Thymidine kinase in rat liver during development.
Ontology highlight
ABSTRACT:
SUBMITTER: Klemperer HG
PROVIDER: S-EPMC1198848 | biostudies-other | 1968 Jul
REPOSITORIES: biostudies-other
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Project description:Cytoplasmic thymidine kinase from cardiac muscle of the rat has been characterized. It has a pH optimum of 9.0 and a K(m) value for thymidine of 1.6mum. The sedimentation coefficient of this enzyme in sucrose gradients is 4.5S, which represents a molecular weight of approx. 69000. Thymidine kinase prepared from cardiac muscle of foetal, neonatal and adult rats is inhibited by dTTP and dTDP; there is neither inhibition nor stimulation by dTMP, dCTP, dATP, dGTP or cyclic AMP. The activity of thymidine kinase in differentiating cardiac muscle of foetal and neonatal rats declines progressively with development, reaching adult values of almost zero by the fifteenth to seventeenth day of postnatal development. This represents a 70-fold decrease in enzyme activity from 3 days before birth to 17 days after birth. The loss of thymidine kinase activity in differentiating cardiac muscle correlates temporally with the cessation of DNA biosynthesis and the loss of cytoplasmic DNA polymerase activity in this tissue.
| S-EPMC1168335 | biostudies-other
Project description:1. The thymidine kinase activity of rat liver is greatly enhanced on addition of phospholipase C to the assay mixture. 2. Most of the thymidine kinase activity of the liver is recovered in the mitochondrial and in the impure ;nuclear' fractions. No activity was detected in purified nuclei prepared in high-density sucrose. 3. A substantial thymidine kinase activity could be detected, with the aid of phospholipase C, in all rat tissues examined.
| S-EPMC1176784 | biostudies-other
Project description:Total adenylate kinase activity was determined in developing rat liver. The activity was 18 units/g wet weight of tissue in foetal liver; this increased to 41 units/g immediately after birth and continued increasing until adult activities of 150 units/g were reached after two weeks. The adenylate kinase activity was separated into four isoenzymes. Only isoenzymes II and III were observed in foetal rat liver. Isoenzyme II activity was 2 units/g in the foetal liver and increased to 25 units/g in adult liver. Adenylate kinase III activity was 20 units/g in the foetal liver and increased to 118 units/g in adult liver. The possible role that adenylate kinase might have in regulating the energy flow in the developing liver cell is discussed.
| S-EPMC1176813 | biostudies-other
Project description:Deficiency in thymidine kinase 2 (TK2) causes mitochondrial DNA depletion. Liver mitochondria are severely affected in Tk2 complete knockout models and have been suggested to play a role in the pathogenesis of the Tk2 knockout phenotype, characterized by loss of hypodermal fat tissue, growth retardation and reduced life span. Here we report a liver specific Tk2 knockout (KO) model to further study mechanisms contributing to the phenotypic changes associated with Tk2 deficiency. Interestingly, the liver specific Tk2 KO mice had a normal life span despite a much lower mtDNA level in liver tissue. Mitochondrial DNA encoded peptide COXI did not differ between the Tk2 KO and control mice. However, the relative liver weight was significantly increased in the male Tk2 KO mouse model. Histology analysis indicated an increased lipid accumulation. We conclude that other enzyme activities can partly compensate Tk2 deficiency to maintain mtDNA at a low but stable level throughout the life span of the liver specific Tk2 KO mice. The lower level of mtDNA was sufficient for survival but led to an abnormal lipid accumulation in liver tissue.
| S-EPMC10553353 | biostudies-literature
Project description:Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of k(cat) = 0.28 s(-1) and K(m)(,ATP) = 140 microm are similar to those of human TK1, the value of K(m)(thymidine) = 48 microm is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.
| S-EPMC2871459 | biostudies-literature
Project description:Activin-betaE mRNA expression was investigated in male and female rats using gel-based and quantitative RT-PCR, in fetal and post-natal liver during development and in a variety of tissues from 200 gm adult animals. Activin-betaE expression was also assessed in rat liver after partial hepatectomy, and after repeated toxic insult. Male Sprague Dawley rats were subjected to partial hepatectomy or sham operations. Samples were collected from the caudate liver lobe during regeneration, from 12 to 240 hr after surgery. Three groups of 5 male rats were treated with CCl(4) for 0 (control), 5 or 10 weeks, to induce liver fibrosis and cirrhosis. Activin-betaE mRNA was predominantly expressed in liver, with much lower amounts of mRNA observed in pituitary, adrenal gland and spleen, in both males and females. Low activin-betaE expression was observed in liver at fetal day 16, with higher levels seen between post-natal days 3 and 35 and a further increase noted by day 47, in both males and females. Liver activin-betaE mRNA concentrations did not change from control values 12-72 hr after PHx, but significantly increased over six fold, 168 hr post-hepatectomy, when liver mass was restored. Activin-betaE mRNA was up-regulated after 5 weeks of CCl(4) treatment, but not after 10 weeks. The changes in activin-betaE mRNA concentrations after liver insult did not always parallel those reported for the activin-betaC subunit, suggesting activin-betaE may have an independent role in liver under certain conditions.
| S-EPMC2702058 | biostudies-literature
Project description:Protein degradation rates for liver subcellular and submitochondrial fractions from neonatal (8-day), weanling (25-day) and adult rats were estimated by the double-isotope method with NaH14CO3 and [3H] arginine as the radiolabelled precursors [Dice, Walker, Byrne & Cardiel (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2093-2097]. Decreased protein degradation rates were found during post-natal development for homogenate, nuclear, mitochondrial, lysosomal and microsomal proteins. A decrease in degradation rates for the immunoisolated subunits of monoamine oxidase and pyruvate dehydrogenase was also observed in neonatal and weanling rats respectively. The results suggest coordinate degradation of the subunits of the multi-subunit enzyme pyruvate dehydrogenase. Pyruvate dehydrogenase has a faster rate of degradation in adult rat liver than does cytochrome oxidase. Data analysis suggests heterogeneity of protein degradation rates in the mitochondrial outer membrane and intermembrane space fractions at each developmental stage but not in the mitochondrial inner membrane or matrix fractions. Results obtained for protein degradation rates in adult rat liver by the method of Burgess, Walker & Mayer [(1978) Biochem. J. 176, 919-926] in general confirmed the results obtained for the adult rat liver by the above method. No evidence of a subunit-size relationship for protein degradation was found for proteins in any subcellular or submitochondrial fraction.
| S-EPMC1162337 | biostudies-other
Project description:1. Lipogenesis, as measured by the incorporation of (14)C-labelled glucose or acetate into fatty acids in liver slices, is high in foetal and adult rat liver but is low in the liver of the suckling rat, especially with glucose as substrate. 2. The rate of synthesis of non-saponifiable lipids from glucose is about 15 times as great in the liver of the 18-day foetus as in adult liver. Activity in the newborn is negligible. 3. Glucose incorporation into fat is strongly concentration-dependent in liver slices from the adult and 2-week-old rat, but less markedly so in liver slices from the foetus. 4. Changes in the activity of hepatic citrate-cleavage enzyme (ATP-citrate lyase) occur in parallel with the changes in the extent of fatty acid formation, supporting the participation of this enzyme in lipogenesis. However, NADP-malate dehydrogenase, a potential source of reduced nucleotide coenzyme for lipogenesis in the adult, could not be detected in foetal rat liver.
| S-EPMC1270349 | biostudies-other
Project description:The ontogeny of rat liver glutathione S-transferase (EC 2.5.1.18) (GSTs) during foetal and postnatal development was investigated. The GSTs are dimers, the subunits of which belong to three multigene families, Alpha (subunits 1, 2, 8 and 10), Mu (subunits 3, 4, 6, 9 and 11) and Pi (subunit 7) [Mannervik, Alin, Guthenberg, Jennsson, Tahir, Warholm & Jörnvall (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7202-7206; Kispert, Meyer, Lalor, Coles & Ketterer (1989) Biochem. J. 260, 789-793]. There is considerable structural homology within each gene family, with the result that whereas reverse-phase h.p.l.c. successfully differentiates individual subunits, immunocytochemical and Northern-blotting analyses may only differentiate families. Enzymic activity, h.p.l.c. and Northern blotting indicated that expression of GST increased from very low levels at 12 days of foetal growth to substantial amounts at day 21. At birth, GST concentrations underwent a dramatic decline and remained low until 5-10 days post partum, after which they increased to adult levels. During the period under study, GST subunits underwent differential expression. The Mu family had a lower level of expression than the Alpha family, and, within the Alpha family, subunit 1 was more dominant in the adult than the foetus. Subunit 2 is the major form in the foetus. Most noteworthy were subunits 7 and 10, which were prominent in the foetus, but present at low levels post partum. Immunocytochemical analysis of the 17-day foetal and newborn rat livers showed marked differences in the distribution of GSTs in hepatocytes. In the 17-day foetal liver Pi greater than Alpha greater than Mu whereas in the newborns Alpha greater than Mu much greater than Pi. Erythropoietic cells were not stained for any of the three GST families. Steady-state mRNA concentrations in the foetus correlated with the relative transcription of the Alpha, Mu and Pi class genes. However, in those genes expressed post partum, namely the Alpha and Mu class, low transcriptional activity was associated with high concentrations of mRNA. This suggests that there is a switch from transcriptional control to post-transcriptional control at birth. GST 7-7 appears to be regulated predominantly by transcription throughout the period of liver development under observation.
| S-EPMC1130909 | biostudies-other
Project description:Discovery of new cancer biomarkers and advances in targeted gene delivery mechanisms have made gene-directed enzyme prodrug therapy (GDEPT) an attractive method for treating cancer. Recent focus has been placed on increasing target specificity of gene delivery systems and reducing toxicity in non-cancer cells in order to make GDEPT viable. To help address this challenge, we have developed an enzymatic switch that confers higher prodrug toxicity in the presence of a cancer marker. The enzymatic switch was derived from the herpes simplex virus thymidine kinase (HSV-TK) fused to the CH1 domain of the p300 protein. The CH1 domain binds to the C-terminal transactivation domain (C-TAD) of the cancer marker hypoxia inducible factor 1?. The switch was developed using a directed evolution approach that evaluated a large library of HSV-TK/CH1 fusions using a negative selection for azidothymidine (AZT) toxicity and a positive selection for dT phosphorylation. The identified switch, dubbed TICKLE (Trigger-Induced Cell-Killing Lethal-Enzyme), confers a 4-fold increase in AZT toxicity in the presence of C-TAD. The broad substrate specificity exhibited by HSV-TK makes TICKLE an appealing prospect for testing in medical imaging and cancer therapy, while establishing a foundation for further engineering of nucleoside kinase protein switches.
| S-EPMC6080848 | biostudies-literature