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)

Polycyclic aromatic hydrocarbons (PAHs) and heavy metals are often environmental cocontaminants that could interact to alter PAH carcinogenicity. The heavy metal, arsenite, and the PAH, benzo[k]fluoranthene, were used as prototypes to investigate, in human HepG2 cells, mechanisms whereby the bioactivation of benzo[k]fluoranthene by human CYP1A1 could be diminished by arsenite-mediated decreases in CYP1A1 induction by benzo[k]fluoranthene. To determine whether arsenite down-regulates CYP1A1 transcription, quantitative real-time reverse transcriptase-polymerase chain reaction assays and luciferase reporter gene expression assays were used with HepG2 cells treated with benzo[k]fluoranthene and arsenite, separately and as a mixture. Benzo[k]fluoranthene (0.5 microM) and arsenite (5 microM) markedly decreased benzo[k]fluoranthene-mediated induction of CYP1A1 mRNA by 45%. Plasmids containing the CYP1A1 promoter region (pHu-1A1-FL) were induced 7.4-fold over vehicle by benzo[k]fluoranthene (0.5 microM), whereas arsenite (1, 2.5, or 5 microM) decreased reporter gene expression by 46%, 45%, and 61%, respectively. The plasmid, pHu-1A1-Delta100-FL, lacked xenobiotic response element (XRE) sites at -1061 and -981 and showed greater responsiveness relative to pHu-1A1-FL, by 1.7-fold. Benzo[k]fluoranthene (0.5 microM) and arsenite (1, 2.5, or 5 microM) decreased reporter gene expression by 0%, 27%, and 39%, respectively, relative to expression levels produced by benzo[k]fluoranthene alone. Arsenite is stable for at least 48 h in the HepG2 cell medium with respect to its ability to diminish CYP1A1 benzo[k]fluoranthene induction. Arsenite did not affect benzo[k]fluoranthene induction directly through XRE sites, nor did it affect the stability of CYP1A1 mRNA. Thus, arsenite affects the transcriptional regulation of the benzo[k]fluoranthene-mediated induction of CYP1A1 and could diminish PAH carcinogenicity by decreasing bioactivation by CYP1A1.
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PMID:Mechanisms of arsenite-mediated decreases in benzo[k]fluoranthene-induced human cytochrome P4501A1 levels in HepG2 cells. 1557 48

In a previous study using fusion of the deregulated lactose promoter lacTp* and reporter genes, we suggested that Lactobacillus casei could initiate de novo protein synthesis during intestinal transit. In order to confirm this finding and extend it to other promoters, we adopted a reverse transcriptase quantitative PCR (RT-QPCR) approach combined with a transcriptional fusion system consisting of luciferase genes under the control of four promoters (ccpA, dlt, ldh, and lacT*) from L. casei DN-114 001. Promoter expression was monitored during cell growth, and variable luciferase activities were detected. In 3-day cultures, all the genetically modified strains survived but without exhibiting luciferase activity. Luciferase mRNA levels determined by RT-QPCR analysis (RNA/CFU) were not significant. The cultures were administered to human-microbiota-associated mice, and the feces were collected 6 h later. L. casei promoters lacTp* and ldhp initiated mRNA synthesis during gastrointestinal transit. The promoters, ccpAp and dltp, exhibited no luciferase activity, nor was de novo-synthesized luciferase mRNA detected in the feces. L. casei seems to adapt its physiology to the gastrointestinal tract environment by modulating promoter activities. The approach (fecal transcriptional analysis) described herein may, moreover, be of value in studying gene expression of transiting bacteria in human fecal specimens.
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PMID:Differential activities of four Lactobacillus casei promoters during bacterial transit through the gastrointestinal tracts of human-microbiota-associated mice. 1574 38

CMKLR1 (chemoattractant-like receptor 1) is a G-protein-coupled receptor implicated in cartilage and bone development and is expressed in organs like the parathyroid gland, brain, and lung. The receptor is also expressed in dendritic cells and in macrophages where it acts as a co-receptor for entry of HIV/SIV isolates into human CD4(+) cells. Recently, a protein named "chemerin" (also known as TIG2) was isolated from human inflammatory fluids and hemofiltrate and found to be the endogenous ligand for CMKLR1. We have previously described the genomic organization of the cmklr1 gene and characterized its promoter in mouse neuroblastoma NB4 1A3 cells. In the present study we identify a second transcript, cmklr1b, in mouse microglia BV2 cells. Cmklr1b is transcribed from an alternative promoter with a transcription start site located 6780 bp downstream of the previously identified exon 1 (cmklr1a). The cmklr1b promoter lacks a TATA box but contains two CCAAT boxes in opposite directions. 5' Deletion analysis of the promoter region in BV2 cells using a luciferase reporter gene assay indicates two regions, between 623-755 bp and 56-125 bp upstream of transcription start site, to be important for promoter function. The proximal promoter region includes both CCAAT boxes, and site-directed mutagenesis separately within these elements revealed that only the forward CCAAT element was important for transcription. Although the forward CCAAT element is essential for transcription electrophoretic mobility shift and super-shift assays demonstrated that both CCAAT elements actually bind nuclear proteins from BV2 cells and identified the binding factor as NFY. Real-time reverse transcriptase-PCR experiments of cmklr1b expression in all-trans retinoic acid (ATRA)- stimulated BV2 cells showed strong up-regulation of receptor transcript. Luciferase reporter gene assay of the promoter in ATRA-stimulated BV2 cells confirmed that transcriptional activity of the cmklr1b promoter is increased by ATRA. However, deletion analysis could not identify an ATRA-responsive element within the promoter region suggesting that gene activation is likely to occur through alternative mechanisms. The results emphasise a possible role of cmklr1 in bone modelling.
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PMID:The mouse chemerin receptor gene, mcmklr1, utilizes alternative promoters for transcription and is regulated by all-trans retinoic acid. 1579 32

In order to obtain indicator cell lines that are exquisitely susceptible to human T-lymphotropic virus type 1 (HTLV-1), luciferase gene driven by HTLV-1 long terminal repeat (LTR) was transfected into lymphocytic H9 cells with neo gene, and cell lines were selected by G418. A cell line (H9/K30luc) was found to produce an extremely high level of luciferase only when co-cultured with HTLV-1 producer MT-2 cells. Both in the absence and presence of a reverse transcriptase (RT) inhibitor azidothymidine, H9/K30luc cells generated similarly high luciferase activity upon co-cultivation with MT-2 cells. To develop an equivalent system for human immunodeficiency virus type 1 (HIV-1), H9/NL432 cells, which are stably infected with HIV-1 and producing a low level of the virus-like MT-2 cells for HTLV-1, were generated. Together with the indicator cell line H9/H1luc for HIV-1 already reported, antiviral effects of some agents on HTLV-1 and HIV-1 could be readily and sensitively evaluated by similar methods. In fact, by using our system, an HIV-1 protease inhibitor, saquinavir, was demonstrated to be highly effective against HIV-1 but not against HTLV-1.
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PMID:Establishment of a biological assay system for human retroviral protease activity. 1589 91

Telomerase activity is suppressed in normal human somatic tissues but is activated in cancer cells and immortal cell lines. The reverse transcriptase (RT) subunit human telomerase reverse transcriptase (hTERT) is the key regulator of telomerase activity. The hTERT promoter contains E-box elements and may allow upstream stimulatory factor (USF), a basic helix-loop-helix (bHLH) leucine zipper family proteins, to bind and regulate the expression. In this study, we investigated whether and how USF effect on hTERT. Through luciferase reporter assays, we found that both USF1 and USF2 possess a comparable effect on the inhibition of hTERT expression. Immunoprecipitation (IP) and immunoblotting (IB) analysis reveal that the suppression of hTERT by USF was not through the interaction of USF with c-myc or mad, nor disturbed the cellular protein levels of those. In gel mobility shift and chromatin immunoprecipitation (CHIP) assays, we found that the USF suppression is through direct binding at the E-box site of hTERT promoter and rendering the effect actively. Analysis on clinical normal and tumor tissues reveal that the expression of USF1 and USF2 was lower in the tumor tissues, correlated with hTERT expression and telomerase activity. Taking together, our results demonstrate that USF is a negative transcriptional repressor for hTERT in oral cancer cells. It is possible that USF lose the inhibitory effect on hTERT expression leading to telomerase reactivation and oral carcinogenesis.
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PMID:Upstream stimulatory factor (USF) as a transcriptional suppressor of human telomerase reverse transcriptase (hTERT) in oral cancer cells. 1601 Jun 90

The development of antiviral drug resistance is an important problem in the treatment of human immunodeficiency virus type 1 (HIV-1) infection. Potent antiretroviral therapy is currently used for treatment, and typically consists of at least two reverse transcriptase (RT) inhibitors. We have previously reported that both drugs and drug-resistant RT mutants can increase virus mutation frequencies. To further assess the contributions of nucleoside RT inhibitors (NRTIs), nonnucleoside RT inhibitors (NNRTIs), and drug-resistant RTs to HIV mutagenesis, a new high-throughput assay system was developed. This assay system was designed to specifically detect frameshift mutations in the luciferase gene in a single virus replication cycle. New drug-resistant RTs were identified that significantly altered virus mutation frequencies. Consistent with our previous observations of NRTIs, abacavir, stavudine, and zalcitabine increased HIV-1 mutation frequencies, supporting the general hypothesis that the NRTIs currently used in antiviral drug therapy increase virus mutation frequencies. Interestingly, similar observations were made with NNRTIs. This is the first report to show that NNRTIs can influence virus mutation frequencies. NNRTI combinations, NRTI-NNRTI combinations, and combinations of drug and drug-resistant RTs led to significant changes in the virus mutation frequencies compared to virus replication of drug-resistant virus in the absence of drug or wild-type virus in the presence of drug. This indicates that combinations of RT drugs or drugs and drug-resistant virus created during the evolution of drug resistance can act together to increase HIV-1 mutation frequencies, which would have important implications for drug therapy regimens. Finally, the influence of drug-resistant RT mutants from CRF01_AE viruses on HIV-1 mutation frequencies was analyzed and it was found that only a highly drug resistant RT led to altered virus mutation frequencies. The results further suggest that high-level drug-resistant RT can significantly influence virus mutation frequencies. A structural model that explains the mutation frequency data is discussed.
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PMID:Human immunodeficiency virus mutagenesis during antiviral therapy: impact of drug-resistant reverse transcriptase and nucleoside and nonnucleoside reverse transcriptase inhibitors on human immunodeficiency virus type 1 mutation frequencies. 1614 Jul 80

Telomerase, a ribonucleoprotein enzyme that functions as a reverse transcriptase, is detected exclusively in immortal cells such as germ cells, stem cells and cancer cells. Telomerase activity is present in almost all human cancers. Telomerase activation is considered to be essential to maintain the integrity of the replicating tumor cell and to establish immortality. Based on this concept antiestrogen should initially regulate estrogen-stimulated telomerase but the enzyme would be expected to be constitutive in tamoxifen-resistant tumor cells. We have studied the estrogen regulation of telomerase in T47D:A18 breast cancer cells with a TRAPEZE Telomerase detection kit. Estradiol significantly increased telomerase activity after a 2-day treatment. Telomerase activity induced by estradiol was up to 10-fold higher within 4 days. Antiestrogens 4-hydroxytamoxifen (4-OHT) and ICI 182,780 were inactive alone and significantly blocked estradiol-stimulated increase in telomerase. These effects were correlated with changes in cell replications and changes in the cell cycle. In contrast, 4-OHT resistant T47D:A18 cells (T47D:A18/4-OHT, cultured in 1 microM 4-OHT for 6 months) grew spontaneously and had no changes in the cell cycle with estrogen treatment. The estrogen receptor (ERalpha) was present and still regulated at an estrogen responsive luciferase reporter gene with estrogen despite the fact that progesterone receptor was not increased in response to estradiol in T47D:A18/4-OHT cells. However, telomerase activity was increased about 40-fold in T47D:A18/4-OHT cells and this was not regulated by ICI 182,780. We conclude that the differential regulation of telomerase gene might be an important transition for tamoxifen resistance in T47D:A18 breast cancer cells.
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PMID:Deregulation of estrogen induced telomerase activity in tamoxifen-resistant breast cancer cells. 1621 Dec 43

Matrix metalloproteinases (MMPs), zinc-dependent proteolytic enzymes, play a pivotal role in tumor metastasis by cleavage of extracellular matrix as well as non-matrix substrates. In this study, we examined the influence of DA-125, a new anthracyclin analog, on the gene expression of MMPs (MMP-2, MMP-9 and MT1-MMP), tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2) and in vitro invasiveness of human fibrosarcoma cells. Dose-dependent decreases of MMPs and TIMPs mRNA levels were observed in DA-125-treated HT1080 human fibrosarcoma cells detected by reverse transcriptase-polymerase chain reaction. Gelatin zymography analysis also showed a significant down-regulation of MMP-2 and MMP-9 expression in HT1080 cells treated with DA-125 compared to controls. In addition, DA-125 inhibited the invasion, motility and cell migration, and colony formation of tumor cells. These data, therefore, provide direct evidence for the role of DA-125 as a potential cancer chemotherapeutic agent, which can markedly inhibit the invasive capacity of malignant cells. Further, to clarify the transcriptional regulatory pathway, we primarily investigated the role of nuclear factor-kappaB (NF-kappaB) in the expression of MMPs by DA-125 in HT1080 cells. Electrophoretic mobility shift assay demonstrated that DA-125 modulates the binding activity of NF-kappaB. Using the luciferase reporter gene assay, a dose-dependent down-regulation of NF-kappaB-mediated luciferase expression was also observed. These results suggest that DA-125 down-regulates MMPs expression in HT1080 cells through the NF-kappaB-mediated pathway.
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PMID:Inhibitory effect of DA-125, a new anthracyclin analog antitumor agent, on the invasion of human fibrosarcoma cells by down-regulating the matrix metalloproteinases. 1627 Dec 63

In this paper we describe construction of a luciferase-based vector, pPL2lux, and use of this vector to study gene expression in Listeria monocytogenes. pPL2lux is a derivative of the listerial integration vector pPL2 and harbors a synthetic luxABCDE operon encoding a fatty acid reductase complex (LuxCDE) involved in synthesis of the fatty aldehyde substrate for the bioluminescence reaction catalyzed by the LuxAB luciferase. We constructed pPL2lux derivatives in which the secA and hlyA promoters were translationally fused to luxABCDE and integrated as a single copy into the chromosome of L. monocytogenes EGD-e. Growth experiments revealed that hlyA was expressed predominantly in the stationary phase in LB medium buffered at pH 7.4, whereas secA expression could be detected in the exponential growth phase. Moreover, the correlation between luciferase activity and transcription levels, as determined by reverse transcriptase PCR, was confirmed using conditions known to lead to repression and activation of hemolysin expression (addition of cellobiose and activated charcoal, respectively). Furthermore, hemolysin expression could be monitored in real time during invasion of an intact monolayer of C2Bbe1 (Caco-2-derived) cells. Finally, hemolysin expression could be detected in the livers, spleens, and kidneys of mice 3 days postinfection. These experiments clearly established the effectiveness of pPL2lux as a quantitative reporter system for real-time, noninvasive evaluation of gene expression in L. monocytogenes.
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PMID:Novel luciferase reporter system for in vitro and organ-specific monitoring of differential gene expression in Listeria monocytogenes. 1659 94

The human equilibrative nucleoside transporter 1 gene (hENT1) is the primary nucleoside transporter for cytosine arabinoside (AraC), a deoxycytidine analog used for treatment of acute leukemias and lymphomas. We screened approximately 1.6 kb upstream of the transcription initiation site of hENT1 for single nucleotide polymorphisms (SNPs) that affect gene expression. We identified one SNP at position -706G>C with a frequency of 21% in whites and 5% in African-Americans. In African-Americans, we observed two SNPs at positions -1345C>G and -1050G>A with allele frequencies of 8% and 19%, respectively. TRANSFAC analysis suggested that -1345C>G and -706G>C may alter transcription factor binding sites. Four naturally occurring haplotypes (CGG, CAG, CGC and GAG) were cloned into a luciferase expression plasmid, transfected into Cos-1 cells, and reporter activity measured at 24 and 48 h. Three haplotypes, CAG, CGC and GAG, respectively, showed average expression that was approximately two-fold (P<0.05), 1.4-fold (P<0.05) and 1.1-fold (P>0.05) higher than lowest expression haplotype CGG at 48 h. When reanalysed as single SNPs, the differences in expression were significant for -1345C>G and -1050G>A genotypes, and not for -706G>C. However, the magnitude of difference was reduced, suggesting that no single SNP completely accounts for the expression differences observed at the haplotype level. By real-time quantitative reverse transcriptase-polymerase chain reaction assay, individuals with CGG/CGC haplotypes showed 1.37-fold higher median expression of hENT1 transcript than those with common CGG/CGG haplotypes. Although not statistically significant (P=0.12), this difference is in the direction predicted by the in vitro data. hENT1 promoter region haplotypes may influence gene expression and alter AraC chemosensitivity.
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PMID:Functional single nucleotide polymorphism haplotypes in the human equilibrative nucleoside transporter 1. 1660 62


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