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)

Association of herpes simplex virus (HSV)-related antigens with chromosomes was demonstrated in human and mouse cells biochemically transformed by HSV that had been irradiated with ultraviolet light. This was accomplished by using peroxidase-anti-peroxidase immunological staining with rabbit antisera that had high neutralizing titers against both HSV-specific thymidine kinase activity and virus infectivity. Antisera-against HSV did not react with chromosomes of uninfected cells nor did normal sera react with any of the constitutents of biochemically transformed cells. Methanol/acetic acid treatment of biochemically transformed cells eliminated their nuclear staining for HSV-related antigens. In vitro binding of HSV-related antigens to chromosomes was demonstrated by incubating soluble antigens from high salt extracts of HSV-infected cells with methanol/acetic acid-fixed chromosomes of biochemically transformed or uninfected cells, followed by exposure to antiserum against HSV and peroxidase-anti-peroxidase staining. There was no staining when soluble extracts from uninfected cells were substituted for those from HSV-infected cells. The results show that cells biochemically transformed and lytically infected by HSV, respectively, contain antigens, which like the Epstein-Barr virus-associated nuclear antigen (EBNA), bind to chromosomes in vivo and in vitro.
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PMID:Binding to chromosomes of herpes simplex-related antigens in biochemically transformed cells. 21 Apr 57

The biochemical characteristics of complex formation in nuclear extracts from mock-infected and herpes simplex virus (HSV)-infected Vero and HeLa cells with a sequence downstream of and adjacent to the promoter for the HSV thymidine kinase gene were studied using the mobility shift electrophoresis assay. This region is bound by host cell proteins, as evidenced by the formation of complexes after incubation in extracts from mock-infected cells. Unique virus-specific complexes form in extracts prepared from infected cells, and these complexes contain ICP4, the major regulatory protein of HSV. Examination of the salt requirements for assembly and the stability of preformed DNA-protein complexes to added salt demonstrate the distinct nature of the complexes that form in each extract. This finding is supported by analyses of the relative association and dissociation rates of these complexes which show that complexes formed in extracts prepared from infected cells are kinetically labile. After depletion with chelators, the divalent cation requirements for complex formation were assayed by supplementation with various metal salts. Addition of Mn2+ restored binding activity in extracts from both mock-infected and infected HeLa cells. Finally, footprinting assays revealed that sequences on each strand throughout this region of the thymidine kinase gene were involved in complex formation only in extracts from mock-infected cells. These experiments suggest that one consequence of virus gene expression is to alter the interaction of cell proteins with virus DNA.
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PMID:Characterization of DNA-protein complex formation in nuclear extracts with a sequence from the herpes simplex virus thymidine kinase gene. 215 38

Both thymidine kinase (TK) and DNA polymerase (DNAp) are present in measurable amounts in human serum. Even though the use of TK as a clinical marker is rapidly increasing there has been no attempt to characterize the serum TK in a wider extent, i.e.; with respect to Mw or other biochemical parameters. Therefore sera with high TK or DNAp activities derived from patients with cytomegalovirus (CMV) infection, B12-deficiency and leukaemia were fractionated by gel exclusion chromatography. The TK activity eluted as two peaks, one major TK activity with an apparent molecular weight (Mw) or 730 kD and one minor TK activity corresponding to a Mw of 58 kD. The amount of TK activity at 58 kD varied between 7 and 23% of total activity, depending on the serum fractionated. The DNAp activity in sera from patients with malignant disease and B12 deficiency eluted as a single peak corresponding to a Mw of 240 kD. A DNAp with a different Mw (greater than 1000 kD) was recovered from 1 of 3 investigated immunosuppressed patients with CMV infection. A similar pattern of enzyme forms was observed when sera were separated by glycerol gradient centrifugation. The effect of high salt and various reaction solution components on the enzymes were studied. The only condition found that affected the molecular forms of TK was the state of reduction. Incubation of sera with high concentrations of dithioerythritol (DTE) (400 mM) prior to separation transferred all serum TK to the 58 kD form, it also converted most of the serum DNAp from the 240 kD form to a smaller form (56 kD) without affecting the total recovery of enzymatic activity. The reaction product from both TK forms was exclusively monophosphate and none of the TK forms could efficiently utilize cytidine triphosphate as phosphate donor. The substrate kinetics of the small serum TK fraction was identical with those of an enzyme with similar size purified from proliferating HeLa cells, indicating that both serum TK activities are forms of TK 1, the proliferation associated cellular isozyme.
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PMID:Molecular forms in human serum of enzymes synthesizing DNA precursors and DNA. 215 79

The molecular forms of estrogen receptor (ER) in estrogen-responsive mouse Leydig cell line (B-1) have been examined in relation to their biological activity. ER was predominantly recovered in the nuclear fraction upon homogenization even after cells were precultured in the absence of E2 and Phenol Red. This unoccupied nuclear ER (ERn) whose hormone binding ability was extremely thermostable could be extracted with 0.4 M KCl. This stability enabled us to determine hydrodynamic parameters in the ligand-free condition. The Stokes radius and sedimentation constant of this ERn in high salt condition were 5.5 nm and 6.0S, respectively, resulting in its molecular weight of 140,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ER labeled with [3H]tamoxifen aziridine gave a single band of 65,000 Da, indicating that this ERn had a oligomer structure similar to that of transformed nuclear ER complexed with estrogen in the putative target cells. Therefore, we further examined the possibility that this ERn in B-1 cells can activate estrogen-responsive genes without any aid from estrogen. Estrogen responsive element-thymidine kinase promoter-chloramphenicol acetyltransferase fusion gene (ERE-tk-CAT) was transfected into B-1 cells. CAT activity was enhanced only in cells stimulated with estrogen. It may be concluded from these results that transformed ERn can be formed in the absence of estrogen but that binding to estrogen may be required in order to exert its biological activity.
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PMID:Identification of unoccupied but transformed nuclear estrogen receptor in cultured mouse Leydig cell. 235 31

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

A series of potential prodrug 5-halouridine 3',5'-cyclic monophosphates (5-X-cUMPs, X = F, Cl, Br, I, 1-4) has been prepared and tested for antitumor activity against murine leukemia L1210/0 and human lymphoblast Raji/0 cells and their deoxythymidine kinase deficient (TK-) counterparts, as well as for antiviral activity in primary rabbit kidney cells infected with herpes simplex virus type 1 or 2, vaccinia virus, or vesicular stomatitis virus. The 5-halopyrimidine bases, nucleosides (5-X-U), and 5'-monophosphates (5-X-UMP) were tested for comparison. 5-F-cUMP (1) showed reasonably potent inhibition of tumor cell proliferation (ID50 = 0.33-1.6 micrograms/mL), while the remaining diesters displayed ID50's ranging from 210 to greater than 1000 micrograms/mL. 5-F-cUMP was 70- to 300-fold less active than 5-F-dU in the same systems. With TK- L1210 cells, 5-F-cUMP was as potent as with the normal (L1210/0) line but was about fourfold less active with TK- Raji cells compared to Raji/0 cells. The 5-X-cUMPs showed little potency as antivirals. A single-crystal X-ray analysis of the ammonium salt of 5-I-cUMP confirmed its structure and showed the conformation of the phosphate ring to be the expected chair. The ribose pucker is near 3(4)T, and the torsion angle about the beta-glycosidic N(1)-C(1') bond is in the syn range (-84.8 degrees).
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PMID:Synthesis, structure, and antitumor and antiviral activities of a series of 5-halouridine cyclic 3',5'-monophosphates. 300 59

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

Present experimental data show that the synthesis of ribosomal protein S1 and PI protein was stimulated greatly by polyamines at the early stage after addition of putrescine in polyamine-requiring mutants of E. coli. No macromolecular synthesis was stimulated at this stage. Polyamine stimulation of the synthesis of these proteins probably plays an important role for cell growth. In polyamine-deficient bovine lymphocytes, protein synthesis became perturbed before RNA and DNA synthesis. Among enzymes concerned with DNA replication, thymidine kinase activity was most strongly influenced by polyamines. The activity in polyamine-deficient cells was only 7% of the level in normal cells. Judging from the amount of thymidine kinase mRNA and its distribution in polysomes, it was concluded that polyamines mainly regulate the synthesis of thymidine kinase at the level of initiation of protein synthesis. A polyamine-free protein synthetic system, established from components of rabbit reticulocytes, consisted of globin mRNA, salt-washed ribosomes, partially purified initiation factors, and pH 5 enzymes. Spermidine added to this system not only lowered the optimal magnesium concentration required for globin synthesis, but it also stimulated the globin synthesis 8- to 10-fold. The optimal spermidine concentration was 0.4 to 0.6 mM, a concentration similar to that in intact rabbit reticulocytes. The ratio of alpha to beta globin chains synthesized in the presence of spermidine and Mg2+ was approximately 1.0, while the ratio in the presence of only Mg2+ was approximately 1.5. The results strongly suggest that polyamines play an important role in rabbit reticulocyte protein synthesis.
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PMID:Regulation of protein synthesis by polyamines. 307 28

Sea urchin early histone genes are active in preblastula embryos; late histone genes are maximally expressed during subsequent stages of embryogenesis. We used the Xenopus laevis oocyte to assay for trans-acting factors involved in this differential regulation. Sea urchin nuclear proteins were prepared by extracting gastrula-stage chromatin successively with 0.45, 1, and 2 M NaCl. We injected three fractions into oocytes along with plasmids bearing sea urchin early and late H2b histone genes. While neither the 0 to 0.45 M nor the 1 to 2 M salt fraction affected H2b gene expression, the 0.45 to 1 M salt fraction stimulated early and late H2b mRNA levels significantly. Late H2b gene expression was stimulated preferentially when the early and late genes were coinjected into the same oocytes. This extract did not stimulate the accumulation of transcripts of injected herpesvirus thymidine kinase genes or of the sea urchin Spec 1 gene, suggesting that the stimulatory activity is not a general transcription factor. We localized the DNA sequence required for the stimulatory effect to a region of the late H2b gene located between -43 and +62 relative to the transcription start site. A component of the 0.45 to 1 M salt wash fraction specifically bound to the 105-base-pair late gene DNA sequence and to the corresponding early gene fragment. The abundance of this binding activity decreased on a per genome basis during early development of the sea urchin.
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PMID:Differential stimulation of sea urchin early and late H2B histone gene expression by a gastrula nuclear extract after injection into Xenopus laevis oocytes. 336 8

Ribonucleotide reductase is an essential enzyme in mammalian DNA replication. In quiescent BHK-21/C13 cells exhibiting a low level of ribonucleotide reductase activity, infection with herpes simplex virus (HSV) resulted in the early induction of an altered ribonucleotide reductase. The extent of the induction was dependent upon the m.o.i. and could be diminished or prevented by u.v. treatment of the viral stock, or by inhibitors of mRNA synthesis or protein synthesis. The induction followed the same course of synthesis as viral thymidine kinase and DNA polymerase, and could thus be classified with them as a beta polypeptide. These results suggested that the new activity was produced as a consequence of the virus genome expression. Comparisons of the properties of ribonucleotide reductase extracted from exponentially growing BHK-21/C13 cells showed that the HSV-induced enzyme differed from the cellular isozyme by its insensitivity to inhibition by dTTP, dATP or araATP and its resistance to high salt concentrations. On the other hand, the virus-induced enzyme and the cellular isozyme exhibited a similar sensitivity to hydroxyurea. Therefore, the reported inhibition of HSV DNA replication by hydroxyurea could be the result of inhibition of both HSV-induced and cellular reductase activities.
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PMID:Characterization of ribonucleotide reductase induction in BHK-21/C13 Syrian hamster cell line upon infection by herpes simplex virus (HSV). 617 49


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