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 extension of mismatched 3'-termini of DNA was implicated as a major determinant that contributes to the low fidelity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). However, HIV-1 RT exhibits variations in its comparative efficiency to extend different 3'-mismatched base pairs that can result either from the differences in the binding capacity of the enzyme to various mispaired DNAs or from differences in the rate of extension of mispairs by a DNA-bound enzyme. In the current study we have examined the interaction of HIV-1 RT with mispaired template-primer 3'-termini, using a gel retardation assay. HIV-1 RT was found to bind mismatched template-primers with purine-pyrimidine (i.e., A . C) and purine-purine (i.e., A . A and A . G) 3'-terminal mispairs to about the same extent. Hence, HIV-1 RT can be considered (in addition to its other basic features) as a 3'-mismatched DNA binding protein. The stability of the complexes formed between HIV-1 RT and the mismatched template-primers tested seems to be unaffected significantly by neighboring sequences and by the presence of the next complementary dNTP. Thus, the dissimilarities observed previously in extension frequencies in the extension of 3'-terminal mismatches are likely to be due to an inherent property of the HIV-1 RT. The fact that HIV-1 RT binds 3'-mismatch-containing template-primers suggests that unextended mismatched DNA can undergo a rebinding process followed by a 3'-mismatch extension, contributing to further understanding of the low fidelity characteristic of HIV-1 RT. It is possible, therefore, that the interaction of the RT with the DNA may constitute an additional suitable target for the development of specific anti-HIV-1 RT drugs.
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PMID:The interaction of the reverse transcriptase of human immunodeficiency virus type 1 with 3'-terminally mispaired DNA. 883 43

HIV-1 replication requires the translocation of viral genome into the nucleus of a target cell. We recently reported the synthesis of an arylene bis(methyl ketone) compound (CNI-H0294) that inhibits nuclear targeting of the HIV-1 genome and thus HIV-1 replication in monocyte cultures. Here we demonstrate that CNI-H0294 inhibits nuclear targeting of HIV-1-derived preintegration complexes by inactivating the nuclear localization sequence of the HIV-1 matrix antigen in a reaction that absolutely requires reverse transcriptase. This drug/reverse transcriptase interaction defines the specificity of its antiviral effect and is most likely mediated by the pyrimidine side-chain of CNI-H0294. After binding to reverse transcriptase, the carbonyl groups of CNI-H0294 react with the nuclear localization sequence of matrix antigen and prevent its binding to karyopherin alpha, the cellular receptor for nuclear localization sequences that carries proteins into the nucleus. Our results provide a basis for the development of a novel class of compounds that inhibit nuclear translocation and that can, in principle, be modified to target specific infectious agents.
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PMID:Critical role of reverse transcriptase in the inhibitory mechanism of CNI-H0294 on HIV-1 nuclear translocation. 887 28

Nucleoside transport may be involved in the regulation of extracellular levels of adenosine, an inhibitory neuromodulator in the central nervous system. Previous reports have provided functional evidence for Na+-dependent nucleoside transport in rat brain. We isolated total RNA from various regions of rat brain and tested for the presence of mRNA for two recently cloned Na+/nucleoside cotransporters using reverse transcriptase PCR (RT-PCR). Messenger RNA for a pyrimidine-selective Na+/nucleoside cotransporter mRNA (rCNT1) was detected in samples from each brain region tested by RT-PCR amplification of a 309-bp DNA product. Southern blot and sequence analysis confirmed that this product was derived from rCNT1 mRNA. A purine-selective Na+/nucleoside cotransporter mRNA (rCNT2, also termed SPNT) was detected throughout brain by amplifying a 235-bp DNA product, the sequence of which was identical to that published. These experiments demonstrate the presence of both rCNT1 and rCNT2 mRNA in rat brain.
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PMID:Demonstration of the existence of mRNAs encoding N1/cif and N2/cit sodium/nucleoside cotransporters in rat brain. 901 95

We report identification of a new human nucleoside transporter protein by molecular cloning and functional expression of its cDNA. Previously, we used expression selection in Xenopus oocytes to isolate a cDNA from rat jejunal epithelium encoding the pyrimidine-selective Na+-dependent nucleoside transporter rCNT1 (Q.-Q. Huang, S. Y. M. Yao, M. W. L. Ritzel, A. R. P. Paterson, C. E. Cass, and J. D. Young. J. Biol. Chem. 269: 17757-17760, 1994). cDNAs for a human homologue of rCNT1, designated hCNT1, have been isolated from human kidney by hybridization cloning and reverse transcriptase polymerase chain reaction amplification strategies. hCNT1 was 83% identical to rCNT1 in amino acid sequence and exhibited the transport characteristics of an Na+-dependent nucleoside transporter with selectivity for pyrimidine nucleosides and adenosine when expressed in Xenopus oocytes. Deoxyadenosine, which undergoes net renal secretion, and guanosine were poor permeants. hCNT1 did, however, transport 3'-azido-3'-deoxythymidine. This is the first demonstration that members of the CNT family exist in human cells and provides evidence of their involvement in the renal transport of physiological nucleosides and nucleoside drugs. The hCNT1 gene was mapped to chromosome 15q25-26.
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PMID:Molecular cloning and functional expression of cDNAs encoding a human Na+-nucleoside cotransporter (hCNT1). 912 15

The anti-HIV-1 and cytotoxic activities of some viral reverse transcriptase inhibitors, namely the analogues of [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-xylo- and -ribofuranosyl]]-3'-spiro-5" -[4"-amino-1",2"-oxathiole 2",2"-dioxide] (TSAO) pyrimidine and pyrimidine modified-nucleotides, are analysed in relation to their physicochemical and molecular properties. The antiviral activities of the compounds are found to be significantly correlated with hydrophobic and electronic properties of the molecules, but no physicochemical parameters were found to be correlated with the cytotoxic effects of the compounds. This difference is exploited to improve the selectivity of the compounds. It is observed that TSAO can provide potent anti-HIV-1 drugs with a disubstituted thymine ring, in which a substituent may be at the N3-position. The disubstitution reduces the cytotoxicity, and substituents' hydrophobicity and electron donating character enhance the antiviral activity.
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PMID:Quantitative structure-activity relationship studies on some viral reverse transcriptase inhibitors acting as anti-HIV-1 agents. 920 86

Nucleoside-diphosphate kinase (NDP kinase) from the matrix space of mitochondria in pigeon liver was purified to homogeneity. Degenerate oligonucleotide primers to the N-terminal sequence of the purified protein and the region containing the active site histidine were used in reverse transcriptase-polymerase chain reaction to obtain a major portion of the coding sequence for the mature protein. The sequences of the C and N termini of the mature protein, and eight residues in the signal peptide, were obtained by rapid amplification of cDNA end procedures. The entire coding sequence of a cytosolic form of NDP kinase was also determined. Both isoforms, which share 53% sequence identity, possess the characteristically conserved regions of known NDP kinases. The mature mitochondrial NDP kinase protein migrates in molecular sieving columns with an apparent molecular mass of about 66 kDa. It shows very high thermal stability even though it lacks the proline residue in the killer of prune loop, and the Tyr/Glu C termini that are important in stabilizing other NDP kinases. The affinity of the mitochondrial isoform for adenine and guanine nucleotides is much higher than for pyrimidine nucleotides, but the enzyme is especially susceptible to substrate inhibition by GDP. Semi-quantitative reverse transcriptase-polymerase chain reaction showed that the relative levels of expression of the mitochondrial isoform are liver > kidney >> heart = brain > breast muscle. The cytosolic isoform is strongly and approximately equally expressed in these same five tissues. This work is the first characterization of a NDP kinase isoform that is found in the matrix space of mitochondria.
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PMID:Characterization and cloning of a nucleoside-diphosphate kinase targeted to matrix of mitochondria in pigeon. 930 28

Platelet basic protein (PBP) is a chemokine family member that is only found in platelets and their precursors megakaryocytes. The PBP gene is physically linked to the gene for another platelet-specific chemokine, platelet factor 4. While the biological basis of platelet factor 4 expression has been pursued by others, the regulatory features controlling the platelet-specific expression of PBP have not been investigated. In this article, we examined the molecular basis by which this megakaryocyte-specific gene is regulated. Transient expression studies of truncated reporter constructs containing from 4.5 to 0.1 kilobases of the functional PBP gene 5'-flanking region, demonstrated that the proximal 0.1 kilobases of the promoter was sufficient for high levels of expression in human erythroleukemia and CHRF-288 cells, two megakaryocytic cell lines. However, none of these constructs was expressed above background levels in HeLa and 293 cells, two non-megakaryocytic cell lines. Further truncation of this promoter suggested that there was an important regulatory element(s) within a pyrimidine-rich tract. Mobility shift analysis of the pyrimidine-rich tract defined a region between -85 and -64 which bound to a nuclear factor(s). This region contains sequences matching the consensus Ets-binding site from -78 to -75 base pairs. In particular, we noted that this site matched a PU.1 consensus sequence known as a PU box. Mobility shift and supershift studies with nuclear extracts as well as recombinant PU.1 protein and anti-PU.1 antibody further confirmed that PU.1 was the specific Ets family factor that bound to this site. Transient expression assays using reporter constructs which contained point mutations that abrogated PU.1 binding also significantly reduced PBP promoter activity in human erythroleukemia and CHRF cells. In addition, while all reporter gene constructs containing PBP promoters were completely inactive in HeLa cells, transactivation experiments using a PU.1 expression construct demonstrated that exogenous expression of PU.1 could increase reporter gene expression up to 8-fold in these cells. Finally, the role of PU.1 in PBP gene expression was compared between wild-type and PU.1-null embryonic stem (ES) cells that were differentiated in vitro into cells that resembled megakaryocytes both morphologically and immunologically. We found that PBP gene expression in the differentiated PU.1(-/-) null ES cells (as determined by semi-quantitative reverse transcriptase-polymerase chain reaction) was more than four times lower than that in the wild-type ES cells, while other platelet-specific genes were expressed equally or similarly in the two ES cell lines. Previous reports have shown that PU.1 is expressed in several hematopoietic lineages, including megakaryocytes. However, the functional role of PU.1 has only been previously demonstrated in the myeloid and lymphoid lineages. Therefore, our studies are the first to show the biological importance of this nuclear factor in the regulated expression of a megakaryocyte-specific gene.
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PMID:Activation of the megakaryocyte-specific gene platelet basic protein (PBP) by the Ets family factor PU.1. 933 92

This article describes several approaches to a selective therapy of virus infections: (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU [brivudin]) for the therapy of herpes simplex virus type 1 and varicella-zoster virus infections: (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC [cidofovir]) for the therapy of various DNA virus (i.e., herpesvirus, adenovirus, papillomavirus, polyomavirus, and poxvirus) infections; 9-(2-phosphonylmethoxyethyl)adenine (PMEA [adefovir]) for the therapy of retrovirus, hepadnavirus, and herpesvirus infections; (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) for the therapy and prophylaxis of retrovirus and hepadnavirus infections; and nonnucleoside reverse transcriptase inhibitors (NNRTIs), such as tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2(IH)-one and -thione (TIBO), 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT), alpha-anilinophenylacetamide (alpha-APA), and 2',5'bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"-oxat hiole- 2",2"-dioxide)pyrimidine (TSAO) derivatives, and thiocarboxanilides for the treatment of human immunodeficiency virus type 1 (HIV-1) infections. For the clinical use of NNRTIs, some guidelines have been elaborated, such as starting treatment with combinations of different compounds at sufficiently high concentrations to effect a pronounced and sustained suppression of the virus. Despite the diversity of the compounds described here and the different viruses at which they are targeted, they have a number of characteristics in common. As they interact with specific viral proteins, the compounds achieve a selective inhibition of the replication of the virus, which, in turn, should be able to develop resistance to the compounds. However, as has been established for the NNRTIs, the problem of viral resistance may be overcome if the compounds are used from the start at sufficiently high doses, which could be reduced if different compounds are combined. For HIV infections, drug treatment regimens should be aimed at reducing the viral load to such an extent that the risk for progression to AIDS will be minimized, if not avoided entirely. This may result in a real "cure" of the disease but not necessarily of the virus infection, and in this sense, HIV disease may be reduced to a dormant infection, reminiscent of the latent herpesvirus infections. Should virus replication resume after a certain time, the armamentarium of effective anti-HIV and anti-herpesvirus compounds now available, if applied at the appropriate dosage regimens, should make the virus return to its dormant state before it has any chance to damage the host. It is unlikely that this strategy would eradicate the virus and thus "cure" the viral infection, but it definitely qualifies as a cure of the disease.
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PMID:In search of a selective antiviral chemotherapy. 933 68

Stavudine (d4T) is a pyrimidine nucleoside analogue used in the treatment of human immunodeficiency virus (HIV) infection. It inhibits viral reverse transcriptase as do zidovudine (AZT), didanosine (ddI), zalcitabine (ddC) and lamivudine (3TC), which comprise the family of nucleoside HIV-reverse transcriptase inhibitors. Stavudine is currently approved by the US Food and Drug Administration for the treatment of patients who have become intolerant to or have failed to response to zidovudine, didanosine or zalcitabine therapy. Oral administration of stavudine results in maximal concentrations within 2 hours and increases linearly as doses increase. The absolute oral bioavailability is high, approaching 100%. There is evidence to suggest that stavudine does not accumulate in the plasma. It distributes into total body water and appears to enter cells by non-facilitated diffusion. Penetration into the cerebrospinal fluid occurs, as does the transfer of the drug across human placental tissue. Stavudine is cleared quickly by both renal and nonrenal processes. The pharmacokinetic properties of stavudine in children are similar to those of adults. The pharmacokinetic parameters of stavudine were not affected by simultaneous administration of didanosine. It appears that stavudine at doses < 2 mg/kg/day is most efficient at increasing CD4 + cell numbers. While stavudine is reported to be less cytotoxic than zidovudine, the principal toxicity in humans is peripheral neuropathy and appears to be related to daily, but not cumulative, doses.
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PMID:Clinical pharmacokinetics of stavudine. 934 3

A series of pyrimidine thioethers was synthesized and evaluated for inhibitory properties against wild-type HIV-1 reverse transcriptase (RT) and an RT carrying the resistance-conferring mutation P236L. Modifications of both the pyrimidine and the functionality attached through the thioether yielded several analogues, which demonstrated activity against both enzyme types, with IC50 values as low as 190 nM against wild-type and 66 nM against P236L RT. Evaluation of a select number of pyrimidine thioethers in cell culture showed that these compounds have excellent activity against HIV-1IIIB-WT and retain good activity against a laboratory-derived HIV-1MF delavirdine-resistant variant.
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PMID:Pyrimidine thioethers: a novel class of HIV-1 reverse transcriptase inhibitors with activity against BHAP-resistant HIV. 974 54


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