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)

Vimentin is one member of the intermediate filament multigene family which exhibits both tissue- and developmental stage-specific expression. In vivo, vimentin is expressed in cells of mesenchymal origin. Previously, we identified both enhancer and promoter elements in the chicken vimentin gene which regulate gene expression in a positive manner. In this report, we have identified a 40-base-pair region at -568 base pairs between the proximal and distal enhancer elements which represses transcriptional activity. This silencer region can also repress the heterologous herpes simplex virus thymidine kinase promoter, which is comparable to the vimentin promoter. In addition, the element is able to function in a position- and orientation-independent manner, and the amount of repression is increased by multiple copies. Here we show by gel retardation assays and DNase I footprinting that this region binds a protein in nuclear extracts from HeLa cells. Southwestern (DNA-protein) blot analysis indicates this protein is approximately 95 kilodaltons in size. Moreover, protein distribution and activity mimic the expression pattern of vimentin during myogenesis, i.e., protein binding increases as vimentin gene expression decreases. The silencer region shares strong sequence similarity with 5'-flanking sequences found in both the human and hamster vimentin genes and with other characterized silencer elements, including the human immunodeficiency virus long terminal repeat, rat growth hormone, chicken lysozyme, and rat insulin genes. Thus, a negative element appears to bind a 95-kilodalton protein involved in regulating the tissue-specific expression of the chicken vimentin gene.
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PMID:A negative element involved in vimentin gene expression. 232 56

Transcription of human immunodeficiency virus type 1 (HIV-1) is regulated by cis-acting DNA elements located in the viral long terminal repeats, by viral transregulatory proteins, and by cellular transcription factors acting in concert to modulate the degree of viral expression. We demonstrate that a DNA fragment corresponding to the central portion of the HIV-1 genome exhibits enhancer activity when cloned upstream of the thymidine kinase promoter of herpes simplex virus. This enhancer is inducible by phorbol 12-myristate 13-acetate in HeLa cells and is independent of its position and orientation with respect to the promoter. We have mapped the activity of the enhancer to two independent domains encompassing nucleotides 4079-4342 (end of the pol gene) and nucleotides 4781-6026 (vif gene and first coding exon of tat). This intragenic enhancer and its subdomains demonstrate cellular specificity because they are only active in specific cell lines. The presence of similar intragenic enhancer elements in other retroviruses suggests that they might be a conserved feature of this family of viruses.
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PMID:Identification and characterization of an enhancer in the coding region of the genome of human immunodeficiency virus type 1. 235 55

Expression of the skeletal troponin I (sTnI) gene is regulated transcriptionally in a muscle-specific fashion. We show here that the region of the sTnI gene between -160 and +61 (relative to the transcription initiation site) is able to direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene is muscle cultures at a level approximately 100 times higher than in fibroblast cultures. RNA analysis demonstrated that transcription of the CAT gene was initiated at the same site as transcription of the endogenous sTnI gene and that CAT activity levels were approximately proportional to CAT mRNA levels. Deletion analysis demonstrated that the region between nucleotides -160 and -40 contained sequences essential for full promoter activity. Surprisingly, 3' deletion analysis indicated that the first exon (-6 to +61) of the sTnI gene was also required for full activity of the sTnI promoter in skeletal muscle cells. Chimeric promoter experiments, in which segments of the sTnI and the herpes simplex virus thymidine kinase promoter were interchanged, indicated that reconstitution of a muscle-specific promoter required inclusion of both the upstream and exon I regions of the sTnI gene. Exon I, and the region immediately upstream, showed DNase protection over sequence motifs related to those found in other genes, including the tar region of human immunodeficiency virus type 1. These results demonstrate that expression of the sTnI promoter in embryonic skeletal muscle cells requires complex interaction between two separate promoter regions, one of which resides within the first 61 transcribed nucleotides of the gene.
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PMID:Muscle-specific activity of the skeletal troponin I promoter requires interaction between upstream regulatory sequences and elements contained within the first transcribed exon. 235 14

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U, azidothymidine) can inhibit human immunodeficiency virus (HIV) replication effectively in the 50-500 nM range [Mitsuya, H., Weinhold, K. J., Furman, P. A., St. Clair, M. H., Nusinoff-Lehrman, S., Gallo, R. C., Bolognesi, D., Barry, D. W. & Broder, S. (1985) Proc. Natl. Acad. Sci. USA 82, 7096-7100]. In contrast, inhibition of the growth of uninfected human fibroblasts and lymphocytes has been observed only at concentrations above 1 mM. The nature of this selectivity was investigated. Azidothymidine anabolism to the 5'-mono-, di-, and -triphosphate derivatives was similar in uninfected and HIV-infected cells. The level of azidothymidine monophosphate was high, whereas the levels of the di- and triphosphate were low (less than or equal to 5 microM and less than or equal to 2 microM, respectively). Cytosolic thymidine kinase (EC 2.7.1.21) was responsible for phosphorylation of azidothymidine to its monophosphate. Purified thymidine kinase catalyzed the phosphorylations of thymidine and azidothymidine with apparent Km values of 2.9 microM and 3.0 microM. The maximal rate of phosphorylation with azidothymidine was equal to 60% of the rate with thymidine. Phosphorylation of azidothymidine monophosphate to the diphosphate also appeared to be catalyzed by a host-cell enzyme, thymidylate kinase (EC 2.7.4.9). The apparent Km value for azidothymidine monophosphate was 2-fold greater than the value for dTMP (8.6 microM vs. 4.1 microM), but the maximal phosphorylation rate was only 0.3% of the dTMP rate. These kinetic constants were consistent with the anabolism results and indicated that azidothymidine monophosphate is an alternative-substrate inhibitor of thymidylate kinase. This conclusion was reflected in the observation that cells incubated with azidothymidine had reduced intracellular levels of dTTP. IC50 (concentration of inhibitor that inhibits enzyme activity 50%) values were determined for azidothymidine triphosphate with HIV reverse transcriptase and with immortalized human lymphocyte (H9 cell) DNA polymerase alpha. Azidothymidine triphosphate competed about 100-fold better for the HIV reverse transcriptase than for the cellular DNA polymerase alpha. The results reported here suggest that azidothymidine is nonselectively phosphorylated but that the triphosphate derivative efficiently and selectively binds to the HIV reverse transcriptase. Incorporation of azidothymidylate into a growing DNA strand should terminate DNA elongation and thus inhibit DNA synthesis.
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PMID:Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase. 243 Feb 86

3'-Azido-2',3'-dideoxyuridine-5'-triphosphate was found to be a potent and highly selective inhibitor of human immunodeficiency virus type 1 and simian immunodeficiency virus reverse transcriptases. The affinity of 3'-azido-2',3'-dideoxyuridine-5'-triphosphate for the reverse transcriptases was similar to that observed for 3'-azido-3'-deoxythymidine-5'-triphosphate. Both compounds were competitive inhibitors with respect to the normal substrate dTTP and served at least 100 times better as substrates than did dTTP. In contrast, cellular DNA polymerase alpha showed an about 60-times-higher preference for dTTP as substrate than for either inhibitor. The phosphorylation of thymidine in human peripheral blood mononuclear cell extracts was inhibited in a competitive manner by both 3'-azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine, with apparent inhibition constants of 290 and 3.4 microM, respectively. The Michaelis-Menten constant, Km, for thymidine was 7.0 microM. 3'-Azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine both served as substrates, with apparent Km values of 67 and 1.4 microM, respectively. The maximal rates of phosphorylation with 3'-azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine were 40 and 30%, respectively, of the rate with thymidine. The different affinities of 3'-azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine for the thymidine kinase and the Km values observed with these compounds as substrates may explain the difference in effects on human immunodeficiency virus type 1 replication in infected peripheral blood mononuclear cells observed when equimolar concentrations of the two compounds are compared.
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PMID:Phosphorylation of 3'-azido-2',3'-dideoxyuridine and preferential inhibition of human and simian immunodeficiency virus reverse transcriptases by its 5'-triphosphate. 248 79

3'-Azido-2',3'-dideoxythymidine (AZT) and 2',3'-didehydro-2',3'-dideoxythymidine (D4T) are potent and selective inhibitors of human immunodeficiency virus replication in MT-4 and ATH8 cells. They are also inhibitory to the replication of murine retroviruses, i.e. Moloney murine sarcoma virus-induced transformation of C3H cells. In MT-4 cells AZT is readily phosphorylated to its 5'-monophosphate, while the 5'-di- and 5'-triphosphates are generated to a 200-600-fold lower extent than the 5'-monophosphate. D4T is phosphorylated in MT-4 cells to its 5'-monophosphate at a 300-600-fold lower extent than AZT. The phosphorylation of AZT in the thymidine kinase-deficient cell line (Raji/TK-) is severely depressed, while D4T phosphorylation is only slightly diminished in Raji/TK- as compared to Raji/0 cells. D4T has a 10-fold lower affinity for phosphorylation by crude MT-4 cell extracts than AZT (Km, 142 and 14 microM, respectively), and the Vmax for phosphorylation of D4T is only 5% that of AZT. D4T is phosphorylated by MT-4 cell extracts about 180-fold less efficiently than AZT (Vmax/Km, 0.06 for D4T, as compared to 11 for AZT), and this is consistent with the differences found in the amounts of phosphorylated products of D4T and AZT formed in intact MT-4 cells. The 5'-triphosphates of AZT and D4T are equipotent in their inhibitory effects on the reverse transcriptases from human immunodeficiency virus and Moloney murine leukemia virus.
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PMID:Differential patterns of intracellular metabolism of 2',3'-didehydro-2',3'-dideoxythymidine and 3'-azido-2',3'-dideoxythymidine, two potent anti-human immunodeficiency virus compounds. 253 71

Herpes simplex virus (HSV) is susceptible to a variety of antiviral compounds, most of which are nucleoside analogues that interfere with DNA metabolism involving the virus enzymes DNA-polymerase and thymidine kinase. Single mutations in the virus genome give rise to resistant mutants following selection in vitro in the presence of a particular drug, and in this respect HSV is similar to several other viruses. Such mutants have been invaluable research tools. HSV is responsible for a variety of lesions which tend to be recurrent, owing to the special ability of the virus to remain latent in and reactivate from neural tissue. The consequences of this upon clinical resistance are discussed in the present review. In fact, clinical resistance in HSV infections has not yet become widespread but does appear to be especially important in immunocompromised patients, including those suffering from AIDS. HSV is proposed as an important model for the investigation of drug resistance in other, more complex organisms, and with respect to antiviral strategies against the human immunodeficiency virus.
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PMID:Persistent herpes simplex virus infection and mechanisms of virus drug resistance. 255 Feb 35

The novel 5-chloro-, 5-bromo-, and 5-iodo-derivatives of 3'-fluoro-2',3'-dideoxyuridine (FddUrd), designated FddCIUrd, FddBrUrd, and FddIUrd, respectively, have been synthesized and evaluated for their antiretrovirus activity against human immunodeficiency virus (HIV) and murine Moloney sarcoma virus. All three 5-halogeno-FddUrd analogues inhibited HIV-1 replication in MT4 cells with an effective dose (ED50) of about 0.2-0.4 microM. However, FddCIUrd was markedly more selective in its anti-HIV-1 activity than FddBrUrd or FddIUrd. The selectivity index of FddCIUrd was similar to that of 3'-azido-2',3'-dideoxythymidine (AZT) when evaluated in parallel (1408 and 1603, respectively). The FddUrd derivatives also had a marked inhibitory effect on HIV-2 replication in MT4 cells and HIV-1 induced antigen expression in HUT-78 cells. However, neither FddUrd nor its 5-halogeno derivatives were inhibitory to Moloney sarcoma virus-induced transformation of murine C3H cells. The anti-HIV-1 activity of FddUrd, FddCIUrd, FddBrUrd, and FddIUrd was reversed by the addition of thymidine and 2'-deoxycytidine. The 5-halogeno-FddUrd analogues had a markedly higher affinity for MT4 thymidine kinase than FddUrd (Ki/Km, 4.0-4.7, as compared with 302 for FddUrd).
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PMID:5-Halogeno-3'-fluoro-2',3'-dideoxyuridines as inhibitors of human immunodeficiency virus (HIV): potent and selective anti-HIV activity of 3'-fluoro-2',3'-dideoxy-5-chlorouridine. 272 68

2',3'-Dideoxy-2',3'-didehydrothymidine (D4T) is a thymidine nucleoside analog which has potent anti-human immunodeficiency virus activity in vitro. We have studied its metabolism in cells to assist in determining its mechanism of action. D4T is metabolized in cells to the mono-, di-, and triphosphate nucleotides. Our data suggest that the initial conversion to the monophosphate is catalyzed by thymidine kinase. This enzyme has an affinity for D4T 600-fold lower than for thymidine and catalyzes the rate-limiting step in production of the triphosphate. Nevertheless, intracellular concentrations of the triphosphate approximately equal to the reported Ki for human immunodeficiency virus reverse transcriptase are attained with extracellular concentrations of free drug as low as 0.05 microM. The pattern of phosphorylation is different from that of 3'-azido-3'-deoxythymidine (AZT), which has an affinity for thymidine kinase equivalent to that of thymidine and is easily phosphorylated. The rate-limiting step in formation of AZT triphosphate is conversion of mono- to diphosphate, and thus the monophosphate accumulates. On removal of D4T or AZT from the media, both triphosphates have an intracellular half-life of about 200 min, and this rate ultimately controls the rate of elimination of the drugs from cells. The differences in metabolism of D4T and AZT observed in vitro may be responsible for the differences in toxicity seen in vitro and in vivo and support the exploration of the clinical utility of D4T as an anti-human immunodeficiency virus agent.
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PMID:Cellular pharmacology of 2',3'-dideoxy-2',3'-didehydrothymidine, a nucleoside analog active against human immunodeficiency virus. 276 35

Zidovudine is a potent in vitro inhibitor of human immunodeficiency virus (HIV) with varying efficacy against other retroviruses. With the exception of Epstein-Barr virus, all non-retroviruses tested so far have been insensitive to inhibition by zidovudine. In vivo, efficacy of zidovudine was demonstrated against Rauscher murine leukemia virus and feline leukemia virus. In both experimental models, infections completely resolved in animals when the drug was administered soon after infection. These results suggest that prompt initiation of zidovudine therapy, following a known exposure to HIV, should be considered. Mechanism studies show that zidovudine is phosphorylated to the monophosphate and diphosphate derivatives by the host cell cytosolic thymidine kinase and thymidylate kinase, respectively. The identity of the enzyme that phosphorylates zidovudine diphosphate is not known, but is believed to be the cellular nucleoside diphosphate kinase. The triphosphate of zidovudine appears to be the active form of the drug. Zidovudine triphosphate competes well with thymidine 5'-triphosphate for binding to the HIV reverse transcriptase and also functions as an alternative substrate. Incorporation of zidovudine monophosphate results in chain termination. However, it is not clear which mechanism, chain termination or competition with thymidine 5'-triphosphate, or a combination of both, is responsible for the inhibition of HIV replication.
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PMID:Spectrum of antiviral activity and mechanism of action of zidovudine. An overview. 304 82


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