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)

We incorporated 3H-labeled thymidine, deoxycytidine, or cytidine into dNTPs and DNA of exponentially growing CEM cells. G1 and S phase cells were separated by centrifugal elutriation, and the size and specific activity of dNTP pools were determined to study the cell cycle-dependent regulation of specific dNTP synthesizing enzymes in their metabolic context. With [3H]thymidine, we confirm the earlier demonstrated S phase specificity of thymidine kinase. Incorporation of radioactivity from [5-3H]deoxycytidine into dCTP occurred almost exclusively in G1 cells. During S phase, de novo synthesis by ribonucleotide reductase was switched on, resulting in a 70-fold dilution of [3H]dCTP, confirming that ribonucleotide reductase is an S phase-specific enzyme, whereas deoxycytidine kinase is not. [5-3H]Cytidine appeared in dCTP almost to the same extent in G1 as in S phase, despite the S phase specificity of ribonucleotide reductase. During S phase, DNA replication greatly increased the turnover of dCTP, requiring a corresponding increase in ribonucleotide reductase activity. During G1, the enzyme maintained activity to provide dNTPs for DNA repair and mitochondrial DNA synthesis. The poor incorporation of isotope from deoxycytidine into DNA earlier led to the suggestion that the nucleoside is used only for DNA repair (Xu, Y-Z., Peng, H., and Plunkett, W. (1995) J. Biol. Chem. 270, 631-637). The poor phosphorylation of deoxycytidine in S phase provides a better explanation.
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PMID:Cell cycle-dependent metabolism of pyrimidine deoxynucleoside triphosphates in CEM cells. 919 7

The Herpesviridae comprise a large class of animal viruses of considerable public health importance. Of the Herpesviridae, replication of herpes simplex virustype-1 (HSV-1) has been the most extensively studied. The linear 152-kbp HSV-1 genome contains three origins of DNA replication and approximately 75 open-reading frames. Of these frames, seven encode proteins that are required for originspecific DNA replication. These proteins include a processive heterodimeric DNA polymerase, a single-strand DNA-binding protein, a heterotrimeric primosome with 5'-3' DNA helicase and primase activities, and an origin-binding protein with 3'-5' DNA helicase activity. HSV-1 also encodes a set of enzymes involved in nucleotide metabolism that are not required for viral replication in cultured cells. These enzymes include a deoxyuridine triphosphatase, a ribonucleotide reductase, a thymidine kinase, an alkaline endo-exonuclease, and a uracil-DNA glycosylase. Host enzymes, notably DNA polymerase alpha-primase, DNA ligase I, and topoisomerase II, are probably also required. Following circularization of the linear viral genome, DNA replication very likely proceeds in two phases: an initial phase of theta replication, initiated at one or more of the origins, followed by a rolling-circle mode of replication. The latter generates concatemers that are cleaved and packaged into infectious viral particles. The rolling-circle phase of HSV-1 DNA replication has been reconstituted in vitro by a complex containing several of the HSV-1 encoded DNA replication enzymes. Reconstitution of the theta phase has thus far eluded workers in the field and remains a challenge for the future.
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PMID:Herpes simplex virus DNA replication. 924 11

During an infection of nonneuronal cells, bovine herpesvirus 1 (BHV-1) gene expression proceeds in a well-defined cascade. Products of immediate-early (IE) genes are expressed first, and they activate expression of early (E) and late (L) genes. Although the same cascade is assumed to occur during an infection of neurons in trigeminal ganglia (TG) of cattle, no experimental data is available to support this hypothesis. Consequently, we analyzed BHV-1 gene expression in bovine TG at 1, 2, 4, 7, and 15 days postinfection (dpi). Infectious virus was detected in ocular swabs from 1 to 7 dpi but not 15 dpi. By reverse transcription (RT)-PCR, IE (bICP4), E (thymidine kinase, ribonucleotide reductase [RR]), L (glycoprotein C, and alpha trans-inducing factor), and dual-kinetic (bICP0 and bICP22) transcripts were analyzed. When cDNA synthesis was primed with random hexamers, IE and E transcripts were detected at the same time. However, full-length and poly(A)+ (FL&P) RR or bICP22 RNAs were detected before FL&P IE RNAs. Furthermore, FL&P IE transcripts were not detected until viral DNA increased in TG. IE transcripts were detected before E or L RNAs when rabbit kidney cells were infected with a low multiplicity of infection and the same RT-PCR detection method was used. These studies suggested that expression of full-length and polyadenylated IE transcripts in trigeminal ganglia was not efficient compared to that of RR and bICP22 transcripts.
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PMID:Analysis of bovine herpesvirus 1 transcripts during a primary infection of trigeminal ganglia of cattle. 926 3

Small DNA viruses (adenoviruses, simian virus 40, or human papillomaviruses) induce S-phase progression but prevent cell division to provide precursors for viral DNA replication. Herpes simplex viruses types 1 or 2 (HSV-1 or HSV-2) contain genes which encode DNA-metabolizing enzymes, for example, ribonucleotide reductase, thymidine kinase and dUTPase, suggesting that S-phase factors are not required for an efficient infection. However, several studies indicated that HSV induces some events that occur during cell-cycle progression. To determine if HSV-2 induces S-phase entry, we examined serum-arrested African green monkey kidney cells (CV-1) after infection. Two hours after infection steady-state levels of the S-phase-specific cyclin, cyclin A, increased. S-phase cyclin-dependent kinase activity (CDK2) was stimulated 10-fold 8 h after infection but decreased at 16 or 24 h after infection. Mitotic CDK activity (CDC2) was not activated after infection, in part due to decreases in CDC2 protein levels and inactivation of enzymatic activity resulting from tyrosine phosphorylation of CDC2. Furthermore, CDK4 activity was not dramatically affected by infection. These studies indicate that HSV-2 infection selectively activates CDK2 after infection but cell-cycle progression does not occur. We hypothesize that infection activates certain components of the cell cycle which enhance viral gene expression and DNA replication.
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PMID:Analysis of cyclin-dependent kinase activity after herpes simplex virus type 2 infection. 940 Sep 86

The present study reports the activity of BILD 1633 SE against acyclovir (ACV)-resistant herpes simplex virus (HSV) infections in athymic nude (nu/nu) mice. BILD 1633 SE is a novel peptidomimetic inhibitor of HSV ribonucleotide reductase (RR). In vitro, it is more potent than ACV against several strains of wild-type as well as ACV-resistant HSV mutants. Its in vivo activity was tested against cutaneous viral infections in athymic nude mice infected with the ACV-resistant isolates HSV type 1 (HSV-1) dlsptk and PAAr5, which contain mutations in the viral thymidine kinase gene and the polymerase gene, respectively. Following cutaneous infection of athymic nude mice, both HSV-1 dlsptk and PAAr5 induced significant, reproducible, and persistent cutaneous lesions that lasted for more than 2 weeks. A 10-day treatment regimen with ACV given topically four times a day as a 5% cream or orally at up to 5 mg/ml in drinking water was partially effective against HSV-1 PAAr5 infection with a reduction of the area under the concentration-time curve (AUC) of 34 to 48%. The effects of ACV against HSV-1 dlsptk infection were not significant when it was administered topically and were only marginal when it was given in drinking water. Treatment under identical conditions with 5% topical BILD 1633 SE significantly reduced the cutaneous lesions caused by both HSV-1 dlsptk and PAAr5 infections. The effect of BILD 1633 SE against HSV-1 PAAr5 infections was more prominent and was inoculum and dose dependent, with AUC reductions of 96 and 67% against infections with 10(6) and 10(7) PFU per inoculation site, respectively. BILD 1633 SE also significantly decreased the lesions caused by HSV-1 dlsptk infection (28 to 51% AUC reduction). Combination therapy with topical BILD 1633 SE (5%) and ACV in drinking water (5 mg/ml) produced an antiviral effect against HSV-1 dlsptk and PAAr5 infections that was more than the sum of the effects of both drugs. This is the first report that a selective HSV RR subunit association inhibitor can be effective against ACV-resistant HSV infections in vivo.
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PMID:Antiviral activity of a selective ribonucleotide reductase inhibitor against acyclovir-resistant herpes simplex virus type 1 in vivo. 966 Sep 95

The expression of seven enzymes involved in the biosynthesis of DNA was measured in HL-60 promyelocytic leukemia cells treated with dimethylsulfoxide (DMSO) or all-trans retinoic acid (RA) to gain information on their role in the termination of proliferation in cells undergoing granulocytic differentiation. The steady-state levels of the mRNAs for topoisomerase I, topoisomerase II. DNA polymerase-alpha, thymidylate synthase, thymidine kinase and hypoxanthine-guanine phosphoribosyltransferase progressively declined from day 3 to day 7 of exposure to the polar solvent or the retinoid suggesting that the expression of these enzymes is coordinately regulated. In contrast, a pronounced difference between the two inducers of differentiation occurred in the expression of the mRNA of the M2 subunit of ribonucleotide reductase, with DMSO causing virtually complete inhibition of the expression of the M2 subunit of the enzyme from day 5 through day 7, with no change in the steady-state levels of the mRNA being produced by retinoic acid. Measurement of the enzymatic activities of two of these catalysts, thymidylate synthase and thymidine kinase, in cells exposed to the two inducers of maturation corroborated the findings at the level of the mRNAs, with corresponding decreases in the activity of these enzymes. The findings collectively demonstrate that the down-regulation of the expression of a relatively wide variety of enzymes involved in DNA replication occurs as late events in the granulocytic differentiation of HL-60 cells, ensuring that cellular replication cannot occur in terminally differentiated cells.
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PMID:Regulation of the expression of enzymes involved in the replication of DNA in chemically-induced granulocytic differentiation of HL-60 leukemia cells. 968 95

The expression of a number of housekeeping enzymes of DNA biosynthesis was measured in HL-60 promyelocytic leukemia cells undergoing monocytic/macrophagic differentiation following treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1alpha,25-dihydroxyvitamin D3 (vitamin D3). Progressive decreases in the steady-state levels of the mRNAs for thymidylate synthase, topoisomerase II, and hypoxanthine guanine phosphoribosyltransferase occurred following exposure to TPA or vitamin D3. In contrast, the steady-state levels of the mRNAs for thymidine kinase, topoisomerase I, and DNA polymerase-alpha did not decrease until days 3-5 of treatment with vitamin D3 and then progressively declined thereafter. The mRNAs for thymidine kinase and topoisomerase I decreased slightly and the mRNA for DNA polymerase-alpha by 30-40%, and then remained constant between days 1 to 3 of treatment with the phorbol ester. The M2 subunit of ribonucleotide reductase exhibited an even greater difference, with no change in the steady-state concentration of mRNA over 3 days of exposure to TPA or vitamin D3. On days 5-7 of treatment with vitamin D3, essentially complete loss of the expression of the mRNA for the M2 subunit of ribonucleotide reductase occurred. Measurement of the enzymatic activities of thymidylate synthase and thymidine kinase in cells exposed to either of the inducers of maturation corroborated the findings at the level of the mRNAs, with corresponding decreases in the activity of these enzymes. The results indicate that the down-regulation of the expression of housekeeping enzymes of DNA replication occurs as late events in HL-60 cells undergoing monocytic/macrophagic differentiation, implying that the decreases in their gene expression are the result of the termination of proliferation rather than an initiating event in the cessation of DNA biosynthesis.
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PMID:Regulation of the expression of enzymes involved in the replication of DNA in chemically induced monocytic/macrophagic differentiation of HL-60 leukemia cells. 968 96

Four independent mutants were isolated from mutagenized cultures of CHO cells by sib selection on the basis of resistance to a low concentration (2.6 x 10(-5) M) of BrdU. All four lines were stable, but all had about 100% of the wild-type (WT) specific activity of thymidine kinase (TK). None of the four yielded derivatives resistant to a high level of BrdU (2 x 10(-4) M) in one step even after mutagenesis, but variants resistant to 4-6 x 10(-5) M BrdU could be isolated at frequencies of about 2 x 10(-5)/cell. At frequencies of 10(-4)-10(-5), the second-step mutants gave colonies resistant to 2 x 10(-4) M BrdU. The second and third steps of resistance were correlated with partial and complete reduction, respectively, in the specific activity of TK, suggesting that the variants may be genotypically heterozygous and homozygous-negative at the tk locus. The first step of BrdU resistance was dominant and appeared to result from a mutation in the gene from ribonucleotide reductase, since in vitro assays on partially purified preparations showed that the reductase activity in mutant cells was less sensitive to BrdUTP than in WT cells.
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PMID:Bromodeoxyuridine resistance in CHO cells occurs in three discrete steps. 973 50

The influence of pre-existing anti-herpes simplex type 1 (HSV-1) immunity on HSV-1 vector-mediated gene transfer to glioma cells was analyzed in this gene marking study using intracranial D74 gliomas in syngeneic Fischer rats. The HSV-1 mutant virus used, hrR3, is defective in ribonucleotide reductase and bears the marker genes E. coli lacZ and HSV-1 thymidine kinase (HSVtk). Initial marker gene expression in tumors 12 h after direct virus injection was reduced in immunized animals to about 15% of that in nonimmunized animals. Marker gene expression in both sets stayed at initial levels for 2 days after intratumoral injection and declined markedly on day 5. Inflammatory infiltrates in the tumor were more prominent in HSV-1-immunized, as compared with nonimmunized animals, at 12 and 24 h, but appeared similar at 2-5 days after injection. By day 10, the immune reaction had subsided in immunized animals and macrophages remained only in nonimmunized animals. In conclusion, gene transfer to brain tumors using a HSV-1 vector was greatly reduced, but not completely abolished, under pre-immunization conditions. Pre-existing antibodies to HSV-1 may also serve a positive role in providing an increased margin of safety in intracranial application of HSV-1 vectors by limiting spread of the virus within the brain and to other tissues.
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PMID:Pre-existing herpes simplex virus 1 (HSV-1) immunity decreases, but does not abolish, gene transfer to experimental brain tumors by a HSV-1 vector. 974 61

Mechanisms responsible for neuroattenuation of herpes simplex virus (HSV) have been defined previously by studies of mutant viruses in cultured cells. The hypothesis that null mutations in host genes can override the attenuated phenotype of null mutations in certain viral genes was tested. Mutants such as those in infected cell protein (ICP) 0, thymidine kinase, ribonucleotide reductase, virion host shutoff, and ICP34.5 are reduced in their capacity to replicate in nondividing cells in culture and in vivo. The replication of these viruses was examined in eyes and trigeminal ganglia for 1-7 d after corneal inoculation in mice with null mutations (-/-) in interferon receptors (IFNR) for type I IFNs (IFN-alpha/betaR), type II IFN (IFN-gammaR), and both type I and type II IFNs (IFN-alpha/beta/gammaR). Viral titers in eyes and ganglia of IFN-gammaR-/- mice were not significantly different from congenic controls. However, in IFN-alpha/betaR-/- or IFN-alpha/beta/gammaR-/- mice, growth of all mutants, including those with significantly impaired growth in cell culture, was enhanced by up to 1,000-fold in eyes and trigeminal ganglia. Blepharitis and clinical signs of infection were evident in IFN-alpha/betaR-/- and IFN-alpha/beta/gammaR-/- but not control mice for all viruses. Also, IFNs were shown to significantly reduce productive infection of, and spread from intact, but not scarified, corneas. Particularly striking was restoration of near-normal trigeminal ganglion replication and neurovirulence of an ICP34.5 mutant in IFN-alpha/betaR-/- mice. These data show that IFNs play a major role in limiting mutant and wild-type HSV replication in the cornea and in the nervous system. In addition, the in vivo target of ICP34.5 may be host IFN responses. These experiments demonstrate an unsuspected role for host factors in defining the phenotypes of some HSV mutants in vivo. The phenotypes of mutant viruses therefore cannot be interpreted based solely upon studies in cell culture but must be considered carefully in the context of host factors that may define the in vivo phenotype.
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PMID:Interferons regulate the phenotype of wild-type and mutant herpes simplex viruses in vivo. 998 81


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