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)

The involvement of protein kinase C (PKC), a 12-O-tetradecanoylphorbol-13-acetate (TPA) receptor, in the transcriptional regulation of TPA-inducible genes was determined. Expression plasmids harboring full-length or kinase domain of PKC alpha and PKC delta (PKC alpha K and PKC delta K) were constructed. Transient transfection of PKC alpha K and PKC delta K into COS cells resulted in approximately 20- and 16-fold increase in phospholipid-, calcium-independent protein kinase activity. To determine the effects of overexpression of PKC alpha K and PKC delta K on the AP-1-mediated TPA-inducible genes, we transfected into COS cells the PKC alpha K or PKC delta K expression plasmids with collagenase chloramphenicol acetyltransferase (CAT) reporter construct containing one TPA responsive element (TRE), or a construct containing five synthetic TRE linked to a thymidine kinase promoter. PKC alpha K or PKC delta K overexpression resulted in a comparable increase (approximately 4-fold) in CAT activity. However, CAT activity was not increased after transfection of PKC constructs with non-TPA responsive thyroid hormone responsive elements CAT construct (delta MTV-TyRE-pCAT). We also found that deletion of the AP-1-like motif in the SV40 promoter abolished the PKC alpha K or PKC delta K-induced activity of luciferase (luc) reporter constructs. Overexpression of full-length PKC delta in COS cells also increased the activity of the CAT construct with TRE after TPA treatment. We determined the effects of overexpression of PKC alpha K and PKC delta K on transcription of the ornithine decarboxylase (ODC) gene, which has a non-AP-1 TRE. Cotransfection of PKC alpha K or PKC delta K expression plasmids with a TPA-inducible ODC luc construct (-72/+130-ODC-luc) into HeLa cells resulted in an increased luc activity. These results indicate that both PKC alpha (calcium dependent) and PKC delta (calcium independent) may mediate the transcription of TPA-inducible genes through both AP-1 and non-AP-1 sequences.
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PMID:Involvement of protein kinase C in the transcriptional regulation of 12-O-tetradecanoylphorbol-13-acetate-inducible genes modulated by AP-1 or non-AP-1 transacting factors. 814 84

Human diploid fibroblast cells lose replicative potential after a certain number of population doublings. We use this experimental system to investigate the role of oxidative damage in cellular aging. Treating cells with H2O2 at < 300 microM did not affect the viability of the majority of cells when judged by morphology, trypan blue exclusion, and protein synthesis. However, the treatment caused a dose-dependent inhibition of DNA synthesis. After a 2-hr treatment with 200 microM H2O2, the cells failed to respond to a stimulus of serum, platelet-derived growth factor, basic fibroblast growth factor, or epidermal growth factor by synthesizing DNA, and the loss of response could not be recovered by 4 days. Subcultivation showed that, as in senescent cells, division of the treated cells was inhibited. The life-time cumulative growth curve showed that the loss of replication due to H2O2 treatment was cumulative and irreversible. The H2O2 treatment decreased the number of the population doublings in the rest of the life span by 35.3 +/- 10.3%. Enzymatic assays indicated that, like the cells in their senescent state, the treated cells were less able to activate ornithine decarboxylase and thymidine kinase. Furthermore, subcultivation after the H2O2 treatment showed that the cells developed the morphology of senescent cells. In conclusion, sublethal treatment of H2O2 "stunned" F65 cells and caused the cells to enter a state resembling senescence.
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PMID:Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. 818 82

Current knowledge of liver regeneration after reduced liver transplantation is limited. Warm ischemia is one component of the reduced liver transplantation procedure that could have an impact on the regenerative response. To study this effect, we performed partial hepatectomy on male Long-Evans rats, with animals divided into four groups: group 1 underwent partial hepatectomy only; group 2 underwent partial hepatectomy and 40 min of ischemia; group 3 underwent partial hepatectomy, 40 min of ischemia and portocaval shunt surgery; and group 4 underwent partial hepatectomy and orthotopic autograft surgery. Group 5 consisted of sham-operated animals. Animals were killed 4, 24, 48, 72 and 96 hr after surgery. Thymidine kinase activity, mitotic index, a liver mass index and ornithine decarboxylase levels were used as parameters of liver regeneration. Aspartate transaminase was recorded. Maximal thymidine kinase and mitotic index were observed in group 1 animals at 24 hr. In groups 2, 3 and 4 maximal thymidine kinase activity and mitotic activity were observed 24 hr later at 48 hr. The magnitude of the peak response in these groups appeared to correlate with the duration of portal venous occlusion, with greatest increases occurring in those groups where portal stasis was most prolonged. The increase in liver mass for these groups was also delayed with respect to group 1 animals. The anticipated peak in ornithine decarboxylase levels was seen at 4 hr in group 1. The ornithine decarboxylase response in the other groups was disorganized, with delay of the recorded peaks.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Liver regeneration after hepatic ischemia and reduced liver autotransplantation in the rat. 842 25

The effect of various components of the transplant procedure upon the regenerative process in reduced liver grafts is not known. In this study, partially hepatectomised rat liver remnants were flushed with 5 ml of either Ringer's lactate, Euro Collins solution or University of Wisconsin solution at 4 degrees C and then 5 ml Ringer's lactate at 4 degrees C. After partial hepatectomy alone, the peak increase in thymidine kinase was measured at 24 hours (36,021 +/- 8,060 disintegrations per minute per milligram protein; the mitotic index was 25 +/- 7). In all the groups in which the remnant was flushed, peak thymidine kinase and mitotic index were measured at 48 hours. The pattern of ornithine decarboxylase activity was disorganised in all groups. Flushing of the liver remnant therefore delays the regenerative response by 24 hours. In large animals, including humans, regeneration appears to commence within the first 5 days after resection. A comparable delay doubling this time might coincide with the onset of rejection and further compromise liver function.
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PMID:Regeneration after in situ flushing of partially hepatectomised rat livers. 854 30

Liver regeneration is an essential component of the reparative process following liver injury and surgical resection. It can be assessed by different tissue-based tests such as liver weights, mitotic counts, DNA contents and synthesis rates, immunohistochemical staining of nuclear antigens, gene expressions and certain protein levels or various serum-based tests that largely consist of specific enzyme determinations or documentation of certain proliferation markers. Although the simplest tissue-based test of liver regeneration is measurement of liver weights, these determinations are influenced by the extent of deposition of various materials not directly related to regeneration, such as lipids, glycogen and blood volumes. Because mitosis constitutes a very short segment of the cell cycle, mitotic counts are infrequently observed by light microscopy. Thymidine and BrdU incorporation into DNA are the reference tools for studying DNA synthesis, but their use requires pre-injection with radioactive isotopes or nucleotides which render them impractical for human studies. Flow cytometry is an accurate and objective method of monitoring hepatic regenerative activity but requires sophisticated equipment that is not generally available in many laboratories. Immunohistochemical staining for nuclear antigens (Ki-67, proliferating cell nuclear antigen [PCNA], DNA polymerase alpha and nucleolar organizer region [NOR] proteins) are acceptable and commonly used methods of monitoring regenerative activity but are subject to inter- and intra-observer variability. Gene expression rates such as Histone-3 mRNA abundance are hampered by the relatively low rates of gene transcription and the need for recombinant DNA technology. Protein and enzyme levels in liver tissues, such as putrescine, ornithine decarboxylase and thymidine kinase, are not precise and are confounded by the nutritional status of the host. While PCNA protein levels measured by immunoblot hold promise as a simple, accurate and reproducible marker of liver regeneration, additional studies are required to determine if this is a valid marker of regenerative activity in various models of hepatic injury and in humans. Of the serum-based determinations: thymidine kinase, ornithine decarboxylase, fibronectin, alpha fetoprotein, and early pregnancy factor offer practical and non-invasive tools to monitor liver regeneration, but the sensitivity and specificity of these tests have yet to be determined. In conclusion, many tissue and serum-based methods have been employed in clinical and experimental studies to assess liver regeneration; however, a gold standard has yet to be identified. Because of the disadvantages inherent in each method, and until a new, more accurate marker is identified, clinicians and scientists should incorporate a minimum of two independent markers in studies of liver regeneration.
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PMID:Liver regeneration: methods for monitoring and their applications. 912 13

Mature, female swine were randomly assigned to one of seven dietary groups. Swine in groups 1-3 were fed a cholesterol-rich diet for 55 days while the remaining groups remained on a basal swine diet. At the end of the cholesterol(Chol)-preloading period the swine in groups 1-7 were placed on menhaden oil (MO) and/or corn oil (CO) as follows: groups 1 and 4, 15% CO (control); groups 2 and 5, 0.75% MO+14.25% CO; groups 3 and 7, 15% MO; and group 6, 7.5% MO+7.5% CO. Animals were killed at the end of the approximately 6-month feeding period and portions of liver, pancreas and colon mucosa were analyzed for both ornithine decarboxylase (ODC) and thymidine kinase (TK) activity while polyamine levels were measured in the liver and pancreas. Statistical analyses were carried out by one-way and two-way ANOVA and by trend analysis. In the pancreas, the highest MO group (group 7) had significantly higher ODC levels when compared with the CO control (group 4) and the next to highest MO group (group 6) (one-way ANOVA)-all non-cholesterol preloaded groups. Using a two-way ANOVA (Chol-by-MO), liver ODC was significantly lower in the CO control when compared with the lowest and highest MO groups (groups 5 and 7, respectively), again in the non-cholesterol-preloaded animals. In the colon, the swine in the Chol-low MO group (group 2) had significantly lower TK activity than the Chol/CO control group (group 1) and Chol/Hi MO group (group 3) (one-way ANOVA) and also had significantly lower activity than all groups except the CO control (group 4) (two-way ANOVA). Liver acetylputrescine in the lowest and highest MO groups (groups 5 and 7, respectively) was significantly higher than in the CO group (group 4). Liver spermidine in the Chol-Hi MO group (group 3) was significantly higher than the Chol-Lo MO group (group 2), while the highest MO group (group 7) had a statistically higher level than the other non-cholesterol groups (groups 4-6) (one-way ANOVA). Liver spermine was significantly higher in the Chol-Hi MO group (group 3) when compared to the CO control (group 1) and the Chol-Lo MO group (group 2) (one-way ANOVA). Pancreatic putrescine in the CO control (group 4) was significantly higher than all other groups (two-way ANOVA) while spermine from the 2 Chol-MO groups (groups 2 and 3) was higher than the Chol-CO control (group 1) (one-way ANOVA). Using trend analysis, liver TK, putrescine and spermidine increased in the non-cholesterol preloaded groups with increasing dietary MO, similar to the increase seen in ODC. Thus, of the three organs studied, only liver responded to menhaden oil with changes in both ODC itself or some of its metabolic engendered products and thymidine kinase; at least for one of the parameters, ODC, change associated with dietary MO was dependent on whether the swine were preloaded with cholesterol.
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PMID:Ornithine decarboxylase and thymidine kinase activities and polyamine levels from selected organs of adult miniature swine receiving three concentrations of dietary menhaden oil. 1152 70

Increasing shortage of cadaveric grafts demands the utilization of living donor and split liver grafts. The purpose of this study was to 1) define the "small-for-size" graft in a pig liver transplant model 2) evaluate pathological changes associated with small-for-size liver transplantation. Pigs were divided into four groups based on the volume of transplanted liver: (a) control group (n=4), 100% liver volume (LV) (b) group I (n=8), 60% LV (c) group II (n=8), 30% LV (d) group III (n=15), 20% LV. Tacrolimus and methyl prednisone were administered as immunosuppression. Animals were followed for 5 days with daily serum biochemistry, liver biopsies on day 3 and 5 for light microscopy, and tissue levels of thymidine kinase (TK) and ornithine decarboxylase (ODC). Liver grafts were weighed pretransplant and at sacrifice. All the recipients of 100%, 60%, and 30% grafts survived. Transplantation of 20% grafts (group III) resulted in a 47% mortality rate. Group III animals showed significantly prolonged prothrombin times (p<0.05), elevated bilirubin levels (p<0.05), and ascites. The rate of regeneration, as indicated by TK activity and graft weight was inversely proportional to the size of the transplanted graft. The severity of the microvascular injury was inversely proportional to graft size and appeared to be the survival-limiting injury. Frank rupture of the sinusoidal lining, parenchymal hemorrhage, and portal vein injury were prominent in group III animals 1 hour following reperfusion. This study established a reproducible large animal model of partial liver grafting; it defined the small-for-size syndrome in this model and described the associated microvascular injury.
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PMID:Porcine partial liver transplantation: a novel model of the "small-for-size" liver graft. 1476 64

We have earlier demonstrated that inhibition of polyamine biosynthesis with difluoromethylornithine (DFMO) can be used to enhance the cytotoxicity of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy in different tumor cell lines. Here, the utility of this treatment combination was tested in vivo in a nude mouse tumor model. First, the effect of DFMO was verified by treating mice bearing subcutaneous 9L rat glioma tumors with 2% DFMO in drinking water. The drug treatment induced almost complete suppression of ornithine decarboxylase activity, and as a result, a strong decrease in intratumoral putrescine and spermidine concentrations, which were normalized 4 days after drug removal. Consequently, the tumors displayed a significant reduction in the proliferation activity that was increased to 20% higher than the normal level at day 4 and returned to normal level 7 days after DFMO removal. Next, 9L tumors with 30% of TK-GFP fusion gene positive cells were induced and the animals were given DFMO and GCV in 2 treatment schemes, with the drug administration periods overlapping either 5 or 2 days. The analysis of tumor size at the end of the treatment revealed that DFMO can enhance HSV-TK/GCV cytotoxicity when the overlap between DFMO and GCV was 5 days, but the result was not significant. However, the 2-day overlap scheme yielded a significantly (p < 0.05, ANOVA) enhanced antitumor effect. In conclusion, the data here confirms that a novel combination of 2 clinically relevant treatment modalities, polyamine deprivation and HSV-TK/GCV suicide gene therapy, can be used synergistically in vivo.
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PMID:In vivo enhancement of herpes simplex virus thymidine kinase/ganciclovir cancer gene therapy with polyamine biosynthesis inhibition. 1638 65

The trypanosomatid protozoan Leishmania tarentolae has been extensively used as a model system for studying causative agents of several tropical diseases and more recently as a host for recombinant protein production. Here we analyze the rates of partial or complete deletions of expression cassettes integrated into small ribosomal RNA and tubulin gene clusters as well as into ornithine decarboxylase gene of L. tarentolae. In approximately 60% of cases gene conversion was responsible for the deletion while in the rest of the cases deletion occurred within the expression cassette. We used this observation to design constitutive and inducible expression vectors that could be stably integrated into the genome and subsequently depleted of the antibiotic resistance genes using thymidine kinase or bleomycin resistance genes as negative selection markers. This enabled us to obtain L. tarentolae strains containing constitutive or inducible markerless expression cassettes. Analysis of the markerless strains demonstrated that although stability varied among clones some were stable for as many as 200 generations. We expect that this approach will be useful for the construction of strains carrying multiple expression cassettes for analysis of trypanosomatid pathogenicity mechanisms and overexpression of multi-subunit protein complexes for biochemical and structural studies.
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PMID:Construction and analysis of Leishmania tarentolae transgenic strains free of selection markers. 1765 88

The role of dietary factors in prevention of chemically-induced cancer was reviewed on two models: i) the role of high fiber diets in prevention of colon cancer and ii) the role of high fat diets in prevention of mammary gland cancer, i) Experiments in colon cancer showed that 20% cellulose content decreased tumor incidence caused by 1,2-dimethylhydrazine (DMH) to 33% compared with 92% of tumors developed in animals fed a fiber-free diet. The tumor-preventive effect of a cellulose diet was accompanied by increased enzyme concentrations, such as ornithine decarboxylase, thymidine kinase and beta-glucuronidase. Corncob fiber (15%), treated with the fungus Pleurotus os., had a significant protective effect against DMH-induced rat colon cancer. This effect was accompanied by activation of some cellular mechanisms, i.e. apoptosis, proliferating cell nuclear antigen (PCNA) and p53 protein synthesis. A high positive correlation was found between tumor grade and p53 protein in the serum (r=0.97) or in the cell cytoplasm (r=0.77), and between tumor grade and PCNA (r=0.81). An inverse relationship was found between tumor grade and apoptosis (r=-0.63). ii) Experiments in mammary gland cancer showed that a 15% olive-oil diet reduced tumor incidence caused by 9,10-dimethyl-1,2-benzanthracene to 30%, compared with 55% in the control group. The antitumor effect of the olive oil diet was connected to its content of monounsaturated fatty acids, such as oleic and palmitic acids. The promotive tumorigenic effects of other high-fat diets (avocado, soybeans) were associated with high content of some polyunsaturated fatty acids (linoleic and alpha-linolenic). We concluded that different diets have different targets. The effect of the same diet depends on its content of anti-tumor substances.
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PMID:The role of dietary factors in prevention of chemically-induced cancers (review). 2152 16


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