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

---

Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An affinity-purified anti-TK1 antibody (pAb1) raised against a synthetic peptide (amino acids K211PGEAVAARKLFAPQ225) corresponding to part of the C-terminus of human cytosolic thymidine kinase (TK1) was produced and characterized by enzyme-linked immunosorbent assay, Western immunoblotting and immunoprecipitation as well as by immunostaining of intact cells. pAb1 recognized a single 25 kDa TK1 polypeptide in extracts of human and rodent cells. The protein was localized to the cytoplasm, as studied by immunohistochemistry and there was no staining in G1/G0 cells or mutant cells lacking TK1 activity, while it was high in S-phase and G2 cells. When series of peptides were tested for antibody binding in which alanine was replacing each of the other amino acids one by one, lysines 211 and 220, proline 212 and glutamic acid 214 were found to be important for antibody reactivity. These results indicate that amino acids 211-214, which may form a turn region, constitute a major recognition site for pAb1, and this structure may also be involved in the cell cycle-dependent modification of TK1, pAb1 is a very useful tool for studies of the cell cycle regulation of TK1, and it may be used to identify and quantify rapidly proliferating cells such as tumor cells.
...
PMID:Characterization of a peptide antibody against a C-terminal part of human and mouse cytosolic thymidine kinase, which is a marker for cell proliferation. 879 83

It has been reported that the polypeptide of thymidine kinase type 1 (TK1) from human and mouse cells can be modified by phosphorylation. Our laboratory has further shown that the level of human TK phosphorylation increases during mitotic arrest in different cell types (Chang, Z.-F., Huang, D.-Y., and Hsue, N.-C. (1994) J. Biol. Chem. 269:21249-21254). In the present study, we demonstrated that a mutation converting Ser13 to Ala abolished the mitotic phosphorylation of native TK1 expressed in Ltk- cells. Furthermore, we expressed recombinant proteins of wild-type and mutated human TK1 with fused FLAG epitope in HeLa cells, and confirmed the occurrence of mitotic phosphorylation on Ser13 of hTK1. By using an in vitro phosphorylation assay, it was shown that wild-type hTK1, but not mutant TK1(Ala13), could serve as a good substrate for Cdc2 or Cdk2 kinase. Coexpression of p21(waf1/cip1), which is a universal inhibitor of Cdk kinases, in Ltk- fibroblasts also suppressed mitotic phosphorylation of hTK1 expressed in this cell line. Thus, Cdc2 or related kinase(s) is probably involved in mitotic phosphorylation on Ser13 of the hTK1 polypeptide. We also found that mutation on Ser13 did not affect the functional activity of hTK1. As the sequences around Ser13 are highly conserved in vertebrate TK1s, we speculate that phosphorylation of Ser13 may play a role in the regulation of TK1 expression in the cell cycle.
...
PMID:Serine 13 is the site of mitotic phosphorylation of human thymidine kinase. 957 53

A monkey kidney cDNA that encodes a nuclear regulatory factor was identified by expression and affinity binding to a synthetic retinoic acid response element (RARE) and was used to isolate human placental and rat germ cell cDNAs by hybridization. The cDNAs encode a 59-kDa protein [nuclear DEAF-1-related (NUDR)] which shows sequence similarity to the Drosophila Deformed epidermal autoregulatory factor-1 (DEAF-1), a nonhomeodomain cofactor of embryonic Deformed gene expression. Similarities to other proteins indicate five functional domains in NUDR including an alanine-rich region prevalent in developmental transcription factors, a domain found in the promyelocytic leukemia-associated SP100 proteins, and a zinc finger homology domain associated with the AML1/MTG8 oncoprotein. Although NUDR mRNA displayed a wide tissue distribution in rats, elevated levels of protein were only observed in testicular germ cells, developing fetus, and transformed cell lines. Nuclear localization of NUDR was demonstrated by immunocytochemistry and by a green fluorescent protein-NUDR fusion protein. Site-directed mutagenesis of a nuclear localization signal resulted in cytoplasmic localization of the protein and eliminated NUDR-dependent transcriptional activation. Recombinant NUDR protein showed affinity for the RARE in mobility shifts; however it was efficiently displaced by retinoic acid receptor (RAR)/retinoid X receptor (RXR) complexes. In transient transfections, NUDR produced up to 26-fold inductions of a human proenkephalin promoter-reporter plasmid, with minimal effects on the promoters for prodynorphin or thymidine kinase. Placement of a RARE on the proenkephalin promoter increased NUDR-dependent activation to 41-fold, but this RARE-dependent increase was not transferable to a thymidine kinase promoter. Recombinant NUDR protein showed minimal binding affinity for proenkephalin promoter sequences, but was able to select DNA sequences from a random oligonucleotide library that had similar core-binding motifs (TTCG) as those recognized by DEAF-1. This motif is also present between the half-sites of several endogenous RAREs. The derived consensus- binding motif recognized by NUDR (TTCGGGNNTTTCCGG) was confirmed by mobility shift and deoxyribonuclease I (DNase I) protection assays; however, the consensus sequence was also unable to confer NUDR-dependent transcriptional activation to the thymidine kinase promoter. Our data suggests that NUDR may activate transcription independently of promoter binding, perhaps through protein-protein interaction with basal transcription factors, or by activation of secondary factors. The sequence and functional similarities between NUDR and DEAF-1 suggest that NUDR may also act as a cofactor to regulate the transcription of genes during fetal development or differentiation of testicular cells.
...
PMID:Characterization of a nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)-related (NUDR) transcriptional regulator protein. 977 84

Crystallographic structures of the ligand-binding domains for the retinoid X (RXR) and estrogen receptors have identified conserved surface residues that participate in dimer formation. Homologous regions have been identified in the human vitamin D receptor (hVDR). Mutating Lys-386 to Ala (K386A) in hVDR significantly reduced binding to glutathione S-transferase-RXRalpha in solution, whereas binding of an I384R/Q385R VDR mutant was almost undetectable. The K386A mutant formed heterodimers with RXRalpha on DR-3 (a direct repeat of AGGTCA spaced by three nucleotides), whereas the I384R/Q385R mutant completely eliminated heterodimer formation. Wild type hVDR effected a 3-fold induction of DR-3-dependent thymidine kinase-luciferase activity in cultured neonatal rat atrial myocytes, an effect that was increased to 8-9-fold by cotransfected hRXRalpha. Induction by K386A, in the presence or absence of RXRalpha, was only slightly lower than that seen with wild type VDR. On the other hand, I384R/Q385R alone displayed no stimulatory activity and less than 2-fold induction in the presence of hRXRalpha. Qualitatively similar findings were observed with the negative regulation of the human atrial natriuretic peptide gene promoter by these mutants. Collectively, these studies identify specific amino acids in hVDR that play a critical role in heterodimer formation and subsequent modulation of gene transcription.
...
PMID:Vitamin D-dependent suppression of human atrial natriuretic peptide gene promoter activity requires heterodimer assembly. 1019 14

Suicide gene therapy systems such as the herpes simplex thymidine kinase/ganciclovir system (TK/GCV) may kill cancer cells by apoptosis through as yet undefined mechanisms. Here we show that TK/GCV treatment induces p53 accumulation and increases cell surface expression of CD95 and tumor necrosis factor receptor, which is likely to involve p53-mediated translocation of CD95 to the cell surface. TK/GCV-induced apoptosis involves CD95-L-independent CD95 aggregation leading to the formation of a Fas-associated death domain protein (FADD) and caspase-8-containing, death-inducing signaling complex. Dominant negative FADD, the caspase-8 inhibitor zIETD-fmk [Z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone], and zVAD-fmk (Z-Val-Ala-Asp-fluoromethylketone) partially abrogate TK/GCV-induced apoptosis. In addition to apoptosis induction, TK/GCV treatment strongly sensitizes for CD95-L-, TNF-, and TNF-related, apoptosis-inducing, ligand (TRAIL)-induced cell death in constitutively resistant cells. These findings may be used to increase the efficacy of TK/GCV and other suicide gene therapy systems for the treatment of cancer.
...
PMID:Herpes simplex virus thymidine kinase/ganciclovir-induced apoptosis involves ligand-independent death receptor aggregation and activation of caspases. 1041 38

With the advent of gene therapy, herpes simplex virus type I (HSV-1) thymidine kinase (TK) has garnered much interest as a suicide gene for cancer ablation. As a means to improve the overall efficacy of the prodrug-gene activation approach, as well as to reduce ganciclovir-mediated toxicity, a large library of mutant thymidine kinases was generated and screened for the ability to enhance in vitro cell sensitivity to the prodrugs, ganciclovir (GCV) and acyclovir (ACV). Enzyme kinetics of one thymidine kinase mutant from this library that contains six amino acid substitutions at or near the active site reveals a distinct mechanism for providing enhanced prodrug-mediated killing in mammalian cells. In in vitro rat C6 cell prodrug sensitivity assays the TK mutant (mutant 30) achieves nanomolar IC50 values with GCV and ACV, in contrast to IC50values of 30 microM and >100 microM, respectively, for wild-type TK. In a mouse xenograft tumor model, growth of mutant 30 expressing tumors is restricted by ganciclovir at a dose at least 10- fold lower than one that impedes growth of wild-type TK-expressing tumors. Furthermore, in the presence of GCV a substantial bystander effect is observable when only 20% of the tumor cells express mutant 30 whereas no restriction in tumor growth is seen in tumors bearing the wild-type TK under the same conditions. The enhanced sensitization to prodrugs conferred by mutant 30 is apparently due to a 35-fold increase in thymidine Km which results in reduced competition between prodrug and thymidine at the active site. This provides mutant 30 a substantial kinetic advantage despite very high Kms for both ganciclovir and acyclovir. Molecular modeling of the mutations within the active site suggests that a tyrosine substitution at alanine 168 (A168) alters thymidine and prodrug interactions by causing catalytically important residues to move. The use of mutant 30 in place of the wild-type TK should provide a more effective gene therapy of cancer.
...
PMID:Enhancement of tumor ablation by a selected HSV-1 thymidine kinase mutant. 1046 66

Mutations in the thymidine kinase (TK) gene of herpes simplex virus (HSV) have been associated with resistance to acyclovir (ACY) and possible recognition of neurotropic strains. We sequenced a 335-bp segment of the TK gene to determine the frequency of mutations in HSV strains recovered from dermal, genital, and cerebrospinal fluid (CSF) specimens (n = 200; 102 HSV type 1 [HSV-1] 98 HSV-2 strains). Four polymorphic sites were detected in HSV-1 strains; C513T, A528G, C575T, and C672T. Among the polymorphisms, only C575T resulted in a change of amino acid sequence (residue 192, Ala-->Val). For HSV-2 strains, only one polymorphism (G420T) which resulted in an amino acid substitution (residue 139, Leu-->Phe) was detected. Phenotypic determination of resistance to ACY by a plaque reduction assay of 48 HSV isolates was not correlated with the sequence results of 11 strains in that 7 of these with genotypic polymorphisms were susceptible to the drug in vitro. In addition, of 32 ACY-resistant HSV strains, 28 (87.5%) had no polymorphisms detected in the 335-bp amplicon of the TK gene. There was no statistical difference in the frequency of polymorphisms according to the source of the specimens. We conclude that the detection of nucleic acid polymorphisms in a previously implicated 335-bp segment of the TK gene cannot be interpreted as indicative of either ACY resistance or neurotropism of HSV strains from dermal, genital, and CSF sources.
...
PMID:Role of genotypic analysis of the thymidine kinase gene of herpes simplex virus for determination of neurovirulence and resistance to acyclovir. 1048 72

It has been shown recently that granulocyte colony-stimulating factor (G-CSF) accelerates and enhances the hepatocyte proliferative capacity of partially hepatectomized rats. In the present study, we investigated the effect of G-CSF administration in a rat model of liver injury and regeneration, induced by thioacetamide (TAA) injection. TAA (300 mg/kg body weight) was injected intraperitoneally in male Wistar rats, and this was followed by administration of either saline (group A) or G-CSF at a dose of 150 microg/kg body weight (group B), 24 hr later. The animals were killed at different time points after TAA treatment and the rate of tritiated thymidine incorporation into hepatic DNA, the activity of the enzyme thymidine kinase (EC 2.7.1.21) in the liver, and the assessment of the mitotic index of hepatocytes, were employed to estimate liver regeneration. The administration of TAA caused severe hepatic injury, recognized histopathologically and by the raised activities of the serum hepatic enzymes aspartate and alanine aminotransferases. The hepatic injury, which peaked 36 hr after TAA injection, was followed by a regenerative process of hepatocytes presenting peaks at time points of 48 and 60 hr (group A). The administration of G-CSF 24 hr after the injection of TAA (group B) caused a statistically significantly increase in the hepatocyte proliferation indices examined (P < 0.001), compared to those found in group A at the same time points. It was concluded that G-CSF administration enhanced the hepatocyte proliferative capacity in this model of liver injury induced by TAA administration.
...
PMID:Effect of granulocyte colony-stimulating-factor administration on tissue regeneration due to thioacetamide-induced liver injury in rats. 1054 47

Herpes simplex virus type 1 (HSV-1) encodes a thymidine kinase (TK) that markedly differs from mammalian nucleoside kinases in terms of substrate specificity. It recognizes both pyrimidine 2'-deoxynucleosides and a variety of purine nucleoside analogs. Based on a computer modeling study and in an attempt to modify this specificity, an HSV-1 TK mutant enzyme containing an alanine-to-tyrosine mutation at amino acid position 167 was constructed. Compared with wild-type HSV-1 TK, the purified mutant HSV-1 TK(A167Y) enzyme was heavily compromised in phosphorylating pyrimidine nucleosides such as (E)-5-(2-bromovinyl)-2'-deoxyuridine and the natural substrate dThd, whereas its ability to phosphorylate the purine nucleoside analogs ganciclovir (GCV) and lobucavir was only reduced approximately 2-fold. Moreover, a markedly decreased competition of natural pyrimidine nucleosides (i.e., thymidine) with purine nucleoside analogs for phosphorylation by HSV-1 TK(A167Y) was observed. Human osteosarcoma cells transduced with the wild-type HSV-1 TK gene were extremely sensitive to the cytostatic effects of antiherpetic pyrimidine [i.e., (E)-5-(2-bromovinyl)-2'-deoxyuridine] and purine (i.e., GCV) nucleoside analogs. Transduction with the HSV-1 TK(A167Y) gene sensitized the osteosarcoma cells to a variety of purine nucleoside analogs, whereas there was no measurable cytostatic activity of pyrimidine nucleoside analogs. The unique properties of the A167Y mutant HSV-1 TK may give this enzyme a therapeutic advantage in an in vivo setting due to the markedly reduced dThd competition with GCV for phosphorylation by the HSV-1 TK.
...
PMID:Selective abolishment of pyrimidine nucleoside kinase activity of herpes simplex virus type 1 thymidine kinase by mutation of alanine-167 to tyrosine. 1109 70

The compounds WHI-05 (5-bromo-6-methoxy-5, 6-dihydro-3'-azidothymidine-5'-[p-methoxyphenyl] methoxyalaninyl phosphate) and WHI-07 (5-bromo-6-methoxy-5, 6-dihydro-3'-azidothymidine-5'-[p-bromophenyl] methoxyalaninyl phosphate) are aryl phosphate derivatives of zidovudine (ZDV) with dual-function anti-human immunodeficiency virus and contraceptive activity. These drugs were rationally designed to bypass the thymidine kinase (TK) dependency of ZDV activation as well as to achieve spermicidal activity. We investigated the TK activity and intracellular metabolism of WHI-05 and WHI-07 in normal human vaginal and cervical epithelial cells as well as sperm. The time- and concentration-dependent intracellular formation of ZDV metabolites following addition of WHI-05 and WHI-07 to normal human vaginal, ectocervical, and endocervical epithelial cells as well as motile sperm was studied by analytical HPLC. Thymidine kinase activity in these cells was determined by the flow cytometric method based on intracellular phosphorylation of the fluorescent nucleoside, 5-amino-2-deoxyuridine-dansyl chloride and by the ability of cell-free extracts to convert [(3)H]thymidine to thymidine monophosphate in comparison to NALM-6, a pre-B leukemia cell line. TK activity of genital tract epithelial cells and sperm was found to be relatively low or lacking. Addition of WHI-05 and WHI-07 to vaginal and cervical epithelial cells resulted in their concentration- and time-dependent conversion to alaninyl ZDV monophosphate (Ala-ZDV-MP) and 5'-ZDV monophosphate as the major metabolites. Studies using motile human sperm also demonstrated the conversion of WHI-05 and WHI-07 to Ala-ZDV-MP. These results demonstrate that human female genital tract epithelial cells and sperm efficiently convert WHI-05 and WHI-07 to bioactive ZDV metabolites despite their TK deficiency.
...
PMID:Thymidine kinase-independent intracellular delivery of bioactive nucleotides by aryl phosphate derivatives of bromo-methoxy zidovudine (compounds WHI-05 and WHI-07) in normal human female genital tract epithelial cells and sperm. 1113 58


<< Previous 1 2 3 4 Next >>

---