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 RACE amplification technology was used on a novel CYP3A-like exon 1 sequence detected during the reverse transcriptase/polymerase chain reaction analysis of human CYP3A gene expression. This resulted in the identification of cDNAs encompassing the complete coding sequence of a new member of the CYP3A gene subfamily, CYP3A43. Interestingly, the majority of the cDNAs identified were characterized by alternative splicing events such as exon skipping and complete or partial intron inclusion. CYP3A43 expression was detected in liver, kidney, pancreas, and prostate. The amino acid sequence is 75% identical to that of CYP3A4 and CYP3A5 and 71% identical to CYP3A7. CYP3A43 differs from CYP3A4 at six amino acid residues, found within the putative substrate recognition sites of CYP3A4, that are known to be determinants of substrate selectivity. The N terminus of CYP3A43 was modified for efficient expression of the protein in Escherichia coli, and a 6X histidine tag was added at the C terminus to facilitate purification. CYP3A43 gave a reduced carbon monoxide difference spectra with an absorbance maximum at 450 nm. The level of heterologous expression was significantly lower than that observed for CYP3A4 and CYP3A5. Immunoblot analyses revealed that CYP3A43 comigrates with CYP3A4 in polyacrylamide gel electrophoresis but does separate from CYP3A5. Monooxygenase assays were performed under a variety of conditions, several of which yielded reproducible, albeit low, testosterone hydroxylase activity. The findings from this study demonstrate that there is a novel CYP3A member expressed in human tissues, although its relative contribution to drug metabolism has yet to be ascertained.
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PMID:cDNA cloning and initial characterization of CYP3A43, a novel human cytochrome P450. 1116 Aug 76

A cDNA was cloned from Japanese monkey liver mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using oligonucleotide primers based on the marmoset cytochrome P450 2D19 (CYP2D19) nucleotide sequence. The full-length cDNA encoded a 497 amino acid protein (designated CYP2D29) that is 96, 91, and 88% homologous to human CYP2D6, cynomolgus monkey CYP2D17, and marmoset monkey CYP2D19, respectively. Yeast cells (Saccharomyces cerevisiae AH-22 strain) transfected with pGYR1 vectors containing the CYP2D29 cDNA were cultured, and microsomal fractions were obtained. Reduced carbon monoxide-difference spectra and western blot analysis using polyclonal antibodies raised against rat CYP2D2 demonstrated that in yeast cell microsomal fractions, the level of CYP2D29 holoenzyme was similar to that of CYP2D6 holoenzyme. However, western blot analysis indicated that the level of CYP2D29 in Japanese monkey liver microsomes might be much higher than that of CYP2D6 in human liver microsomes. Japanese monkey liver microsomes exhibited much higher activities than did human liver microsomes, expressed as nmol/min/mg protein, for debrisoquine (DB) 4-hydroxylation and bufuralol (BF) 1"-hydroxylation (typical reactions catalyzed by CYP2D6), whereas recombinant CYP2D29 activity, expressed as nmol/min/nmol CYP, was similar to that of CYP2D6 for DB and BF hydroxylation. In kinetic analyses, the K(m) value of CYP2D29 for DB 4-hydroxylation was much lower than that of Japanese monkey liver microsomes, whereas the K(m) value of CYP2D6 for DB 4-hydroxylation was similar to that of human liver microsomes. In contrast, K(m) values for BF 1"-hydroxylation were similar for Japanese monkey and human liver microsomes and yeast cell microsomal fractions expressing recombinant CYP2D29 or CYP2D6. These results suggest that the properties of Japanese monkey CYP2D29 are similar to those of human CYP2D6, but their populations and/or some other factors in liver microsomes may cause the difference in microsomal DB 4-hydroxylase activities between Japanese monkeys and humans.
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PMID:Complementary DNA cloning and characterization of cytochrome P450 2D29 from Japanese monkey liver. 1223 13

Prior studies have shown that acidification due to hypercarbia protects endothelial cells from serum deprivation-induced apoptosis. However, the mechanism(s) responsible for the antiapoptotic effect of acidification is still unclear. cDNA array screening was performed on human umbilical vein endothelial cells cultured in a bicarbonate medium equilibrated either with 5% CO2 (pH 7.4) or with 20% CO2 (pH 7.0). Tyrosine kinase receptor Axl expression was 3.3-fold higher after 6 hours at pH 7.0 compared with pH 7.4; this modulation was confirmed by reverse transcriptase-polymerase chain reaction (3.0+/-0.9-fold, P<0.03; n=3), Northern blot (3.6+/-0.1-fold, P<0.0003; n=3), and Western blot (10+/-1.8-fold, P<0.004; n=3). In a time-course study, both Northern and Western blot analyses showed that the most marked difference in Axl expression between pH 7.4 and pH 7.0 occurred after 24 to 48 hours. Furthermore, Axl phosphorylation was enhanced at pH 7.0. Axl ligand, the survival factor growth arrest-specific gene 6 product (Gas6), was released into the conditioned medium, and by Western blot analysis, similar amounts of protein were found at pH 7.0 and 7.4. Full-length Axl cDNA overexpression reduced serum deprivation-induced apoptosis by 64.4+/-11.9% in human umbilical vein endothelial cells cultured at pH 7.4 compared with mock-transfected cells (P<0.0004). Furthermore, overexpression of either soluble Axl or antisense Gas6 mRNA partially reverted the protective effect of acidification, increasing approximately 2.5-fold the number of apoptotic cells at pH 7.0 (control 19.3+/-2.7%, soluble Axl 48.9+/-9.7%, P<0.001; antisense Gas6 49.3+/-14.3%, P<0.03). In conclusion, Gas6/Axl signaling may play an important role in endothelial cell survival during acidification. The full text of this article is available at http://www.circresaha.org.
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PMID:Acidification prevents endothelial cell apoptosis by Axl activation. 1236 94

Syntheses of [carbonyl-11C]2-(2-benzoylphenoxy)-N-phenylacetamide, a radiolabeled inhibitor of human immunodeficiency virus type 1 (HIV 1) reverse transcriptase, were achieved by applying palladium-mediated cross-coupling reactions with insertion of [11C]carbon monoxide. Our interest was focused for the present on a comparison of the Stille and Suzuki methods, using trimethylphenylstannane or phenylboronic acid as alternative coupling reagents, respectively. The Suzuki variant gave a much higher amount of [11C]CO radioactivity trapped in the reaction mixture, but a significant loss of product occurred due to adsorption phenomena on the potassium carbonate present in the heterogeneous reaction mixture. The labeled product was isolated in only 20% yield (based on trapped [11C]CO, not corrected for decay). According to Stille, the reaction provided a product that could be isolated more easily but it did not increase the final yield of the target compound due to a low trapping efficiency for [11C]CO. Both methods were performed in an overall synthesis time of 30min, starting from [11C]CO2, and gave a product with a specific radioactivity of at least 30GBq/micromol. The Stille method as well as the Suzuki reaction allowed the synthesis of a radiochemically pure product in aqueous acetonitrile.
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PMID:Syntheses of [carbonyl-11C]2-(2-benzoylphenoxy)-N-phenylacetamide from [11C]carbon monoxide by the Suzuki and the Stille reactions. 1243 42

To investigate the expression of the HO-1 gene in PC12 cells in hypoxic environment and gain further insight to the role of HO-1 in cerebral ischemia, PC12 cells were exposed to hypoxia environment (95% N2, 5% CO2) for 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h respectively. The level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR); the volume of COHb in the media were measured spectrophotometrically and the cGMP concentration of PC12 cell extracts was determined by radioimmunoassay. We found that after exposure to hypoxia for 1 h, 4 h, 8 h, 12 h, 24 h, HO-1 mRNA increased by 3%, 4%, 17%, 31% 36% as compared with that in control group respectively (P < 0.01 or P < 0.05); the COHb increased by 12%, 29%, 59%, 88%, 94% as compared with that in control group respectively (P < 0.01 or P < 0.05), and the cGMP concentration were 2.2, 3.4, 5.2, 8.1, 10.9-fold as that of the control group (P < 0.01). We are led to conclude that hypoxia induced the expression of HO-1 gene, the production of endogenous CO, and the concentration of cGMP was elevated as well.
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PMID:Induction of the expression of heme oxygenase gene in PC12 cells by hypoxia. 1267 63

The cyanobacterial CO2-concentrating mechanism (CCM) allows photosynthesis to proceed in CO2-limited aquatic environments, and its activity is modulated in response to inorganic carbon (Ci) availability. Real-time reverse transcriptase-PCR analysis was used to examine the transcriptional regulation of more than 30 CCM-related genes in Synechococcus sp. strain PCC7942 with an emphasis on genes encoding high-affinity Ci transporters and carboxysome-associated proteins. This approach was also used to test hypotheses about sensing of Ci limitation in cyanobacteria. The transcriptional response of Synechococcus sp. to severe Ci limitation occurs rapidly, being maximal within 30 to 60 min, and three distinct temporal responses were detected: (a). a rapid, transient induction for genes encoding carboxysome-associated proteins (ccmKLMNO, rbcLS, and icfA) and the transcriptional regulator, cmpR; (b). a slow sustained induction of psbAII; and (c). a rapid sustained induction of genes encoding the inducible Ci transporters cmpABCD, sbtA, and ndhF3-D3-chpY. The Ci-responsive transcripts investigated had half-lives of 15 min or less and were equally stable at high and low Ci. Through the use of a range of physiological conditions (light and Ci levels) and inhibitors such as 3-(3,4-dichlorophenyl)-1,1dimethylurea, glycolaldehyde, dithiothreitol, and ethoxyzolamide, we found that no strict correlation exists between expression of genes known to be induced under redox stress, such as psbAII, and the expression of the Ci-responsive CCM genes. We argue that redox stress, such as that which occurs under high-light stress, is unlikely to be a primary signal for sensing of Ci limitation in cyanobacteria. We discuss the data in relation to current theories of CO2 sensing in cyanobacteria.
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PMID:Inorganic carbon limitation induces transcripts encoding components of the CO(2)-concentrating mechanism in Synechococcus sp. PCC7942 through a redox-independent pathway. 1464 30

Rhinoviruses are major causes of morbidity in patients with respiratory diseases; however, their modes of transmission are controversial. We investigated detection of airborne rhinovirus in office environments by polymerase chain reaction technology and related detection to outdoor air supply rates. We sampled air from 9 A.M. to 5 P.M. each workday, with each sample run for 1 work week. We directly extracted RNA from the filters for nested reverse transcriptase-polymerase chain reaction analysis of rhinovirus. Nasal lavage samples from building occupants with upper respiratory infections were also collected. Indoor carbon dioxide (CO2 concentrations were recorded every 10 minutes as a surrogate for outdoor air supply. To increase the range of CO2 concentrations, we adjusted the outdoor air supply rates every 3 months. Generalized additive models demonstrated an association between the probability of detecting airborne rhinovirus and a weekly average CO2 concentration greater than approximately 100 ppm, after controlling for covariates. In addition, one rhinovirus from a nasal lavage contained an identical nucleic acid sequence similar to that in the building air collected during the same week. These results suggest that occupants in buildings with low outdoor air supply may have an increased risk of exposure to infectious droplet nuclei emanating from a fellow building occupant.
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PMID:Detection of airborne rhinovirus and its relation to outdoor air supply in office environments. 1516 7

Hypoxia causes dysfunction of excitatory and inhibitory neurotransmission, often resulting in encephalopathy, seizures or myoclonus. We evaluated the effects of hypoxia on GABAA receptor (GABAAR) function and expression in an in vitro model of neuronal hypoxia. NT2-N cells, derived from the human NT2 teratocarcinoma cell line, were exposed to < or =1% O2 for 8 h and then used immediately for experiments or allowed to recover under normoxic conditions (95% air/5% CO2) for 24, 48 or 96 h. Hypoxic treatment did not cause obvious morphological changes or cell death. In whole-cell patch-clamp recordings, the GABA current EC50 was unchanged, however, maximal GABA-evoked currents changed in a biphasic manner. Maximal GABA currents were significantly increased immediately after hypoxia, but were significantly reduced after 48 h normoxic recovery, and then returned to baseline after 96 h recovery. Maximal potentiation of 10 microM GABA currents by diazepam was increased 48 h after hypoxia, but potentiation by zolpidem was decreased. Barbiturate enhancement and zinc inhibition of GABA currents were unchanged. Semiquantitative reverse transcriptase (RT)-PCR showed decreased alpha1, alpha5, beta2 and gamma2 subunit mRNA after hypoxia. Hypoxic exposure altered GABAAR physiology and subunit mRNA expression, which may correlate with symptoms observed after hypoxia in vivo.
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PMID:Hypoxia alters GABAA receptor function and subunit expression in NT2-N neurons. 1497 87

Shoalwater Bay military training area (SWBTA), 2,713 km2 of land located 50-80 km north of Rockhampton, Queensland, Australia, is used by Australian and allied forces for training purposes. Between March 1998 and February 2000, monthly collections of mosquitoes at 15 sites were conducted using carbon dioxide-baited traps to study the seasonal occurrence of mosquitoes and Ross River virus (RRV) and Barmah Forest virus (BFV) in mosquitoes. A total of 72,616 mosquitoes, comprising 3,897 pools were collected, and 2,428 pools were tested using a reverse transcriptase-polymerase chain reaction. A total of 15 pools of mosquitoes were positive for virus, 10 RRV and five BFV. Blood meals from an additional 763 mosquitoes were tested by a gel diffusion assay, and the majority (96%) of those identified were from kangaroo, which was the most common mammal in the study area. The results indicate that Culex annulirostris Skuse and Ochlerotatus vigilax (Skuse) are the main vectors of RRV at SWBTA.
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PMID:Occurrence of Ross River virus and Barmah Forest virus in mosquitoes at Shoalwater Bay military training area, Queensland, Australia. 1498 54

A field-scale experiment with a complete randomized block design was performed to study the degradation of buried oil on a shoreline over a period of almost 1 year. The following four treatments were examined in three replicate blocks: two levels of fertilizer treatment of oil-treated plots, one receiving a weekly application of liquid fertilizer and the other treated with a slow-release fertilizer; and two controls, one not treated with oil and the other treated with oil but not with fertilizer. Oil degradation was monitored by measuring carbon dioxide evolution and by chemical analysis of the oil. Buried oil was degraded to a significantly greater extent in fertilized plots, but no differences in oil chemistry were observed between the two different fertilizer treatments, although carbon dioxide production was significantly higher in the oil-treated plots that were treated with slow-release fertilizer during the first 14 days of the experiment. Bacterial communities present in the beach sediments were profiled by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene fragments and 16S rRNA amplified by reverse transcriptase PCR. Similarities between the DGGE profiles were calculated, and similarity matrices were subjected to statistical analysis. These analyses showed that although significant hydrocarbon degradation occurred both in plots treated with oil alone and in the plots treated with oil and liquid fertilizer, the bacterial community structure in these plots was, in general, not significantly different from that in the control plots that were not treated with oil and did not change over time. In contrast, the bacterial community structure in the plots treated with oil and slow-release fertilizer changed rapidly, and there were significant differences over time, as well as between blocks and even within plots. The differences were probably related to the higher concentrations of nutrients measured in interstitial water from the plots treated with slow-release fertilizer. Bacteria with 16S rRNA sequences closely related (>99.7% identity) to Alcanivorax borkumensis and Pseudomonas stutzeri sequences dominated during the initial phase of oil degradation in the plots treated with slow-release fertilizer. Field data were compared to the results of previous laboratory microcosm experiments, which revealed significant differences.
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PMID:Bacterial community dynamics and hydrocarbon degradation during a field-scale evaluation of bioremediation on a mudflat beach contaminated with buried oil. 1512 9


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