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: UMLS:C0679427 (myeloblastosis)
982 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The action of 3'-azido-3'-deoxythymidine 5'-triphosphate (N3dTTP) on DNA strand elongation catalyzed by human immunodeficiency virus type 1 reverse transcriptase was evaluated in comparison with human DNA polymerase alpha and proliferating cell nuclear antigen-independent DNA polymerase delta. Sequencing gel analysis demonstrated that the human immunodeficiency virus 1 reverse transcriptase preferentially incorporated N3dTTP into the T sites of the growing DNA strands and caused chain termination in a dose-dependent manner. This effect was observed even when the N3dTTP concentration was 0.3 microM, 100-fold less than dTTP. Studies with reverse transcriptases from avian myeloblastosis virus and Moloney murine leukemia virus showed that N3dTTP was also efficiently incorporated into DNA by these enzymes and terminated DNA strand elongation. In contrast, human DNA polymerases alpha and delta did not incorporate detectable amounts of N3dTTP into the DNA and were not inhibited by 300 microM N3dTTP. The selective incorporation of the chain-terminating nucleotide by the viral reverse transcriptases appears to be a molecular basis for the positive therapeutic index of 3'-azido-3'-deoxythymidine.
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PMID:Selective action of 3'-azido-3'-deoxythymidine 5'-triphosphate on viral reverse transcriptases and human DNA polymerases. 169 49

Rubromycins, a class of quinone antibacterials, were discovered to selectively inhibit human immunodeficiency virus-1 (HIV-1) RNA-directed DNA polymerase (reverse transcriptase) (RT) activity more potently than cellular DNA polymerase alpha. beta- and gamma-rubromycin each inhibited equipotently HIV-1 RT and avian myeloblastosis virus RT, in a concentration-dependent manner, and were significantly weaker as inhibitors of calf thymus DNA polymerase alpha. These agents inhibited HIV-1 RT reversibly, were competitive with respect to template.primer, and were noncompetitive with respect to TTP. Dixon analyses yielded HIV RT Ki values of 0.27 +/- 0.014 and 0.13 +/- 0.012 microM for beta- and gamma-rubromycin, respectively. Similarly, using DNA polymerase alpha, the Ki values were 25.1 +/- 4.3 and 3.9 +/- 0.6 microM for beta- and gamma-rubromycin, respectively. Because these agents were toxic to noninfected human T lymphoid cells using concentrations at or above 6 microM, HIV-1 infectivity studies were carried out at 0.8-6 microM. At these concentrations, which are below the range expected to provide protection, no significant antiviral activity was observed. Although beta- and gamma-rubromycins did not possess sufficient HIV RT inhibitory potency or selectivity versus mammalian DNA polymerase to demonstrate antiviral activities, these studies support the hypothesis that specific molecules containing quinone functional groups can selectively inhibit viral polymerase activities over cellular polymerase activities. In addition, these studies suggest that rubromycins may be lead structures for the development of more potent and selective agents.
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PMID:Inhibition of human immunodeficiency virus-1 reverse transcriptase activity by rubromycins: competitive interaction at the template.primer site. 169 17

The genome of human immunodeficiency virus (HIV) and especially the envelope gene are mutated with unusually high frequency during in vivo replication. Recent studies indicate that HIV reverse transcriptase (RT) is unusually error prone and that the number of generated mutations is disproportionately high within repetitive base sequences. To study the ability of recombinant and wild-type HIV RT to traverse specific homo-oligomeric stretches, we used bacteriophage M13 DNA templates that contain different oligo(purine) and oligo(pyrimidine) inserted tracts. The progress of HIV RT along these templates was potently inhibited from further progression only at a (dA)16 insert. Comparison with other polymerases indicates that the almost complete blockage of polymerization beyond an oligo(dA) insert is unique to HIV RT and Moloney murine leukemia virus RT, which has high sequence homology with HIV RT. The extent of termination of HIV RT at the oligo(dA) run is not affected by alterations in the concentration of KCl, Mg2+, dNTP, or by a decrease in pH. Obstruction of HIV RT opposite the oligo(dA) insert is not alleviated by moving the primer position further upstream from the oligo(dA) insert. Lastly, HIV RT purified directly from virions is also specifically arrested at an oligo(dA) tract. Competition experiments indicate that the concentration of active HIV RT in the presence of M13(dA)16 DNA is similar to that observed in the presence of M13(dG)16 DNA. In addition, preincubation of M13(dA)16 DNA with HIV RT does not subsequently inhibit avian myeloblastosis virus RT from successfully traversing the (dA)16 insert. Therefore, it appears that the blockage of chain elongation of HIV RT at the (dA)16 insert is not the result of trapping the enzyme at this site.
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PMID:Synthesis of DNA by human immunodeficiency virus reverse transcriptase is preferentially blocked at template oligo(deoxyadenosine) tracts. 169 89

2-Chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) was compared with dATP as a substrate for DNA synthesis by bacterial and viral DNA polymerases in vitro. Lengths of chain extension and DNA synthesis pause sites were determined by comparison with products generated by dideoxynucleotide sequencing methods on the same end-labeled primer/template duplex after high-resolution polyacrylamide gel electrophoresis. Reverse transcriptase (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) incorporated CldATP efficiently. DNA strand elongation continued past most chloroadenine (ClA) insertion sites but resulted in shorter chains than when dATP was inserted. Phage T4 DNA polymerase incorporated CldATP least efficiently; Klenow fragment of Escherichia coli DNA polymerase I and modified T7 DNA polymerase (Sequenase) showed intermediate ability to utilize the analogue. Incorporation of several consecutive ClA residues into the replicating strand dramatically reduced the ability of Sequenase, Klenow fragment, and T4 DNA polymerases to continue strand elongation. In the absence of the corresponding normal deoxyribonucleoside triphosphate during DNA synthesis, ClA was frequently misincorporated as thymine, cytosine, or guanine by both AMV RT and HIV-1 RT but rarely, if at all, by Klenow fragment, Sequenase, and T4 DNA polymerase. Except T4, for most DNA polymerases, CldATP at 10-20-fold molar excess over dATP was not a strong competitive inhibitor of dATP, as judged by the amount of strand extension and polymerase pause sites during DNA synthetic reactions. Our results indicate that the degree of strand extension in the presence of CldATP, the number and location of polymerase pause sites, and the amount of misincorporation of the analogue are both polymerase- and sequence-dependent.
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PMID:Effects of 2-chloro-2'-deoxyadenosine 5'-triphosphate on DNA synthesis in vitro by purified bacterial and viral DNA polymerases. 170 19

The functional interaction between the RNA-dependent DNA polymerase and the RNase H activities of reverse transcriptases (RTs) were examined using a 272 nucleotide long plasmid-derived RNA transcript primed in a specific location. Properties of the avian myeloblastosis virus (AMV) RT, the human immunodeficiency virus RT and the Moloney murine leukemia virus RT were examined. All three enzymes formed stable complexes with the primer-template with half-lives ranging from about 16 to 41 s. Each enzyme synthesized full-length primer extension products and cleaved the RNA template at least once during DNA synthesis. Polymerization was then assayed in the presence of challenger RNA that effectively sequestered RTs after one round of processive DNA synthesis. This assay allowed measurement of the number of endonucleolytic cleavages catalyzed by the RT during one encounter with the primer-template. Results indicated that each of the three RTs cut the transcript before dissociating from the primer-template, whether or not deoxynucleoside triphosphates were present to allow synthesis. During synthesis, the extent of RNA degradation differed among the RTs, with AMV-RT generating mostly large segments of RNA-DNA hybrid, and virtually no small RNA cleavage products. Human immunodeficiency virus and Moloney murine leukemia virus-RT generated more small degradation products than AMV-RT, but still left much of the potentially degradable hybrid undigested. Results demonstrate that the RNase H function is much less active than the polymerization function during processive DNA synthesis and that the activities are not strictly coupled.
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PMID:Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled. 170 86

Inhibition of human immunodeficiency virus reverse transcriptase is currently considered a useful approach in the prophylaxis and intervention of acquired immunodeficiency syndrome (AIDS), and natural products have not been extensively explored as inhibitors of this enzyme. We currently report that the reverse transcriptase assay developed for the detection of the enzyme in virions involving polyadenylic acid.oligodeoxythymidylic acid (poly rA.oligo dT) and radiolabeled thymidine 5'-triphosphate (TTP), can be applied as a simple method for screening the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) inhibitory potential of natural products. As reported herein, 156 pure natural products have been examined in this system. Benzophenanthridine alkaloids such as faragaronine chloride [1] and nitidine chloride, which are known inhibitors of avian myeloblastosis virus reverse transcriptase, demonstrated potent activity in the HIV-1 RT system, and 1 (IC50 10 micrograms/ml) was adopted as a positive-control substance. Additional inhibitors found were columbamine iodide [2] and other protoberberine alkaloids, the isoquinoline alkaloid O-methylpsychotrine sulfate [3], and the iridoid fulvoplumierin [4]. A number of indolizidine, pyrrolizidine, quinolizidine, indole, and other alkaloids, as well as compounds of many other structural classes, were tested and found to be inactive. A total of 100 plant extracts have also been evaluated, and 15 of these extracts showed significant inhibitory activity. Because tannins and other polyphenolic compounds are potent reverse transcriptase inhibitors, methods were evaluated for the removal of these from plant extracts prior to testing. Polyphenolic compounds were found to be responsible for the activity demonstrated by the majority of plant extracts. After appropriate tannin removal procedures were established, the bioassay system was shown to be generally applicable to both pure natural products and plant extracts. The method also proved useful in directing an isolation procedure with Plumeria rubra to yield fulvoplumierin [4] as an active compound (IC50 45 micrograms/ml).
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PMID:Evaluation of natural products as inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. 171 Jun 53

We have examined the properties of reverse transcriptases (RTs) required for strand transfer synthesis on poly(rA). In this process, a primer is elongated on one template and then switches to other templates for additional elongation until it is much longer than the templates on which it was made. Models of retrovirus replication require the RT to catalyze two distinct strand transfers. Additionally, they propose that the RT ribonuclease H (RNase H) activity is involved in both transfers. RTs from human immunodeficiency virus (HIV), avian myeloblastosis virus, and murine leukemia virus differ in molecular mass and subunit composition. However, they all catalyzed strand transfer synthesis on (rA)300, generating characteristically long products. An RNase H-deficient enzyme, HIV-RTRD, catalyzed strand transfer synthesis to the same degree as native HIV-RT, indicating that a functional RNase H activity is not required. Additionally, N-ethylmaleimide, which inhibits RNase H but not polymerase activity of HIV-RT, did not diminish strand transfer synthesis. Highly processive DNA synthesis by each RT was found to be required for the strand transfer reaction. RNase H- murine leukemic virus RT has a structural modification that not only eradicates RNase H, but also makes the polymerase much less processive for DNA synthesis. However, conditions that allow this modified enzyme to bind repeatedly to the same primer during synthesis, i.e. conditions that simulate higher processivity, allow strand transfer synthesis. Catalysis of strand transfer synthesis is not a property of all DNA polymerases, since the Klenow fragment of Escherichia coli DNA polymerase I is unable to catalyze this reaction even if high processivity is simulated. These results suggest that strand transfer synthesis relies on an unidentified functional activity present in RTs.
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PMID:Requirements for the catalysis of strand transfer synthesis by retroviral DNA polymerases. 171 74

Two model substrates were prepared to examine the mechanism of tRNA-primer excision catalyzed by reverse transcriptase associated ribonuclease H (RT-RNase H). The first model substrate contained sequences from the HIV genome and was designed to be structurally similar to the DNA-extended tRNA created by initiation of minus-strand DNA synthesis during retroviral replication. The DNA-extended RNA was a template and was annealed to a DNA oligonucleotide that primed reverse transcription of the RNA in the template. The second model substrate was structurally similar the first substrate but contained sequences unrelated to the HIV viral genome. The RT-RNase H catalyzed excision of the RNA from the template of the two model substrates was examined. Human immunodeficiency virus (HIV) and Moloney murine leukemia virus RT-RNase H hydrolyzed the substrates to leave a single ribonucleotide 5'-phosphate at the 5'-terminus of the model DNA genome. In contrast, avian myeloblastosis virus RT-RNase H hydrolyzed the phosphodiester bond at the DNA-RNA junction. These hydrolytic specificities were not highly dependent on substrate sequence. The importance of these specificities to retroviral integration is discussed. Additional data indicated that the HIV polymerase and RNase H active sites are separated by a distance equivalent to the length of a 15-nucleotide RNA-DNA heteroduplex.
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PMID:Human immunodeficiency virus reverse transcriptase ribonuclease H: specificity of tRNA(Lys3)-primer excision. 171 59

The binding of substrates to recombinant reverse transcriptase from human immunodeficiency virus (HIV) and the natural enzyme from avian myeloblastosis virus (AMV) has been examined by analyzing both the ribonuclease H and the RNA-dependent DNA polymerase activities. With 3'-end-labeled globin mRNA hybridized to (dT)15 as the substrate in the ribonuclease H reaction, the enzymes partially deadenylated the mRNA in a distributive manner. Under these conditions, there was a rapid initial burst followed by a prolonged, but much slower, steady-state rate. The biphasic reaction made possible determinations of kinetic constants as follows: values for Km, KD, and kcat were, respectively, 27 nM, 11 nM, and 5 x 10(-3) s-1 for the HIV enzyme and 30 nM, 9 nM, and 5 x 10(-3) s-1, respectively, for the avian enzyme. These constants were used to derive other parameters: The rate of association of the template-primer with reverse transcriptase was approximately 2 x 10(5) M-1 s-1, and the rate of dissociation was approximately 2 x 10(-3) s-1, regardless of the source of the enzyme. The rate of release of the product was essentially equivalent to the value of kcat indicated above for each of the enzymes. The polymerase reaction was evaluated under processive conditions of synthesis; values of Km and kcat of approximately 6 nM and approximately 2.5 s-1, respectively, for the human enzyme, and approximately 10 nM and approximately 2 s-1, respectively, for the avian enzyme were observed. The interaction of substrates with HIV reverse transcriptase was characterized further with the aid of ribonucleoside-vanadyl complexes. These complexes inhibited the polymerase and ribonuclease H activities of the enzyme competitively with respect to globin mRNA.(dT)15. Values of Ki ranging from 1 to 3 mM were obtained. With respect to deoxyribonucleoside triphosphate substrates in the polymerase reaction, mixed inhibition was observed. Deoxyribonucleoside triphosphates had no effect on kinetic parameters governing the ribonuclease H activity of the HIV enzyme but apparently facilitated the formation of active enzyme. These data fit a model in which one template-primer binding site serves both the polymerase and the ribonuclease H catalytic sites.
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PMID:Reverse transcriptase from human immunodeficiency virus: a single template-primer binding site serves two physically separable catalytic functions. 171 23

Psychotrine dihydrogen oxalate and O-methylpsychotrine sulfate heptahydrate (MP), the salts of isoquinoline alkaloids from ipecac, were found to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT). We currently report the results of additional studies designed to characterize the mechanism of inhibition facilitated by MP. The inhibition was noncompetitive with respect to TTP and uncompetitive with respect to poly(rA) and oligo(dT)12-18 (4:1) at low template-primer concentrations but competitive at high concentrations (greater than 200 microM). Identical non-Michaelis-type kinetics were observed when activated DNA was used as the template. The biphasic nature of the double-reciprocal plots and Hill coefficients of less than 1 indicate that MP functions as an allosteric inhibitor of the enzyme which appears to possess multiple active sites that interact in a cooperative (negative) fashion in the presence of the inhibitor. MP was selective for the recombinant HIV-1 RT (p66) utilizing poly(rA) and oligo(dT)12-18 (4:1) as template-primer. Greater inhibition was observed with this template primer as compared with other natural and synthetic template-primers tested. MP had significantly less effect on avian myeloblastosis virus RT as well as mammalian or bacterial DNA and RNA polymerases. Other members of the ipecac class of alkaloids, e.g. emetine hydrochloride, were inactive against all of these enzymes, including HIV-1 RT. Conversely, MP did not inhibit in vitro protein synthesis, a property manifested by all the other ipecac alkaloids tested. Studies conducted with structural analogs revealed that the imine functionality at positions 1' and 2' of MP is the key structural requirement for HIV-1 RT inhibitory activity. Therefore, MP appears to possess unique structural properties that enable interaction with HIV-1 RT in a manner that can be differentiated from other polymerases. Use of these alkaloids for the definition of this viral enzyme-specific topology may lead to the development of therapeutically useful chemotherapeutic agents.
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PMID:Psychotrine and its O-methyl ether are selective inhibitors of human immunodeficiency virus-1 reverse transcriptase. 172 Oct 50


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