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)

We here presented evidence that a 94-kDa glucose-regulated protein (GRP94) was associated with hepatitis B viral (HBV) polymerase in the human liver cell HepG2 and this association could be applied even in Escherichia coli. We investigated the role of GRP94 in the expression and stabilization of HBV polymerase in Escherichia coli by coexpression of the two proteins. The affinity column-purified glutathione S-transferase-tagged HBV polymerase (GST-P, 130 kDa) showed a proper molecular size and reverse transcriptase activity on several exogenous templates and was sensitive to specific inhibitors. The GST-P was associated with the maltose-binding protein-tagged GRP94 (MBP-GRP94, 130 kDa) using analyses by an affinity chromatography, native gel electrophoresis and glycerol gradient centrifugation. However, nondenaturing and partially denaturing activity gel analyses showed two active bands of approximately 260 kDa and approximately 130 kDa, respectively. Furthermore, in the presence of the encapsidation signal RNA template (HBV epsilon RNA), the approximately 260-kDa active band was gradually converted to approximately 130 kDa, which implies that HBV polymerase was dissociated from the chaperone GRP94 and bound preferentially to the HBV epsilon RNA. These results suggested that the chaperone GRP94 was necessary for the stabilization and production of HBV polymerase as an active form.
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PMID:Expression of stable hepatitis B viral polymerase associated with GRP94 in E. coli. 1096 39

Schistosoma mansoni p14 gene encodes an eggshell precursor that is expressed only in vitelline cells of mature female worms in response to a male stimulus. The upstream region of the p14 gene contains several potential cis-acting regulatory sequences. We used the upstream region of the p14 gene as bait in a yeast-one-hybrid screen of a S. mansoni cDNA library to identify interacting proteins. We report the identification and characterization of a cDNA (S. mansoni PUR-alpha (SmPUR-alpha)) encoding a protein homologous to single-stranded DNA transcription activator PUR-alpha, that binds to the p14 upstream region and activates transcription of the HIS3 reporter gene in yeast. SmPUR-alpha has a predicted molecular mass of 30 kDa and shares an overall homology of 63% with mammalian PUR-alpha. The DNA binding domain of SmPUR-alpha is highly conserved. We show by gel shift assays that GST-SmPUR-alpha binds to oligonucleotides comprising the p14 upstream region. SmPUR-alpha binds preferentially to single-stranded DNA and also binds RNA. Unlike the mammalian homologue, SmPUR-alpha exhibits little specificity for the PUR element GGn, but shows strong preference for a sequence containing alternating pyrimidines. Our data support that SmPUR-alpha is a single-copy gene and through reverse transcriptase-polymerase chain reaction and in situ hybridization, we show that SmPUR-alpha is constitutively transcribed in many cell types and thus likely plays a role as a general transcription activator in schistosomes.
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PMID:Identification and functional characterization of a member of the PUR-alpha family from Schistosoma mansoni. 1107 Dec 90

Smad proteins are essential intracellular signal transducers of the transforming growth factor-beta (TGF-beta) superfamily. The TGF-beta superfamily signals through phosphorylation and activation of R-Smad proteins, receptor-regulated Smads, by heteromeric complexes of ligand-specific type I and type II serine/threonine kinase receptors. R-Smads receive a signal from the activated receptor complex and transmit it to the nucleus. A cDNA was isolated that encodes a 649-amino acid protein found to be homologous to members of R-Smad subfamily with highest homology scored to clawed African frog and human Smad2. The Schistosoma mansoni homologue (SmSmad2) was overexpressed in bacteria as a Sj26-GST fusion protein and used to raise specific antibodies. The IgG fraction of the immunized rabbit serum identified 70- and 72-kDa protein bands in Western analysis of schistosome extracts. Treatment with alkaline phosphatase removed the 72-kDa band, which indicates that this band represents the phosphorylated form of schistosome Smad2. SmSmad2 was localized in the subtegument, parenchymal cells, and sex organs in both male and female worm cryosections. Similar results were also obtained from the analysis of the Smad2 mRNA distribution pattern revealed by in situ hybridization of adult worm pair paraffin sections. SmSmad2 mRNA levels were determined by reverse transcriptase-polymerase chain reaction in different mammalian host developmental stages and found to be constitutively expressed. SmSmad2 was also found to interact with a previously identified SmTbetaR-I, a serine/threonine type I kinase receptor. Furthermore, SmSmad2 was shown to undergo phosphorylation by constitutively active forms of SmTbetaR-I in vitro. In addition, SmSmad2 localized in the nuclei of mink lung epithelial cells upon treatment with TGF-beta(1). These data indicate that the SmSmad2 responds to the TGF-beta signals by interaction with receptor I, which phosphorylates it, whereupon it translocates into the nucleus presumably to regulate target gene transcription and consequently elicit a specific TGF-beta effect.
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PMID:Identification and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor-beta signal transducer. 1115 51

Differential effects of partial hepatectomy (PH) and carbon tetrachloride (CCl(4)) administration on induction of glutathione S-transferase placental form (GST-P)-positive foci were investigated in a model for detection of initiation activity. Firstly, we surveyed cell proliferation kinetics and fluctuation in cytochrome P450 (CYP) mRNA levels by means of relative-quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and CYP 2E1 apoprotein amount by immunoblotting (experiment I) after PH or CCl(4) administration. Next, to assess the interrelationships among cell proliferation, fluctuation of CYPs after PH or CCl(4) administration and induction of liver cell foci, the non-hepatocarcinogen, 1,2-dimethylhydrazine (DMH) was administered to 7-week-old male F344 rats and initiated populations were selected using the resistant hepatocyte model (experiment II). In experiment I, the values of all CYP isozyme mRNAs after PH or CCl(4) administration were drastically decreased at the 12-h time point. From 72 h, mRNAs for all CYP isozymes began increasing, with complete recovery after 7 days. The CYP 2E1 apoprotein content in the PH group fluctuated weakly, whereas in the CCl(4) group it had decreased rapidly after 12 h and was still low at the 48 h point. In experiment II, induction of GST-P-positive foci was related to cell kinetics in the PH group, with about a 6-h time lag between time for carcinogen administration giving greatest induction of GST-P-positive foci and peaks in bromodeoxyuridine (BrdU) labeling, presumably due to the necessity for bioactivation of DMH. With CCl(4) administration, induction of foci appeared dependent on the recovery of CYP 2E1. In conclusion, PH was able to induce cell proliferation with maintenance of CYP 2E1, therefore being advantageous for induction of liver cell foci in models to detect initiation activity.
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PMID:Differential effects of partial hepatectomy and carbon tetrachloride administration on induction of liver cell foci in a model for detection of initiation activity. 1167 51

Genome sequencing of C. trachomatis serovar D revealed the presence of three putative open reading frames (ORFs), CT145 (Pkn1), CT673 (Pkn5), and CT301 (PknD), encoding eukaryote-like serine/threonine kinases (Ser/Thr kinases). Two of these putative kinase genes, CT145 and CT301, were PCR amplified from serovar L2, cloned, and sequenced. Predicted translation products of the ORFs showed the presence of conserved kinase motifs at the N terminus of the proteins. CT145 and CT301 (encoding Pkn1 and PknD, respectively) were expressed in Escherichia coli as GST fusion proteins. In vitro kinase assays with Escherichia coli-derived glutathione S-transferase fusion proteins showed autophosphorylation of Pkn1 and PknD, indicating that they are functional kinases. Gene expression analysis of these kinase genes in Chlamydia by reverse transcriptase PCR indicated expression of these kinases at the early mid phase of the developmental cycle. Immunoprecipitated native chlamydial Pkn1 and PknD proteins also showed autophosphorylation in an in vitro kinase assay. Phosphoamino acid analysis by thin-layer chromatography confirmed that Pkn1 and PknD are phosphorylated on both serine and threonine residues. Interaction of Pkn1 and PknD with each other as well as interaction of Pkn1 with inclusion membrane protein G (IncG) was demonstrated by using a bacterial two-hybrid system. These interactions were further suggested by phosphorylation of the proteins in in vitro kinase assays. This report is the first description of the existence of functional Ser/Thr kinases in Chlamydia. The results of these findings should lead to a better understanding of how Chlamydia interact and interfere with host signaling pathways, since kinases represent potential mediators of the intimate host-pathogen interactions that are essential to the intracellular life cycle of Chlamydia.
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PMID:Identification of two eukaryote-like serine/threonine kinases encoded by Chlamydia trachomatis serovar L2 and characterization of interacting partners of Pkn1. 1450 Apr 99

We tried to identify a novel marker characteristic for rat hepatocellular preneoplastic and neoplastic lesions, undetectable by well established cytochemical markers. Glutathione S-transferase placental (GST-P)-negative hepatocellular altered foci (HAF), hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC) were generated by two initiation-promotion models with N-nitrosodiethylamine (NDEN) and peroxisome proliferators, Wy-14,643 and clofibrate. Total RNAs isolated from laser-microdissected GST-P-negative HAF (amphophilic cell foci) and adjacent normal tissues were applied to microarray analysis. As a result, five up-regulated genes were identified, and further detailed examinations of the gene demonstrating most fluctuation, ie, that for alpha(2)-macroglobulin (alpha(2)M) were performed. In reverse transcriptase-polymerase chain reaction, alpha(2)M mRNA was overexpressed not only in amphophilic GST-P-negative HAF but also in amphophilic GST-P-negative HCA and HCC. In situ hybridization showed accumulation of alpha(2)M mRNA to be evenly distributed within GST-P-negative HAF (predominantly amphophilic cell foci). Distinctive immunohistochemical staining for alpha(2)M could be consistently demonstrated in GST-P-negative HAF, HCA, and HCC induced not only by peroxisome proliferators but also N-nitrosodiethylamine alone. Thus our findings suggest that alpha(2)M is an important novel cytochemical marker to identify hepatocellular preneoplastic and neoplastic lesions, particularly amphophilic cell foci, undetectable by established cytochemical markers and is tightly linked to rat hepatocarcinogenesis.
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PMID:alpha(2)-Macroglobulin: a novel cytochemical marker characterizing preneoplastic and neoplastic rat liver lesions negative for hitherto established cytochemical markers. 1550 19

Telomerase is a cellular reverse transcriptase that elongates the single-stranded chromosome ends and oligonucleotides in vivo and in vitro. In Saccharomyces cerevisiae, Est2p (telomerase catalytic subunit) and Tlc1 (telomerase RNA template subunit) constitute the telomerase core complex. We co-overexpressed GST (glutathione S-transferase)-Est2p and Tlc1 in S. cerevisiae, and reconstituted the telomerase activity. The GST-Est2p-Tlc1 complex was partially purified by ammonium sulphate fractionation and affinity chromatography on glutathione beads, and the partially purified telomerase did not contain the other two subunits of the telomerase holoenzyme, Est1p and Est3p. The purified recombinant GST-Est2p-Tlc1 telomerase core complex could specifically add nucleotides on to the single-stranded TG(1-3) primer in a processive manner, but could not translocate to synthesize more than one telomeric repeat. The purified telomerase core complex exhibited different activities when primers were paired with the Tlc1 template at different positions. The procedure of reconstitution and purification of telomerase core enzyme that we have developed now allows for further mechanistic studies of the functions of other subunits of the telomerase holoenzyme as well as other telomerase regulation proteins.
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PMID:Characterization of recombinant Saccharomyces cerevisiae telomerase core enzyme purified from yeast. 1581 5

Lung is a target organ for the toxicity of inhalated compounds. The respiratory tract is frequently exposed to elevated concentrations of these compounds and become the primary target site for toxicity. Occupational, accidental or prolonged exposure to a great variety of chemicals may result in acute or delayed injury to cells of the respiratory tract. Nevertheless, lung has a significant capability of biotransforming such compounds with the aim of reducing its potential toxicity. In some instances, the biotransformation of a given compound can result in the generation of more reactive, and frequently more toxic, metabolites. Indeed, lung tissue is known to activate pro-carcinogens (i.e. polycyclic aromatic hydrocarbons or N-nitrosamines) into more reactive intermediates that easily form DNA adducts. Lungs express several enzymes involved in the metabolising of xenobiotics. Among them, cytochrome P450 enzymes are major players in the oxidative metabolism as well metabolic bioactivation of many organic toxicants, including pro-carcinogens. Xenobiotic-metabolising P450 enzymes are expressed in bronchial and bronchiolar epithelium, Clara cells, type II pneumocytes, and alveolar macrophages Individual CYP isoforms have different patterns of localisation within pulmonary tissue. With the aid of sensitive techniques (i.e. reverse transcriptase-polymerase chain reaction, RT-PCR) it has become possible to detect CYP1A1, CYP1B1, CYP2A6, CYP2B6, CYP2E1 and CYP3A5 mRNAs in lung cells. Less conclusive results have been obtained concerning CYP2Cs, CYP2D6 and CYP3A4. CYP3A5 protein appears to be widely present in all lung samples and is localised in the ciliated and mucous cells of the bronchial wall, bronchial glands, bronchiolar ciliated epithelium and in type I and type II alveolar epithelium. Lung cells also express Phase II enzymes such as epoxide hydrolase, UGT1A (glucuronyl transferase) and GST-P1 (glutathione S-transferase), which largely act as detoxifying enzymes. A key question concerning organ-specific chemical toxicity is whether the actual target has the capacity to activate (or efficiently inactivate) chemicals. Results of several studies indicate that the different xenobiotic-metabolising CYPs, present in the human lung and lung-derived cell lines, likely contribute to in situ activation of pulmonary toxins, among them, pro-carcinogens. Some CYPs, in particular CYP1A, are polymorphic and inducible. Interindividual differences in the expression of these CYPs may explain the different risk of developing lung toxicity (possibly cancer), by agents that require metabolic activation. Few cell lines, principally A549, have been used with variable success as an experimental model for investigating the mechanisms of toxicity. Although RT-PCR analysis has evidenced the presence of the major human pulmonary CYP mRNAs, the measurable P450 specific activities are, however, far below those present in human lungs. Detection of the toxicity elicited by reactive metabolites requires the use of metabolically competent cells; consequently, better performing cells are needed to ensure realistic in vitro prediction of toxicity. Genetic manipulation of lung-derived cells allowing them to re-express key biotransformation enzymes appear to be a promising strategy to improve their functionality and metabolic performance.
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PMID:Metabolism and bioactivation of toxicants in the lung. The in vitro cellular approach. 1609 27

A cDNA encoding an omega-class glutathione S-transferase of the silkmoth, Bombyx mori (bmGSTO), was cloned by reverse transcriptase-polymerase chain reaction. The resulting clone was sequenced and deduced for amino acid sequence, which revealed 40, 40, and 39% identities to omega-class GSTs from human, pig, and mouse, respectively. A recombinant protein (rbmGSTO) was functionally overexpressed in Escherichia coli cells in a soluble form and purified to homogeneity. rbmGSTO was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation. This enzyme was shown to have high affinity for organophosphorus insecticide and was present abundantly in silkmoth strain exhibiting fenitrothion resistance. These results indicate that bmGSTO could be involved in the increase in level of insecticide resistance for lepidopteran insects.
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PMID:Biochemical properties of an omega-class glutathione S-transferase of the silkmoth, Bombyx mori. 1902 97

We sought to evaluate the molecular markers involved in breast tumorigenesis in a rat model that mimics many essential elements of human breast cancer. Female Sprague-Dawley rats were divided into two groups. Animals in group 1 were given a single dose of 7,12-dimethylbenz[a]anthracene (DMBA) (20 mg/rat) dissolved in 1 ml of sesame oil by intragastric intubation. Group 2 animals received basal diet and served as control. We analyzed DMBA-induced changes in the expression of CYP isoforms (CYP1A1 and 1B1) involved in DMBA metabolism, markers of oxidative stress (4HNE, HEL, and 8-OHdG), cell survival and proliferation (PCNA, NF-kappaB-p50, NF-kappaB-p65, GST-P, and p53), apoptosis (Bcl-2, Bax, caspases, Apaf-1, cytochrome C, and Fas), invasion (uPA, MMP-2, MMP-9, TIMP-2, and RECK), and angiogenesis (VEGF, VEGF-R1, HIF-1alpha, and PLGF) by immunohistochemical localization, Western blot, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The present study demonstrates increased carcinogen metabolism, oxidative stress, cell proliferation, together with apoptosis evasion, invasion, metastasis, and neovascularization that may confer a selective growth advantage to DMBA-induced mammary tumors. Aberrant expression of multiple molecules in key signaling pathways in Sprague-Dawley rat mammary tumors renders this model as an important tool for monitoring carcinogenic progression and chemointervention.
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PMID:Evaluation of molecular markers in a rat model of mammary carcinogenesis. 1972 28


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