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 presence and abundance of 5-HT1A and 5-HT2A receptor mRNAs in post mortem human hippocampus was investigated using a novel quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) technique using cyclophilin mRNA as an internal standard. 5-HT1A and 5-HT2A receptor mRNAs were each co-amplified with varying dilutions of cyclophilin primers, and their abundance expressed as a ratio of cyclophilin mRNA. Using this technique in combination with quantitative autoradiography we have investigated the effect of aging on hippocampal 5-HT1A and 5-HT2A receptor mRNA abundance and binding site densities. There was a significant negative correlation between hippocampal 5-HT1A receptor binding site densities and age and a similar trend for 5-HT1A receptor mRNA abundance. Neither 5-HT2A receptor binding site densities nor mRNA abundance were affected by age. Both 5-HT1A and 5-HT2A receptor binding site densities in individual subjects correlated significantly with abundance of their encoding mRNA. This study demonstrates that 5-HT1A and 5-HT2A receptor mRNAs can be measured in small samples of human brain. Combining studies of mRNA with those directed at binding sites will help reveal mechanisms underlying changes in expression of these receptors in various neuropsychiatric disorders.
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PMID:Detection and quantitation of 5-HT1A and 5-HT2A receptor mRNAs in human hippocampus using a reverse transcriptase-polymerase chain reaction (RT-PCR) technique and their correlation with binding site densities and age. 752 88

SDZ NIM 811 is a cyclosporin A analog that is completely devoid of immunosuppressive capacity but exhibits potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity. The mechanism of action of SDZ NIM 811 is clearly different from those of all other anti-HIV agents described so far. In cell-free assays, it is not an inhibitor of reverse transcriptase, protease, integrase, and it does not interfere with Rev or Tat function. SDZ NIM 811 does not down-regulate CD4 or inhibit fusion between infected and uninfected, CD4-expressing cells. p24 production from chronically HIV-infected cells is not impaired either. To elucidate the mode of action of SDZ NIM 811, we performed DNA PCR analysis in HIV-1 IIIB-infected MT4 cells in one cycle of virus replication. The effects of SDZ NIM 811 on the kinetics of viral DNA synthesis, appearance of two-long terminal repeat circles (2-LTR circles), and integration of DNA were studied. SDZ NIM 811 inhibited 2-LTR circle formation in a concentration-dependent manner, which is indicative of nuclear localization of preintegration complexes. Half-maximal inhibition was achieved at 0.17 microgram/ml; this concentration is close to the 50% inhibitory concentrations (0.01 to 0.2 microgram/ml) for viral growth inhibition. As expected, integration of proviral DNA into cellular DNA was also inhibited by SDZ NIM 811. Analysis of the viral particles produced by SDZ NIM 811-treated, chronically infected cells revealed amounts of capsid proteins, reverse transcriptase activity, and viral RNA comparable to those of the untreated control. However, these particles showed a dose-dependent reduction in infectivity (50% inhibitory concentration of 0.028 microgram/ml) which indicates that the assembly process is also impaired by SDZ NIM 811. Gag proteins are postulated to play a role not only in assembly but also in early steps of viral replication, e.g., nuclear localization of the preintegration complex. Recently, it was reported that HIV-1 Gag protein binds to cyclophilin A, the intracellular receptor for cyclosporin A. Interference with Gag-cyclophilin interaction may be the molecular basis for the antiviral activity of cyclosporin A and its analogs.
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PMID:Mode of action of SDZ NIM 811, a nonimmunosuppressive cyclosporin A analog with activity against human immunodeficiency virus type 1 (HIV-1): interference with early and late events in HIV-1 replication. 781 48

The high sensitivity of reverse transcriptase-polymerase chain reaction for detecting low copy number mRNA transcripts has been standardized to analyze the mRNA profiles of catalase, glutathione peroxidase, CuZn-superoxide dismutase and aldose reductase, with respect to the housekeeping gene cyclophilin, in rat lenses cultured in hyperglycemic (50mM glucose) or oxidative (100 microM H2O2) media for 24, 40 and 60 hr. In response to hyperglycemia mRNA expression of catalase appeared to be inhibited at 24 hr but attained normal levels by 40 hr. On the other hand, mRNA levels of catalase were higher than normal between 40 and 60 hr in the presence of H2O2. Glutathione peroxidase mRNA abundance although enhanced in response to both hyperglycemia as well as H2O2-induced stress, displayed opposite trends with time-an increase from 24-60 hr due to hyperglycemia and a decrease to normal by 60 hr in the presence of H2O2. In contrast, CuZn-superoxide dismutase was inhibited at 50 mM glucose achieving baseline levels by 60 hr, while H2O2 elicited an induction at 24 hr which waned to basal levels by 60 hr. Interestingly, aldose reductase was unaffected by hyperglycemia but showed an appreciable increase with time upon exposure of the lens to H2O2. The role of these enzymes in cataractogenesis with regard to their respective mRNA levels is discussed.
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PMID:Semi-quantitation of mRNA by polymerase chain reaction. Levels of oxidative defense enzymes and aldose reductase in rat lenses cultured in hyperglycemic or oxidative medium. 873 24

1. After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM gamma-aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor alpha 1, alpha 6 and beta 2,3 subunit protein expression and alpha 1 and alpha 6 subunit steady-state mRNA levels, was examined using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. 2. GABA exposure for 2 days decreased alpha 1 (35 +/- 10%, mean +/- s.e.mean), beta 2,3 (21 +/- 9%) and alpha 6 (28 +/- 10%) subunit protein expression compared to control levels. The GABA-mediated reduction in alpha 1 subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 microM). 3. GABA exposure for 8 days increased alpha 1 (26 +/- 10%, mean +/- s.e.mean) and beta 2,3 (56 +/- 23%) subunit protein expression over control levels, whereas alpha 6 subunit protein expression remained below control levels (by 38 +/- 10%). However, after 10 days GABA exposure, alpha 6 subunit protein expression was also increased over control levels by 65 +/- 29% (mean +/- s.e.mean). 4. GABA exposure did not change the alpha 1 or alpha 6 subunit steady-state mRNA levels over and 8 day period, nor did it alter the expression of cyclophilin mRNA over 1-8 days. 5. These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi-phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve post-transcriptional and/or post-translational processes.
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PMID:The effect of GABA stimulation on GABAA receptor subunit protein and mRNA expression in rat cultured cerebellar granule cells. 896 48

GluR2 is the key subunit of heteromeric AMPA-preferring glutamate receptors. GluR2 mRNA has been shown by in situ hybridization histochemistry to be decreased in the hippocampal formation in schizophrenics. Here, a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to investigate GluR2 expression further and to examine the relative abundance of its alternatively spliced mRNA isoforms ('flip' and 'flop') in 11 schizophrenics and 11 matched controls. Compared to the controls, schizophrenics showed reduced expression of both isoforms relative to cyclophilin mRNA, but a greater loss of the flop isoform led to a higher flip:flop ratio. These differences were observed having controlled for the confounding effects of brain pH and age upon the mRNAs. We also found that the abundance of GluR2 mRNA correlates with that of the encoded subunit. This study has confirmed that, in schizophrenia, hippocampal GluR2 mRNA is reduced, and indicates that GluR2 subunits are composed of a higher proportion of the flip variant. These data extend the evidence for glutamatergic dysfunction in the disease. They suggest that signal transduction through hippocampal AMPA receptors is impaired in schizophrenia both by an overall loss of GluR2 expression, and by the change in flip:flop ratio which is predicted to alter the desensitization kinetics of the remaining GluR2 subunits.
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PMID:GluR2 glutamate receptor subunit flip and flop isoforms are decreased in the hippocampal formation in schizophrenia: a reverse transcriptase-polymerase chain reaction (RT-PCR) study. 903 Jul 2

The 5alpha-reduced metabolites of testosterone, including dihydrotestosterone, are considered the primary regulators of epididymal function. Two genes encode two 5alpha-reductase isozymes. We examined 5alpha-reductase type 1 and type 2 mRNA tissue distribution and relative abundance in cynomolgus monkey (Macaca fascicularis) testicular and epididymal tissues using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). mRNA extracted from monkey tissues including the testis (T) and the proximal caput (PCp), the caput (Cp), the midcorpus (Co), and the distal cauda (Cd) epididymis was reverse transcribed to produce cDNAs. 5alpha-reductase type 1 and 2 cDNAs were subsequently coamplified with the housekeeping gene, cyclophilin, in a PCR spiked with 33P-dCTP. Relative abundance was reported as the cpm ratios of type 1 or type 2/cyclophilin mRNA. Semiquantitative RT-PCR results indicated that type 1 mRNA was most abundant in the testis (0.48 +/- 0.06) and significantly decreased distally along the monkey epididymis (PCp: 0.29 +/- 0.04; Cp: 0.29 +/- 0.04; Co: 0.21 +/- 0.03; Cd: 0.07 +/- 0.01) (P < 0.001). Type 2 mRNA was undetectable in the testis but was present throughout the epididymis at uniform levels (PCp: 1.6 +/- 0.2; Cp: 1.4 +/- 0.3; Co: 1.6 +/- 0.2; Cd: 1.5 +/- 0.2). These data demonstrate that 5alpha-reductase type 1 mRNA is differentially expressed but of low abundance along the nonhuman primate epididymis, whereas 5alpha-reductase type 2 gene expression is uniform.
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PMID:Regional distribution of 5alpha-reductase type 1 and type 2 mRNA along the nonhuman primate (Macaca fascicularis) epididymis. 943 32

Alzheimer's disease (AD) has been hypothesized to be associated with oxidative stress. In this study, the expression of key oxidative stress-handling genes was studied in hippocampus, inferior parietal lobule, and cerebellum of 10 AD subjects and 10 control subjects using reverse transcriptase-polymerase chain reaction (RT-PCR). The content of Mn-, Cu,Zn-superoxide dismutases (Mn- and Cu,Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GSSG-R) mRNAs, and the "marker genes" (beta-actin and cyclophilin) mRNAs was determined. This study suggests that gene responses to oxidative stress can be significantly modulated by the general decrease of transcription in the AD brain. To determine if the particular oxidative stress handling gene transcription was induced or suppressed in AD, the "oxidative stress-handling gene/beta-actin" ratios were quantified and compared with control values in all brain regions studied. The Mn-SOD mRNA/beta-actin mRNA ratio was unchanged in all regions of the AD brain studied, but an increase of the Cu,Zn-SOD mRNA/beta-actin mRNA ratio was observed in the AD inferior parietal lobule. The levels of peroxidation handling (CAT, GSHPx, and GSSG-R) mRNAs normalized to beta-actin mRNA level were elevated in hippocampus and inferior parietal lobule, but not in cerebellum of AD patients, which may reflect the protective gene response to the increased peroxidation in the brain regions showing severe AD pathology. The results of this study suggest that region-specific differences of the magnitude of ROS-mediated injury rather than primary deficits of oxidative stress handling gene transcription are likely to contribute to the variable intensity of neurodegeneration in different areas of AD brain.
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PMID:The expression of key oxidative stress-handling genes in different brain regions in Alzheimer's disease. 1009 42

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

Although our understanding of effects of space flight on human physiology has advanced significantly over the past four decades, the potential contribution of stress at the cellular and gene regulation level is not characterized. The objective of this ground-based study was to evaluate stress gene regulation in cells exposed to altered gravity and environmentally suboptimal conditions. We designed primers to detect message for both the constitutive and inducible forms of the heat shock protein, HSP-70. Applying the reverse transcriptase-polymerase chain reaction (RT-PCR), we probed for HSP-70 message in human acute T-cell leukemia cells, Jurkat, subjected to three types of environmental stressors: (1) altered gravity achieved by centrifugation (hypergravity) and randomization of the gravity vector in rotating bioreactors, (2) serum starvation by culture in medium containing 0.05% serum, and (3) temperature elevation (42 degrees C). Temperature elevation, as the positive control, significantly increased HSP-70 message, while centrifugation and culture in rotating bioreactors did not upregulate heat shock gene expression. We found a fourfold increase in heat shock message in serum-starved cells. Message for the housekeeping genes, actin and cyclophilin, were constant and comparable to unstressed controls for all treatments. We conclude that gravitational perturbations incurred by centrifugal forces, exceeding those characteristic of a Space Shuttle launch (3g), and culture in rotating bioreactors do not upregulate HSP-70 gene expression. In addition, we found RT-PCR useful for evaluating stress in cultured cells.
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PMID:Regulation of heat shock protein message in Jurkat cells cultured under serum-starved and gravity-altered conditions. 1067 23

Chronic hypertension is associated with remodeling of small arteries. There is evidence that the high pressure itself may cause these structural changes, but the responsible mechanisms are not clearly defined. Previously we showed that pressure-induced c-fos expression in intact cannulated rat mesenteric small arteries was inhibited by genistein, a general tyrosine kinase inhibitor. The purpose of this study was to further unravel the underlying signal transduction mechanisms, and we particularly tested the involvement of src tyrosine kinases and extracellular signal-regulated kinase (ERK). Rat mesenteric small arteries were cannulated in a dual-vessel chamber. After a 60-minute equilibration period, the pressure in 1 artery was increased to 140 mm Hg, while the other artery remained at 90 mm Hg. Semiquantitative reverse transcriptase-polymerase chain reaction was used to determine c-fos expression, and Western blotting was used to examine levels of ERK phosphorylation. The involvement of src and ERK was tested with the inhibitors herbimycin A (1 micromol/L), PP1 (10 micromol/L), PP2 (10 micromol/L), and PD98059 (30 micromol/L). One-hour exposure to 140 mm Hg increased the c-fos/cyclophilin ratio 3.6-fold, from 0.29+/-0.07 to 1.06+/-0.25. All the tested inhibitors suppressed the pressure-induced increase of c-fos expression. A 5-minute exposure period to 140 mm Hg increased ERK phosphorylation, and this was abolished in the presence of PP1. The results suggest that pressure-induced c-fos expression in intact cannulated rat mesenteric small arteries may be mediated, at least in part, by src tyrosine kinases and ERK.
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PMID:Src tyrosine kinases and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases mediate pressure-induced c-fos expression in cannulated rat mesenteric small arteries. 1124 24


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