ADP-ribosylation of histones and NAD-pyrophosphorylase activity of chicken liver nuclei during induction of DNA damage

The rate of [14C]NAD incorporation into chicken liver nuclear histones was studied under conditions of DNA damage by N-methyl-N-nitrosourea and pancreatic DNAase I. With an increase in N-methyl-N-nitrosourea concentration from 8.5 X 10(-2) to 34.0 X 10(-2) mM, the ADP ribosylation of histones increases by 20% as compared to the control. In DNAase I-treated nuclei, the binding by histones of [14C]NAD sharply increases, reaching its maximum (18.3 X 10(-8) mM) at 30% cleavage of DNA. When 50% of DNA was cleaved, the rate of [14C]NAD incorporation into the histones was 8.0 X 10(-8) mM as compared to 6.1 X 10(-8) mM/mg protein in control samples. The poly(ADPR)polymerase activity was increased in both cases. It was shown that the NAD-pyrophosphorylase activity in chicken liver nuclei treated with N-methyl-N-nitrosourea does not differ from the control one, while in DNAase I-treated nuclei the maximum of the NAD-pyrophosphorylase activity was achieved, as well as the maximum of [14]NAD incorporation into the histones within the range of DNA damage of 25-35%, being equal to 37 X 10(-8) mM NAD/min/mg protein as compared to 26.0 X 10(-8) mM/min/mg protein in the control. At different degrees of DNA damage, the average length of the poly-ADP-ribose chain did not practically alter, thus suggesting the increase in the number of polymer binding sites in the histones.     

Increase in the activity of nuclear DNA polymerases in the tissues of mice subjected to long-term gamma irradiation

Activity of nuclear DNA-polymerase in the liver, lung and spleen tissues of mice subjected to long-term chronic gamma-irradiation (1.3 mGy/h) has been investigated. Chronic gamma-irradiation with a cumulative dose of 1.7 Gy during 55 days raises DNA polymerase activity in the irradiated tissue nuclei. Analysis of DNA-polymerase activity in the liver nuclei have demonstrated that this increase is connected with activation of DNA-polymerase beta.     

Enzymes of glycogen metabolism in chick embryo liver]

At all stages of ontogenesis glycogen phosphorylase (EC 2.4.1.1) from liver chick embryos in represented by an isoenzyme whose properties are close to those of isoenzyme IL or F. Total enzyme activity (a+b forms) from the 8th day of development up to hatching gradually increases 1.5-fold, a practically complete activation of enzyme being observed by the end of embryogenesis. Phosphorylase b possesses high catalytic activity in the presence of 1 mM AMP and it activated by protamine and 0.2 M Na2SO4. Glycogen synthetase (EC 2.4.1.11) has a constant Km(UDFG) value during ontogenesis. This value is about 5.10(-4) M in the presence of 10 mM glucose-6-phosphate, both for I- and D-forms of enzyme. The total enzyme activity reaches its maximum on the 17th postembryonic day and is decreased more than 6-fold thereafter. In the course of embryogenesis the I/D ratio is increased from 0.2 on the 8th day of development up to 0,45 during extensive accumulation of glycogen and falls down to 0.33 before hatching. Glycogen biosynthesis in embryonic liver is wellcorrelated with the increase in the I/D ratio, i.e. the increase of the active form of enzyme. The proportion of granular glycogen in embryonic liver is increased from 15% up to 90% of total glycogen content between the 8th and 14th days of development. The activity of glycogen synthetase contained in granular glycogen is increased from 40% in the 8-day-old embryos up to 90% in the 18-day-old ones. The activity of phosphorylase is found in granular glycogen only on the 12th day of embryogenesis and reaches its maximum (80% of total enzyme activity) only on the 19th days of development. It is concluded that in the adult chicken liver the embronic enzymes--glycogen phosphorylase and glycogen synthetase--are retained.     

Gamma- and UV-induced DNA synthesis in neurons of the rat neocortex

Nuclear DNA synthesis in neocortex neurons of neonatal 14- and 60-day rats after in vitro irradiation of isolated sections was estimated by the incorporation of a labeled precursor into DNA. gamma- and UV-radiation increased the rate of DNA synthesis in the cells of animals of all studied age groups. However, the level of the UV-induced synthesis sharply dropped during the postnatal ontogenesis while gamma-radiation-induced synthesis decreased slightly. The peculiarities revealed in the repair DNA synthesis seem to be influenced by the process of postnatal differentiation of a neuron accompanied by the nucleosome length shortening and the decrease in the DNA-polymerase alpha content.     

NAD participation in protecting DNA from damaging factors

It was found that autolytic degradation of DNA during the isolation of nuclei under conditions of an in situ stimulated NAD biosynthesis is decreased to 2.5% in comparison with 5.2% in control. The increase in the NAD-pyrophosphorylase activity and the intranuclear content of NAD was almost 3-fold, whereas the amount of the acid-soluble DNA material was decreased 2-fold. The degree of DNA damage was increased up to 23% after induction with ethidium bromide. Under these conditions a preliminary in vivo stimulation of NAD biosynthesis by nicotinamide diminished the amount of the acid-soluble material. The levels of intranuclear NAD as well as the NAD-pyrophosphorylase and poly-(ADP-riboso)polymerase activities were essentially affected.     

ADP-ribosylation of histones of the chicken liver nucleus at different rates of glycohydrolase hydrolysis of NAD

The rate of incorporation of nicotinamide-[adenosine-U-14C]adenine dinucleotide [( Ado-U-14C]NAD) into histones and the poly(ADPR) polymerase activity of chromatin suggest that the NAD-dependent ADP-ribosylation of histones depends on the rate of NAD hydrolysis by glycohydrolase in chicken liver nuclei. With a rise in the NAD-glycohydrolase activity after treatment of nuclei with Triton X-100 the synthesis of poly(ADP-ribose) via the poly(ADPR)polymerase reaction is augmented, as a result of which the rate of [Ado-U-14C]NAD incorporation into total histones is increased. On the contrary, the decrease of NAD-glycohydrolase hydrolysis after treatment of nuclei with SDS lowers the poly(ADPR)polymerase activity and [Ado-U-14C]NAD incorporation into histones. Under these conditions, i. e. different rates of glycohydrolase hydrolysis of NAD in the nuclei, some redistribution of [Ado U-14C]NAD incorporation into individual histones occurs.     

ADP-ribosyltransferase in isolated nuclei from sea-urchin embryos.

The activity of ADP-ribosyltransferase in nuclei isolated from sea-urchin embryos was estimated by the incorporation of [adenosine-14C]NAD+ into the acid-insoluble fraction. Hydrolysis of this acid-insoluble product by snake venom phosphodiesterase yielded radioactive 5'-AMP and phosphoribosyl-AMP. The incorporation of [14C]-NAD+ was inhibited by 3-aminobenzamide and nicotinamide, potent inhibitors of ADP-ribosyltransferase. [14C]NAD+ incorporation into the acid-insoluble fraction results from the reaction of ADP-ribosyltransferase. The optimum pH for the enzyme in isolated nuclei was 7.5. The enzyme, in 50 mM-Tris/HCl buffer, pH 7.5, containing 0.5 mM-NAD+ and 0.5 mM-dithiothreitol, exhibited the highest activity at 18 degrees C in the presence of 14 mM-MgCl2. The apparent Km value for NAD+ was 25 microM. The activity of the enzyme was measured in nuclei isolated from the embryos at several stages during early development. The activity was maximum at the 16-32-cell stage and then decreased to a minimum at the mesenchyme blastula stage. Thereafter its activity slightly increased at the onset of gastrulation and decreased again at the prism stage.     

Dependence of ADP-ribosylation of histones of chicken liver nuclei on the intranuclear content of NAD

The dependence of ADP-ribosylation of chicken liver nuclear histones on NAD concentration in the nuclei was studied under conditions of stimulation of coenzyme synthesis by the nicotinamide and nicotinic acid as well as upon addition of various concentrations of the [Ado-U-14C]NAD nuclei to the incubation mixture. In the first case, the rate of [Ado-U-14C]NAD incorporation into the histones was decreased due to the dilution of the label by the de novo synthesized NAD. The amount of the latter formed under effects of nicotinic acid and nicotinamide increased, correspondingly, from 2,2 X 10(-5) mmol up to 4.1 X 10(-5) and 7.0 X 10(-5) mmol per mg of nuclear protein. The incorporation of [Ado-U-14C]NAD into the histones decreased from 12.0 X 10(-8) mmol after incubation of liver slides with nicotinic acid and nicotinamide down to 8.0 X 10(-8) and 7.0 X 10(-8) mmol, respectively. With a rise in the concentration of exogenous [Ado-U-14C]NAD, the level of ADP-ribosylation of nuclear histones increased, the plot [14C]NAD incorporation at the labeled coenzyme concentration of 25 X 10(-7) mM/mg of histone had a plateau. Changes in the labeled substrate concentration brought about corresponding changes in the average length of the histone-linked poly-(ADP-ribose) chain.     

Effect of poly(ADP-ribose) formation on DNA synthesis and DNA fragmentation in nuclei of rat liver and rat ascites hepatoma AH-130 cells

To elucidate the role of poly(ADP-Rib) in the nucleus, DNA synthesis and DNA fragmentation were studied in isolated nuclei of rat liver and rat ascites hepatoma AH-130 cells. Liver and hepatoma cell nuclei formed the same amount of poly(ADP-Rib) per mg of nuclear DNA from NAD. Preincubation of liver nuclei with NAD repressed DNA polymerase activity to 30% of that of the control, but preincubation of hepatoma cell nuclei with NAD did not affect DNA polymerase activity. It was also found that incubation of liver nuclei with NAD prevented the fragmentation of nuclear DNA which occurred without NAD. Incubation of hepatoma cell nuclei with or without NAD did not result in fragmentation of DNA. The role of endonuclease in primer formation for DNA synthesis is discussed.     

Regulatory properties of 14 day embryo and adult hen heart AMP-deaminase

Chromatography on phosphocellulose column revealed changes in the elution profile of chicken heart AMP-deaminase during ontogenesis. The extracts from the heart of adult hen and 14 day-old embryo displayed a single peak of the enzyme activity at a slightly different elution volume, whereas in the heart extract of 1 day-old chicken two molecular forms of adenylate deaminase have been eluted. The kinetic and regulatory properties of the purified adult hen heart AMP-deaminase were studied and compared with those of the corresponding enzyme from 14 day-old embryo heart. Both enzymes exhibited a slightly sigmoid-shaped plot of the reaction rate versus substrate concentration, which shifted to hyperbolic form when ATP or ADP were added into the incubation medium. The enzymes were strongly activated by ATP, less efficiently by ADP and the activatory effect was enhanced at low substrate concentration. Orthophosphate inhibited both enzymes but this inhibition was more potent for the embryo heart enzyme. Palmitoyl-CoA inhibited adult hen but not the embryo heart AMP-deaminase. The data presented indicate that the differences also in the regulatory properties of the molecular forms studied do exist and correspond with the ontogenetic differences observed previously (Kaletha and Skladanowski (1981) Experientia 37, 232-234) concerning the effect of temperature on the chicken heart adenylate deaminase.     

Differential regulation of DNA synthesis in heterokaryons between chicken erythrocytes and culture cells with various proliferative potentials

The regulation of DNA synthesis in heterokaryons between chicken erythrocytes and culture cells of various proliferative potential was studied. The following regularities were found: 1) Both immortalized and non-immortalized cells can efficiently reactivate DNA synthesis in erythrocyte nuclei. 2) Erythrocytes drastically inhibit the entry of non-malignant culture cell nuclei into the S-period, not acting upon DNA synthesis. 3) The inhibitory action is characteristic of erythrocytes from different stages of chicken ontogenesis (from 5-day-old embryos to the adult bird). 4) Malignant cells are completely refractory to the inhibitory action of erythrocytes. The ability of erythrocytes to inhibit the onset of replication in heterokaryons may be connected with the mechanisms of maintaining these terminally differentiated cells in a non-proliferating state.     

Phosphorylation of brain and liver ribosomal proteins during early development of chick embryo

32P-ortophosphate was introduced intraamnionally into 5, 9, 12, 15 and 19 day-old chick embryos. After 4 hours the specific activity of brain and liver ribosomal proteins and their fractions were determined. It was found that the specific activities of those in both tissues were highest at the early stages of development, and they declined rapidly to reach the lowest value at day 19. The observed differences between brain and liver ribosomal proteins are consistent with an unequal rhythm of ontogenesis.     

Changes in subfraction composition of chicken lysine-rich histone during ontogenesis]

Lysine-rich histone isolated from different chicken tissues was separated electrophoretically into 4-5 subfractions. The subfrations reffered to as 1, 2, 3, and 4, occur in each the tissue studied, erythrocyte lysine-rich histone containing an additional subfraction 1a. F1 histone from mitotically active tissues (intestinal mucosa, thymus, testes) has a higher content of subfraction 2, while the same histones from mototically inactive tissues (liver, heart, brain) contain an elevated amount of subfraction 3. F1 histone isolated from liver, brain and heart of 21-day embryo has much more of subfraction 2, than the same histone of adult animal. During the chicken development from hatching till maturation the content of subfraction 2 in these organs decreases, and the content of subfraction 3 increases. The rate of this change in liver corresponds to the rate of DNA synthesis. In F1 histone of erythrocytes the content of subfraction 4 falls down during the post hatching ontogenesis.     

Intracellular NAD+ content and ADP-ribose polymerase activity of serum-stimulated baby hamster kidney fibroblasts

We have examined a number of events relating to ADP-ribose metabolism during serum-stimulated growth of BHK-21/C13 fibroblasts. Both the intracellular NAD+ content and the ADP-ribose polymerase activity were found to increase after serum stimulation of cells that were previously arrested by growth in low-serum medium. NAD+ content increased about two-fold, reaching a maximum of 4.2 nmol/microgram of DNA 8 hr after serum steK-21/C13 fibroblasts. Both the intracellular NAD+ content and the ADP-ribose polymerase activity were found to increase after serum stimulation of cells that were previously arrested by growth in low-serum medium. NAD+ content inreased about two-fold, reaching a maximum of 4.2 nmol/microgram of DNA 8 hr after serum step-up. The polymerase exhibited a sharp rise in activity, reaching a peak at about 5 hr after step-up; the activity declined below initial values by 10 hr, and then increased again to reach a plateau at 20 hr. We also report evidence which suggests a possible effect of ADP-ribosylation on the activity of DNA-dependent RNA polymerase I. The activity of this enzyme is diminished in isolated nuclei, and in a subsequent (NH4)2SO4 extract, when the nuclei are incubated with NAD+, the substrate for poly(ADP-ribose) polymerase. This inhibitory effect on the RNA polymerase is abolished when nuclei are incubated also with nicotinamide, a powerful inhibitor of the poly(ADP-ribose) polymerase.     

DNA and DNA-polymerase activity in chicken brain regions during ontogeny

Abstract—

• 1 The DNA content of cerebral hemispheres, optic lobes, cerebellum and remainder was determined in chicken brains from the 11th day of embryonic life to 6 weeks after hatch. Each region showed a characteristic pattern of variation during development. The cerebellum showed the most rapid and the optic lobes the least rapid rate of DNA increase during the period studied. The concentration of DNA within these regions decreased continuously with age except for that of the cerebellum which passed through a maximum just before hatching.
• 2 The nature of the DNA-polymerase activity in soluble extracts from these brain regions seemed to be similar to the properties reported for this enzyme activity in other vertebrate tissues. Glycerol was stimulatory and denatured DNA was preferred to native DNA as primer. The requirements for magnesium ions and DNA were absolute. The requirement for deoxynucleoside triphosphates indicated this to be a replicative rather than a terminal addition enzyme. At nearly every age the level of enzyme activity was highest in extracts from the embryonic cerebellum.
• 3 The particulate fraction from brain homogenates decreased the DNA-polymerase activity observed in soluble brain extracts. Data are presented which indicate that this inhibition was the result of dephosphorylation of the deoxynucleoside triphosphate substrates by an ATPase in the brain particulate fraction whose activity increases during ontogeny.

     

DNA-polymerase activity of the liver of adult and old rats

The DNA-polymerase activity was determined in the cytosol of the intact and regenerating liver of adult and old rats under conditions of free passage of enzymes from nuclei and mitochondria. The DNA-polymerase activity of the intact liver is significantly increased in adult rats. The regeneration results in about 2-fold and 10-fold increase of the activity in the liver of adult and old rats, respectively. As a result, the DNA-polymerase activity in the regenerating liver of old rats significantly increased as compared to that of adult rats. The revealed age-related changes in the DNA-polymerase activity of the liver do not correlate with the decrease in the replication rate in the process of aging.     

The effect of a low dose of ionizing radiation on the activity of cAMP-dependent phosphodiesterase and protein kinase of chicken thymus in ontogenesis

Dynamics of changes in activities of cAMP-dependent enzymes, phosphodiesterase and protein kinase, were studied in the thymus of intact and irradiated, prior to incubation, (0.05 Gy) chicken embryos and chicks. The changes observed were wave-like. In determining phosphodiesterase activity of irradiated chicken thymocytes during ontogenesis the values were obtained that correlated with the cAMP level and adenylate cyclase activity. It was also shown that the increase in the rate of cAMP-dependent phosphorylation under the effect of low-level radiation corresponded to the cyclic nucleotide content of a cell at different development stages.     

Intranuclear distribution of mouse liver nuclear DNA polymerase

Adult mouse liver nuclei and their subfractions corresponding to heterochromatin, nucleoli, membranes, and euchromatin were studied for DNA-polymerase activity. The intact nuclei and the two heavy nuclear fractions contained rather low activity while the two light fractions (membranes and euchromatin) had no activity at all. In the two heavy fractions, the activity was stimulated by β-mercaptoethanol and depressed by p-hydroxymercuribenzoate and by omission of one or more nucleotides. A nuclease activity, detected in the intact nuclei, may also be present in the nuclear subfractions. DNA-polymerase activity in the heavy fractions of mouse liver nuclei is discussed in relation to other published results.     

Quantitative isolation of oligo- and polyadenosine-diphosphoribosylated proteins by affinity chromatography from livers of normal and dimethylnitrosamine-treated Syrian hamsters. In vivo and in vitro metabolism of the homopolymer

Polyadenosine- and adenosine-diphosphoribosylated proteins of hamster liver were quantitatively isolated with the aid of m-aminophenyl boronic acid glutaryl hydrazide polyacrylamide affinity resin by selective adsorption at pH 8.2 and elution at pH 4.0. Polymer-free proteins, DNA, and RNA are readily separated from adenosine-diphosphoribosylated proteins. The total quantity of proteins that is covalently modified by the homopolymer is 14.3 micrograms/mg of DNA or 37.4 micrograms/g of liver in controls and 38.7 micrograms/mg of DNA or 116 micrograms/g of liver in dimethylnitrosamine-treated hamsters. Polymer content increases from 9 to 15 nmol/mg of DNA to 42 to 118 nmol/mg of DNA following treatment with dimethylnitrosamine. Pulse labeling with [14C]ribose results in a parallel doubling in dimethylnitrosamine-treated animals of the specific activities of adenosine- diphosphoribose and NAD+ and of the [14C]ribose content of polyadenosine-diphosphoribose of chain length between 20 and 40, indicating chain elongation of pre-existing larger polymers. Two groups of proteins that are isolated as polyadenosine-diphosphoribose adducts are increased significantly after treatment with dimethylnitrosamine, one minor component of a mass between 100-112 X 10(3) daltons, and a major group exhibiting a mass of 158-162 X 10(3) daltons. Polyadenosine-diphosphoribose synthetase activity of isolated hepatic nuclei is increased by 32-37% after dimethylnitrosamine treatment, and since the change in glycohydrolase activity is negligible relative to the increase in synthetase, the augmentation of polyadenosine-diphosphoribosylated proteins can be explained by the increased synthetase of nuclei. The molecular size distribution of DNA in liver nuclei of control and dimethylnitrosamine-treated hamsters is indistinguishable.     

Ultrastructure of the hypophysiotropic region of the hypothalamus in early ontogenesis in rats]

The results of electron microscopic studies have shown that the 16--18 days old rat embryos already have in the hypophysiotropic area some structures necessary for the realization of neuroendocrine regulations. In the arcuate nuclei, the neurosecretory cells differentiate which are capable to synthesize specific neurosecretory granules of 800--1,000 A in diameter. In the median eminence, the primary portal capillaries develop with which tanicytes and a few axon terminals make contact. One can see in the tanicytes the signs of active transport and accumulation of electron dense polymorphic material. All these phenomena are strengthen during the subsequent development. Hence, several days are before birth the neurosecretory and glial elements of the embryos show the signs of functional activity which strengthen during ontogenesis and are expressed most distinctly in the adult animals.