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 effects of 3-methylcholanthrene (3-MC) and pyridine on rat renal cytochrome P450 (CYP) 1A1 and 1A2 mRNA expression have been examined by Northern-blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR), followed by Southern-blot analysis of the PCR products. Northern-blot hybridization and RT-PCR analyses of kidney poly(A)+ RNA revealed that 3-MC treatment produced a time-dependent increase in the renal CYP1A1 and 1A2 mRNA levels, with CYP1A1 and 1A2 mRNA levels maximally increased at 24 and 18 hr, respectively, after treatment. These data were confirmed via RT-PCR analysis using a subsaturating level of cDNA template and by Southern-blot analysis of the PCR products. This approach served as the foundation for examining the effects of pyridine on CYP1A1 and 1A2 expression in renal tissue. RT-PCR analysis of renal CYP1A1 and 1A2 poly(A)+ RNA levels after treatment with pyridine (200 mg/kg/day for 3 consecutive days) revealed that CYP1A1 mRNA levels were maximally elevated approximately 10-fold after pyridine treatment for 2 consecutive days, whereas CYP1A2 mRNA levels were maximally elevated approximately 3-fold at 24 hr after treatment. The mRNA levels of glyceraldehyde 3-phosphate dehydrogenase, which served as an internal control, remained constant after 3-MC or pyridine treatment. These results show that expression of CYP1A1 and 1A2 mRNAs is enhanced in renal tissue after exposure to 3-MC or pyridine, and that constitutive expression of CYP1A1 seems to be greater than that of CYP1A2 in renal tissue.
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PMID:3-Methylcholanthrene and pyridine effects on CYP1A1 and CYP1A2 expression in rat renal tissue. 749 48

In view of the potential role of the cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) biotransformation enzymes in the metabolism of protoxicants in the circulatory system, we examined CYP and mEH expression in several primary cultures of human umbilical vein endothelial cells (HUVEC), each established from a different individual. Total RNA was isolated from untreated cells and cells 72 hr after exposure to dimethyl sulfoxide (DMSO), Arochlor 1254 (PCB), and beta-naphthoflavone (beta NF). Specific mRNA transcripts were examined by Northern blotting and reverse transcriptase-coupled polymerase chain reaction (RT/PCR) analyses. CYP2E1, CYP3A, and CYP1A2 mRNAs were not detectable in any of the cultures by Northern blot analysis with radiolabeled oligomer probes; however, CYP1A1 mRNA was detected using this procedure in HUVEC cultures exposed to beta NF for 72 hr. Using RT/PCR, constitutive levels of CYP1A1, CYP1A2, CYP2E1, and CYP3A gene expression in HUVEC cultures were evident; however, constitutive CYP2B6 mRNA was not detected. Constitutive CYP1A2 transcript levels were detected in four of six HUVEC cultures, but levels varied between individual cultures. CYP1A2 mRNA levels were also increased in HUVEC cultures exposed to PCB and beta NF. No increases in the levels of CYP2E1 and CYP3A mRNAs were observed in HUVEC cells subsequent to PCB or beta NF exposures. Constitutive CYP2E1 transcript levels were present in all HUVEC cultures examined and varied among individuals. All HUVEC cultures examined for mEH activity exhibited constitutive levels of mEH which varied 40% between individual cultures and produced on average, 1.51 pmol benzo[a]pyrene 4,5-dihydrodiol per milligram protein per minute of reaction. Thus, these results demonstrate that human endothelial cells express CYP and mEH gene products and suggest that these enzymes may play important roles in determining metabolic fates for circulating protoxicants.
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PMID:Expression of cytochrome P450s and microsomal epoxide hydrolase in primary cultures of human umbilical vein endothelial cells. 750 67

A sensitive assay of multiple mRNAs by reverse transcriptase-polymerase chain reaction was adopted to study the hormonally regulated expression of steroidogenic enzymes in primary rat granulosa cells in culture. As little as 15-60 ng total RNA prepared from cultured cells were reverse transcribed in the presence of pd(T)6, and polymerase chain reaction was conducted in the presence of specific oligonucleotide pairs designed to identify cDNAs of steroidogenic enzymes. In combination with Northern blot analysis of cholesterol side-chain cleavage cytochrome P450 (P450scc) message, it is shown that a novel protein kinase inhibitor, tyrphostin AG18, arrests the FSH-induced accumulation of P450scc mRNA. This inhibition is dose dependent (IC50, 15 microM) and reversible. The addition of 80 microM AG18 to cells containing high levels of P450scc mRNA caused a rapid decline of the cytochrome message (t 1/2, 5 h), similar to the effect of 30 micrograms/ml alpha-amanitin. However, concomitant addition of the two drugs did not accelerate the mRNA degradation process, suggesting that AG18 does not affect message stabilization. Tyrphostin AG18 did not affect mRNA species that are not FSH inducible, such as the ribosomal protein L19, or the constitutively expressed low levels of steroid 5 alpha-reductase mRNA. Moreover, even the extremely high levels of P450scc mRNA in granulosa-lutein cells, being cAMP independent and terminally differentiated a few hours after LH surge, were not affected by the addition of AG18 in culture. In contrast, two additional key and FSH-inducible steroidogenic enzymes, i.e. aromatase cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase-I, were inhibited by AG18 at their mRNA levels. These results suggest that an as yet undetermined tyrosine kinase pathway is involved in the cAMP-dependent signal transduction pathway of FSH action, so that the presence of AG18 does not allow FSH induction of gene expression to occur.
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PMID:Tyrosine kinase inhibitor AG18 arrests follicle-stimulating hormone-induced granulosa cell differentiation: use of reverse transcriptase-polymerase chain reaction assay for multiple messenger ribonucleic acids. 751 96

Epidemiological evidence suggests that the presence of human papillomaviruses (HPV), when combined with smoking behaviors, considerably enhances the risk of developing oral, cervical, vulvar, and/or anal carcinomas. It is well established that the cytochrome P450 (CYP), microsomal epoxide hydrolase (mEH), and other biotransformation enzymes are important modulators of the bioactivation and detoxification of many environmental chemicals, including constituents of tobacco smoke such as certain nitrosamines and polycyclic aromatic hydrocarbons (PAH). Since there is little information regarding oral and cervical epithelial-specific expression of these genes, established primary and HPV-immortalized oral and cervical epithelial cell lines were analyzed for morphology, mRNA and protein expression patterns of specific CYPs and mEH. Primary human oral and cervical epithelial cells were immortalized using retroviral infection with HPV-16 E6/E7 genes. Primary human keratinocyte cells were immortalized by transfection of HPV-18 and made tumorigenic with nitrosomethylurea treatment. Expression profiles for mEH, CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP3A, and CYP2E1 were evaluated in these cultures in the presence or absence of a PAH inducer, using reverse transcriptase-coupled polymerase chain reaction analysis. mEH gene expression was evident in all cultures, while CYP2A6 mRNA was not detected in any of the cell lines, regardless of culture conditions. CYP2E1 mRNA expression was greatest in the oral epithelial cultures and detectable in all other epithelial cultures except for the HPV-18 immortalized keratinocyte cell line. Elevated levels of CYP2D6 mRNA existed in both oral epithelial cell lines and the HPV-16 immortalized cervical epithelial cells when compared to the other cell lines examined. CYP1A1 and CYP1A2 mRNAs were detected in all the cells and several cultures were inducible by PAH exposure. To corroborate the RT/PCR data, Western immunoblotting experiments were conducted on selected samples. Using these methods, CYP1A1 and CYP2E1 proteins were detected in primary and HPV-immortalized oral and cervical epithelial cultures. These data indicate that both primary and HPV immortalized cells appear to express certain biotransformation enzymes necessary for the activation of tobacco-specific nitrosamines and PAHs. Although the overall impact of HPV gene infection on expression of these systems remains to be fully elucidated, as in vitro system is characterized which should prove useful in examining interactive mechanisms of HPV with xenobiotic activation in the etiology of squamous cell carcinomas.
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PMID:Expression of cytochrome P450 and microsomal epoxide hydrolase in cervical and oral epithelial cells immortalized by human papillomavirus type 16 E6/E7 genes. 761 6

Most of the cytochrome P450 (CYP) genes are expressed in an uneven, zonated pattern in the liver. Factors regulating this regionally restricted expression are not well known. In this study we have analysed cell lysates obtained from opposite zones of rat liver by infusing digitonin to the perfused liver to study the zonation of CYP1A1 and CYP1A2 induction. 3-Methylcholanthrene induced CYP1A1 protein in perivenous cells, while a low dose of beta-naphthoflavone caused periportal induction. Analysis of CYP1A1 mRNA from cell lysates by reverse transcriptase-coupled polymerase chain reaction (RT-PCR) and in situ hybridization experiments both demonstrated that this inducer-specific differently localized effect occurred at the pretranslational level. A corresponding difference in the regional pattern of CYP1A2 induction was seen: induction by beta-naphthoflavone reversed the constitutive perivenous pattern into a periportal CYP1A2 mRNA pattern while induction after 3-methylcholanthrene treatment was more panacinar. Attempts to identify the regiospecific factors involved were made by comparing the in vitro induction of CYP1A1 by beta-naphthoflavone and 3-methylcholanthrene in hepatocytes isolated from the periportal and perivenous region. However, after isolation, induction seemed to be independent of the source of the cells. Our results demonstrate the existence in the liver of regionally acting factors that mediate the induction of CYP1A1 and 1A2 in a local and inducer-specific fashion. These factors could be Ah receptor associated binding proteins operating in vivo, but no longer in isolated cells.
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PMID:Pretranslational induction of cytochrome P4501A enzymes by beta-naphthoflavone and 3-methylcholanthrene occurs in different liver zones. 781

Exposures of cultured whole rat conceptuses during organogenesis to 3-methylcholanthrene (MC; 0.025-25 microM), 5,6-benzoflavone (BNF; 5-100 microM) or benz[a]anthracene (BA; 5-100 microM) were effected by placement of each of these "MC-type" inducing agents in the culture medium at the time of explantation on day 9.5 of gestation. Conceptuses were then cultured for 48 hr and evaluated on day 11.5 for increased expression of inducible conceptal cytochrome P450 (P450). The three agents each elicited concentration-dependent increases in 7,8-benzoflavone (ANF)-inhibitable ethoxyresorufin O-deethylase (EROD) activities and increased P4501A1 mRNA as detected by primer-specific reverse transcriptase-polymerase chain reaction (RT-PCR) in cell-free preparations of the treated, cultured conceptuses. At effective inducing concentrations, dysmorphogenic or other embryotoxic effects were not detectable. At 20 microM concentrations, the three agents exhibited roughly equal induction that was approximately equivalent in magnitude (6- to 13-fold) to that achieved previously with exposures to MC in utero. Additions to the culture medium of 2.5 to 10 microM concentrations of dexamethasone (DEX) did not alter significantly the magnitude of MC-elicited induction in vitro. Repeated full-length sequencing of an RT-PCR-amplified cDNA revealed a coding region sequence identical to that reported for the P4501A1 sequence from adult rat liver. The results provide a basis for investigations, in the absence of maternal influences, of the regulation of mammalian conceptal P4501A1 in intact tissues during organogenesis, a gestational period critical in terms of the dysmorphogenic and other embryotoxic effects of foreign organic chemicals. The results are also pertinent to studies of embryotoxicity, particularly to the transplacental carcinogenicity, mutagenicity and dysmorphogenicity of P4501A1 substrates.
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PMID:Induction in vitro and complete coding region sequence of cytochrome P4501A1 cDNA from cultured whole rat conceptuses during early organogenesis. 798 Jun 50

Expression of human cytochrome P450 (CYP) genes in human adult and fetal liver were studied using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. In adult liver mRNA of CYPs 1A1, 1A2, 2A6/2A7, 2B6/2B7, 2C8-19, 2D6, 2E1, 3A3/3A4 and 3A7 were detected while CYPs 2F1 and 4B1 were absent. In fetal liver mRNA of CYPs 2C8, 2D6, 3A3/3A4 and 3A7 were found but all other forms studied were undetectable. The results provide a comprehensive qualitative picture of the expression of CYP genes in families CYP1 through CYP4 in human adult and fetal liver.
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PMID:Expression of xenobiotic-metabolizing cytochrome P450 forms in human adult and fetal liver. 804 31

Accurate human risk assessment requires sensitive methods to evaluate dose-response relationships, especially following low level exposures. We have developed a reverse transcriptase polymerase chain reaction (RT-PCR) method to quantitative cytochrome P450-1A1 (CYP1A1) mRNA levels in human blood lymphocytes. Many polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene, and chlorinated PAH such as polychlorinated dibenzodioxins, dibenzofurans and biphenyls induce CYP1A1 expression through activation of an endogenous protein, the Ah receptor. Using a quantitative competitive RT-PCR method that included a synthetic internal standard we determined copy numbers of CYP1A1 mRNA in resting as well as mitogen-stimulated human blood lymphocytes. In mitogen-stimulated human blood lymphocytes assay variation was approximately 10% for measurement of this low expression gene and mRNA levels correlated well with ethoxyresorufin-O-deethylase (EROD) activity. The expression of mRNA was induced 20-fold upon culturing human lymphocytes with 10 nM TCDD. In nonstimulated, uninduced lymphocytes CYP1A1 levels are extremely low (1000 copies mRNA/10(4) cells) and cannot be measured by EROD activity. Studies of CYP1A1 mRNA expression in chemically-exposed populations are in progress.
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PMID:CYP1A1 mRNA levels as a human exposure biomarker: use of quantitative polymerase chain reaction to measure CYP1A1 expression in human peripheral blood lymphocytes. 822 45

The purpose of the present experiments was to examine dose-response relationships for induction of hepatic mRNA following a single administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats. The induction of cytochrome P450-1A1 (CYP1A1) mRNA is compared to other "dioxin-responsive" genes including UDP-glucuronosyltransferase I, plasminogen activator inhibitor 2, and transforming growth factor alpha using a sensitive reverse transcriptase-polymerase chain reaction-based method. Sample-to-sample variability in amplification is a concern in using polymerase chain reaction to quantitate biological responses. However, in the present study recombinant RNA templates were synthesized to use as internal standards in both the reverse transcription and the polymerase chain reaction steps. The induction of CYP1A1 mRNA was extremely sensitive to TCDD treatment with increases observed at doses as low as 1 ng/kg body weight. The induction of CYP1A1 mRNA correlated highly (R2 > 0.90) with an increase in ethoxyresorufin-o-deethylase activity, a CYP1A1-associated enzyme activity. However, induction of CYP1A1 mRNA levels was detected at lower TCDD doses than was ethoxyresorufin-o-deethylase activity, reflecting the greater sensitivity of the reverse transcription-polymerase chain reaction approach to detect transcriptional activation of the CYP1A1 gene. UDP-glucuronosyltransferase I mRNA was increased over control (5-fold) but required 1000-times higher TCDD doses (1 microgram/kg) to result in a significant increase than did CYP1A1. Plasminogen activator inhibitor 2 and transforming growth factor alpha mRNA, both previously shown to be induced by TCDD in human keratinocytes, were not increased in rat liver. Hence, these studies reaffirm that TCDD acts through classical receptor mechanisms with gene-to-gene differences in responsiveness. The reverse transcription-polymerase chain reaction method developed to measure mRNA for dioxin-responsive genes in rat liver will allow for measuring multigene and tissue responses to TCDD and other xenobiotics with high sensitivity, reproducibility, and adaptability and should increase our understanding of various dose-response relationships.
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PMID:Dioxin-responsive genes: examination of dose-response relationships using quantitative reverse transcriptase-polymerase chain reaction. 826 64

Most carcinogens are bioactivated by cytochrome P450s (CYPs) and these enzymes within target cells are closely related to susceptibility to cancer. Since extrahepatic CYPs occur typically at much lower levels, the existence and the role of CYP in extrahepatic tissues have been difficult to assess. In this study, we modified the reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate the relative quantities of CYP 2E1 mRNA in human endometrium. Total RNA from human endometrium was reverse-transcribed and co-amplified by PCR in the same tube containing both primer pairs of CYP 2E1 and beta-actin. The CYP 2E1 and beta-actin PCR products were 298 and 600 bp, respectively. The restriction enzyme MboI digested these two products to the predicted size for DNA fragments, demonstrating that both PCR products were specific and CYP 2E1 mRNA exists in human endometrium. CYP 1A1 mRNA was also examined, but could not be detected clearly. Adding [alpha-32P]dCTP to the reaction mixture made it possible to quantify the relative yield of the CYP 2E1 PCR product in comparison with the beta-actin product. The ratio of the yield of the CYP 2E1 PCR product to the beta-actin PCR product could be calculated at a point of 25 cycles of amplification. This ratio and serum estradiol levels were correlated positively (r = 0.654; p < 0.05), but no relationship to serum progesterone levels was observed.
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PMID:Positive correlation between cytochrome P450 2E1 mRNA level and serum estradiol level in human uterine endometrium. 826 3


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