Studies on the hyperplasia (''regeneration'') of the rat liver following partial hepatectomy. Changes in lipid peroxidation and general biochemical aspects.

Using the experimental model of partial hepatectomy in the rat, we have examined the relationship between cell division and lipid peroxidation activity. In rats entrained to a regime of 12 h light/12 h dark and with a fixed 8 h feeding period in the dark phase, partial hepatectomy is followed by a rapid regeneration of liver mass with cycles of synchronized cell division at 24 h intervals. The latter phenomenon is indicated in this study by pulses of thymidine kinase activity having maxima at 24 h, 48 h and 72 h after partial hepatectomy. Microsomes prepared from regenerating livers show changes in lipid peroxidation activity (induced by NADPH/ADP/iron or by ascorbate/iron), which is significantly decreased relative to that in microsomes from sham-operated controls, again at 24 h, 48 h and 72 h after the operation. This phenomenon has been investigated with regard to possible underlying changes in the content of microsomal fatty acids, the microsomal enzymes NADPH:cytochrome c reductase and cytochrome P-450, and the physiological microsomal antioxidant alpha-tocopherol. The cycles of decreased lipid peroxidation activity are apparently due, at least in part, to changes in microsomal alpha-tocopherol content that are closely associated in time with thymidine kinase activity.     

Synthesis and antiviral evaluation of 1-O-hexadecylpropanediol-3-P-acyclovir: efficacy against HSV-1 infection in mice

We synthesized, 1-O-hexadecylpropanediol-3-P-acyclovir, an orally bioavailable lipid prodrug of acyclovir and evaluated it for in vitro and in vivo activity against herpes simplex virus infections. Although 1-O-hexadecylpropanediol-3-P- acyclovir was less active in vitro than acyclovir, on a molar basis it was 2.4 times more active orally in preventing mortality from acute HSV-1 infection in mice. In vitro, 1-O-hexadecylpropanediol-3-P-acyclovir was also more active than acyclovir in a thymidine kinase negative mutant strain of HSV-1 (DM21) and had somewhat higher activity in cytomegalovirus infection in vitro due to it's ability to bypass thymidine kinase.     

Assessment of cell damage caused by spontaneous lipid peroxidation in rabbit spermatozoa

Damage to the plasma membrane of rabbit epididymal spermatozoa during spontaneous lipid peroxidation was examined by means of trypan blue uptake and expression of activity of the intracellular enzymes, lactate dehydrogenase and pyruvate kinase. Both the dye uptake and the expression of enzyme activity probe cell damage from lipid peroxidation as loss of integrity of the plasma membrane. A linear correlation was obtained between trypan blue staining of the cells and malondialdehyde production, a quantifiable measure of the extent of lipid peroxidation. At the point of trypan blue staining of all cells, 0.5 nmol malondialdehyde/10(8) cells was produced. This is the same amount produced at the point of complete loss of motility and superoxide dismutase activity. We have defined this as the "lipoperoxidative lethal end point." Expression of lactate dehydrogenase and pyruvate kinase activities increased with time of aerobic incubation. In the high Na+ medium, NTP, in which lipid peroxidation is slow, there is a linear correlation between increase in expressed enzyme activities and malondialdehyde production. But in the high K+ medium, KTP, in which lipid peroxidation is rapid, there is an initial rapid rise in expressed enzyme activity over 3 h, followed by a slower increase. Activities of rabbit sperm lactate dehydrogenase, pyruvate kinase, and flagellar ATPase were unaffected by aerobic incubations for up to 48 h, double the incubation period used for the assay of enzymatic activities for the first two. The activity of glyceraldehyde-3-phosphate dehydrogenase decreased during aerobic incubation, the time course matching the loss of motility. The subcellular distribution of lactate dehydrogenase in rabbit spermatozoa was determined: 4% in the mitochondrial matrix, 10% in the plasma membrane and 85% in the cytosolic compartment.     

Association of thymidylate kinase activity with pyrimidine deoxyribonucleoside kinase induced by herpes simplex virus.

Thymidine kinase derived from LMTK+ does not exhibit thymidylate kinase activity. However, protein isolated by affinity column chromatography from thymidine kinase-deficient mouse cells (LMTK-) infected by herpes simplex virus type 1 shows thymidylate kinase activity in addition to thymidine kinase and deoxycytidine kinase activities. The virus-induced multifunctional enzyme has a molecular weight of 85,000, whereas the molecular weight of thymidylate kinase from uninfected LMTK- mouse cells is 71,000. The virus-induced enzyme has a Km for thymidine of 0.8 micromolar, and for thymidylate of 25 micromolar, and for thymidylate of 25 micromolar; the ratio of Vmax for thymidylate kinase to thymidine kinase is 1.7. When subjected to isoelectric focusing, thymidylate kinase activity is not separated from thymidine kinase activity, and even though four peaks of activity are observed they have a constant ratio of thymidylate kinase to thymidine kinase activity. The isoelectric points (pI) of these four peaks are 4.8, 5.8, 6.2, and 6.6, respectively. Thymidylate kinase, derived from uninfected cells when subjected to isoelectric focusing, separates into a major component with an isoelectric point at pH 8.2 and a minor component at pH 7.7. Although thymidine and thymidylate kinase activities derived from the virus-infected cells cannot be separated either by affinity column chromatography, glycerol density gradient centrifugation, or isoelectric focusing, there is a differential rate of inactivation when the enzyme is subjected to incubation at 37 degrees, with thymidylate kinase activity being more labile than thymidine kinase activity.     

Thymidine kinase and deoxyribonucleic acid metabolism in growing and regenerating livers from rats on controlled feeding schedules

Rats accustomed to eating during the first 8h of a daily 12h dark period re-established about 80% of intact liver weight, protein and DNA within 4 days following partial hepatectomy; further increases were not observed. Liver thymidine kinase activity and thymidine incorporation into liver DNA exhibited marked daily oscillations during liver regeneration. Maximum values were observed near the end of the dark period both in intact growing rats and in rats partially hepatectomized 2h before the end of the dark period. The time of day of surgery affected thymidine kinase activity and thymidine incorporation into DNA at specific times following partial hepatectomy. This seriously affects the interpretation of reports of experiments where the time of day of killing has been held constant and time of surgery varied. Highly significant correlation coefficients were observed for thymidine incorporation before killing versus thymidine kinase activity at time of killing and for thymidine versus orotic acid incorporation into DNA of livers from rats partially hepatectomized 2h before the end of the dark period and killed at 12h intervals. Thymidylate phosphatase activity returned to the normal amount at a rate similar to that for liver protein. Thymidylate phosphatase did not affect the validity of the thymidine kinase assay. The relationship of [(14)C]orotic acid to [(3)H]thymidine incorporation into liver DNA varied with the time of day, with the age of the rat and during the regeneration of the liver.     

Utilization of exogenous thymidine by Chlamydia psittaci growing in the thymidine kinase-containing and thymidine kinase-deficient L cells.

The incorporation of [3H]thymidine into the deoxyribonucleic acid (DNA) of Chlamydia psittaci (strain 6BC) growing in thymidine kinase (adenosine 5'-triphosphate-thymidine 5'-phosphotransferase, EC 1.7.1.21)-containing L cells, L(TK+), and thymidine kinase-deficient L cells, LM(TK-), was examined by autoradiography. Label was detected over C. psittaci inclusions in L(TK+) but not LM(TK-) cells. No evidence for a chlamydia-specific thymidine kinase activity in either L(TK+) or LM(TK-) cells was obtained. Entry of [3H]thymidine into the DNA of C. psittaci growing in L(TK+) cells was quantitated by measuring label in purified C. psittaci. It was 265 times less efficient than entry into infected host cell DNA. It is concluded that low levels of exogenous thymidine are incorporated into the DNA of C. psittaci and that this incorporation is dependent on a fully competent host thymidine kinase activity. Evidence also is presented that L cells possess at least two thymidine kinase activities, both of which are capable of supplying thymidylate precursors for nuclear DNA synthesis.     

Ornithine decarboxylase and thymidine kinase activity in tissues of prolactin-treated rats: effect of hypophysectomy

The activities of ornithine decarboxylase and thymidine kinase were determined in tissues of young intact and hypophysectomized rats at various times after treatment with prolactin. In both types of animals, ornithine decarboxylase activity increased in liver, kidney, spleen and adrenal of prolactin treated rats. Thymidine kinase activity increased only in liver and spleen of intact rats. Increase in the kinase activity was smaller, and occurred later than the change in ornithine decarboxylase. In hypophysectomized animals, thymidine kinase activity increased in spleen, but not in liver, following prolactin treatment.     

Isolation of mutants of bacteriophage T4 unable to induce thymidine kinase activity. II. Location of the structural gene for thymidine kinase.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.     

Characterization of new regulatory mutants of bacteriophage T4. II. New class of mutants.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.     

A mammalian cell mutant with temperature-sensitive thymidine kinase.

We have isolated a mutant clone from mouse FM3A cells with temperature-sensitive defects both in cytokinesis and in thymidine kinase enzyme activity. The clone, designated tsCl.B59, was isolated after mutagenesis at 33 degrees C followed by exposure to cytosine arabinoside at 39 degrees C. It was derived from a thymidine kinase deficient, 5-bromodeoxyuridine-resistant clone (S-BUCl.42) which was originally derived from wild-type clone H-5 of FM3A cells. The temperature-sensitive mutant clone grows normally at 33 degrees C, but not at 39 degrees C, where it exhibits an increased frequency of multinucleate cells due to defective cytokinesis. Unlike the parental S-BUCl.42 cells, which have negligible thymidine kinase activity and are unable to incorporate 3H-thymidine, the mutant in corporates substantial amounts of 3H-thymidine at 33 degrees C, although its thymidine kinase activity remains lower than that of wild-type H-5 cells. When cultures of tsCl.B59 cells are transferred to 39 degrees C, incorporation of 3H-thymidine decreases markedly. The decrease has been shown to be due to thermolability of the thymidine kinase in tsCl.B59 cells.     

Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene.

We have obtained 42 active artificial mutants of HSV-1 thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) by replacing codons 166 and 167 with random nucleotide sequences. Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene. The spectrum of active mutations indicates that neither Ile166 nor Ala167 is absolutely required for thymidine kinase activity. Each of these amino acids can be replaced by some but not all of the 19 other amino acids. The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 (a strain lacking thymidine kinase activity) in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions. E. coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine (less than 1 microgram/mL), but are unable to grow in the presence of high amounts of thymidine. On the other hand, E. coli KY895 harboring low-activity plasmids can grow at a high concentration of thymidine (greater than 50 microgram/mL) in the media. The high-activity plasmids also have an enhanced [3H]dT uptake. The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake. Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP-dependent and -independent protein phosphokinase activity in subcellular fractions of synchronously growing leydig I-10 cells.

The Leydig I-10 tumor cell line was synchronized by the double thymidine block method using 1.0 mM thymidine. Protein phosphokinase activity of subcellular fractions was determined at various times throughout the cell cycle. Microsomal cAMP-independent kinase activity increased in G2 and decreased during the S and G1 phases. Except for relatively small increases during the G1 and late S phases, microsomal cAMP-dependent kinase activity remained unchanged throughout most of the cycle. In the lysosomal-mitochondrial fraction, cAMP-dependent and cAMP-independent protein kinase activity increased during the S phase. Independent kinase activity peaked again during G1, while the dependent kinase became depressed. Phosphokinase activity increased in the nuclear fraction in late G2 and during mitosis, and was due to increases in both cAMP-independent and cAMP-dependent kinase activity. Cytosol cAMP-dependent kinase activity increased in G2 and during mitosis; cAMP-independent kinase activity showed some increased activity during late G2 and mitosis. These temporal variations in the subcellular kinase activities throughout the cell cycle may act to phosphorylate subcellular protein substrates in a cell cycle-specific fashion.     

Design, synthesis and enzymatic activity of highly selective human mitochondrial thymidine kinase inhibitors

Highly selective arabinofuranosyl nucleosides, which inhibit the mitochondrial thymidine kinase (TK-2) without affecting the closely related herpes simplex virus type 1 thymidine kinase (HSV-1 TK), varicella-zoster virus thymidine kinase (VZV-TK), cytosolic thymidine kinase (TK-1) or the multifunctional Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK), have been obtained. SAR studies indicate a close relation between the length of the substituent at the 2' position of the arabinofuranosyl moiety and the inhibitory activity.     

Physarum thymidine kinase. A step or a peak enzyme depending upon temperature of growth.

The variations of thymidine kinase or ATP:thymidine 5'-phosphotransferase (EC 2.7.1.21) during the cell cycle of Physarum polycephalum plasmodia have been studied at two extreme physiological temperatures: 22 degrees C and 32 degrees C. At 22 degrees C the enzyme activity increases near mitosis and stays constant during late S and G2 phases, exhibiting the typical pattern of a 'step enzyme'. But at 32 degrees C thymidine kinase activity goes through a maximum 1 h 30 min after mitosis and decreases during the subsequent phases as expected for a 'peak enzyme'. The rate of enzyme degradation and/or inactivation, measured in the presence of metabolic poisons (cycloheximide or dinitrophenol), appears to follow a simple exponential function with a half-life of approximately 3 h and 1 h at 22 degrees C and 32 degrees C respectively. The effect of growth temperature on the decrease of thymidine kinase activity can account entirely for the differences in the pattern of enzyme activity at the two extreme temperatures. Tentative calculations indicate that the rate of enzyme synthesis is nearly constant during the cell cycle except near mitosis, where it is temporarily increased. The results suggest the existence of a regulatory mechanism able to modulate the rate of synthesis of thymidine kinase during the cell cycle.     

Identification of true thymidine kinase in Tetrahymena pyriformis ST.

Thymidine kinase is present in the cytoplasm (outside mitochondria) of Tetrahymena pyriformis. Previous workers have been unable to find a specific thymidine kinase activity in this organism. The cytoplasm of Tetrahymena contained a thymidine phosphorylating activity which was ATP dependent, was stimulated by Mg²⁺, and was inhibited by dTTP. This activity was also partly inhibited by dCTP. Although the mitochondrial fraction also exhibited ATP-dependent phosphorylation, it is not stimulated by Mg²⁺ and not significantly inhibited by dTTP. Nucleoside phosphotransferase activity is detectable both in cytoplasmic and mitochondrial fractions, although it is not clear whether they represent separate enzymes. Nucleoside phosphotransferase activity is inhibited both by NaF and by ATP. Thymidine kinase and nucleoside phosphotransferase activities were separated by polyacrylamide gel electrophoresis, establishing the presence of both enzymes in this organism. Both crude mitochondrial lysate and postmitochondrial supernatant samples exhibited similar gel electrophoretic patterns for thymidine kinase and nucleoside phosphotransferase activities. The former, however, exhibited a relatively small peak of thymidine kinase migrating at the same rate as that of the postmitochondrial supernatant. A separate peak of thymidine kinase was not found in the mitochondria of Tetrahymena.     

Microsomal lipid peroxidation: mechanisms of initiation. The role of iron and iron chelators

The role of iron and iron chelators in the initiation of microsomal lipid peroxidation has been investigated. It is shown that an Fe3+ chelate in order to be able to initiate enzymically induced lipid peroxidation in rat liver microsomes has to fulfill three criteria: (a) reducibility by NADPH; (b) reactivity of the Fe2+ chelate with rat liver microsomes has to fulfill three criteria: (a) reducibility by NADPH; (b) reactivity of the Fe2+ chelate with O2; and (c) formation of a relatively stable perferryl radical. NADH can support lipid peroxidation in the presence of ADP-Fe3+ or oxalate-Fe3+ at rates comparable to those obtained with NADPH but requires 10 to 15 times higher concentrations of the Fe3+ chelates for maximal activity. The results are discussed in relation to earlier proposed mechanisms of microsomal lipid peroxidation.     

Azacytidine induces reversion of thymidine kinase deficiency in Friend erythroleukemia cells

Cells of the Friend erythroleukemia cell line show a high frequency of variants which have lost thymidine kinase activity. We have exposed a thymidine kinase-deficient clone of this cell line to a series of concentrations of azacytidine and found a dose-dependent induction of thymidine kinase-positive revertants. Maximum reversion occurred with 0.9 micrograms/ml azacytidine where the frequency of revertants amongst survivors was increased by a factor of 10(6) compared to that of control cultures. Revertants were found to have varying levels of thymidine kinase activity. We conclude that DNA methylation of one or both alleles of the thymidine kinase gene is largely responsible for the instability of this gene in Friend erythroleukemia cells.     

The effect of hyperbaric oxygenation on the antioxidant status of Xenopus laevis after its preliminary adaptation to oxygen]

We studied the level of lipid peroxidation and the activity of antioxidant enzymes (superoxide dismutase and catalase) in various tissues of adult Xenopus laevis after an initial exposure to hyperbaric oxygenation at the developmental stage 38. We have found that irrespective to the mode of treatment, the level of lipid peroxidation and activity of antioxidant enzymes in the brain, lungs, and blood of these animals were higher as compared to control animals. We demonstrate that, after the exposure of adult animals to hyperoxia, if they were earlier subjected to hyperbaric oxygenation (0.2 MPa) at stage 38, there was no intensification of lipid peroxidation or changes in the activity of superoxide dismutase and catalase. In adult animals initially subjected to hyperbaric oxygenation at the same stage of development but at the pressure--0.7 MPa, the second exposure to hyperoxia led to a drastic intensification of lipid peroxidation in the brain; in some animals, an increased level of lipid peroxidation products in the lungs was observed.