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 family Poxviridae contains two subfamilies: the Entomopoxvirinae (poxviruses of insects) and the Chordopoxvirinae (poxviruses of vertebrates). Here we present the first characterization of the genome of an entomopoxvirus (EPV) which infects the North American migratory grasshopper Melanoplus sanguinipes and other important orthopteran pests. The 236-kbp M. sanguinipes EPV (MsEPV) genome consists of a central coding region bounded by 7-kbp inverted terminal repeats and contains 267 open reading frames (ORFs), of which 107 exhibit similarity to previously described genes. The presence of genes not previously described in poxviruses, and in some cases in any other known virus, suggests significant viral adaptation to the arthropod host and the external environment. Genes predicting interactions with host cellular mechanisms include homologues of the inhibitor of apoptosis protein, stress response protein phosphatase 2C, extracellular matrixin metalloproteases, ubiquitin, calcium binding EF-hand protein, glycosyltransferase, and a triacylglyceride lipase. MsEPV genes with putative functions in prevention and repair of DNA damage include a complete base excision repair pathway (uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and an NAD+-dependent DNA ligase), a photoreactivation repair pathway (cyclobutane pyrimidine dimer photolyase), a LINE-type reverse transcriptase, and a mutT homologue. The presence of these specific repair pathways may represent viral adaptation for repair of environmentally induced DNA damage. The absence of previously described poxvirus enzymes involved in nucleotide metabolism and the presence of a novel thymidylate synthase homologue suggest that MsEPV is heavily reliant on host cell nucleotide pools and the de novo nucleotide biosynthesis pathway. MsEPV and lepidopteran genus B EPVs lack genome colinearity and exhibit a low level of amino acid identity among homologous genes (20 to 59%), perhaps reflecting a significant evolutionary distance between lepidopteran and orthopteran viruses. Divergence between MsEPV and the Chordopoxvirinae is indicated by the presence of only 49 identifiable chordopoxvirus homologues, low-level amino acid identity among these genes (20 to 48%), and the presence in MsEPV of 43 novel ORFs in five gene families. Genes common to both poxvirus subfamilies, which include those encoding enzymes involved in RNA transcription and modification, DNA replication, protein processing, virion assembly, and virion structural proteins, define the genetic core of the Poxviridae.
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PMID:The genome of Melanoplus sanguinipes entomopoxvirus. 984 59

Biotransformation of all-trans-retinol (t-ROH) and all-trans-retinal (t-RAL) to all-trans-retinoic acid (t-RA) in human prenatal hepatic tissues (53-84 gestational days) was investigated with HPLC using human adult hepatic tissues as positive controls. Catalysis of the biotransformation of t-ROH by prenatal human cytosolic fractions resulted in accumulation of t-RAL with minimal t-RA. Oxidations of t-ROH catalyzed by prenatal cytosol were supported by both NAD+ and NADP+, although NAD+ was a much better cofactor. In contrast, catalysis of the oxidation of t-RAL to t-RA appeared to be solely NAD+ dependent. Substrate Km values for conversions of t-ROH to t-RAL and of t-RAL to t-RA were 82.4 and 65.8 microM, respectively. At concentrations of 10 and 90 mM, ethanol inhibited the conversion of t-ROH to t-RAL by 25 and 43%, respectively, but did not inhibit the conversion of t-RAL to t-RA significantly. In contrast, acetaldehyde reduced the conversion of t-RAL to t-RA by 25 and 87% at 0.1 and 10 mM respective concentrations. Several alcohols and aldehydes known to be generated from lipid peroxides also exhibited significant inhibition of t-RA biosynthesis in human prenatal hepatic tissues. Among the compounds tested, 4-hydroxy-2-nonenal (4-HNE) was highly effective in inhibiting the conversion of t-RAL to t-RA. A 20% inhibition was observed at a concentration of only 0.001 mM, and nearly complete inhibition was produced at 0.1 mM. Human fetal and embryonic hepatic tissues each exhibited significant CYP2E1 expression as assessed with chlorzoxazone 6-hydroxylation, a highly sensitive western blotting technique, and reverse transcriptase-polymerase chain reaction (PCR) (RT-PCR), suggesting that lipid peroxidation can be initiated via CYP2E1-catalyzed ethanol oxidation in human embryonic hepatic tissues. In summary, these studies suggest that ethanol may affect the biosynthesis of t-RA in human prenatal hepatic tissues directly and indirectly. Ethanol and its major oxidative metabolite, acetaldehyde, both inhibit the generation of t-RA. Concurrently, the CYP2E1-catalyzed oxidation of ethanol can initiate lipid peroxidation via generation of a variety of free radicals. The lipid peroxides thereby generated could then be further converted via CYP2E1-catalyzed reactions to alcohols and aldehydes, including 4-HNE, that act as potent inhibitors of t-RA synthesis.
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PMID:Inhibition of human prenatal biosynthesis of all-trans-retinoic acid by ethanol, ethanol metabolites, and products of lipid peroxidation reactions: a possible role for CYP2E1. 1007 87

Sulfolipids of photosynthetic bacteria and plants are characterized by their unique sulfoquinovose headgroup, a derivative of glucose in which the 6-hydroxyl group is replaced by a sulfonate group. These sulfolipids have been discussed as promising anti-tumor and anti-HIV therapeutics based on their inhibition of DNA polymerase and reverse transcriptase. To study sulfolipid biosynthesis, in particular the formation of UDP-sulfoquinovose, we have combined computational modeling with biochemical methods. A database search was performed employing the derived amino acid sequence from SQD1, a gene involved in sulfolipid biosynthesis of Arabidopsis thaliana. This sequence shows high similarity to other sulfolipid biosynthetic proteins of different organisms and also to sugar nucleotide modifying enzymes, including UDP-glucose epimerase and dTDP-glucose dehydratase. Additional biochemical data on the purified SQD1 protein suggest that it is involved in the formation of UDP-sulfoquinovose, the first step of sulfolipid biosynthesis. To understand which aspects of epimerase catalysis may be shared by SQD1, we built a three-dimensional model of SQD1 using the 1.8 A crystallographic structure of UDP-glucose 4-epimerase as a template. This model predicted an NAD(+) binding site, and the binding of NAD(+) was subsequently confirmed by enzymatic assay and mass spectrometry. The active-site interactions together with biochemical data provide the basis for proposing a reaction mechanism for UDP-sulfoquinovose formation.
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PMID:Prediction of the active-site structure and NAD(+) binding in SQD1, a protein essential for sulfolipid biosynthesis in Arabidopsis. 1046 38

We studied 11beta-hydroxysteroid dehydrogenase activities in the renal cell line LLC-PK1 and the effects of different steroids on them. Cortisol was oxidized in the presence of NAD as well as NADP, reflecting the presence of two different 11beta-HSD forms. Enzyme kinetics for cortisol 11beta-oxidation were: Vmax = 5.9 pmol/(min x mg), Km = 0.2 microM with NAD, and Vmax = 4.5 pmol/(min x mg), Km = 1.0 microM with NADP. Interestingly, no reverse reaction was observed when using cortisone and NADPH as substrate and cosubstrate, respectively. Exposure of cells to a variety of steroids had different effects on cortisol 11beta-oxidation rates with NADP compared to those with NAD. Dexamethasone initially (3-60 min of exposure) decreased the NAD-dependent 11beta-HSD activity to about 60%, which was no longer evident after 2 h or longer. By contrast, the 11beta-oxidation of cortisol with NADP increased by dexamethasone treatment of the cells, after a lagtime of about 2 h, and this effect was still evident after 32 h. The increase of 11beta-HSD activity with NADP by dexamethasone was concentration dependent (estimated EC50:125 nM). The antiglucocorticoid RU486 did not antagonize dexamethasone induction. Exposure of cells for 19 h to 1 microM cortisol, cortisone, progesterone, and estradiol also increased NADP-dependent cortisol 11beta-oxidation, but had no effect on the NAD-dependent 11beta-HSD activity. Immunoblot and reverse transcriptase-polymerase chain reaction experiments failed to detect any 11beta-HSD 1 protein or mRNA in these cells. Our observations suggest that in LLC-PK1 cells, two forms of 11beta-HSD exist, which differ in cosubstrate dependency, kinetics for cortisol, and modulation by steroids. Whereas the NAD-dependent form seems identical to renal 11beta-HSD 2, the NADP-dependent 11beta-HSD possibly resembles an as yet unknown third isoform.
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PMID:Characterization of 11beta-hydroxysteroid dehydrogenase activities in the renal cell line LLC-PK1: evidence for a third isoform? 1078 27

Two different amplification products, termed c1 and c2, showing a high similarity to glutamate dehydrogenase sequences from plants, were obtained from Asparagus officinalis using two degenerated primers and RT-PCR (reverse transcriptase polymerase chain reaction). The genes corresponding to these cDNA clones were designated aspGDHA and aspGDHB. Screening of a cDNA library resulted in the isolation of cDNA clones for aspGDHB only. Analysis of the deduced amino acid (aa) sequence from the full-length cDNA suggests that the gene product contains all regions associated with metabolic function of NAD glutamate dehydrogenase (NAD-GDH). A first phylogenetic analysis including only GDHs from plants suggested that the two GDH genes of A. officinalis arose by an ancient duplication event, pre-dating the divergence of monocots and dicots. Codon usage analysis showed a bias towards A/T ending codons. This tendency is likely due to the biased nucleotide composition of the asparagus genome, rather than to the translational selection for specific codons. Using principal coordinate analysis, the evolutionary relatedness of plant GDHs with homologous sequences from a large spectrum of organisms was investigated. The results showed a closer affinity of plant GDHs to GDHs of thermophilic archaebacterial and eubacterial species, when compared to those of unicellular eukaryotic fungi. Sequence analysis at specific amino acid signatures, known to affect the thermal stability of GDH, and assays of enzyme activity at non-physiological temperatures, showed a greater adaptation to heat-stress conditions for the asparagus and tobacco enzymes compared with the Saccharomyces cerevisiae enzyme.
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PMID:Cloning of two glutamate dehydrogenase cDNAs from Asparagus officinalis: sequence analysis and evolutionary implications. 1079 19

In mammals cyclic ADP-ribose (cADPR), a universal calcium mobilizer from intracellular stores, is generated from NAD(+) at the outer cell surface by the multifunctional ectoenzyme CD38 and by related ADP-ribosyl cyclases. Recently, influx of extracellular cADPR has been observed in 3T3 murine fibroblasts, where it elicits Ca(2+)-mediated enhancement of proliferation. Here we addressed the nature and the properties of cADPR influx into CD38(-) 3T3 cells, which showed pleiotropic mechanisms of both equilibrative and concentrative transport. Based on selective inhibitors or experimental conditions (e.g. abrogation of Na(+)-dependent active symport processes and transient transfection experiments) and on reverse transcriptase-polymerase chain reaction analysis of transcripts in 3T3 fibroblasts and comparatively in HeLa cells, we identified cADPR-transporting activities with specific nucleoside transporters (NT), both equilibrative (ENT2) and concentrative (CNT2 and a nitrobenzylthioinosine (NBMPR)-inhibitable NT). A reciprocal inhibition relationship was observed between inosine and cADPR fluxes across these NT species. Concentrative (but not equilibrative) transport of nanomolar extracellular cADPR took place in CD38(-) 3T3 cells co-cultured for 48 h in transwells on feeders of CD38-transfected, cADPR-generating 3T3 fibroblasts. These results suggest possible, hitherto unrecognized, correlations between ectocellular metabolism of nucleotides/nucleosides and cADPR-mediated regulation of intracellular calcium homeostasis.
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PMID:Equilibrative and concentrative nucleoside transporters mediate influx of extracellular cyclic ADP-ribose into 3T3 murine fibroblasts. 1236 85

Abacavir (ZIAGEN) is a reverse transcriptase inhibitor marketed for the treatment of HIV-1 infection. A small percentage of patients experience a hypersensitivity reaction indicating immune system involvement and bioactivation. A major route of metabolism for abacavir is oxidation of a primary betagamma unsaturated alcohol to a carboxylic acid via an aldehyde intermediate. This process was shown to be mediated in vitro by human cytosol and NAD, and subsequently the alphaalpha and gamma2gamma2 human isoforms of alcohol dehydrogenase (ADH). The alphaalpha isoform effected two sequential oxidation steps to form the acid metabolite and two isomers, qualitatively reflective of in vitro cytosolic profiles. The gamma2gamma2 isozyme generated primarily an isomer of abacavir, which was minor in the alphaalpha profiles. The aldehyde intermediate could be trapped in incubations with both isozymes as an oxime derivative. These metabolites can be rationalized as arising via the aldehyde which undergoes isomerization and further oxidation by the alphaalpha enzyme or reduction by the gamma2gamma2 isozyme. Non-extractable abacavir protein residues were generated in cytosol, and with alphaalpha and gamma2gamma2 incubations in the presence of human serum albumin (HSA). Metabolism and residue formation were blocked by the ADH inhibitor 4-methyl pyrazole (4-MP). The residues generated by the alphaalpha and gamma2gamma2 incubations were analyzed by SDS-PAGE with immunochemical detection. The binding of rabbit anti-abacavir antibody to abacavir-HSA was shown to be dependent on metabolism (i.e. NAD-dependent and 4-MP sensitive). The mechanism of covalent binding remains to be established, but significantly less abacavir-protein residue was detected with an analog of abacavir in which the double bond was removed, suggestive of a double bond migration and 1,4 addition process.
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PMID:The metabolic activation of abacavir by human liver cytosol and expressed human alcohol dehydrogenase isozymes. 1239 60

Uridine diphospho-D-glucuronate carboxy-lyase (UDP-D-xylose synthase; EC 4.1.1.35), which catalyzes the conversion of UDP-D-glucuronate to UDP-D-xylose, was purified to apparent homogenity from pea (Pisum sativum L.) seedlings. The pH optimum for enzyme activity was around 5-6, and the activity was not affected by exogeneously supplied NAD+ and NADH. The purified enzyme had a molecular weight of 250 kDa and consisted of 42 kDa polypeptides. Based on the amino acid sequence, a probe (400 bp) was prepared with degenerate primers by a reverse transcriptase-PCR. Using this probe, a clone encoding 346 amino acid residues was screened from a pea cDNA library. The recombinant protein expressed in Escherichia coli catalyzed conversion of UDP-D-glucuronate to UDP-D-xylose, confirming that the isolated clone encoded UDP-D-glucuronate carboxy-lyase.
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PMID:Purification and cDNA cloning of UDP-D-glucuronate carboxy-lyase (UDP-D-xylose synthase) from pea seedlings. 1246 Nov 25

An early key event in the activation of neutrophil granulocytes is Ca(2+) influx. Members of the transient receptor potential (TRP) channel family may be held responsible for this. The aim of the present study is to analyse the expression pattern of TRP mRNA and identify characteristic currents unambiguously attributable to particular TRP channels. mRNA was extracted from human neutrophils, isolated by gradient centrifugation and also by magnetically labelled CD15 antibodies. The presence of mRNA was demonstrated using reverse transcriptase-PCR in neutrophils (controlled to be CD5-negative) as well as in human leukaemic cell line 60 (HL-60) cells, for the following TRP species: the long TRPC2 (LTRPC2), the vanilloid receptor 1, the vanilloid receptor-like protein 1 and epithelial Ca(2+) channels 1 and 2. TRPC6 was specific for neutrophils, whereas only in HL-60 cells were TRPC1, TRPC2, TRPC3, melastatin 1 and melastatin-related 1 found. Patch-clamp measurements in neutrophils revealed non-selective cation currents evoked by intracellular ADP-ribose and by NAD(+). Both these modes of activation have been found to be characteristic of LTRPC2. Furthermore, single-channel activity was resolved in neutrophils and it was indistinguishable from that in LTRPC2-transfected HEK-293 cells. The results provide evidence that LTRPC2 in neutrophil granulocytes forms an entry pathway for Na(+) and Ca(2+), which is regulated by ADP-ribose and the redox state.
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PMID:Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD. 1256 54

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


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