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)

Understanding peroxidase function in plants is complicated by the lack of substrate specificity, the high number of genes, their diversity in structure and our limited knowledge of peroxidase gene transcription and translation. In the present study we sequenced expressed sequence tags (ESTs) encoding novel heme-containing class III peroxidases from Arabidopsis thaliana and annotated 73 full-length genes identified in the genome. In total, transcripts of 58 of these genes have now been observed. The expression of individual peroxidase genes was assessed in organ-specific EST libraries and compared to the expression of 33 peroxidase genes which we analyzed in whole plants 3, 6, 15, 35 and 59 days after sowing. Expression was assessed in root, rosette leaf, stem, cauline leaf, flower bud and cell culture tissues using the gene-specific and highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR). We predicted that 71 genes could yield stable proteins folded similarly to horseradish peroxidase (HRP). The putative mature peroxidases derived from these genes showed 28-94% amino acid sequence identity and were all targeted to the endoplasmic reticulum by N-terminal signal peptides. In 20 peroxidases these signal peptides were followed by various N-terminal extensions of unknown function which are not present in HRP. Ten peroxidases showed a C-terminal extension indicating vacuolar targeting. We found that the majority of peroxidase genes were expressed in root. In total, class III peroxidases accounted for an impressive 2.2% of root ESTs. Rather few peroxidases showed organ specificity. Most importantly, genes expressed constitutively in all organs and genes with a preference for root represented structurally diverse peroxidases (< 70% sequence identity). Furthermore, genes appearing in tandem showed distinct expression profiles. The alignment of 73 Arabidopsis peroxidase sequences provides an easy access to the identification of orthologous peroxidases in other plant species and will provide a common platform for combining knowledge of peroxidase structure and function relationships obtained in various species.
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PMID:Structural diversity and transcription of class III peroxidases from Arabidopsis thaliana. 1247 2

Cultured asparagus (Asparagus officinalis L. cv Y6) cells induced to regenerate into whole plants through somatic embryogenesis secreted a 38-kD protein into cell walls. The full-length cDNA sequence of this protein (Asparagus officinalis peroxidase 1 [AoPOX1]) determined by reverse transcriptase-polymerase chain reaction showed similarity with plant peroxidases. AoPOX1 transcripts were particularly abundant during early somatic embryogenesis. To evaluate the in vivo function of AoPOX1 protein, purified recombinant AoPOX1 protein was reacted with a series of phenolic substrates. The AoPOX1 protein was effective in the metabolism of feruloyl (o-methoxyphenol)-substituted substrates, including coniferyl alcohol. The reaction product of coniferyl alcohol was fractionated and subjected to gas chromatography-mass spectrometry analysis and (1)H-nuclear magnetic resonance analysis, indicating that the oxidation product of coniferyl alcohol in the presence of AoPOX1 was dehydrodiconiferyl alcohol. The concentration of dehydrodiconiferyl alcohol in the cultured medium of the somatic embryos was in the range of 10(-8) M. Functions of the AoPOX1 protein in the cell differentiation are discussed.
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PMID:Expression and function of cell wall-bound cationic peroxidase in asparagus somatic embryogenesis. 1269 35

An analysis of apoplastic extensin cross-linking activity in vegetative organs of Lupinus albus indicated that leaves contained the highest specific activity. Assays of peroxidases fractionated from this material demonstrated that this activity could be largely attributed to a soluble and apoplastic 51-kDa peroxidase, denoted LEP1. Relative to other purified peroxidases, LEP1 demonstrates high extensin cross-linking activity and can be classified as an extensin peroxidase (EP). Optimal conditions for the in vitro oxidation of other phenolic substrates included 1.5-3.0 mm peroxide at pH 5.0. EP activity of LEP1 was low under these conditions but optimal and substantially higher with 100 microm peroxide and neutral pH, suggesting that physiological changes in pH and peroxide in muro could heavily influence the extensin cross-linking activity of LEP1 in vivo. Analysis of LEP1 glycans indicated 11-12 N-linked glycans, predominantly the heptasaccharide Man3XylFucGlcNAc2, but also larger structures showing substantial heterogeneity. Comparative assays with horseradish peroxidase isoform C and peanut peroxidases suggested the high level of glycosylation in LEP1 may be responsible for the high solubility of this EP in the apoplastic space. A full-length cDNA corresponding to LEP1 was cloned. Quantitative reverse transcriptase-PCR demonstrated LEP1 induction in apical portions of etiolated hypocotyls 30-60 min after exposure to white light, prior to the onset of growth inhibition. Comparative modeling of the translated sequence indicated an unusually unobstructed equatorial cleft across the substrate access channel, which might facilitate interaction with extensin and confer higher EP activity.
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PMID:A biochemical and molecular characterization of LEP1, an extensin peroxidase from lupin. 1288 82

Pigs were experimentally inoculated with Glentorf, Lelystad/97, and Alfort/187: representative low, moderate, and high virulent strains of classical swine fever virus (CSFV). Animals were tested for viremia using virus isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) assays run under routine diagnostic conditions. The virus was detected in the peripheral blood by virus isolation and RT-PCR assays of all Glentorf- and Lelystad/97-infected pigs beginning at 3 days postinoculation (dpi) and in all Alfort/187-infected pigs beginning at 2 dpi. Viremia, as determined by virus isolation, remained detectable in Lelystad/97- and Alfort/187-infected pigs until the last animal within each cohort was euthanized on days 12 and 7 postinoculation, respectively. In contrast, the virus could be isolated from the blood of all Glentorf-infected pigs between 3 and 7 dpi but not from 10 to 21 dpi when the experiment was concluded. Viremia, as determined by RT-PCR, became apparent in Alfort/187-infected pigs at 2 dpi and in Glentorf- and Lelystad/97-infected pigs at 3 dpi. All pigs, regardless of the CSFV strain used, remained RT-PCR positive until they were euthanized. Tonsils were harvested from all the pigs and frozen sections tested for the presence of the CSFV antigen using polyclonal pestivirus and monoclonal CSFV horseradish peroxidase (HRPO) conjugates. Immunostaining reactions were positive for all the Alfort/187- and Lelystad/97-infected pigs. By contrast, tonsils from the Glentorf-infected pigs gave negative to equivocal results. These data suggest that an RT-PCR assay performed on blood may be the best test when dealing with pigs infected with low virulent strains of CSFV.
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PMID:Comparison of reverse transcriptase-polymerase chain reaction, virus isolation, and immunoperoxidase assays for detecting pigs infected with low, moderate, and high virulent strains of classical swine fever virus. 1505 64

A drug composition consisting of nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) is commonly used in AIDS therapy. A major difficulty encountered with the therapeutic composite involves the emergence of drug-resistant viruses, especially to the PIs, regarded as the most effective drugs in the composition. We present a novel bioelectronic means to detect the appearance of mutated HIV-1 exhibiting drug resistance to the PI saquinavir. The method is based on the translation of viral RNA, the association of cleaved or uncleaved Gag polyproteins at an electrode surface functionalized with the respective antibodies, and the bioelectronic detection of the Gag polyproteins associated with the surface. The bioelectronic process includes the association of anti-MA or anti-CA antibodies, the secondary binding of an antibody-horseradish peroxidase (HRP) conjugate, and the biocatalyzed precipitation of an insoluble product on the electronic transducers. Faradaic impedance measurements and quartz crystal microbalance analyses are employed to follow the autoprocessing of the Gag polyproteins. The method was applied to determine drug resistance in infected cultured cells and also in blood samples of consenting AIDS patients. The method described here is also applicable to the determination of drug effectiveness in AIDS patients and to screening of the efficiency of newly developed drugs.
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PMID:Electronic transduction of HIV-1 drug resistance in AIDS patients. 1523 52

The two COX (cyclo-oxygenase) isoenzymes COX-1 and -2 catalyse the initial step in the conversion of arachidonic acid into PG (prostaglandin) hormones. The identification of an mRNA transcript encoding a splice variant of human COX-1 was reported more than a decade ago [Diaz, Reginato and Jimenez (1992) J. Biol. Chem. 267, 10816-10822], yet catalytic activity and tissue expression of the corresponding spliced protein remained uncharacterized. The splice variant lacks amino acids 396-432, corresponding to the last 37 amino acids of exon 9 of the gene encoding COX-1. These amino acids form a loop at one side of the peroxidase active site of the protein. We expressed the full-length and spliced COX-1 cDNAs in COS-7 and Sf9 insect cells, and determined the PG-forming activity using incubations with radiolabelled arachidonic acid and HPLC analyses. When expressed in either system, abundant PG formation was observed with the full-length COX-1, whereas the spliced protein did not form any detectable product. Peroxidase activity was readily detected in microsomes prepared from COS-7 cells transfected with COX-1 but not with the splice variant. In reverse transcriptase-PCR experiments, we detected the mRNA for the alternatively spliced and full-length COX-1 in human brain, tonsil and colon tissue, yet we were unable to detect expression of the spliced protein in the same tissues using immunoprecipitation and Western-blot analyses. We conclude that, whereas the mRNA transcript for the spliced COX-1 is present in various human tissues, the corresponding protein is either not formed or subject to rapid proteolytic degradation.
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PMID:Human cyclo-oxygenase-1 and an alternative splice variant: contrasts in expression of mRNA, protein and catalytic activities. 1536 Oct 66

Nitric oxide (NO) derived from inducible NO synthase has been implicated in cardiac rejection. However, little is known about the role of the reactive nitrogen species peroxynitrite. We examined the protective actions of a peroxynitrite decomposition catalyst, WW85, in an experimental model of acute cardiac rejection. Heterotopic, abdominal transplantation of rat donor hearts was performed. Groups included isografts, allografts, or allografts treated with WW85, cyclosporine, or cyclosporine + WW85. We determined graft survival, histological rejection, and graft function (by in situ sonomicrometry). Intragraft biochemical analysis of cytokines and proapoptotic and antiapoptotic gene expression using reverse transcriptase-polymerase chain reaction were determined. Treatment with WW85 or cyclosporine alone prolonged graft survival, improved graft function, and decreased histological rejection. Graft survival was further significantly (P < 0.001) enhanced by combination treatment. A decrease was also shown in nitrotyrosine, poly(ADP-ribose) polymerase (PARP) activation, and lipid peroxide formation by WW85 that was potentiated when given in combination with cyclosporine. Benefits could not be ascribed to changes in intragraft myeloperoxidase activity. Only combination therapy produced significant decreases in inflammatory cytokine gene expression, suggesting that WW85 acted primarily downstream of these stimuli. In general, WW85 had no direct action on expression of the proapoptotic gene, Fas ligand; however, WW85 given alone or with cyclosporine enhanced expression of antiapoptotic genes Bcl-2 and Bcl-xL. Collectively, these findings suggest a protective action of the peroxynitrite decomposition catalyst WW85 on graft rejection that is independent of any action on leukocyte sequestration and cytokine gene expression. Rather, effects seem to be downstream on decreased protein nitration, decreased lipid peroxidation, and decreased PARP activation.
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PMID:Protective mechanisms of a metalloporphyrinic peroxynitrite decomposition catalyst, WW85, in rat cardiac transplants. 1578 53

The objective of this study is to investigate the anti-inflammatory effect of hydroxyethylpuerarin on focal brain ischemia injury in rats and to explore its mechanisms of action. After 24 h of reperfusion following 2 h of cerebral ischemia, the infiltration of neutrophils was observed by myeloperoxidase (MPO) activity determination, the expression of intercellular adhesion molecule-1 (ICAM-1) was observed by western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, and the nuclear translocation and DNA binding activity of nuclear factor-kappaB (NF-kappaB) were observed by western blot and electrophoretic mobility shift assay (EMSA). The results showed that hydroxyethylpuerarin could obviously inhibit the MPO activity and ICAM-1 expression following 2 hours of ischemia with 24 hours of reperfusion. The nuclear translocation and DNA binding activity were also decreased by hydroxyethylpuerarin treatment. These results suggested that hydroxyethylpuerarin could inhibit neutrophil-mediated inflammatory response after brain ischemia reperfusion in rats. This effect may be mediated by down-regulation of ICAM-1 and NF-kappaB activity.
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PMID:Anti-inflammatory effect of hydroxyethylpuerarin on focal brain ischemia/reperfusion injury in rats. 1580 53

The aim of this longitudinal observational study was to investigate and describe the spectrum of messenger ribonucleic acid (mRNA) expression of multiple inflammatory markers in circulating leukocytes after major orthopaedic surgery. We studied ten elective arthroplasty patients perioperatively on the orthopaedic ward, and eight healthy volunteers for a comparison group. Venous blood specimens were collected preoperatively, and 6, and 24 hours postoperatively, together with 6- and 24-hour postoperative wound drain specimens. The mRNA of 21 different inflammatory mediators was measured by real-time reverse transcriptase Polymerase Chain Reaction. Comparisons were made with the venous blood of eight healthy comparison subjects. There were significant differences (P<0.01) between preoperative specimens and normal comparisons (i.e. higher MPO, PDGF, TREM and IRAKM; lower mtHSP) reflecting the effects of chronic inflammation associated with osteoarthritis. There were significant increases (P<0.01) in expression of IL-8, MPO, IL-1beta, TREM, MMP9, and C5aR in circulating blood at 24 hours postoperatively, but not at six hours. There was no significant decrease in expression of any inflammatory mediator. There was no statistical difference in inflammatory mediator expression between drain specimens and venous specimens taken at the same time. We conclude that, in uncomplicated orthopaedic surgical patients, there was up-regulation of some cytokine mRNAs at both the local and systemic levels during the first day after surgery. We observed no evidence of immune compartmentalization, and found no evidence for innate immune paresis within the first day after surgery.
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PMID:Messenger RNA expression of multiple immune mediators in leukocytes from elective orthopaedic surgical patients. 1595 15

KC is a mouse homolog of human chemokine gro-alpha (CXCL1), expression of which is increased in liver diseases. We show that activated, but not quiescent, hepatic stellate cells (HSCs) express KC. Hepatic stellate cells constitutively express the KC receptor, CXCR2. Addition of recombinant KC to HSCs undergoing activation in culture increases secretion and processing of Type I collagen. Overexpression of endogenous KC in the mouse liver could be achieved by an intraperitoneal injection of CCl(4), followed after 24 hrs by an injection of recombinant KC into circulation. This protocol resulted in about a 14-fold increase in concentration of KC protein in the liver. Overexpression of KC was associated with upregulation of the mRNA for CXCR2 and MIP-2 and with necrosis and increased synthesis of Type I collagen. This suggests that KC has a direct hepatotoxic effect, which led to a massive liver necrosis after 48 hrs. No accumulation of neutrophils was seen in the livers as judged by histology and reverse transcriptase-polymerase chain reaction analysis of myeloperoxidase mRNA. Autostimulation of KC and CXCR2 expression by recombinant KC protein in the mice with preexisting liver injury indicates a positive feedback regulation. Such regulation and direct hepatotoxicity of KC with increased collagen synthesis represent novel findings about the role of KC/ gro-alpha in liver pathology.
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PMID:Direct hepatotoxic effect of KC chemokine in the liver without infiltration of neutrophils. 1611 8


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