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)

In vitro and in vivo studies were done on a herpes simplex virus type 2 strain recovered from a patient on acyclovir (ACV) which was ACV resistant but expressed thymidine (dThd) kinase (EC 2.7.1.21) activity. Plaque-purified clones derived from the original clinical sample were heterogeneous with respect to plaque size and drug susceptibility. The heterogeneity of this viral mixture was also evident from varied 125I-labeled 5-iodo-2'-deoxycytidine autoradiographic patterns and from varied expression of dThd kinase-associated phosphorylating activities. Four clones from this mixture were 1-beta-D-arabinofuranosylthymine (ara-T) susceptible and ACV resistant. Extracts of cells infected with these clones catalyzed the phosphorylation of ara-T but little of ACV. The virus-coded dThd kinase was purified from one of these clones to determine whether its substrate specificity was altered. The amount of virus-coded dThd phosphorylating activity with the cell extracts was estimated to be sevenfold lower with the resistant clone than with the MS strain of herpes simplex virus type 2. The dThd kinase eluted from a dThd-agarose affinity column under the same conditions with extracts from both sources and substrate saturations of both enzymes by acyclic nucleoside analog phosphate acceptors were classical hyperbolic functions. However, there were significant differences in the kinetic parameters of substrates between the two enzymes. Apparent Km (Km') values for dThd, deoxycytidine, ara-T, ACV, and the acyclic guanosine analog 9-[[2-hydroxyl-1-(hydroxymethyl)ethoxy]methyl]guaine (BW B759U) were 2- to 60-fold higher with the variant enzyme than with the enzyme from laboratory strain MS. Comparing these two enzymes, relative maximal phosphorylation rates (Vm) were eightfold lower for ACV but unchanged for BW B759U. In contrast, the relative rates for deoxycytidine and ara-T were eight- and twofold higher, respectively. The surprisingly good substrate activity with BW B759U compared with that of ACV (Vm/Km' = 0.39 versus 0.01) coincided with susceptibility of the ACV-resistant virus to BW B759U. This clinical variant retained its pathogenicity for mice and was only moderately less neurovirulent than wild-type virus. Although such mutants have the potential to induce illness less responsive to therapy, the recurrence from which the isolate was obtained was typical for this patient in severity and duration. Since this episode, the patient has been treated successfully with ACV.
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PMID:Clinical isolate of herpes simplex virus type 2 that induces a thymidine kinase with altered substrate specificity. 282 90

The administration in ovo of hydrocortisone-21-phosphate caused, in chick embryo liver, a reduction of the number of hepatocytes which can be isolated from 1 mg dry weight of liver and a marked increase of their size. Moreover, the treatment diminished the incorporation of thymidine into acid-insoluble fraction in these cells whilst it augmented the content of protein, RNA, DNA and the level of thymidine kinase/cell. These effects were highest at 8-10 days, then declined with the age, disappearing after 18th day of incubation. Similar effects were obtained by injecting other glucocorticoids or ACTH. Combined treatment with metopirone abolished the effects found with ACTH, but did not modify the action of hydrocortisone. These findings suggest that glucocorticoids interfere with the proliferative cycle of hepatocytes by inhibiting the mitotic phase and favouring the production of abnormally large cells.
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PMID:Morphological and biochemical effects of glucocorticoids in chick embryo hepatocytes during development. 283 58

Substrate cycles constructed from a deoxyribonucleoside kinase and a deoxyribonucleotidase contribute to the metabolism of deoxyribonucleotides in cultured cells. The two enzymes catalyze in opposite directions the irreversible interconversion between a deoxyribonucleoside and its 5'-phosphate. Depending on the balance between the two reactions the net result of the cycle's activity will be synthesis or degradation of the deoxyribonucleotide, and favor import or export of the deoxyribonucleoside. With genetically changed hamster cells (V79 and CHO) deficient in either deoxycytidine or thymidine kinase we now quantify by kinetic isotope flow experiments the contributions of the two kinases to the function of the respective cycles. For each, loss of the relevant kinase was accompanied by an increased degradation of the deoxynucleotide, a slower rate of DNA synthesis, and a longer generation time for the mutant cells. The size of the corresponding deoxyribonucleoside triphosphate pool was apparently not decreased.
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PMID:Effects of deoxycytidine and thymidine kinase deficiency on substrate cycles between deoxyribonucleosides and their 5'-phosphates. 283 22

Dietary fibers may tend to enhance or inhibit chemically induced experimental colon cancer, depending on the particular fiber consumed. This study examined the relationship between colonic thymidine kinase enzyme activity and mucin histochemistry and the reported effects of various dietary fibers on chemically induced colon carcinogenesis. Fiber-supplemented diets containing fibers reported to inhibit (wheat bran) or enhance (guar gum, carrageenan) chemically induced colon carcinogenesis in the rat were selected. Four groups of male Fischer 344 rats consumed 10% wheat bran, 5% guar gum, 5% carrageenan, or fiber-free diets ad libitum for 4 weeks. At the completion of the treatment period, the distal 12 cm of colonic mucosa was scraped off and homogenized for determination of thymidine kinase activity, and a 0.5-cm section of midcolon was processed by the high-iron diamine/Alcian blue method for mucin histochemistry. Final animal weights did not differ significantly among groups. Thymidine kinase enzyme specific activity (mumole thymidine phosphate formed x 10(6)/min/mg protein, means +/- SEMs) was not significantly different in the fiber-free, wheat bran, and guar gum groups (10.98 +/- 1.50, 7.41 +/- 1.09, and 9.11 +/- 2.04, respectively) but was markedly elevated at 41.84 +/- 4.65 in the carrageenan group (alpha less than 0.001). Mucin histochemistry failed to reveal any significant differences among dietary groups.
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PMID:Alterations in colonic thymidine kinase enzyme activity induced by consumption of various dietary fibers. 284 79

Antiviral activities of five nucleoside analogs against the VR-3 and WT-34 strains of herpes simplex virus type 1 (HSV-1) were investigated in Vero and human embryo lung fibroblast (HEL) cells. In HEL cells, the compounds showed antiviral activities against both strains of HSV-1, but in Vero cells, the antiviral activities of the compounds were reduced in proportion to their antiviral indexes (the 50% inhibitory dose [ID50] for cell growth divided by the 50% plaque reduction dose for virus). The ratio of the ID50 in Vero cells to the ID50 in HEL cells was larger in VR-3-infected cells than in WT-34-infected cells. The following results were obtained. (i) Thymidine kinase (TK; EC 2.7.1.21) activity in the VR-3- or WT-34-infected Vero cells was about half that in VR-3- or WT-34-infected HEL cells. Induction of viral TK was especially low in the VR-3-infected Vero cells. (ii) The ID50 of the plaque reduction assay in hypoxanthine, aminopterin, and thymidine medium revealed that the activity of cellular thymidylate synthetase (EC 2.1.1.45) was important in viral replication in VR-3-infected Vero cells. (iii) The VR-3-infected cells required larger thymidine and thymidine phosphate pools for viral replication than the WT-34-infected cells did, although uptake of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil into infected cells was equal for both strains. (iv) In the VR-3-infected Vero cells, the quantity of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil triphosphate was smaller than that in VR-3-infected HEL cells and WT-34-infected Vero and HEL cells.
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PMID:Efficacies of antiherpesvirus nucleosides against two strains of herpes simplex virus type 1 in Vero and human embryo lung fibroblast cells. 284 37

A plasmid carrying a promoterless herpes simplex virus thymidine kinase gene was transfected via calcium phosphate precipitation into LM (tk-) mouse fibroblast cells. The transfected gene was efficiently expressed, as the transfected cells grew perfectly well in selective hypoxanthine-aminopterin-thymidine medium, suggesting that the thymidine kinase-coding region became linked to a promoterlike element on integration into the recipient genome. To investigate the structure of the surrogate promoter, we first isolated the integrated gene from a genomic library. The nucleotide sequence of the DNA adjacent to the thymidine kinase-coding sequence was then determined. We found, first, that the integration of the transfected DNA apparently occurred by a blunt end ligation mechanism involving no obvious sequence similarities between integrated and recipient DNA and, second, that the 5'-flanking region included a TATA box, two CCAAT boxes, and a GC box element. However, the TATA box motif and the most proximal CCAAT box appeared to be sufficient for full promoter activity, as determined by the transfection efficiencies of appropriate plasmid constructs. Except for these canonical promoter elements, the surrogate promoter had no obvious similarities to known thymidine kinase gene promoters.
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PMID:Insertional activation of a promoterless thymidine kinase gene. 285 Apr 88

The 5'-PAA and 5'-PFA phosphate esters of 5-bromo-2'-deoxyuridine (BUdR) were synthesized and their antiherpes virus activity was evaluated in vitro. Both compounds showed activity of the same order as the parent nucleoside, BUdR, against HSV-2 but were 4 to 12 times less potent against HSV-1. The 5'-PAA phosphate ester of BUdR (Ro 21-9875) was also active against varicella-zoster virus (VZV) and human cytomegalovirus (HCMV). The 5'-PAA phosphate ester of 5-bromovinyl-2'-deoxyuridine (BVdU) was also synthesized and showed good antiviral activity against HSV-1 only (ID50 = 1.3 mg/l). Further evaluation against selected mutants (TK- or PAAr) indicated a requirement for the expression of the virus-coded thymidine kinase (TK) for the antiviral activity of Ro 21-9875. Kinetic studies revealed non-competitive mixed inhibition of the viral enzyme by this compound. This suggests that it may have some intrinsic TK mediated activity though breakdown to its component parts is undoubtedly a significant contributing factor.
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PMID:Antiviral activity of 5'-PAA and 5'-PFA phosphate esters of 2'-deoxyuridines. 294 89

It has been demonstrated that certain alternating purine and pyrimidine sequences may assume a left-handed Z-DNA conformation. In order to evaluate the possibility that Z-DNA is involved in the modulation of gene expression, we examined the ability of various synthetic DNA polymers to affect the transfection of herpes simplex virus thymidine kinase (HSVtk) gene in Ltk- cells using the DNA-calcium phosphate cotransfection technique. We found that potential Z-DNA forming polymers such as, poly(dG-m5dC) X poly(dG-m5dC) and poly(dG-dC) X poly(dG-dC), cotransfected with the tk gene decreased the level of Tk+ transformed colonies. In contrast, cotransfection of the tk gene with polymers which do not assume Z-conformation such as, poly(dG) X poly(dC) or poly(dA-dT) X poly(dA-dT) showed no effect on the number of colonies formed. About 50% inhibition of the Tk+ colony formation was obtained by 0.4 micrograms of poly(dG-m5dC) X poly(dG-m5dC), or by 2 micrograms of poly(dG-dC) X poly(dG-dC). DNA uptake into Ltk- cells was not significantly affected by any of these polymers. Approximately 20-42 base pairs (bp) long alternating dG-dC sequence linked at either the 5'-end or 3'-end of tk gene were cloned into plasmids. These recombinant plasmids, however, showed no remarkable effect upon the transfection of Ltk- cells. The DNAs of Tk+ colonies obtained by transfecting these recombinant plasmids were digested with BssH II and analyzed by Southern blotting. We demonstrated that the dG-dC sequences proximal to the tk gene were integrated into cellular DNA. All the presented results indicate that only larger polymers with the potential to assume a Z-DNA conformation may affect tk gene transfection either by inhibiting transcription or more probably by affecting the stable integration of the tk gene into the host chromosome.
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PMID:Inhibition of the herpes simplex virus thymidine kinase gene transfection in Ltk- cells by potential Z-DNA forming polymers. 299 54

The cellular tandemization of the herpes simplex virus (HSV) thymidine kinase (tk) gene was studied in tk- mouse fibroblasts after gene transfer by microinjection into the nucleus or by calcium phosphate-mediated transfection. Three different DNA substrates, designed to yield simple integration patterns, were used: a gel-purified 3.6-kb Bam HI fragment containing the HSV tk gene; the same fragment self-ligated; and the 3.6-kb fragment ligated to a Bam HI-cleaved subset of genomic mouse DNA. The genomic DNA of six independently isolated transformed cell lines was analyzed by Southern blotting and the structure of the tk-specific DNA was studied. The data suggest that modifications (mutations, deletions, recombination events, and recircularization, etc.) of the input DNA fragment occur early after its introduction into the cell. Subsequently these structures are multiplied in a directional manner, generating larger arrays of DNA with distinct and regularly repeated areas. These concatemers can eventually be integrated into the host genome.
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PMID:Studies on cellular tandemization of herpes simplex virus thymidine kinase DNA. 299 72

New multisubstrate-type inhibitors of the deoxynucleoside kinases have been synthesized, tested for their specificity as soluble inhibitors of enzymes from Lactobacillus acidophilus, and used to construct media for affinity chromatography. Each inhibitor was a deoxynucleoside 5'-adenosine 5"'-P1,P4-tetraphosphate (abbreviated dNp4A, where dN represents a dAdo, dCyd, dGuo, or dThd moiety linked through its 5'-hydroxyl to the terminal phosphate of adenosine tetraphosphate). At micromolar concentrations, each inhibitor strongly and specifically inhibited the corresponding deoxynucleoside kinase. Each of the four Lactobacillus deoxynucleoside kinase activities was selectively retained on its homologous dNp4A-Sepharose affinity medium. The activity was eluted on addition of the respective dNp4A with up to 70% recovery and 300-500-fold purification (relative to an ammonium sulfate fraction). Whereas dThd kinase was retained only by the dTp4A column, a portion of the dAdo kinase activity was retained, along with all the dCyd kinase or dGuo kinase, on dCp4A- or dGp4A-Sepharose, respectively, and coeluted with these activities. Conversely, all three activities were quantitatively retained on dAp4A-Sepharose, without competition from either dCyd or dGuo, and were eluted simultaneously upon addition of dAp4A. These observations further confirm the understanding that this organism employs paired, and presumably bifunctional, kinases, namely dCyd/dAdo kinase and dGuo/dAdo kinase, along with a separate thymidine kinase.
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PMID:Multisubstrate analogs for deoxynucleoside kinases. Use in novel affinity media applied to resolution of Lactobacillus enzymes. 299 85


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