechanisms. The aim of this study was to evaluate, in isolated working rat hearts, the effect of acute simvastatin administration on endothelial and inducible NO-synthase (eNOS and iNOS) mRNA and on myocytic apoptosis after ischemia-reperfusion. We used isolated working rat hearts submitted to 15 min global, no-flow, normothermic ischemia and 180 min reperfusion. To detect myocytic apoptosis we used DNA agarose gel electrophoresis and Tunel technique; eNOS and iNOS expression were evaluated by multiplex reverse transcriptase-polymerase chain reaction; glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as standard. The eNOS and iNOS mRNAs were expressed as G3PDH/eNOS and G3PDH/iNOS densitometric ratio (BioRad Gel Doc 1000). Hearts were divided into four groups: A) hearts excised and used as histological controls; B) untreated hearts submitted to ischemia and reperfusion; C) actinomicin D-treated (1.5 mg/kg) hearts, perfused with 25 microM simvastatin, subjected to ischemia and reperfusion; D) hearts treated with simvastatin 25 microM and submitted to ischemia and reperfusion. In Group B we evidenced a significant myocytic apoptotic damage, reduced in groups C and D. In Group B an increase in G3PDH/eNOS ratio vs Group A was detected; in Group D a reduction in G3PDH/eNOS ratio vs Group B occurred; no significant changes were observed between groups C and D. As for G3PDH/iNOS ratio, it was significantly increased in Group D with respect to groups A and B. Our data suggest that simvastatin in acute may modulate NO-synthase mRNA expression (induction of eNOS mRNA by means of post-transcriptional mechanisms and inhibition of iNOS postischemic overexpression) and reduce myocytic apoptosis.
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PMID:[Simvastatin and ischemia-reperfusion damage: its effects on apoptotic myocyte death and on the endothelial expression of nitric-oxide synthetase in an experimental model of the isolated rat heart]. 1018 33

Glutathione S-transferase (GST) M1 is a member of the GST mu family of cytosolic enzymes that have been hypothesized to catalyze the conjugation of glutathione to a large number of hydrophobic substances, including carcinogens such as polynuclear aromatic hydrocarbons present in tobacco smoke, leading to their excretion. Epidemiologic and experimental evidence suggests that the risk of cervical cancer is related to both human papillomavirus (HPV) infection and cigarette smoking. We compared the enzymatic activities and mRNA levels of GSTs in GSTM1-positive human cervical keratinocytes (HCKs) that had been transfected with HPV16 with those in the parental cells. The GSTM1 activity toward the substrate trans-stilbene oxide was 5- to 7-fold lower than in the parental cells. The relative mRNA level in HCK transfected with HPV16 E6/E7, as quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) with normalization against endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, was 6% that of the parental cells. It was 16 and 82%, respectively, in cells that were transfected with HPV16 E6 alone or HPV16 E7 alone. When quantified by competitive RT-PCR using an exogenous nuclease-resistant synthetic cyclophilin RNA transcript as control, the mRNA level in HCK transfected with HPV16 E6 was approximately 10-fold lower that that in the parental cells. It was approximately 5- to 7-fold lower in the HPV16 E7 or HPV16 E6/E7 cells. Our results suggest that viral infections, through the modulation of cellular xenobiotic-metabolizing enzymes, may play a role in the ability of cells to handle environmental carcinogens.
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PMID:Decreased expression of glutathione S-transferase M1 in HPV16-transfected human cervical keratinocytes in culture. 1022 2

Exogenous prostaglandin E2 (PGE2) given by inhalation almost completely abrogates aspirin-induced asthma and the accompanying increase in cysteinyl-leukotrienes production. Cyclooxygenase (COX) may be present in cells in both constitutive (COX-1) and inducible (COX-2) forms. To increase the production of the potentially protective endogenous PGE2, COX-2 should be upregulated. We hypothesize that an abnormal regulation of COX-2 will predispose patients with asthma to develop aspirin-intolerant asthma/rhinitis (AIAR). We therefore examined the expression of COX-2 messenger RNA (mRNA) in healthy nasal mucosa (n = 11) and in nasal polyps from both patients with AIAR (n = 8) and those with aspirin-tolerant asthma/rhinitis (ATAR) (n = 20). After total mRNA extraction, COX-1 and COX-2 mRNA expression were measured using a reverse transcriptase (RT)-semiquantitative PCR technique. Hybrid primers of COX-1. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or COX-2. GAPDH were used to create PCR products that were cloned and used as internal standard controls in the competitive PCR reaction. Results are presented as mean +/- standard error of 10(6) molecules of mRNA/micrograms of total RNA. No differences in COX-1 mRNA expression were found between nasal mucosa and nasal polyps from both patients with ATAR and those with AIAR. However, COX-2 mRNA expression in nasal polyps from the AIAR group (0.38 +/- 0.10) was markedly and significantly lower than in polyps from the ATAR group (2.93 +/- 0. 52, sevenfold, p < 0.0001) and nasal mucosa (2.10 +/- 0.54, sixfold, p < 0.01). These findings suggest that an inadequate COX-2 regulation may be involved in AIAR.
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PMID:Cyclooxygenase-2 mRNA is downexpressed in nasal polyps from aspirin-sensitive asthmatics. 1039 Apr 14

Recent research suggests that antidepressants exert their clinical action in depression via the restoration of glucocorticoid receptor (GR) function with a subsequent normalization of the altered feed-back regulation of the hypothalamic-pituitary adrenocortical (HPA) system. We, therefore, studied the effects of amitriptyline, a standard antidepressant, and of the glucocorticoid dexamethasone, which has recently been reported to possess antidepressive properties, on glucocorticoid receptor mRNA (GR-mRNA) derived from blood cells of healthy male volunteers. Whole blood samples were exposed in vitro for 24 h to amitriptyline and dexamethasone, the mRNA was extracted, transcripts of the 'house-keeping gene' glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the GR-gene were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and semiquantitatively determined by subsequent densitometry. In a concentration of 10 nM, amitriptyline induced a significant increase in GR-mRNA (GR/GAPDH ratio) to 186 +/- 31% of the control condition, while a concentration of 10 microM of amitriptyline resulted in an increase of GR-mRNA (GR/GAPDH ratio) to 165 +/- 36%. Dexamethasone also up-regulated blood cell GR-mRNA (GR/GAPDH ratio) levels at a concentration of 10 nM to 184 +/- 29%, whereas an incubation with 10 microM apparently resulted in toxic effects on blood cells with a decreased amount of total mRNA samples recovered. In conclusion, we here show an increase of GR-mRNA in human blood cells after treatment with amitriptyline and dexamethasone, pointing to a direct action of these substances on GR-gene expression in a human system.
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PMID:Regulation of glucocorticoid receptor-mRNA in human blood cells by amitriptyline and dexamethasone. 1040 68

Chlamydia trachomatis is an obligate intracellular eubacteria that is dependent on a eukaryotic host cell for a variety of metabolites. For years, it has been speculated that chlamydiae are energy parasites, totally dependent on their host cell for ATP and other high-energy intermediates. To determine whether C. trachomatis contains functional enzymes that produce energy or reducing power, four enzymes involved in glycolysis or the pentose phosphate pathway, specifically pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase, were cloned, sequenced and expressed as recombinant proteins in Escherichia coli. The deduced amino acid sequences obtained show high homology to other pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase enzymes. In contrast to numerous other bacterial species, chlamydial glycolytic genes are not arranged in an operon, but are dispersed throughout the genome. Results from reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicate that all four genes are maximally expressed in the middle of the chlamydial developmental cycle. The chlamydial genes are capable of complementing mutant E. coli strains lacking the respective enzyme activities. In vitro enzyme analysis indicates that recombinant chlamydial enzymes expressed in E. coli are active and, interestingly, recombinant chlamydial pyruvate kinase is not regulated allosterically by fructose 1,6 bisphosphate or AMP, as found with other bacterial pyruvate kinases. In summary, identification and characterization of these glucose-catabolizing enzymes indicate that chlamydia contains the functional capacity to produce its own ATP and reducing power.
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PMID:Glucose metabolism in Chlamydia trachomatis: the 'energy parasite' hypothesis revisited. 1041 34


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