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)

Injury to motor neurons associated with mutant Cu,Zn-superoxide dismutase (SOD1)-related familial amyotrophic lateral sclerosis (FALS) results from a toxic gain-of-function of the enzyme. The mechanisms by which alterations to SOD1 elicit neuronal death remain uncertain despite intensive research effort. Analysis of the cellular proteins that are differentially expressed in the presence of mutant SOD1 represents a novel approach to investigate further this toxic gain-of-function. By using the motor neuron-like cell line NSC34 stably transfected with wild-type, G93A, or G37R mutant human SOD1, we investigated the effects of mutant human SOD1 on protein expression using proteomic approaches. Seven up-regulated proteins were identified as argininosuccinate synthase, argininosuccinate lyase, neuronal nitric-oxide synthase, RNA-binding motif protein 3, peroxiredoxin I, proteasome subunit beta 5 (X), and glutathione S-transferase (GST) Alpha 2. Seven down-regulated proteins were identified as GST Mu 1, GST Mu 2, GST Mu 5, a hypothetical GST Mu, GST Pi B, leukotriene B(4) 12-hydroxydehydrogenase, and proteasome subunit beta5i (LMP7). GST assays demonstrated a significant reduction in the total GST activity of cells expressing mutant human SOD1. Proteasome assays demonstrated significant reductions in chymotrypsin-like, trypsin-like, and post-glutamylhydrolase proteasome activities. Laser capture microdissection of spinal cord motor neurons from human FALS cases, in conjunction with reverse transcriptase-PCR, demonstrated decreased levels of mRNA encoding GST Mu 1, leukotriene B(4) 12-hydroxydehydrogenase, and LMP7. These combined approaches provide further evidence for involvement of alterations in antioxidant defenses, proteasome function, and nitric oxide metabolism in the pathophysiology of FALS.
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PMID:Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways. 1247 80

Oxaliplatin (L-OHP) is a new platinum analogue that has shown antitumour activity against colon cancer both in vitro and in vivo and is now used in the chemotherapeutic treatment of metastatic colon and rectal cancer. L-OHP like cisplatin (CDDP), is detoxified by glutathione (GSH)-related enzymes and forms platinum (Pt)-DNA adducts lesions that are repaired by the nucleotide excision repair system (NER). We investigated the cytotoxicity and the pharmacology of L-OHP and CDDP on a panel of six colon cell lines in vitro. We showed that GSH and glutathione S-transferase (GST) activity were not correlated to oxaliplatin cytotoxicity. Pt-DNA adducts formation and repair were correlated with CDDP, but not with L-OHP cytotoxicity. The determination of ERCC1 and XPA expression, two enzymes of the NER pathway, by reverse transcriptase-polymerase chain reaction (RT-PCR), demonstrated that ERCC1 expression was predictive of L-OHP sensitivity (r(2)=0.67, P=0.02) and XPA level after oxaliplatin exposure was also correlated to L-OHP IC(50) (r(2)=0.5; P=0.04). The knowledge of such correlations could help predict the sensitivity of patients with colon cancer to L-OHP.
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PMID:Cellular determinants of oxaliplatin sensitivity in colon cancer cell lines. 1250 67

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

Isoforms of metallothionein in the digestive gland of control and experimentally Cd-exposed mussels (Mytilus edulis) (200 microg L(-1) Cd2+ and 400 microg L(-1) Cd2+; 20 days) were studied using the reverse transcriptase-polymerase chain reaction. In addition, glutathione S-transferase (GSTpi) primers were designed to evaluate the reduction in the antioxidant defense systems (glutathione) accompanying the aging process in the same organisms. Following experimental exposure, an accumulation of Cd was observed in the digestive gland of exposed mussels, both adults and juveniles, up to 500 times higher than in the control. An induction of the dimeric form MT20 II was detected in 400 microg L(-1) exposed mussels, as well as a visible inhibition of the monomeric form MT10 IV. After 20 days of exposure juveniles expressed increased GSTpi compared with adults. Results reveal individual variation of both metallothioneins and GSTpi expression among control and Cd2+-exposed mussels of different ages. The ecotoxicological significance of MT utilization in biomonitoring of seawater for trace metals has been considered in light of these results.
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PMID:Cadmium induction of metallothionein isoforms in juvenile and adult mussel (Mytilus edulis). 1499 21

We recently showed that zerumbone, a sesquiterpene found in subtropical ginger, suppresses colonic tumor marker formation in rats and induces apoptosis in colon cancer cell lines. In our present study, the anti-tumor initiating and promoting activities of zerumbone in mouse skin were evaluated using a conventional 2-stage carcinogenesis model. A single topical pretreatment to mouse skin (2 micromol) 24 hr before application of dimethylbenz[a]anthracene (0.2 micromol) markedly suppressed tumor incidence by 60% and the number of tumors by 80% per mouse. Repeated pretreatment (16 nmol) twice weekly during the post-initiation phase reduced the number of 12-O-tetradecanoylphorbol-13-acetate (TPA, 1.6 nmol)-induced tumors by 83% as well as their diameter by 57%. Multiple reverse transcriptase (RT) PCR experiments revealed that zerumbone (2 micromol) enhanced the mRNA expression level of manganese superoxide dismutase, glutathione peroxidase-1, glutathione S-transferase-P1 and NAD(P)H quinone oxidoreductase in the epidermis, but not that of cytochrome p450 1A1 or 1B1. Further, it diminished TPA-induced cyclooxygenase-2 protein expression and phosphorylation of extracellular signal-regulated kinase 1/2, while pretreatment(s), in either the priming or activation stage or both, reduced double TPA application-induced hydrogen peroxide formation and edema induction by 29% to 86%, respectively. Histologic examination revealed that pretreatment(s) with zerumbone suppressed leukocyte infiltration and reduced proliferating cell nuclear antigen-labeling indices. Together, our results indicate that zerumbone is a promising agent for the prevention of both tumor initiating and promoting processes, through induction of anti-oxidative and phase II drug metabolizing enzymes as well as attenuation of proinflammatory signaling pathways.
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PMID:Zerumbone, a sesquiterpene in subtropical ginger, suppresses skin tumor initiation and promotion stages in ICR mice. 1512 79

Sphingosine 1-phosphate (S1P) is a lipid agonist that regulates smooth muscle cell (SMC) and endothelial cell functions by activating several members of the S1P subfamily of G-protein-coupled Edg receptors. We have shown previously that SMC differentiation is regulated by RhoA-dependent activation of serum response factor (SRF). Because S1P is a strong activator of RhoA, we hypothesized that S1P would stimulate SMC differentiation. Treatment of primary rat aortic SMC cells with S1P activated RhoA as measured by precipitation with a glutathione S-transferase-rhotekin fusion protein. In SMC and 10T1/2 cells, S1P treatment up-regulated the activities of several transiently transfected SMC-specific promoters, and these effects were inhibited by the Rho-kinase inhibitor, Y-27632. S1P also increased smooth muscle alpha-actin protein levels in SMC but had no effect on SRF binding to the smooth muscle alpha-actin CArG B element. Quantitative reverse transcriptase-PCR showed that S1P treatment of SMC or 10T1/2 cells did not increase the mRNA level of either of the recently identified SRF co-factors, myocardin or myocardin-related transcription factor-A (MRTF-A). MRTF-A protein was expressed highly in SMC and 10T1/2 cultures, and importantly the effects of S1P were inhibited by a dominant negative form of MRTF-A indicating that S1P may regulate the transcriptional activity of MRTF-A. Indeed, S1P treatment increased the nuclear localization of FLAG-MRTF-A, and the effect of MRTF-A overexpression on smooth muscle alpha-actin promoter activity was inhibited by dominant negative RhoA. S1P also stimulated SMC growth by activating the early growth response gene, c-fos. This effect was not attenuated by Y-27632 but could be inhibited by the MEK inhibitor, UO126. S1P enhanced SMC growth through ERK-mediated phosphorylation of the SRF co-factor, Elk-1, as measured by gel shift and Elk-1 activation assays. Taken together these results demonstrate that S1P activates multiple signaling pathways in SMC and regulates proliferation by ERK-dependent activation of Elk-1 and differentiation by RhoA-dependent activation of MRTF-A.
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PMID:Sphingosine 1-phosphate stimulates smooth muscle cell differentiation and proliferation by activating separate serum response factor co-factors. 1529 66

Pi-class glutathione S-transferase (GSTP1), located on chromosome 11q13, codes for a phase II metabolic enzyme that detoxifies reactive electrophilic intermediates. The protein also interacts with steroid hormones in the human body. The role of GSTP1 in endometrial carcinoma has not been reported. In this study, we aimed at determining the expression of GSTP1 in relation to the epigenetic and genetic changes of the gene in endometrial carcinoma. The GSTP1 protein and mRNA expression was assessed by immunohistochemistry on tissue microarray and quantitative real-time reverse transcriptase-polymerase chain reaction, respectively. Its methylation status was studied by methylation-specific polymerase chain reaction and bisulfite sequencing. Possible mutations in coding region of GSTP1 were assessed by cDNA sequencing. Ninety-seven cases of endometrial carcinoma with available tissue blocks and clinical data were studied. Our results showed that 68.0% (66 of 97) of the cases showed reduced protein expression while 64% (16 of 25) showed reduced mRNA expression; 30.9% (30 of 97) of the cases demonstrated methylated alleles in at least one of the six methylation-specific polymerase chain reaction reactions. The methylation status significantly correlated with reduced protein expression (P = 0.008) and reduced mRNA expression (P = 0.003). Methylation at non-CpG sites including CpCpG trinucleotides and CpT dinucleotides were also observed. cDNA sequencing did not reveal genetic alterations in coding region of the gene. The extent of myometrial invasion was found to be significantly correlated with both the methylation status (P = 0.009) and the protein expression (P = 0.036) of the GSTP1 gene. We postulated that hypermethylation of the GSTP1 gene promoter region may act as a dynamic regulation mechanism contributing to reduced GSTP1 expression, which is associated with myometrial invasion potential of the endometrial carcinoma.
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PMID:Promoter methylation and differential expression of pi-class glutathione S-transferase in endometrial carcinoma. 1568 69

Nucleostemin (NS) is a novel protein that promotes the proliferation of stem cells and cancer cells. The aim of this study is to prepare specific monoclonal antibodies against nucleostemin as a tool in nucleostemin research. To generate the antibodies, the cDNA coding nucleostemin was cloned from human gastric cancer cell line NCI-N87 with reverse transcriptase-polymerase chain reaction (RT-PCR). The partial cDNA fragment was inserted into prokaryotic expression vector pGEX-4T1 to make glutathione S-transferase (GST) fusion protein in Escherichia coli, and the whole-length cDNA fragment was cloned into vector pcDNA3 for expression in mammalian cells. The spleen cells, from the BALB/c mouse immunized with the GST-nucleostemin fusion protein, were used to prepare the monoclonal antibody with hybridoma technique. After screening with enzyme-linked immunosorbent assay (ELISA) and subcloning, six specific monoclonal antibodies against nucleostemin were obtained. Moreover, they also worked well in Western blotting, immunoprecipitation, and immunohistochemistry. These antibodies will be helpful in elucidating biological functions and the molecular mechanism of nucleostemin.
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PMID:Preparation and characterization of monoclonal antibodies against nucleostemin, a protein that controls cell proliferation in stem cells and cancer cells. 1578 7

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

This study focused on glutathione S-transferase (GST), one of the detoxification enzymes, from the silkworm, Bombyx mori (GSTT1). A cDNA encoding a putative GST was amplified by reverse transcriptase-polymerase chain reaction and sequenced. The deduced amino acid sequence revealed 59%, 57% and 56% identities to theta-class GSTs of Musca domestica, Anopheles gambiae and Drosophila melanogaster, respectively. GSTT1 was also estimated to be close to those GSTs in a phylogenetic tree. Recombinant GST (rGSTT1) was functionally overexpressed in Escherichia coli in a soluble form, purified to homogeneity, and characterized. The pH-optimum of rGSTT1 was broad from pH 4 to 9 and rGSTT1 retained more than 75% of its original activity after incubation at pH 5-11. Incubation for 30 min at temperatures below 50 degrees C also affected the activity insignificantly. The Michaelis constant for 1-chloro-2,4-dinitrobenzene was 0.48 mM.
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PMID:Cloning, expression and characterization of theta-class glutathione S-transferase from the silkworm, Bombyx mori. 1595 May 11


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