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)

The metabolisms of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (2'NDG) and its cyclic phosphate, 9-[(2-hydroxy-1,3,2-dioxophosphorinan-5-yl) oxymethyl]guanine P-oxide (2'-nor-cGMP), were compared in cultures of primary rabbit kidney cells infected with herpes simplex virus type 1 (HSV-1). 2'-Nor-cGMP was taken up by the cells essentially intact, after which it was opened to the acyclic monophosphate and phosphorylated further, ultimately to the triphosphate. Formation of the triphosphate was independent of HSV thymidine kinase expression, unlike what is observed with 2'NDG. In addition, there was a direct correlation between the antiviral activity of 2'NDG and the level of triphosphate formed in HSV-1-infected cells, whereas such a correlation was absent with 2'-nor-cGMP. In vivo experiments indicated that only a small percentage of free 2'NDG was formed in the bloodstream of mice after oral administration of 2'-nor-cGMP. Incubation of 2'-nor-cGMP with crude extracts of HSV-1-infected or uninfected HeLa cells resulted in the direct production of 2'NDG triphosphate. The possibility that the triphosphate of 2'NDG produced from 2'-nor-cGMP was the enantiomer of the triphosphate made from 2'NDG by viral and cellular kinases was investigated and disproved. Taken together, these data indicate that (i) 2'-nor-cGMP does not act simply as a prodrug of 2'NDG, (ii) 2'-nor-cGMP does not require viral thymidine kinase for its activity, and (iii) 2'-nor-cGMP may have an additional, triphosphate-independent mode of action.
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PMID:Comparison of the modes of antiviral action of 2'-nor-deoxyguanosine and its cyclic phosphate, 2'-nor-cyclic GMP. 301 13

We have demonstrated the presence of an Epstein-Barr virus (EBV)-coded thymidine kinase (TK) by producing biochemically transformed, TK-positive mammalian cell lines using either microinjection of whole EBV virions or calcium phosphate-mediated transfection of the SalI-B restriction endonuclease fragment of EBV DNA. Analysis of these cell lines showed that: (i) EBV DNA was present in the cell lines, (ii) sequences from the SalI-B restriction endonuclease fragment of EBV were expressed, (iii) a TK activity was present and (iv) a protein with antigenic cross-reactivity with the herpes simplex virus (HSV) TK was produced. The identity of the EBV TK gene was determined by demonstrating that a recombinant plasmid, which expressed the protein product of the BXLF1 open reading frame as a fusion protein, could complement TK- strains of E. coli. A comparison of the predicted amino acid sequences of the TK proteins of EBV and HSV-1 revealed significant regions of homology.
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PMID:Identification of an Epstein-Barr virus-coded thymidine kinase. 301 75

9-[(2-Hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide (2'-nor-cGMP), the cyclic phosphate of 2'-nor-deoxyguanosine (2'-NDG) was synthesized by phosphorylation of 2'-NDG and evaluated for antiherpetic activity in cell cultures and in animal protection studies. 2'-nor-cGMP was effective in cell culture against both thymidine kinase deficient and wild-type herpes simplex virus type 1 strains and also against herpes simplex virus type 2. The anti-herpes activity of 2'-nor-cGMP against thymidine kinase deficient HSV-1 was confirmed by animal protection studies. Also, in comparative cell culture protection studies, the ED50 (microM) of 2'-nor-cGMP was approximately 10-fold lower than that of 2'-NDG against three strains of varicella zoster virus. In addition, 2'-nor-cGMP was effective orally in preventing HSV-1 orofacial infection and HSV-2 genital infection of mice. Topical therapeutic applications of 2'-nor-cGMP prevented orofacial HSV-1 lesion development in mice and development of HSV-2 genital lesions in guinea pigs. Subcutaneous application of 2'-nor-cGMP to intracerebral HSV-1 challenged weanling mice significantly prolonged survival. These studies indicate that 2'-nor-cGMP is not dependent on viral thymidine kinase for its antiviral activity and is highly effective in preventing experimental HSV infections.
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PMID:Efficacy of 2'-nor-cyclicGMP in treatment of experimental herpes virus infections. 302 43

We investigated the sites of integration of exogenous DNA fragments introduced by DNA-mediated gene transfer. Mouse Ltk- cells were transformed with the herpes simplex virus thymidine kinase gene and pBR322 DNA by the calcium phosphate precipitation method. Some of the integrated exogenous DNA sequences were recovered from the stable tk+ transformants in the form of plasmids that were capable of propagation in bacteria. Four plasmids derived from two cloned cell lines were analyzed in detail by nucleotide sequencing and hybridization techniques. These plasmids contained a total of seven cellular-exogenous DNA junctions. In all cases, there was no sequence homology between the exogenous and cellular DNA sequences adjacent to the joining sites, and no specific exogenous or cellular sequences occurred at the junctions. Rearrangement or deletion of Ltk- DNA was always associated with the integration of exogenous DNA. All of the assignable cellular sequences at the junctions were repetitive sequences. Two of these sequences were from the MIF-1 repetitive sequence family, and a third consisted of a 40-base pair simple copolymer of alternating deoxyadenosine-deoxythymidine. Our results suggest that repetitive sequences are relatively favorable sites for the integration of exogenous DNA.
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PMID:Foreign DNA introduced by calcium phosphate is integrated into repetitive DNA elements of the mouse L cell genome. 302 4

Replication of equine herpesvirus type 1 (EHV-1) was sensitive to 9-(1,3-dihydroxy-2-propoxymethyl)guanine(DHPG) but relatively resistant to E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). Likewise, plaque formation by EHV-1 was inhibited by DHPG, but not by BVDU. Plaque formation by a thymidine kinase-negative (tk-) mutant of EHV-1 was not inhibited by DHPG. In order to investigate biochemical mechanisms determining the differential sensitivity of EHV-1 to these drugs, the EHV-1-encoded thymidine kinase enzyme activity (TK)1 was partially purified from EHV-1-infected cells and analyzed. The EHV-1-induced enzyme utilized both ATP and CTP as phosphate donors and differed in relative electrophoretic mobility from the TKs of mock-infected and HSV-1-infected cells. Phosphorylation of 3H-dThd by the EHV-1 TK was inhibited by AraT, IdUrd, BVDU, and DHPG. The EHV-1 TK phosphorylated 125I-dCyd and 3H-ACV. The results indicate that EHV-1 encodes a pyrimidine deoxyribonucleoside kinase with broad nucleoside substrate specificity. These observations suggest that the failure of BVDU to inhibit EHV-1 replication is not attributable to an inability of the EHV-1 TK to phosphorylate BVDU, but may result from the incapacity of the viral TK to convert BVDU monophosphate to the triphosphate or from lack of inhibitory effect of BVDU triphosphate on viral DNA polymerase reactions.
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PMID:Phosphorylation of nucleoside analogs by equine herpesvirus type 1 pyrimidine deoxyribonucleoside kinase. 302 47

The title diesters (11-15; halo substituents F, Cl, Br, I) were prepared by DCC-induced cyclization of the precursor 5'-monophosphate or direct halogenation of the 2'-deoxyuridine 3',5'-cyclic monophosphate. Antitumor activities of 11-15 in cell systems (L1210 and Raji/0) were compared to those of the corresponding nucleosides and 5'-monophosphates. Thus, the 5-F- and 5-CF3-2'-deoxyuridines proved to be highly active derivatives [ID50 values (microgram/mL) for L1210, 0.002 and 0.06, respectively], with the 5'-monophosphates showing comparable potencies. The corresponding 3',5'-cyclic monophosphate diesters were 20-30 times less potent but nonetheless highly cytostatic. All derivatives including 11-15 had greatly increased ID50 values for the thymidine kinase deficient (TK-) L1210 and Raji cells. The 3',5'-cyclic diesters (11-15) evidently are not efficient prodrug sources of the nucleoside 5'-monophosphates in TK- cells. They also proved to be 100- to 2000-fold less efficient inhibitors of L1210 thymidylate synthetase than were the 5'-monophosphates. The 5-substituted 2'-deoxyuridines and their 5'-monophosphates were potent inhibitors of herpes simplex virus (MIC50 mostly 0.07-10 micrograms/mL) and vaccinia virus (MIC50 0.07-0.2 microgram/mL), with antiviral activity decreasing in the order 5-I, 5-Br greater than 5-CF3 greater than 5-Cl greater than 5-F. The 3',5'-cyclic monophosphates (11-15) were for the most part 10- to 40-fold less active than the 5'-monophosphates in the virus assay systems (e.g., MIC50 for the 5-Br and 5-I derivatives ranged 1-20 micrograms/mL). By contrast 11-15 were considerably more potent inhibitors of vaccinia virus growth (MIC50 0.4-2 micrograms/mL). As the neutral 3',5'-cyclic methyl phosphate triesters (16-18), the 5-I and 5-Br compounds were less potent in antiviral and cytostatic agents than the 3',5'-cyclic diesters, while the 5-iodo benzyl triester was in several cases as active as the 3',5'-cyclic diester. The title compounds (11-15) appear to require extracellular hydrolysis to the nucleoside before functioning as antitumor or antiviral agents.
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PMID:Synthesis and antitumor and antiviral properties of 5-halo- and 5-(trifluoromethyl)-2'-deoxyuridine 3',5'-cyclic monophosphates and neutral triesters. 302 28

The biological activities of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-fluorouracil (2'F-ara-FU), 1-(3'-deoxy-3'-fluoro-beta-D-arabinofuranosyl)-5-fluorouracil (3'F-ara-FU) and 1-beta-D-arabinofuranosylthymine (ara-T) were compared in human cytomegalovirus (HCMV)-infected and noninfected human fibroblasts. 2'F-ara-FU inhibited HCMV plaque formation (ED50, 16 microM for AD 169 strain) at lower concentrations than 3'F-ara-FU (ED50, 100 microM for AD 169). These nucleoside analogues are expected to be phosphorylated to their 5'-phosphate forms by cellular thymidine kinase in HCMV-infected cells. The thymidine kinase activities in the virus-infected and noninfected cells were compared. Cellular thymidine kinase was increased in the virus-infected cells and showed better phosphorylation of 2'F-ara-FU than did 3'F-ara-FU. HCMV DNA polymerase was purified using affinity column chromatography, and the inhibitory effect of the 5'-triphosphate derivatives of 2'F-ara-FU (2'F-ara-FUTP) and 3'F-ara-FU (3'F-ara-FUTP) against viral and host DNA polymerase alpha was examined. No significant difference in the effectiveness of inhibition was observed between viral DNA polymerase and host polymerase alpha. However, viral polymerase incorporated 2'F-ara-FUTP into newly synthesized DNA, whereas polymerase alpha did not utilize 2'F-ara-FUTP as a substrate. Thus, viral polymerase differs from host polymerase alpha in its recognition and utilization of 2'F-ara-FUTP. This difference may be important to the design of selective antiviral agents for HCMV.
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PMID:A proposed mechanism for the selective inhibition of human cytomegalovirus replication by 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-fluorouracil. 303 45

Treatment of chick embryos in ovo with hydrocortisone-21-phosphate (a single dose of 150 micrograms) caused a marked reduction of retinal thymidine kinase activity 24 h later. The inhibitory effect was highest (65-70%) in 8-10-day-old embryos and declined with age, disappearing after day 15. It was accompanied by a reduction in thickness of the retinal layers. Adrenocorticotropic hormone (ACTH) treatment (10 micrograms daily for 2 days) also produced an age-dependent inhibitory effect on retinal thymidine kinase, whereas treatment with a single dose of 200 micrograms of metopirone, a compound that prevents the 11 beta-hydroxylation of steroid molecules in the adrenal glands, impeded the decrease in thymidine kinase activity that normally occurs in chick embryo retina after day 9 of development. In addition, metopirone prevented the inhibition exerted by ACTH on thymidine kinase activity but had no effect on the action of hydrocortisone.
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PMID:Influence of hydrocortisone on chick embryo retina development. 303 47

To initially determine the effect that base-pair mismatch has on homologous recombination in mammalian cells, we have studied genetic recombination between thymidine kinase (tk) gene sequences from herpes simplex virus 1 and 2. These tk genes are approximately 81% homologous at the nucleotide level. We observed that, in mouse LTK- cells, intrachromosomal recombination between type 1 and type 2 tk sequences is reduced by a factor of at least 1000 relative to the rate of intrachromosomal recombination between homologous type 1 tk sequences. In sharp contrast, the rate of intermolecular or intramolecular extrachromosomal recombination between the heterologous tk sequences introduced by calcium phosphate or microinjection was reduced only by a factor of 3 to 15 compared with extrachromosomal homologous tk crosses. Our results suggest differences between the mechanisms of extrachromosomal and intrachromosomal recombination in mammalian cells.
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PMID:Differential effects of base-pair mismatch on intrachromosomal versus extrachromosomal recombination in mouse cells. 303 44

The biological synthesis and purification of 5-[125I]iododeoxyuridine monophosphate (IdUMP) are described. The specificity of IdUMP as substrate in the thymidylate monophosphate kinase (TMPK) assay is demonstrated, and a 100-fold gain in sensitivity as compared to the conventional TMPK assay is shown. TMPK measurements of isozymes derived from herpes simplex virus (HSV)-infected cells, uninfected cells, and tumor biopsies were performed. The results showed a significant difference in dependence of phosphate donor concentration present for TMPK activity from HSV-infected cells compared to the corresponding activity from uninfected cells, while only a minor difference in pH optima was observed for these enzyme activities. The increased sensitivity made it possible to detect and quantify HSV TMPK-blocking antibodies (ab) present in human sera. Sera from HSV ab-positive individuals were found to block the two HSV TMPKs to varying degrees and with different specificities. The immunological relationship between the TMPK and thymidine kinase (TK) induced by HSV-1 and HSV-2, respectively, was studied by comparing the capacities of different sera to block the two enzymatic activities. The results showed that the capacity to block HSV-1 TK and TMPK was proportional for all of the sera studied, while sera that preferentially blocked only the HSV-2 TMPK or HSV-2 TK were found. It was concluded that the HSV-2 TMPK and TK activities are less related than the corresponding activities for HSV-1 and that the HSV-2 enzyme activities are mediated by different catalytic sites.
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PMID:An optimized thymidylate kinase assay, based on enzymatically synthesized 5-[125I]iododeoxyuridine monophosphate and its application to an immunological study of herpes simplex virus thymidine-thymidylate kinases. 303 45


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