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)

3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma- diphosphate and 2'-deoxythymidine-5'-(alpha-methylphosphonyl)-beta, gamma- diphosphate have been synthesized. Both compounds are incorporated into DNA chains during catalysis by reverse transcriptases of human immunodeficiency (HIV) and avian myeloblastosis (AMV) viruses, DNA polymerase beta from rat liver, terminal deoxynucleotidyl transferase from calf thymus and (at a very low rate) is by E. coli DNA polymerase I, Klenow fragment. The first compound is a termination substrate while the second is capable of multiple incorporation into the DNA chains. For instance, reverse transcriptase catalysis resulted in the appearance of 8 residues of second compound. DNA polymerases alpha and epsilon from human placenta incorporated none of the above compounds into DNA chains, although an inhibition of DNA synthesis by both compounds was observed with all enzymes mentioned. The 3'----5'-exonuclease activity of DNA polymerase I, Klenow fragment, hydrolyzed DNA fragments containing phosphonomethyl internucleoside groups, while such DNA fragments were resistant to the E. coli exonuclease III.
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PMID:Formation of phosphonester bonds catalyzed by DNA polymerase. 137 65

HIV infection is associated with qualitative and functional immune deficiencies. It has been shown that the in vitro infection of CD4+ cells with HIV was associated with sustained elevation of cAMP and cGMP. In the present report the role of cAMP on HIV replication in MT-4 cells was investigated. The MT-4 cells were infected with HIV (strain 3b), in the presence or absence of agents that increase intracellular levels of cAMP, through different mechanisms. At selected times postinfection, HIV replication was measured by reverse transcriptase activity or HIV P24Ag in culture supernatants. Forskolin (FK, an activator of adenylate cyclase 1-100 microM), Isobutyl-methylxanthine (IBMX, a phosphodiesterase inhibitor, which indirectly increases intracellular levels of cAMP, 30-100 microM) and dibutyryl (db) cAMP (0.1-10 microM) enhanced HIV replication, in a dose-dependent manner. FK, IBMX, and db cAMP enhanced HIV replication by 2- to 10-fold, 4- to 7-fold, and 2- to 6-fold, respectively. Intracellular levels of cAMP were measured by radioimmunoassay and were also enhanced. Since cAMP exerts its catalytic effects through activation of protein kinase (PK) A the effect of H-8 (a specific inhibitor of the cAMP dependent PK A) on HIV replication was simultaneously examined. The H8 at doses of 0.1 to 10 microns inhibited HIV replication by 25 to 99.9%. Moreover H9 inhibited HIV replication in peripheral blood mononuclear cells by more than 90%. The replication of HIV appears to be a cAMP-dependent event, and PK A could possibly be a target for the development of anti-HIV therapies.
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PMID:Human immunodeficiency virus replication: modulation by cellular levels of cAMP. 138

The mode of action of the RNase H activity from HIV-1 was analyzed with a purified recombinant p66/p51 reverse transcriptase RT/RNase H protein and RNA-DNA hybrid consisting of RNA harboring the polypurine tract (ppt) and three complementary synthetic DNA oligonucleotides. Upon incubation of this preformed RNA-DNA hybrid with the p66/p51 RT/RNase H, a cleavage pattern is observed that indicates endonucleolytic RNase H activity with some sequence specificity for the next to last nucleotide of the 3'-end of the ppt RNA and one cut within the ppt. The RNase H avoids cleavage of G or A stretches. During RNA-directed DNA synthesis the RNA is hydrolyzed in a concerted action of RT and RNase H whereby the RNase H exhibits endonuclease as well as 3'-5'-exonuclease activity. The distance between the active centers of the RT and RNase H corresponds to 18 base pairs of the RNA-DNA hybrid. Plus-strand DNA-directed DNA synthesis initiates exactly at the next to last nucleotide of the 3'-end of the ppt RNA by means of the RNase H activity.
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PMID:Interaction of HIV-1 ribonuclease H with polypurine tract containing RNA-DNA hybrids. 170 2

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a potent and selective inhibitor of retrovirus (i.e., human immunodeficiency virus) replication in vitro and in vivo. Uptake of PMEA by human MT-4 cells and subsequent conversion to the mono- and diphosphorylated metabolites (PMEAp and PMEApp) are dose-dependent and occur proportionally with the initial extracellular PMEA concentrations. Adenylate kinase is unable to phosphorylate PMEA. However, 5-phosphoribosyl-1-pyrophosphate synthetase directly converts PMEA to PMEApp with a Km of 1.47 mM and a Vmax that is 150-fold lower than the Vmax for AMP. ATPase, 5'-phosphodiesterase, and nucleoside diphosphate kinase are able to dephosphorylate PMEApp to PMEAp, albeit to a much lower extent than the dephosphorylation of ATP. PMEApp has a relatively long intracellular half-life (16-18 hr) and has a much higher affinity for the human immunodeficiency virus-specified reverse transcriptase than for the cellular DNA polymerase alpha (Ki/Km: 0.01 and 0.60, respectively). PMEApp is at least as potent an inhibitor of human immunodeficiency virus reverse transcriptase as 2',3'-dideoxyadenosine 5'-triphosphate. Being an alternative substrate to dATP, PMEApp acts as a potent DNA chain terminator, and this may explain its anti-retrovirus activity.
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PMID:Intracellular metabolism and mechanism of anti-retrovirus action of 9-(2-phosphonylmethoxyethyl)adenine, a potent anti-human immunodeficiency virus compound. 170 39

cDNA was synthesized on Ig kappa-L-chain mRNA isolated from mouse plasmocytoma MOPC 21 using avial myeloblastosis RNA-dependent DNA polymerase. The cDNA was used as a matrix for second DNA chain synthesis using 5'-exonuclease free DNA-polymerase I (Klenoff fragment). Hairpin DNA having double length of initial cDNA was obtained without added exogenous primer. SI nuclease treatment of the hairpin DNA results in reducing the DNA length twice, as shown by electrophoresis in denaturing conditions. The fact, that S1 nuclease resistance of the hairpin DNA is 75% shows high complementarity between first and second DNA chain. The product length obtained after S1 nuclease treatment was shown to be heterogenous. The maximal length of the product, reaching at least 900 base pairs, is near to be initial mRNA length (without the poly(A)-fragment).
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PMID:[Synthesis of double-stranded DNA on light immunoglobulin chain matrix RNA]. 615 6

The regulation of murine mammary tumor virus (MuMTV) production by mammotropic hormones, hormonomimetic substances, and cyclic nucleotides was investigated. The virus produced in control and treated mammary tumor cell cultures was quantitated by measuring the supernatant reverse transcriptase activity in exogenous reaction using poly(rC).oligo(dG) as template-primer. Two days after exposure, the synthetic glucocorticoid, dexamethasone (DXMT), increased spontaneous MuMTV production at optimal concentration (0.1 mumol) up to ten times. Dibutyryl derivative of cyclic AMP had no effect on spontaneous MuMTV production, whereas the drug potentiated suboptimal concentrations of the glucocorticoid. Natural prostaglandins, potent agonists of adenylate cyclase catalyzing intracellular synthesis of cyclic AMP, enhanced both basal (up to five times) and DXMT-stimulated (up to 1.6 times) MuMTV replication. The MuMTV-stimulating activity of prostaglandins decreased in the order of PGA1 greater than PGE1 greater than PGB1 greater than PGF2 alpha. Prostaglandins can be replaced partially by norepinephrine and isoproterenol by enhancing the DXMT-mediated MuMTV stimulation, whereas these drugs remained without effect on spontaneous MuMTV production. Theophylline, an antagonist of cAMP-phosphodiesterase converting cAMP to AMP, enhanced the virus-stimulating activity of DXMT as well as of prostaglandins. The enhancement of MuMTV production by adenylate cyclase agonists do not correlate absolutely with the estimates of intracellular cAMP levels, since the highest amounts of cAMP has been repeatedly observed in cells treated with PGE1 and norepinephrine. The results indicate that besides hormones, other hormone-like substances and cyclic nucleotides may be involved in the complex mechanism of hormone-regulated MuMTV genome expression.
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PMID:Role of natural prostaglandins in the control of murine mammary tumor virus expression. 628 Dec 84

Synthesis of 2',3'-dideoxyuridine 5'-triphosphate analogues with fluorescent and biotin residues at C5 of uracil base was carried out. The substrate properties of these analogues were studied with AMV, M-MLV, and HIV reverse transcriptase. All 5-derivatives studied were shown to be incorporated into the 3'-terminus of oligonucleotide. The stability of oligodeoxyribonucleotides terminated with ddUTP analogues modified at the 5-position of the pyrimidine residue to the exonuclease action of phosphodiesterase I and Klenow enzyme was more than 1000 times higher than that of nonterminated oligonucleotides.
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PMID:[Derivatives of ddUTP, modified at the 5-position of uridine, as substrate terminators of reverse transcriptase. Hydrolysis of oligonucleotides, terminated by these analogs, by phosphodiesterase I]. 751 82

Saccharomyces cerevisiae mtDNA polymerase, isolated as a single 135-kDa recombinant polypeptide, showed high processivity and a capacity of use poly(dA).oligo(dT), poly(rA).oligo(dT), or primed bacteriophage M13 DNA as a template. In a primer extension assay, the enzyme exhibited an intrinsic 3'-5'-exonuclease activity. By optimizing the polymerization reaction conditions, apparent Km and Vmax values could be determined for the incorporation of dTTP, 2'-3'-dideoxy-TTP (ddTTP), 3'-azido-TTP (AZTTP), 3'-fluoro-TTP, dCTP, 2'-3'-dideoxy-CTP, and didehydro(d4)CTP. The yeast mtDNA polymerase used ddTTP, 3'-fluoro-TTP, and ddCTP almost as efficiently as natural deoxynucleoside trisphosphates. Both 3'AZTTP and d4CTP were each significantly less efficient as substrates. Overall, the kinetic data with mtDNA polymerase were very similar to those of the recombinant human immunodeficiency virus reverse transcriptase control. Terminally incorporated AZTTP or ddTTP was not removed by the 3'-5' exonuclease activity of mtDNA polymerase. This may explain the inhibition of mtDNA replication observed in anti-human immunodeficiency virus treatment with dideoxynucleoside analogs for their effects of mtDNA polymerase could be of value in future rational drug design.
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PMID:Efficient incorporation of anti-HIV deoxynucleotides by recombinant yeast mitochondrial DNA polymerase. 764 50

Seven days after activation with concanavalin A and irradiated spleen cells, murine CD4+ T cells were re-stimulated with ionomycin and phorbol 12-myristate 13-acetate (PMA). IL-2 and IL-4 were determined in the supernatant. When cholera toxin, forskolin together with phosphodiesterase inhibitors or dibutyryl-cAMP were added at the time of re-stimulation, a dose-dependent increase of IL-4 and IL-5 release was noted. IL-2 was down-regulated as reported before. The up-regulation of IL-4 and the down-regulation of IL-2 correlated with an increase of IL-4 mRNA and a decrease of IL-2 mRNA as determined by semi-quantitative reverse transcriptase polymerase chain reaction. Similar results were found with prostaglandin E2 using PMA and ionomycin or plate-bound anti-CD3 antibody as re-stimulants. These results suggest that, in activated CD4+ T cells, cAMP-elevating agents induce a switch of lymphokine production towards a Th2-like phenotype through regulation at the transcriptional level. This is supported by the fact that complex formation between a synthetic nuclear factor of activated T cells (NF-AT) binding site from the IL-2 promoter and nuclear extracts was decreased when cholera toxin was added to re-activated CD4+ T cells, suggesting that cholera toxin and cAMP down-regulate IL-2 expression via decreased NF-AT binding. Finally, since IL-4 has been reported to amplify IL-4 release from activated CD4+ T cells, the autoinduction of IL-4 may very well function via cAMP.
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PMID:cAMP up-regulates IL-4 and IL-5 production from activated CD4+ T cells while decreasing IL-2 release and NF-AT induction. 781 41

A human cDNA clone encoding autotaxin, a tumor cell motility-stimulating protein, reveals that this protein is an ecto/exo-enzyme with significant homology to the plasma cell membrane differentiation antigen PC-1. ATX is a 125-kDa glycoprotein, previously isolated from a human melanoma cell line (A2058), which elicits chemotactic and chemokinetic responses at picomolar to nanomolar concentrations. Affinity-purified antipeptide antibodies to the ATX peptide, ATX-102, were employed to screen an A2058 cDNA expression library made in lambda gt11. The partial cDNA sequence which was obtained was then extended by utilizing reverse transcriptase on total cellular RNA followed by polymerase chain reaction amplification. The isolated cDNA clone contained 3251 base pairs, and the mRNA message size was approximately 3.3 kilobases. The deduced amino acid sequence of autotaxin matched 30 previously sequenced peptides and comprised a protein of 915 amino acids. Data base analysis of the ATX sequence revealed a 45% amino acid identity (including 30 out of 33 cysteines) with PC-1, a pyrophosphatase/type I phosphodiesterase expressed on the surface of activated B cells and plasma cells. ATX, like PC-1, was found to hydrolyze the type I phosphodiesterase substrate p-nitrophenyl thymidine-5'-monophosphate. Autotaxin now defines a novel motility-regulating function for this class of ecto/exo-enzymes.
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PMID:cDNA cloning of the human tumor motility-stimulating protein, autotaxin, reveals a homology with phosphodiesterases. 798 64


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