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 have studied the effect of sodium n-butyrate on the expression of specific genes. For this purpose, tk-ts13 cells (a thymidine kinase-deficient mutant originating from Syrian hamster cells) were microinjected or transfected with pC2, a plasmid containing the entire SV40 genome and the herpes simplex virus thymidine kinase gene (HSV-TK), cloned in pBR322. As a measure of the expression of these two genes, one of which is spliced (SV40) and the other one (HSV-TK) which is not, we have taken the protein levels (amount of T antigen for SV40 and incorporation of [3H]thymidine for HSV-TK) and the levels of RNA (by dot blot hybridization). The expression of the microinjected genes was inhibited when tk-ts13 cells were exposed to butyrate, actinomycin D, cycloheximide, and mitomycin C, but not when the cells were treated with insulin or dexamethasone. Further studies showed that a decrease in the percentage of T-positive cells occurs at lower concentrations of butyrate than a decrease in the levels of specific mRNA. In tk-ts13 cells transfected with pC2 and treated with butyrate at a concentration of 3 mM, SV40 mRNA levels are not decreased but the percentage of T-positive cells is decreased 50%. At 5 mM, the amount of T antigen/cell is decreased a further 40%. These results indicate that butyrate may have at least two sites of action, one at the level of mRNA amount and a second at the level of protein amount. In addition, our studies show that the use of microinjected or transfected genes offers certain unique possibilities for studies on the effects of environmental manipulations on gene expression.
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PMID:Effect of butyrate on the expression of microinjected or transfected genes. 298 77

As part of our study of antiherpetic acyclonucleosides, we synthesized a cyclic GMP analog, 9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide, sodium salt (2'-nor-cGMP), and discovered its potent and broad spectrum anti-DNA-viral activities. 2'-Nor-cGMP inhibits the replication of many DNA viruses, including herpes simplex virus, human cytomegalovirus, vaccinia, SV40, and adenovirus, but does not inhibit RNA viruses. In plaque reduction studies this potent antiviral agent is also approximately 10-fold more potent than 9-(1,3-dihydroxy-2-propoxymethyl)guanine (2'NDG) against varicella-zoster virus and inhibits cell transformation by bovine papilloma virus. Unlike 2'NDG, the potent activity of 2'-nor-cGMP against herpes virus is not dependent upon the action of virus-specified thymidine kinase. Intercellular metabolism of 2'-nor-cGMP produced small amounts of 2'NDG triphosphate which were insufficient to account for the antiviral activity observed, implying that this potent anti-DNA-viral agent operates by a mechanism different from that of known acyclonucleosides.
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PMID:2'-Nor-cGMP: a seco-cyclic nucleotide with powerful anti-DNA-viral activity. 298 34

Previous work from our laboratory suggested that the selective inhibition of Epstein-Barr virus (EBV) replication by 1-beta-D-arabinofuranosylthymine in human lymphoid cell lines involved the induction of a new thymidine kinase (TK) able to phosphorylate the thymidine analog. We further characterized this enzyme induced in various EBV-positive cell lines after viral genome activation with a combination of sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate. The following results confirmed the existence of an EBV-specific deoxypyrimidine kinase: induction of EBV-related TK was connected with the appearance of viral early antigens in EBV-carrying cells; unexpected behaviors of the enzyme activity upon different fractionating treatments led to the conclusion that EBV-induced TK was extracted as a complex molecular form, larger than other known cellular or viral isozymes; enzymatic properties distinguished EBV-induced TK from host lymphoid cell isozymes but made it resemble other herpesvirus-specific deoxypyrimidine kinases, i.e., by partial inhibition by dTTP or ammonium sulfate, insensitiveness to dCTP, and nonstringent specificity for normal TK substrates. Genetic evidence is required to definitively ensure that EBV-specific TK actually is virus coded in EBV-transformed human lymphoid cells.
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PMID:Characterization of an Epstein-Barr virus-induced thymidine kinase. 300 13

Several "sugar" modified acyclic nucleoside analogues related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 2) were synthesized and evaluated for antiviral activity. The preparation generally involved the condensation of the acetoxymethyl ether of alcohols 6c-g and 10-12a with diacetylguanine to give adducts 7c-g and 14-16, which were then deprotected to afford analogues 9c-g and 17-19. Alternatively, alcohols 12a and 13a were converted to iodides via their tosylates 12b and 13b and then reacted with the sodium salt of guanine to afford, after deprotection, analogues 22 and 23. A crossed aldol-Cannizzaro reaction on aldehyde 27 readily afforded 28, which was deprotected to give analogue 29. An in vitro assay against HSV-1 showed that all compounds tested were less active than DHPG, though several were good substrates for the viral thymidine kinase. The more promising acyclic nucleosides 9c, 19, and 29 were evaluated in a mouse encephalitis model and proved ineffective at preventing death at a dose of 20 mg/kg.
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PMID:Synthesis and anti-herpes-virus activity of acyclic 2'-deoxyguanosine analogues related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine. 301 63

We have demonstrated that X-rays induce mutations at 4 of 5 genetic loci. 2 of these loci, which code for a mRNA synthesis factor (resistance to 5,6-dichlororibofuranosylbenzimidazole) and tubulin (resistance to podophyllotoxin), are "small-marker" loci, in that they theoretically respond only to mutations which eliminate a toxin-binding site while leaving the major function of the protein intact. Thus mutations induced by X-rays in these two loci are most likely due to base-pair substitution-type alterations. X-Rays did not induce mutations in the Na+/K+ ATPase (resistance to ouabain), another small-marker locus. Two other loci, hypoxanthine guanine phosphoribosyl transferase (resistance to 6-thioguanine) and thymidine kinase (resistance to trifluorothymidine), are "whole-gene" targets in that they theoretically respond to a wide variety of mutagenic changes. X-Rays induced dose-dependent increases in mutant fraction at both of these loci. Ethyl methanesulfonate (EMS), an agent thought to produce mutations primarily through a base-pair substitution mechanism, induced mutations at all genetic loci tested. The pattern of mutations at the small-marker loci induced by EMS was different than that induced by X-rays, suggesting that the specificities of the mutagens and/or of the loci are different.
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PMID:X-rays mutate human lymphoblast cells at genetic loci that should respond only to point mutagens. 301 57

We measured the response of jejunal sodium (Na) absorption to neutral amino acid (L-alanine) and to dipeptides (L-alanyl-L-alanine, glycylsarcosine) in normal piglets and in piglets with acute viral diarrhea after experimental infection with transmissible gastroenteritis (TGE) virus. In the TGE jejunum villi were blunted, crypts were deepened, and the epithelium was composed of relatively undifferentiated cells with reduced disaccharidase, decreased sodium-potassium-stimulated ATPase, and elevated thymidine kinase activities. The response of Na absorption to a maximal concentration of L-alanine (20 mM) or D-glucose (30 mM) was significantly blunted in TGE jejunum in Ussing chambers. However, the addition of L-alanine together with D-glucose caused a significantly greater increment of Na absorption than either L-alanine or D-glucose alone in control and TGE tissue. The effect of Na absorption of the dipeptide L-alanyl-L-alanine (10 mM), which was rapidly hydrolyzed by control and TGE mucosa, was similar to that of L-alanine (20 mM), while glycylsarcosine, a poorly hydrolyzed dipeptide, did not change net Na absorption in the jejunum. Our data support the concept of separate carrier systems for neutral amino acid and hexose in the crypt-type intestinal epithelium characterizing viral enteritis. We speculate that a sodium-cotransporting amino acid, if added to oral glucose-electrolyte solutions, could benefit oral rehydration therapy in acute viral diarrhea; neither of the dipeptides tested here can be expected to enhance absorption to any greater extent than its constituent amino acids.
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PMID:Alanine enhances jejunal sodium absorption in the presence of glucose: studies in piglet viral diarrhea. 301 59

Dose-response experiments show that the presence of 300 microM cicloxolone sodium (CCX) or 500 microM carbenoxolone sodium (CBX) during the HSV replication cycle reduced the infectious virus yield by 10,000- to 100,000-fold: CCX is the more potent anti-herpes agent. HSV-2 replication was consistently more severely restricted by either drug than was that of HSV-1. The ED50 values obtained for either drug against HSV-1 or HSV-2 correlate well with data from dose-response curves. CCX, and to a lesser extent CBX, can be cytotoxic but the degree of cytotoxicity varies between cell lines and is also affected by the physiological state of the cells. Triterpenoid drugs exhibit some activity against virus particles in suspension but the effect is small and contributes little to the overall antiviral effect. The drugs appear to be active throughout the replication cycle. In contrast to all other anti-herpesvirus agents in clinical use the triterpenoid compounds do not appear to act directly to block virus DNA synthesis. HSV mutants resistant to ACG and PAA, or lacking a thymidine kinase gene, appear as sensitive as wild-type virus to CCX inhibition. HSV growth in the presence of the drugs resulted in a lower number of assembled virus particles but reduced to a much greater extent the infectious virus yield: thus the progeny virus quality is greatly diminished. Thermolability of progeny virus correlated well with this diminution of quality in increasing CCX concentration. SDS PAGE analysis of the structural proteins of virus particles made in cells treated with 300 microM CCX revealed numerous differences in the relative intensities of protein bands, which is in keeping with the changed quality of the drug-produced virus. SDS PAGE analysis of the polypeptides induced in drug treated infected cells revealed two effects; some polypeptides were synthesised in reduced amounts and the nuclear/cytoplasmic distribution of certain proteins was affected. Post-translational processing by glycosylation and sulphation of both cellular and HSV induced proteins was strongly inhibited by the triterpenoid drugs, while phosphorylation of only a few polypeptides appeared to be affected.
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PMID:The antiviral activity against herpes simplex virus of the triterpenoid compounds carbenoxolone sodium and cicloxolone sodium. 302 56

To clone the gene for the human Na+/H+ antiporter, we first constructed a stable mouse LTK- cell line (LAP1) lacking Na+/H+ antiport activity. Second, we devised a selective technique based on acid killing that specifically sorts out cells expressing low levels of Na+/H+ antiport activity from a population of antiporter-deficient cells (AP-). LAP1 cells (TK- and AP-) were cotransformed with human genomic DNA and the thymidine kinase (TK) gene. TK+ transformants, first selected, were submitted to acid loading. The rare transformants that survived (frequency, 2-8 X 10(-7) expressed Na+/H+ antiport activity (AP+). We found that: transformation with mouse LAP1 DNA did not give rise to AP+ transformants; transformation of LAP1 cells with DNA from an altered Na+/H+ antiporter hamster variant led to AP+ transformants expressing the altered Na+/H+ antiporter of the DNA donor; human repeated sequences were present in all primary, secondary, and tertiary mouse AP+ transformants; six identical EcoRI human DNA fragments (55 kilobase pairs of the human genome) cosegregated with the Na+/H+ antiport activity in secondary and tertiary transformants. These results strongly suggest that we have stably expressed the structural gene for the human Na+/H+ antiporter in mouse cells.
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PMID:Functional expression of a human Na+/H+ antiporter gene transfected into antiporter-deficient mouse L cells. 302 40

In the relatively undifferentiated jejunal mucosa occurring in piglet viral enteritis, we measured the response of transepithelial Na+ and Cl- fluxes in vitro to raised intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. At the acute 40-h stage of transmissible gastroenteritis (TGE), luminal membrane markers, sucrase and lactase, and a basolateral jejunal epithelial membrane marker Na+-K+-ATPase, were significantly decreased in activity, while a proliferative marker, thymidine kinase, was significantly enriched; these enzyme characteristics are typical of enterocytes isolated from crypts of other species. As expected, control piglet jejunum in short-circuited Ussing chambers after theophylline (10 mM) developed significant net secretory Na and Cl fluxes primarily due to significant antiabsorptive effects (delta JNa m----s = 3.48 +/- 0.52, delta JCl m----s = 2.59 +/- 0.28). Furosemide (10(-4) M), an inhibitor of electroneutral NaCl cotransport, produced antiabsorptive effects (delta JNa m----s = 2.53 +/- 0.31, delta JCl m----s = 2.58 +/- 0.28) in control jejunum that were not significantly different from those seen in response to theophylline. TGE jejunum, however, responded to theophylline not by an antiabsorptive effect but by significant electrogenic Cl- secretion (delta JCl s----m = 1.59 +/- 0.48); furosemide had no effect on ion fluxes in TGE tissue. Control and TGE jejunal mucosal homogenates did not differ in their basal or theophylline-stimulated levels of cAMP. We conclude that the relatively undifferentiated small intestine occurring in acute TGE does not generate either a cAMP-mediated antiabsorptive effect or a furosemide-mediated antiabsorptive effect.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Absence of a cAMP-mediated antiabsorptive effect in an undifferentiated jejunal epithelium. 303 40

A modified-live pseudorabies virus (PRV) vaccine, designated PRV(dlg92/d1tk), with deletions in the thymidine kinase (tk) and glycoprotein-gIII (g92) genes, was derived from the PRV (Bucharest [BUK]-d13) vaccine strain. The vaccine virus also contained a deletion in glycoprotein gI. Despite 3 deletions, PRV(dlg92/d1tk) replicated to high titers in cell culture from 30 C to 39.1 C. Enzyme assays and autoradiography revealed that PRV(dlg92/d1tk) did not induce a functional tk activity in infected tk- RAB(BU) cells (rabbit skin). Rabbit skin cells were infected with PRV(dlg92/d1tk), with vaccine strains derived from BUK or Bartha K strains of PRV or with the virulent Illinois (ILL), Indiana-Funkhauser (IND-F), and Aujeszky (Auj) strains of PRV and were labeled with [3H]mannose from 4 or 5 to 24 hours after infection to investigate whether these viruses induced the synthesis of glycoprotein gIII. Nonionic detergent extracts were prepared and immunoprecipitated with antisera from pigs vaccinated with tk(-)-PRV(BUK-d13) or tk+-Bartha K, pigs vaccinated with tk+-PRV(BUK) strains and then challenge exposed to tk+-PRV(IND-F), naturally infected domestic or feral pigs, and pigs vaccinated with tk-)-PRV(dlg92/d1tk). Mouse monoclonal antibodies against PRV glycoproteins gIII, gp50, and gII were also studied. After immunoprecipitation, labeled PRV-specific proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography. The PRV glycoprotein-gII complex, but not glycoprotein gIII, was synthesized in PRV(dlg92/d1tk)-infected cells. Glycoprotein gII and gIII were made in cells infected with PRV vaccine strains BUK, Bartha K, and BUK-d13 and with virulent PRV strains ILL, IND-F, and Auj. Cells infected with PRV(dlg92/d1tk) and with PRV strains ILL, IND-F, Auj, Bartha K, BUK, and BUK-d13, excreted into the cell culture medium a highly sulfated glycoprotein gX of about 90 kilodaltons. Antibodies to glycoprotein gIII were not detected in the sera of pigs inoculated with PRV(dlg92/d1tk), but were found in all other swine sera.
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PMID:Second-generation pseudorabies virus vaccine with deletions in thymidine kinase and glycoprotein genes. 303 72


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