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:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recombinant vaccinia viruses have been proposed as live vaccines against a variety of infectious diseases, including AIDS (acquired immune deficiency syndrome). Objections have been concerned primarily with side effects of the vaccinia virus vector itself. Recently it has been shown that inactivation of the vaccinia virus thymidine kinase gene or deletion of certain other non-essential genes is associated with a marked reduction in pathogenicity. Nevertheless, the ability of vaccinia virus to produce a progressive infection in immunodeficient individuals remains a most serious problem. Indeed, an incident of this type in a vaccinated man seropositive for human immunodeficiency virus was recently reported. We have used immunodeficient athymic nude mice to establish a model of disseminated vaccinia virus infection, and to demonstrate a novel approach to virus attenuation which involves insertion of a gene encoding human interleukin-2 into the genome of vaccinia virus vectors.
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PMID:Prevention of vaccinia virus infection in immunodeficient mice by vector-directed IL-2 expression. 311 19

The activities of CDP reductase and thymidine kinase in 10(6) to 5 X 10(6) phytohemagglutinin (PHA)-stimulated lymphocytes isolated from 2 to 5 ml of peripheral blood of individual subjects were measured. The activities of CDP reductase (pmol/h/10(7) cells) and thymidine kinase (nmol/h/10(7) cells) were high in infants, 698 +/- 307 and 64.2 +/- 20.2, constant in subjects of 1-40 years old, 401 +/- 181 and 38.1 +/- 15.3, and low in persons of more than 80 years old, 121 +/- 113 and 22.3 +/- 17.8, respectively. The ratio of thymidine kinase to CDP reductase activity increased with age, indicating that dependency on the salvage pathway of DNA synthesis in lymphocytes increased with age. The activities of CDP reductase and thymidine kinase were reduced in patients with the hyperimmunoglobulin E syndrome, congenital cytomegalovirus infection, anhidrotic ectodermal dysplasia with hyperimmunoglobulin A, Bloom's syndrome, immunodeficiency with hyperimmunoglobulinemia, and Down's syndrome. The clinical symptoms of these diseases seem to be due to impaired DNA synthesis of PHA-stimulated lymphocytes, but the degrees of reduction of enzyme activities were generally greater than that of thymidine incorporation in these patients.
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PMID:Cytidine 5'-diphosphate reductase and thymidine kinase activities in phytohemagglutinin-stimulated lymphocytes of normal subjects of various ages and patients with immunodeficiency. 638 37

Plasmids were constructed whereby the expression of a reporter gene, either the cDNA corresponding to the secreted form of human alkaline phosphatase (SEAP) or the herpes simplex virus type 1 (HSV1) thymidine kinase (tk) gene, was rendered dependent upon the expression of the human immunodeficiency virus type 1 (HIV1) tat and rev proteins. The SEAP or tk genes were placed between HIV1 splice donor and acceptor sites. One SEAP construct carried a series of alternating splice donor and acceptor sites. In all cases, the rev response element mapped within an intron. Despite such mimicry of the HIV1 genome, residual expression of the reporter gene in the absence of tat and rev was observed. These results, as well as non-specific T-cell recruitment, suggest limits to the specificity of using HIV-activated toxic gene expression to kill HIV-infected cells.
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PMID:Residual expression of reporter genes in constructs mimicking HIV genome organization. 748 Oct 89

Retroviruses evolve at rapid rates, which is presumably advantageous for responding to selective pressures. Understanding the basic mutational processes involved during retroviral replication is important for comprehending the ability of retroviruses to escape immunosurveillance and antiviral drug treatment. Moreover, since retroviral vectors are important vehicles for somatic cell gene therapy, knowledge of the mechanism of retroviral variation is critical for anticipating untoward mutational events occurring during retrovirus-medicated gene transfer. The focus of this report is to examine the spectrum of genomic rearrangements arising during a single cycle of Moloney murine leukemia virus (MoMLV) vector virus replication. An MoMLV vector containing the herpes simplex virus thymidine kinase (tk) gene was constructed. MoMLV vector virus was produced in packaging lines, and target cells were infected. From a total of 224 mutant proviruses analyzed, 114 had gross rearrangements readily detectable by Southern blotting. The remaining proviruses were of parental size. PCR and DNA sequence analysis of 73 of the grossly rearranged mutant proviruses indicated they resulted from deletions, combined with insertions, duplications, and complex mutations that were a result of multiple genomic alterations in the same provirus. Complex hypermutations distinct from those previously described for spleen necrosis virus and human immunodeficiency virus were detected. There was a correlation between the mutation breakpoints and single-stranded regions in the predicted viral RNA secondary structure. The results also confirmed that the tk gene is inactivated at an average rate of about 8.8% per cycle of retroviral replication, which corresponds to a rate of mutation of 3%/kbp.
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PMID:Genetic rearrangements occurring during a single cycle of murine leukemia virus vector replication: characterization and implications. 749 12

L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides.
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PMID:Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate. 754 86

Human immunodeficiency virus (HIV) reverse transcriptase substitutes for temperature-sensitive DNA polymerase I (Pol Its) in Escherichia coli, providing a screen for anti-HIV reverse transcriptase nucleoside analogs in bacteria. Since phosphorylation of nucleosides in E. coli is limited to thymidine and its derivatives, we coexpressed herpes simplex virus thymidine kinase, an enzyme that phosphorylates a wide variety of nucleoside analogs, together with HIV reverse transcriptase. Coexpression of herpes simplex virus thymidine kinase and HIV reverse transcriptase rendered Pol Its cells sensitive to dideoxycytidine. Studies with different nucleoside analogs indicate that this bacterial screening system is able to select and identify nucleoside analogs that specifically target HIV reverse transcriptase.
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PMID:A screen in Escherichia coli for nucleoside analogs that target human immunodeficiency virus (HIV) reverse transcriptase: coexpression of HIV reverse transcriptase and herpes simplex virus thymidine kinase. 754 49

3'-Azido-2',3'-dideoxy-5-iodouridine (AzIdUrd) and 3'-azido-2',3'-dideoxy-5-bromouridine (AzBdUrd), previously shown to be potent and selective inhibitors of human immunodeficiency virus replication in vitro were minimally toxic to the uninfected human lymphoid cell line H9 (IC50 = 197 and 590 microM, respectively). Both compounds strongly inhibited the incorporation of [3H]thymidine but not [3H]deoxyadenosine into DNA, and we observed no significant inhibition of [3H]uridine incorporation into RNA or [3H]amino acid incorporation into protein. Exposure of H9 cells to AzIdUrd or AzBdUrd (100 microM, 24 hr) and pulse-labeling with [3H]thymidine resulted in approximately 80% reduction in levels of tritiated dTMP, dTDP, and dTTP relative to control. [125I]AzIdUrd was phosphorylated rapidly in H9 cells with the monophosphate accounting for over 90% of total soluble radioactivity. A relatively low but stable level of AzIdUTP was maintained over a 12-hr period. [125I]AzIdUrd was phosphorylated by a cell free extract of H9 cells at a rate approximately three times that of thymidine and its phosphorylation was inhibited by excess thymidine. AzIdUrd was found to be a competitive inhibitor of cytosolic thymidine kinase with a Ki of 2.63 microM and AzIdUMP a weak competitive inhibitor of thymidylate kinase with a Ki of 55.3 microM. Both AzIdUTP and AzBdUTP were potent competitive inhibitors of HIV-1 reverse transcriptase (Ki = 0.028 and 0.043 microM, respectively) and relatively poor inhibitors of H9 cell DNA polymerase alpha (Ki = 42.0 and 42.7 microM, respectively). Thus, the high therapeutic index of these compounds is due to the sensitivity of the viral reverse transcriptase, coupled with the relative insensitivity of the host cell DNA polymerase alpha.
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PMID:Metabolism and mode of selective inhibition of human immunodeficiency virus replication by 3'-azido-2',3'-dideoxy-5-iodouridine and 3'-azido-2',3'-dideoxy-5-bromouridine. 767 40

Activation of the anti-human immunodeficiency virus (HIV) compound 3'-azido-3'-deoxythymidine (AZT) is dependent on its 5'-phosphorylation by cellular nucleoside and nucleotide kinases. Azidothymidine 5'-triphosphate (AZTTP) is considered to be the metabolite responsible for both the anti-HIV effect of AZT, via inhibition of reverse transcriptase, and cytoxicity by interference with cellular DNA polymerases. During the characterization of AZT metabolism in cultured human T-lymphoblastoid CEM cells, a spontaneously occurring variant cell line, CEM/Ag-1, was found that showed approximately 10-fold resistance to AZT growth inhibition as compared to wild type (wt) cells (EC50 = 2 mM as compared to 350 microM for wt cells). CEM/Ag-1 cells had a 3-fold reduced capacity to accumulate azidothymidine monophosphate (AZTMP) compared to wt cells whereas similar levels of AZTTP were found in both cell lines. The intracellular half-life of AZTMP was approximately 70 min in both wt and CEM/Ag-1 cells. A 3-fold lower specific activity of cytoplasmic thymidine kinase was observed in CEM/Ag-1 extracts as compared to wt. The reduced thymidine kinase activity was not correlated to a decreased level of thymidine kinase mRNA. Syncytium formation of CEM/Ag-1 cells infected with HIV-2 as well as HIV-1 antigen production was inhibited at the same concentrations of AZT (approx. 0.01 microM) as were HIV-1 and HIV-2 infected wt cells. Thus, minor decreases in cellular thymidine kinase levels may markedly affect the cytoxicity of AZT but have no major effect on the antiviral activity of AZT. Our results strongly suggest that AZTMP is responsible for a major part of the growth inhibitor effects, while AZTTP mainly mediates the antiviral activity of AZT.
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PMID:Cytotoxicity of 3'-azido-3'-deoxythymidine correlates with 3'-azidothymidine-5'-monophosphate (AZTMP) levels, whereas anti-human immunodeficiency virus (HIV) activity correlates with 3'-azidothymidine-5'-triphosphate (AZTTP) levels in cultured CEM T-lymphoblastoid cells. 770 41

Long term administration of 3'-azidothymidine (AZT) for the treatment of AIDS has led to detrimental clinical side effects in some patients, the biochemical causes of which are still being delineated. Base-substituted, azido-nucleotide photoaffinity analogs have routinely proven to be effective tools for identifying and characterizing nucleotide-utilizing enzymes. Therefore, we have synthesized 5-azido-3'-azido-2',3'-dideoxyuridine, which is a potential photoaffinity analog of two human immunodeficiency virus drugs, AZT and 3'azido-2',3'-dideoxyuridine. A partially purified herpes simplex virus type 1 thymidine kinase and [gamma-32P]ATP were used to make an AZT monophosphate analog, [32P]5-azido-3'-azido-2',3'-dideoxyuridine monophosphate. The photoaffinity properties of this analog were initially tested with herpes simplex virus type 1 thymidine kinase. Photoaffinity labeling of this enzyme was saturable (half-maximal, 30 microM) and could be specifically inhibited by AZT, AZT monophosphate, thymidine, and thymidine monophosphate. Photolabeling of rat liver microsomal membranes was also done, and several membrane proteins that interact with AZT monophosphate were identified. The antiviral and cytotoxic activities of 5-azido-3'-azido-2',3'-dideoxyuridine were determined using human immunodeficiency virus, type 1 strain IIIB and an AZT drug-resistant strain in human T lymphocyte H9 cells.
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PMID:Synthesis of a photoaffinity analog of 3'-azidothymidine, 5-azido-3'-azido-2',3'-dideoxyuridine. Interactions with herpesvirus thymidine kinase and cellular enzymes. 777 17

This article reviews uses of metabolic suicide genes in gene therapy. Suicide genes encode novel nonmammalian enzymes that can convert a relatively nontoxic prodrug into a highly toxic agent. Cells genetically transduced to express such genes essentially commit metabolic suicide in the presence of the appropriate prodrug. Three metabolic suicide genes are described: herpes simplex thymidine kinase, Escherichia coli cytosine deaminase and varicella zoster thymidine kinase. Transfer and expression of these genes into mammalian cells is described. Preclinical models of suicide gene therapy of cancer and human immunodeficiency virus are discussed, and several clinical trials employing suicide genes are described.
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PMID:Metabolic suicide genes in gene therapy. 780 80


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