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)

Renal cell carcinomas (RCC) contain tumour infiltrating lymphocytes (TIL) but these are essentially immunosuppressed in that they do not generate effective antitumour immune responses in vivo. These TIL comprise predominantly alpha beta T cells, although gamma delta T cells are also present. The repertoire of gamma delta T cells in RCC, however, has not been fully investigated. To identify the gamma delta T cell populations infiltrating RCC, this study has characterised the gamma delta T cell receptor (TCR) repertoire expression in these tumours and compared this to autologous normal kidney and autologous peripheral blood. A semi-quantitative reverse transcriptase/polymerase chain reaction technique was used for amplification of rearranged TCR V-C mRNA transcripts. Primers specific for the four human TCR V gamma and six V delta subfamilies were used, each in conjunction with a primer specific for either the C gamma or C delta region. The specificity of the PCR products was confirmed by Southern blotting and hybridisation with an internal C region probe. A densitometry score was assigned to each DNA band and the level of V gene expression was determined as ratio of C delta gene expression. The gamma delta TCR expression in each sample was determined as a ratio of C delta: glyceraldehyde phosphate dehydrogenase densitometry score. This demonstrated that TCR C delta gene expression was significantly higher in RCC compared to normal kidney (P < 0.019), suggesting a selective infiltration of gamma delta T cells into the tumour. Furthermore, we observed differences in the TCR V gamma and V delta repertoires between RCC and peripheral blood. V gamma l expression was significantly decreased (P < 0.043) whereas there was an over-representation of V gamma 4 transcripts (P < 0.028) in RCC compared to blood. A significant reduction in expression of both V delta 1 (P < 0.028) and V delta 3 (P < 0.051) was also observed within kidney tumour compared to peripheral blood. These findings show that expression of the gamma delta TCR repertoire in RCC differs from that in peripheral blood and normal kidney.
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PMID:Characterisation of gamma delta T cells in renal cell carcinoma patients by polymerase chain reaction analysis of T cell receptor transcripts. 911 81

The expression of the mitochondrial benzodiazepine receptor gene was assayed by a semi-quantitative non-radioactive reverse transcriptase polymerase chain reaction (RT-PCR) assay. The level of amplified mitochondrial benzodiazepine receptor mRNA was expressed as a ratio of either glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or beta-actin mRNA co-amplified in the same RT-PCR assay. The relative amounts of mitochondrial benzodiazepine receptor RNA in several rat tissues were found to be similar to the previously reported relative amount of mitochondrial benzodiazepine receptor binding sites. The level of these binding sites has also been reported to be altered by stress stimuli. In this study we specifically measured the effect of stress on the mRNA levels of the mitochondrial benzodiazepine receptor as an alternative method to the binding assay in an attempt to understand the mechanism by which stress alters binding. Sprague-Dawley male rats were either forced to swim for 15 min in 18 degrees C water or restrained in a plastic cylinder for 45 min either once, or twice daily for 7 days. Neither the swim stress, nor acute or chronic restraint stress, caused a measurable statistically significant relative change in mitochondrial benzodiazepine receptor mRNA in the adrenal gland, kidney, testis and olfactory bulb. However, daily treatment of rats for 7 days with 4 mg/kg of dexamethasone caused a significant decrease in mitochondrial benzodiazepine receptor gene expression in adrenal glands. This finding and the measurement of the relative levels of mitochondrial benzodiazepine receptor mRNA in the various tissues indicate that mitochondrial benzodiazepine receptor density is regulated to some extent at the gene expression level. However, the lack of detectable stress-induced changes in mRNA levels for this receptor seem to indicate that either mRNA changes were below detectable levels or that other mechanisms may be involved in the previously reported stress-induced changes of mitochondrial benzodiazepine receptor density. Because the focus of this work was on the regulation of mitochondrial benzodiazepine receptor gene expression, ligand binding studies to determine changes in receptor densities were not performed.
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PMID:Dexamethasone, but not stress, induce measurable changes of mitochondrial benzodiazepine receptor mRNA levels in rats. 927 84

Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in the glycolytic pathway. Since its transcript levels do not vary in most experimental conditions, it has been often used as a control in northern blot or reverse transcriptase-polymerase chain reaction analysis. We have cloned and sequenced a gene encoding glyceraldehyde-3-phosphate dehydrogenase (Tthgapdh) from Tetrahymena thermophila cDNA library and determined whether the Tthgapdh mRNA is a loading control in gene expression studies of T. thermophila cell. The open reading frame encoded a protein of 341 amino acid residues (36.8 kDa) containing a nicotinamide adenine dinucleotide-binding domain and a catalytic domain, which was highly similar to those of other organisms. Its mRNA levels at different growth stages were examined by northern blot analysis. The fragment of the isolated cDNA was hybridized to a 1.3-kb mRNA transcript. There was a marked increase in Tthgapdh mRNA level at the mid-exponential phase, followed by a gradual decrease. Therefore, much caution should be made to use Tthgapdh mRNA as an internal standard for northern blot analysis in Tetrahymena.
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PMID:Cloning and sequencing of a cDNA encoding glyceraldehyde-3-phosphate dehydrogenase from Tetrahymena thermophila: growth-associated changes in its mRNA expression. 930 12

Nitric oxide (NO), a potent and versatile free radical, is synthesized in leukocytes by the inducible form of NO synthase (iNOS). In this study, leukocytes in pregnant mouse uterus were investigated for expression of the iNOS gene. Inducible NOS mRNA, which was identified by reverse transcriptase polymerase chain reaction, was high relative to an invariant mRNA, glyceraldehyde-3-phosphate dehydrogenase, in midgestation uteri (gestation days [g.d.] 10, 12, and 14) but was low in late-gestation uteri (g.d. 16 and 18). Inducible NOS protein, identified immunohistochemically in paraformaldehyde-fixed uteri taken from g.d. 6 through 18 using rabbit antibodies generated to mouse carboxyl terminus iNOS peptides, was prominent in a few myometrial mast cells at early stages and was strongly expressed from g.d. 6 through g.d. 14 in myometrial macrophage-like cells. Inducible NOS protein was first detected in uterine (u) natural killer (NK) cells at g.d. 8. Signals peaked in this lineage at g.d. 10 and declined thereafter. Uterine leukocytes cultured in vitro expressed the iNOS gene; a hybridoma cell line derived from mouse uNK cells (GWM1-2) contained iNOS mRNA, and cells migrating from mouse metrial gland explants included iNOS/ leukocytes. Large, granular iNOS + uNK cells were absent from the uteri of homologously mated pregnant TgE26 mice, an NK cell-deficient transgenic mouse strain, but immunoreactive iNOS was detectable in trophoblast, a cell lineage that did not contain immunoreactive iNOS in NK cell-competent Swiss-Webster mice. In TgE26 mothers gestating normal embryos, the same pattern was observed. Collectively, the results of this study demonstrate that iNOS is present in mouse uterine leukocytes including mast cells, macrophage-like cells, and uNK cells, and suggest that in the absence of uNK cells, the placenta synthesizes iNOS. These findings are consistent with the postulate that leukocyte NO contributes importantly to events associated with successful pregnancy that are likely to include relaxation of vascular smooth muscle.
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PMID:Expression of the inducible nitric oxide synthase gene in mouse uterine leukocytes and potential relationships with uterine function during pregnancy. 931 87

The purpose of this study was to develop a method by which endothelial cell nitric oxide synthase (ecNOS) mRNA expression could be measured in single coronary resistance arteries and to test the hypothesis that ecNOS gene expression is upregulated by exercise training. Yucatan miniature swine were randomly assigned to exercise-trained (ET; n = 5) or sedentary (Sed; n = 4) groups for 16 wk. Individual coronary resistance arteries (50-100 microns) were dissected, frozen in liquid nitrogen, and homogenized in a LiCl buffer, mRNA was isolated from each vessel, and ecNOS gene expression was assessed using reverse transcriptase (RT)-polymerase chain reaction (PCR) standardized by coamplifying ecNOS with glyceraldehyde 3-phosphate dehydrogenase (GAPHD). The ecNOS-to-GAPDH amplicon ratio was significantly greater in coronary resistance arteries isolated from ET pigs than in Sed controls. On the basis of these data, it is concluded that RT-PCR can be used on single coronary resistance arteries to assess cell-specific mRNA expression and that ecNOS gene expression is upregulated by exercise training in porcine coronary resistance arteries.
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PMID:Induction of nitric oxide synthase mRNA in coronary resistance arteries isolated from exercise-trained pigs. 943 89

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

The type XVII collagen alpha 1 chain has been identified as a component of the type I hemidesmosome, and is thus thought to play a role in extracellular matrix (ECM) maintenance and signal transduction between the cell and the ECM. We examined the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart by Northern blot analysis and determined the sequential changes of its expression in different developmental stages of the heart using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Northern blotting: Total RNA was extracted from 10 adult mouse hearts by the guanidine/cesium method. Hybridization was performed with mouse cDNA for alpha 1 (XVII) collagen. RT-PCR: Total RNA was extracted from 7 embryos, 4 neonates and 8 adult mice. Reverse transcription was performed using oligo-dT primer and MMLV. Amplification was carried out in alpha 1 (XVII) collagen and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH served as an internal control. Northern blotting revealed a 5.6 kb signal that was identical to that of the alpha 1 (XVII) of skin and transformed keratinocyte reported previously. The sequences of the PCR products were also identical to those reported. The normalized expression ratios of alpha 1 (XVII) were 0.91 +/- 0.20 in the embryonic heart, 0.36 +/- 0.20 in the neonatal heart and 0.96 +/- 0.21 in the adult heart. In conclusion, we identified the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart, suggesting that the type I hemidesmosome is located in the heart. The results of the RT-PCR at different developmental stages of the heart suggest that type XVII collagen contributes not only to cardiogenesis in the embryonic stage but also to maintenance of architecture and function in the adult heart.
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PMID:Expression of type XVII collagen alpha 1 chain mRNA in the mouse heart. 968 29

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


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