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)

Metallothioneins (MT) are cytosolic proteins rich in cysteine which play a physiological role in metal ion homeostasis. Heat shock proteins (HSPs) are expressed in various organs in response to different stress stimuli. The purpose of the present study was to examine the intrahepatic distribution of MT and HSP-27, -70 and -90 in two different experimental models of acute liver injury and regeneration, induced by either thioacetamide, or carbon tetrachloride administration in male Wistar rats. Toxicological endpoints and markers of hepatocellular regeneration were assessed at various time points following toxin administration. The enzymatic activities of aspartate and alanine aminotransferases in serum, and histological findings in the liver were used to estimate toxin-induced injury. Tritiated thymidine incorporation into hepatic DNA, liver thymidine kinase activity and hepatocyte mitotic index were used to estimate liver regeneration. MT and HSPs were detected immunohistochemically. At the time of maximum liver injury, moderate MT and intense HSPs expression was prominent in hepatocytes in the vicinity of necrotic areas. At the time of maximum hepatocellular proliferation, intense MT and HSP-90 staining was evident in all hepatocytes, while at the same time, mild HSP-27 and HSP-70 immunoreactivity was noted. Our findings indicate that the differential distribution of MT and HSPs in the liver after toxin-induced injury, in common with the observed pattern of staining, reflect liver proliferating capacity.
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PMID:Metallothionein and heat shock protein expression during acute liver injury and regeneration in rats. 1115 44

Metallothioneins (MTs), are low molecular weight proteins, mainly implicated in metal ion detoxification. In the present study, we investigated the expression of hepatic MT in a rat model of injury and regeneration, induced by carbon tetrachloride (CCl(4)) administration. A single intraperitoneal injection of 1 ml CCl(4)/kg body weight was performed in male Wistar rats, killed at different time points post-administration. The enzymatic activities of aspartate and alanine aminotransferases in serum were determined, in addition to the liver histological findings, to estimate hepatotoxicity. The rate of tritiated thymidine incorporation into hepatic DNA, the enzymatic activity of thymidine kinase in liver tissue and the assessment of the mitotic index in hepatocytes, were used as indices of regeneration. MT was detected immunohistochemically in liver tissue sections. CCl(4) administration caused severe hepatic injury, followed by regeneration. MT expression became prominent as early as 12 h after the administration of CCl(4), in the nuclei of hepatocytes, while at 24 and 36 h intense cytoplasmic staining for MT appeared in the hepatocytes in the vicinity of necrotic areas. The peak of hepatocyte proliferative capacity, occurring at 48 h post-CCl(4) administration coincides with the maximum nuclear and cytoplasmic MT expression. At further time points MT expression presented a decreasing trend. Induction of MT expression was observed in the liver after a single administration of CCl(4), being more prominent at the time of maximum hepatocellular proliferation, participating actively in the replication of hepatocytes.
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PMID:Induction of metallothionein in the liver of carbon tetrachloride intoxicated rats: an immunohistochemical study. 1129 62

The imaging of apoptosis represents an attractive diagnostic goal in the area of tumor therapy, degenerative diseases and organ transplantation. Since caspases play a key role during the early period of the intracellular signal cascade of cells undergoing apoptosis we considered benzyloxycarbonyl-Val-Ala-DL-Asp(O-methyl)-fluoromethyl ketone [Z-VAD-fmk], a pan-caspase inhibitor, as a potential apoptosis imaging agent. Applying the Tl(TFA)(3)/[131I]iodide method Z-VAD-fmk was successfully labeled at the benzyloxycarbonyl protecting group. The success of radioiodination, however, depended on the presence of carrier iodide resulting in specific radioactivities of 2.6 GBq/micromol and the formation of a mixture of the 2- and 4-iodophenyl derivative (61%) which could not be separated by HPLC. Uptake measurements were performed with Morris hepatoma cells (MH3924Atk8) which showed expression of the Herpes Simplex Virus thymidine kinase (HSVtk) gene. Apoptosis was induced by treatment of the cells with 25 microM ganciclovir. The TUNEL assay revealed 1.3 +/-0.3 and 23 +/-1.1% apoptotic cells immediately and 24 h after therapy, respectively. A two-fold increase of [131I]IZ-VAD-fmk uptake was found at the end of treatment with the HSVtk/suicide system which constantly remained elevated for the following 4 hours. The slow cellular influx and lack of uptake saturation of [131I]IZ-VAD-fmk are evidence for simple diffusion as transport mechanism. In addition, the absolute cellular uptake of [131I]IZ-VAD-fmk was found to be low. This quality was related to the rather high lipophilicity of [131I]IZ-VAD-fmk causing unspecific binding to macromolecules in the medium. Instead of using an inhibitor, synthetic caspase substrates are currently investigated which may accumulate in the apoptotic cell by metabolic trapping thereby enhancing the imaging signal.
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PMID:Investigation of a potential scintigraphic marker of apoptosis: radioiodinated Z-Val-Ala-DL-Asp(O-methyl)-fluoromethyl ketone. 1157

We report the design, synthesis and antiviral evaluation of a number of lipophilic, masked phosphoramidate derivatives of the antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), designed to act as membrane soluble prodrugs of the free nucleotide. The phosphoramidate derivatives of BVDU that contain L-alanine exhibited potent anti herpes simplex virus type 1 and varicella-zoster virus activity but lost marked activity against thymidine kinase-deficient virus strains. The phosphoramidate derivative bearing the amino acid alpha,alpha-dimethylglycine showed poor activity in all cell lines tested. It appears that successful kinase bypass by phosphoramidates is highly dependent on the nucleoside analogue, amino acid and ester structure, as well as the cell line to which the drugs are exposed.
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PMID:Synthesis and antiviral evaluation of phosphoramidate derivatives of (E)-5-(2-bromovinyl)-2'-deoxyuridine. 1190 Mar 48

The expression level of human thymidine kinase (hTK) is regulated in a cell-cycle-dependent manner. One of the mechanisms responsible for the fluctuation of TK expression in the cell cycle can be attributed to protein degradation during mitosis. Given the facts that cell-cycle-dependent proteolysis is highly conserved in all eukaryotes and yeast cells are an excellent model system for protein-degradation study, here we report on the use of Saccharomyces cerevisiae and Schizosaccharomyces pombe to investigate the degradation signal and mechanism required for hTK degradation. We found that the stability of hTK is significantly reduced in mitotic yeasts. Previously, we have observed that Ser-13 is the site of mitotic phosphorylation of hTK in HeLa cells [Chang, Huang and Chi (1998) J. Biol. Chem. 273, 12095-12100]. Here, we further provide evidence that the replacement of Ser-13 by Ala (S13A) renders hTK stable in S. pombe and S. cerevisiae. Most interestingly, we demonstrated that degradation of hTK is impaired in S. cerevisiae carrying a temperature-sensitive mutation in the proteasomal gene pre1-1 or the Skp1-Cullin-1/CDC53-F-box (SCF) complex gene cdc34 or cdc53, suggesting the contribution of the SCF-mediated pathway in hTK degradation. As phosphorylation is a prerequisite signal for SCF recognition, our results implied that phosphorylation of Ser-13 probably contributes to the degradation signal for hTK via the SCF-mediated proteolytic pathway.
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PMID:Degradation of human thymidine kinase is dependent on serine-13 phosphorylation: involvement of the SCF-mediated pathway. 1243 75

We report the first mutational study of thymidine kinase 1 (TK1) performed in human solid tumors. We sequenced cDNAs representing the complete coding region of TK1 in human breast (n=22) and colorectal (n=26) cancer. Codon 106 near the ATP binding site constantly differed (ATG --> GTG; Met --> Val) from the one deposited by Bradshaw and Deininger in the Genbank database (Accession number NM_003258). Silent polymorphisms at codon 11 (CCC --> CCT; Pro --> Pro) and codon 75 (GCG --> GCA; Ala --> Ala) were frequently detected in tumors as well as in normal tissues. In breast cancer the two polymorphisms were observed in 63.6% of the samples analyzed. No significant association could be found between polymorphisms and TK activity. In colorectal cancer the incidence of the two changes was 73.1% and 69.2%, respectively. Interestingly, one colon cancer with high cytosolic TK activity displayed two missense mutations located in and near the putative phosphorylation site by tyrosine kinase (s) (TAT --> CAT; Tyr --> His) and by cAMP-, cGMP-dependent protein kinase (TAC --> TGC; Tyr --> Cys), respectively; adjacent normal mucosa showed no mutation. This may open new avenues that imply TK1 activity in tumor cell proliferation.
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PMID:Mutation analysis in the coding sequence of thymidine kinase 1 in breast and colorectal cancer. 1269 56

A major obstacle in gene-therapy protocols is T-cell-mediated destruction of transgene-expressing cells. Therefore new approaches are needed to prevent rapid clearance of transduced cells. We exploited the Gly-Ala repeat (GAr) domain of the Epstein-Barr virus nuclear antigen-1, since the GAr prevents cytotoxic T-lymphocyte-epitope generation. Here we show that three different enzymes (viz. the E. coli LacZ gene encoded beta-galactosidase, firefly luciferase, and HSV1 thymidine kinase) fused with the GAr retained their function. Moreover, linking GAr with beta-galactosidase successfully prevented recognition of GAr-LacZ-expressing cells by beta-galactosidase-specific CTL. Nonetheless, vaccination with a GAr-LacZ adenovirus or with an allogeneic cell line expressing GAr-LacZ resulted in the induction of beta-gal-specific CTL. This demonstrates that the GAr domain does not inhibit cross presentation of antigens, but only affects breakdown of endogenously synthesized proteins. These data demonstrate how the GAr domain can be exploited to create immuno'stealth' genes by hiding transgene products from CTL-mediated immune attack.
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PMID:Creation of immune 'stealth' genes for gene therapy through fusion with the Gly-Ala repeat of EBNA-1. 1456 61

Mitochondrial deoxyribonucleic acid depletion syndromes are autosomal recessive disorders characterized by a reduction of the amount of mitochondrial deoxyribonucleic acid, which impairs the synthesis of respiratory chain complexes. Mutations in the deoxyguanosine kinase and polymerase gamma genes have been identified in hepatocerebral forms, whereas thymidine kinase 2 gene mutations have been found in patients with isolated myopathy, encephalomyopathy, or spinal muscular atrophy. Mutations in the gene encoding the beta subunit of the adenosine diphosphate-forming succinyl-coenzyme A synthetase have also been reported in a family. In this report, the clinical, molecular, morphologic, and biochemical features of five children from two independent families with an infantile encephalomyopathy are characterized. The affected children manifested muscle mitochondrial deoxyribonucleic acid depletion and three novel thymidine kinase 2 gene mutations. They consist of a homozygous substitution resulting in Ala to Val change at the highly conserved position 181 of thymidine kinase in the first family, and two heterozygous substitutions in the second family: a Cys to Trp change at residue 108 and a Leu to Pro change at residue 257 of the enzyme. Common clinical features associated with these TK2 mutations are a normal early developmental phase followed by psychomotor regression, encephalopathy often with epileptic seizures, and myopathy with features of a progressive dystrophic process.
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PMID:New mutations in TK2 gene associated with mitochondrial DNA depletion. 1650 86

Herpes simplex virus type 1 (HSV) thymidine kinase (TK) has been widely used in suicide gene therapy for the treatment of cancer due to its broad substrate specificity and the inability of the endogenous human TK to phosphorylate guanosine analogs such as ganciclovir (GCV). The basis of suicide gene therapy is the introduction of a gene that encodes a prodrug-activating enzyme into tumor cells. After administration, the prodrug is selectively converted to a toxic drug by the suicide gene product thereby bringing about the eradication of the cancer cells. A major drawback to this therapy is the low activity the enzyme displays towards the prodrugs, requiring high prodrug doses that result in adverse side effects. Earlier studies revealed two HSV TK variants (SR39 and mutant 30) derived by random mutagenesis with enhanced activities towards GCV in vitro and in vivo. While these mutants contain multiple amino acid substitutions, molecular modeling suggests that substitutions at alanine 168 (A168) may be responsible for the observed increase in prodrug sensitivity. To evaluate this, site-directed mutagenesis was used to individually substitute A168 with phenylalanine or tyrosine to reflect the mutations found in SR39 and mutant 30, respectively. Additionally, kinetic parameters and the ability of these mutants to sensitize tumor cells to GCV in comparison to wild-type thymidine kinase were determined.
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PMID:The role of herpes simplex virus-1 thymidine kinase alanine 168 in substrate specificity. 1894 76

Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-type dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K(m) values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.
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PMID:Extending thymidine kinase activity to the catalytic repertoire of human deoxycytidine kinase. 1915 29


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