Binding of beryllium to nuclear acidic proteins.

In vitro beryllium (Be) binding to rat liver nuclei has been reassessed (KAss = 2.0 X 10(6) M: n = 17 nmol Be/mg protein). Be also binds to rat liver nucleoli (KAss approx. 4 X 10(6) M: n = 10 nmol Be/mg protein). Examination of rat liver chromatin fractionated on a hydroxyapatite column shows that Be does not bind to histone or to the non-histone protein eluted by 0.05 M sodium phosphate. Be is strongly bound to the non-histone proteins eluted by 0.2 M sodium phosphate (KAss = 1.1 X 10(6) M: n = 55 nmol Be/mg protein) and also to the same extent to the fraction containing DNA which is subsequently eluted from the column. Evidence is provided that the latter binding is not due to DNA. The fractions containing the Be-binding proteins also contain the proteins which are phosphorylated to the greater extent.     

Age-dependent variation of rates of polyadenosine-diphosphoribose synthesis by cardiocyte nuclei and the lack of correlation of enzymatic activity with macromolecular size distribution of DNA

Cardiocyte nuclei from neonate (5-day-old) and from adult (90-day-old) male Wistar rats were selectively isolated by a technique that eliminates interference by noncardiocyte nuclei (Jackowski, G., and Liew, C. C. (1980) Biochem. J. 188, 363-373). Polyadenosine-diphosphoribose synthetase activity of cardiocyte nuclei of neonates was ten times greater than in nuclei of adult rats, as calculated from initial velocity linear rate measurements. The molecular size of DNA extracted from cardiocyte nuclei of neonates was significantly larger (peak at 54 S) than DNA of cardiocyte nuclei of adults (peak at 33 S) as determined by alkaline sucrose density gradient ultracentrifugation (Knopf, K. W., and Weissbach, A. (1977) Biochemistry 16, 3190-3194). Comparison of the molecular sizes of DNA extracted from whole cardiac tissue with DNA isolated from cardiocyte nuclei shows that no DNA fragmentation takes place during the process of isolation of nuclei. Polyadenosine-diphosphoribose glycohydrolase activity was about 30% higher in cardiocyte nuclei of neonates than in adults, but the activity represents only 10% of the rate of the synthetase. Results demonstrate that polyadenosine-diphosphoribose metabolism in differentiated tissues is not directly related to the molecular size of DNA.     

Investigations on myo-inositol-1-phosphate synthase from the wild type and the inositol-dependent mutant of Neurospora crassa.

The inositol-dependent mutant of Neurospora crassa lacks inositol-1-phosphate synthetase activity. This defect can be revorted by the addition of high-molecular DNA isolated from the wild type. To elucidate the biochemical background of inositol dependence, inositol-1-phosphate synthetase was studied. A method has been developed fro the isolation of the enzyme from the wild type strain in 10 mg scale by salt fractionation, gel filtration and ion-exchange chromatography. The specific activity of the purified enzyme is 4750 U/mg protein and its purity has increased about 100-fold. Polyacrylamide gel electrophoresis indicated that, in addition to the main enzymatically active band, several accompanying proteins occur in very small amount. The molecular weight of the enzyme is 225,000 daltons. Probably it consists of four subunits, two with a molecular weight of 64,000 daltons and another two of 50,000 daltons. An enzymatically inactive protein has been isolated from the mutant with the same procedure as that of the enzyme; it migrated at gel electrophoreis similarly to the enzyme. It may be supposed that the isolated protein is the defective enzyme molecule.     

Accumulation of O6-methylguanine in non-target-tissue deoxyribonucleic acid during chronic administration of dimethylnitrosamine.

1. BD-IV rats were given labelled dimethylnitrosamine (2 mg/kg) by stomach tube on weekdays (Monday to Friday) for up to 24 weeks. The rats killed after 2, 4, 8, 16 and 24 weeks of treatment (72 h after the final dimethylnitrosamine gavage) and DNA was isolated from the pooled livers, kidneys and lungs. Purine bases were released from the DNA by mild acid hydrolysis and separated by Sephadex G-10 chromatography. 2. Throughout the experiment, the content of 7-methylguanine in liver DNA was approx. 16 times that in kidney and lung. The amount of this product increased in the DNA of all three tissues up to 16 weeks, but by 24 weeks had decreased by 20% in the liver and 46% in the other tissues. 3. O6-Methylguanine was not detected in liver DNA, but was easily measured in kidney and lung DNA after 4 weeks of dimethylnitrosamine administration. The amount of O6-methylguanine in kidney and lung DNA increased relative to that of 7-methylguanine, and by 24 weeks was 60% of the 7-methylguanine content in both tissues. 4. Incorporation of radioactive C1 breakdown products of dimethylnitrosamine into normal purines in DNA increased continuously in all three tissues. 5. The results are discussed with respect to the specific hepatocarcinogenic effect of chronic administration of dimethylnitrosamine and the possible contribution of increased DNA repair and DNA synthesis.     

A possible mechanism of inhibition of protein synthesis by dimethylnitrosamine

1. The incorporation of [¹⁴C]leucine into liver proteins of rats was measured in vivo at various times after treatment of the animals with dimethylnitrosamine and was correlated with the state of the liver ribosomal aggregates. Inhibition of incorporation ran parallel with breakdown of the aggregates. 2. Inhibition of leucine incorporation into protein and breakdown of ribosomal aggregates were not preceded by inhibition of incorporation of [¹⁴C]orotate into nuclear RNA of the liver. 3. Evidence was obtained of methylation of nuclear RNA in the livers of rats treated with [¹⁴C]dimethylnitrosamine. 4. Zonal centrifugation analysis of radioactive, nuclear, ribosomal and transfer RNA from livers of rats treated with [¹⁴C]dimethylnitrosamine revealed labelling of all centrifugal fractions to about the same extent. 5. It is suggested that methylation of messenger RNA might occur in the livers of dimethylnitrosamine-treated rats and the possible relation of this to inhibition of hepatic protein synthesis is discussed.     

Effects of adrenaline pretreatment on in vitro binding of 125I-triiodothyronine to nuclear receptor, intracellular distribution of endogenous triiodothyronine and activities of alfa-glycerophosphate dehydrogenase and malic enzyme in rat liver

Especially coated adrenaline tablets (A) or placebo tablets (P) which release linearly the hormone were implanted in male Wistar rats. Six hours later animals were sacrificed and kinetic parameters of T3-125I binding to nuclear receptor, intracellular distribution of endogenous T3 and activities of alfa-GPD and ME were investigated. The association constant values (Ka) of nuclear receptor were increased after pretreatment with 7.5, 15 and 45 mg A tablets and were 1.07, 1.35 and 1.48 X 10(9) M-1 vs 0.85 X 10(8) M-1 value seen after P. The maximal binding capacity (MBC) values decreased after pretreatment with the same doses of A and were 0.044, 0.036 and 0.025 pmol T3/100 micrograms DNA vs. 0.065 pmol T3/100 micrograms DNA in P pretreated. Adrenaline pretreatment significantly increased the amount of endogenous T3 present in liver nuclei while the amount of T3 present in cytosol decreased. Activity of mitochondrial alfa-GPD was increased after 15 and 45 mg of A. Significant rise of activity of cytosol ME was seen only after pretreatment with 45 mg of A.     

Hyperoxia enhances lung and liver nuclear superoxide generation

Porcine lung and liver nuclei generated superoxide (O-2) at a rate which increased with increasing oxygen concentration. NADH-dependent O-2 generation increased from 0 to 2.21 +/- 0.11 nmol/min per mg protein for lung nuclei and from 0.16 +/- 0.09 to 1.34 +/- 0.14 nmol/min per mg protein for liver nuclei, when oxygen concentration increased from 0 to 100%. NADPH-dependent O-2 generation increased similarly in liver nuclei (from 0.20 +/- 0.09 to 1.20 +/- 0.12 nmol/min per mg protein), while lung nuclei produced only 0.45 +/- 0.09 nmol/min per mg protein at 100% oxygen. NADH and NADPH had an additive effect on O-2 generation by liver nuclei, yielding 2.58 +/- 0.21 nmol/min per mg protein at 100% oxygen. Very little or no superoxide dismutase activity was present in washed nuclear preparations. The oxygen-dependence of nuclear O-2 generation shows that nuclear-derived partially reduced species of oxygen may affect nuclear function during hyperoxia or other metabolic situations where overproduction of oxygen radicals is problematic.     

Stimulation of 2',5'-oligoadenylate synthetase activity in sheep endometrium during pregnancy, by intrauterine infusion of ovine trophoblast protein-1, and by intramuscular administration of recombinant bovine interferon-alpha I1.

In Expt 1, activity of 2',5'-oligoadenylate (2',5'-A) synthetase in endometrium collected on Day 16 (oestrus is Day 0) from the uterine horn ipsilateral to the corpus luteum was greater (P less than 0.001) for pregnant (135.5 +/- 1.72 nmol/mg protein/h) than for cyclic ewes (58.5 +/- 0.99 nmol/mg protein/h). In pregnant ewes, activity of 2',5'-A synthetase in endometrium collected from the contralateral uterine horn (119.5 +/- 1.72 nmol/mg protein/h) did not differ from that of the ipsilateral horn. In Expt 2, three ovariectomized ewes were treated with progesterone for 10 days and then with oestrogen for 2 days. Activity of 2',5'-A synthetase on Day 13 was 18% greater (P less than 0.10) in endometrium collected from the uterine horn receiving infusions of 30 micrograms ovine trophoblast protein-1 (oTP-1) twice a day on Days 10, 11 and 12(57.7 +/- 0.22 nmol/mg protein/h) than from the uterine horn receiving control infusions of serum protein (SP; 48.8 +/- 0.22 nmol/mg protein/h). In Expt 3, activity of 2',5'-A synthetase on Day 15 was not significantly greater in endometrium collected from the uterine horn of cyclic ewes receiving infusions of 30 micrograms oTP-1 twice a day on Days 12, 13 and 14 (46.5 +/- 0.37 nmol/mg protein/h) than in endometrium from the uterine horn receiving infusions of SP (38.2 +/- 0.37 nmol/mg protein/h). When results of Expt 2 and Expt 3 were combined, intrauterine infusion of oTP-1 increased (P less than 0.05) activity of 2',5'-A synthetase in endometrium by 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylated purines in the deoxyribonucleic acid of various Syrian-golden-hamster tissues after administration of a hepatocarcinogenic dose of dimethylnitrosamine.

1. DNA was extracted from livers, kidneys and lungs of Syrian golden hamsters at various times (up to 96h) after injection of a hepatocarcinogenic dose of [14C]dimethylnitrosamine. Purine bases were released from the DNA by mild acid hydrolysis and separated by Sephadex G-10 chromatography. 2. At 7h after dimethylnitrosamine administration liver DNA was alkylated to the greatest extent, followed by that of lung and kidney, the values for which were 8 and 3% respectively of those for liver. 3. The O6-methylguanine/7-methylguanine ratios were initially the same in all three organs and in the liver DNA of rats under similar conditions of dose. 4. O6-Methylguanine was the most persistent alkylated purine in all three hamster tissues. There was evidence for excision of 7-methyl-guanine, the highest activity for this being present in the liver. 5. Detectable amounts of the minor products 3-methyladenine, 1-methyladenine, 3-methylguanine and 7-methyladenine were present in most hamster tissues, and their individual rates of loss from liver DNA were determined. 6. Ring-labelling of the normal purines in DNA was highest in the liver, followed closely by the lung (80% of that in liver) whereas the kidney had very low incorporation (3% of that in liver). 7. The results are discussed with respect to the hepatotoxicity of dimethylnitrosamine, the miscoding potential of the various alkylation products and the induction of liver tumours in hamsters.     

Formation and subsequent removal of O6-methylguanine from deoxyribonucleic acid in rat liver and kidney after small doses of dimethylnitrosamine

1. The amounts of 7-methylguanine and O⁶-methylguanine present in the DNA of liver and kidney of rats 4h and 24h after administration of low doses of dimethylnitrosamine were measured. 2. O⁶-Methylguanine was rapidly removed from liver DNA so that less than 15% of the expected amount (on the basis of 7-methylguanine found) was present within 4h after doses of 0.25mg/kg body wt. or less. Within 24h of administration of dimethylnitrosamine at doses of 1mg/kg or below, more than 85% of the expected amount of O⁶-methylguanine was removed. Removal was most efficient (defined in terms of the percentage of the O⁶-methylguanine formed that was subsequently lost within 24h) after doses of 0.25–0.5mg/kg body wt. At doses greater or less than this the removal was less efficient, even though the absolute amount of O⁶-methylguanine lost during 24h increased with the dose of dimethylnitrosamine over the entire range of doses from 0.001 to 20mg/kg body wt. 3. Alkylation of kidney DNA after intraperitoneal injections of 1–50μg of dimethylnitrosamine/kg body wt. occurred at about one-tenth the extent of alkylation of liver DNA. Removal of O⁶-methylguanine from the DNA also took place in the kidney, but was slower than in the liver. 4. After oral administration of these doses of dimethylnitrosamine, the alkylation of kidney DNA was much less than after intraperitoneal administration and represented only 1–2% of that found in the liver. 5. Alkylation of liver and kidney DNA was readily detectable when measured 24h after the final injection in rats that received daily injections of 1μg of [³H]dimethylnitrosamine/kg for 2 or 3 weeks. After 3 weeks, O⁶-methylguanine contents in the liver DNA were about 1% of the 7-methylguanine contents. The amount of 7-methylguanine in the liver DNA was 10 times that in the kidney DNA, but liver O⁶-methylguanine contents were only twice those in the kidney. 6. Extracts able to catalyse the removal of O⁶-methylguanine from alkylated DNA in vitro were isolated from liver and kidney. These extracts did not lead to the loss of 7-methylguanine from DNA. 7. The possible relevance of the formation and removal of O⁶-methylguanine in DNA to the risk of tumour induction by exposure to low concentrations of dimethylnitrosamine is discussed.     

Developmental pattern of poly (ADP-ribose) synthetase and NAD glycohydrolase in the brain of the fetal and neonatal rat

Poly (ADP-ribose) synthetase and NAD glycohydrolase were examined in nuclear fractions from rat brain at sequential times during late fetal and the first two weeks of neonatal life. In whole brain, both enzymes were demonstrable at all stages of development, but followed separate patterns. Activity of the synthetase which was greatest in fetal life, fell steadily with fetal maturation from 3.90±0.06 nmol/mg DNA at 16 days, to reach a nadir of 1.36±0.09 nmol/mg DNA on the 4th postnatal day. Subsequently it underwent a non sustained neonatal rise reaching a peak of 2.46±0.07 nmol/mg DNA on the 8th day. By contrast, NAD glycohydrolase activity increased steadily throughout late fetal and during the first two weeks of neonatal life, from 12.77±0.40 nmol/mg DNA on day 16 of gestation to 25.80±.95 nmol/mg DNA on neonatal day 12. In neonatal cerebellum the activity of poly (ADP-ribose) synthetase was greater at 8 than at 4 days, could be stimulated with graded concentrations of sonicated DNA up to 100 g, but was inhibited by higher concentrations of DNA and by all concentrations of exogenous histone. In an in vitro culture system of fetal rat brain cells, the activity of poly (ADP-ribose) synthetase increased steadily over six days. Cycloheximide 10^–3 M completely inhibited the activity of this enzyme. NAD glycohydrolase activity increased progressively in vitro, and after 6 days in cycloheximide (10^–3 M), the cultures contained significantly greater levels of enzyme activity. It is suggested that changing activities of poly (ADP-ribose) synthetase and NAD glycohydrolase could both provide potential markers for brain cell differentiation in this system.     

In vivo distribution of dimethylnitrosamine metabolites in mouse liver and their binding to micrococcal nuclease sensitive and resistant chromatin

Mice were injected i.p. with a single dose (5 mg/kg body wt) of [14C]dimethylnitrosamine and killed at time intervals between 15 and 120 min. Isolated nuclei were incubated with micrococcal nuclease and the chromatin separated into a 1100 g pellet P1 and supernatant fraction S1. The incorporation of 14C from [14C]dimethylnitrosamine into chromatin was significantly higher in the P1 than the S1 fraction. The purine bases of the P1 DNA showed a lower methylation than those of the S1 DNA. In contrast, radioactivity of the proteins was higher in the P1 than the S1 fraction. It is concluded that the open structures of the S1 chromatin were preferentially attacked by the hepatotoxin leading to a high 14C-labelling of the DNA relative to that of the proteins.     

Repair replication of DNA in the intact animal following treatment with dimethylnitrosamine and with methyl methanesulphonate, studied by fractionation of nuclei in a zonal centrifuge.

To relate repair of lesions in DNA with carcinogenesis, it is necessary to study repair in the intact animal under conditions which will and will not induce cancer. The methods used to study the replications stage of repair in isolated cells are not suitable for use in the intact animal. A method is presented which depends on the fact that during cell replication the nuclei enlarge, and may be separated from nuclei of non-replicating cells by rate zonal centrifugation on a sucrose gradient in a zonal rotor. Thus incorporation of [3H]thymidine as a result of de novo synthesis of DNA in replicating nuclei can be separated from incorporation due to repair synthesis in non-replicating nuclei. Treatment of animals with dimethylnitrosamine or with methyl methanesulphonate produced a "repair-type" profile, which contrasted with that given by liver nuclei from untreated animals, or from animals in which DNA synthesis had been reduced by treatment with cycloheximide, a compound which is not known to cause direct damage to DNA. Evidence is presented which suggests that the method is a rapid sensitive test for the occurrence of repair replication of some kind in the liver of the intact animal.     

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.     

Cellular injury and carcinogenesis. The effect of a protein-free high-carbohydrate diet on the metabolism of dimethylnitrosamine in the rat

1. Rats fed on a protein-free high-carbohydrate diet for 7 days metabolized dimethylnitrosamine at only 55% the rate of rats fed on a commercial diet. 2. Dimethylnitrosamine was metabolized by liver slices from rats fed on the protein-free diet at less than half the rate attained by slices from rats fed on a commercial diet. But kidney slices from these rats metabolized dimethylnitrosamine at the same rate as kidney slices from rats on a commercial diet. 3. Methylation by dimethylnitrosamine (70mg/kg body wt.) of N-7 of guanine of the liver RNA and DNA of rats fed on a protein-free diet was only slightly higher than in rats fed on a normal diet given 27mg/kg body wt. In contrast, the methylation by dimethylnitrosamine of guanine in kidney nucleic acids of these rats was three times that in the rats fed on a normal diet. 4. In rats fed on a protein-free diet the incidence of kidney tumours produced by a single dose of dimethylnitrosamine is increased.     

The absorption and metabolism in rats of small oral doses of dimethylnitrosamine. Implication for the possible hazard of dimethylnitrosamine in human food.

1. Groups of rats were given one dose of the carcinogen dimethylnitrosamine by gastric intubation. The dose was varied between 10mg/kg body wt. and 1 microgram/kg body wt. 2. The dose was rapidly absorbed. 3. The methylation of liver DNA resulting from the administration of this carcinogen was proportional to dose. This suggests that small doses are absorbed from the gut with no more loss than large doses. 4. As the dose was decreased there was a disproportionately greater decrease in the alkylation of kidney DNA, and when the dose was less than 40 microgram/kg body wt. the methylation of kidney DNA was no longer detectable. This possibly explains why small amounts of dimethylnitrosamine in the diet do not induce kidney tumours. 5. Comparison of the relative alkylation of liver DNA and kidney DNA resulting from an oral and from an intravenous dose of dimethylnitrosamine suggest that small amounts of dimethylnitrosamine absorbed into the portal blood from the gut are completely metabolized by the liver and do not enter the general circulation. 6. The implications of these results for the possible hazard of dimethylnitrosamine in human food is discussed.     

The effect of inhibition of RNA synthesis by actinomycin D on the population of basic polypeptides of the 30S unclear ribonucleoprotein particles

In rats injected intraperitoneally with actinomycin D (2 mg/kg body weight) 12 h earlier, the yield of the 30S ribonucleoprotein particles isolated from liver nuclei by extraction with 0.1 M NaCl at pH 8.0 decreased by 60 per cent. The protein-to-RNA ratio of these particles increased to 32:1 from the ratio 4.4:1 found in the same particles isolated from the nuclei of liver of control rats. The particles isolated from the liver nuclei of rats injected with actinomycin D were depleted of all charge isomers of the two most prominent polypeptides (33,000 and 39,000 daltons) present in the particles of liver of control animals. The most abundant protein in these particles was a 43,000 dalton polypeptide. This polypeptide is the least prominent of the 3 major polypeptides present in the control particles. The same charge isomers of the 43,000 dalton polypeptide were present in the nuclear ribonucleoprotein particles isolated from the liver of control animals and from the liver of animals treated with actinomycin D 12 h earlier. In control animals the nuclear ribonucleoprotein monoparticles isolated from kidney contained 3 major polypeptides of the same molecular weight with the same distribution of their charge isomers as were present in the particles isolated from liver nuclei. The injection of actinomycin D 12 h earlier was without any effect on the protein composition of the 30S nuclear ribonucleoprotein particles of rat kidney.     

Interaction of dexamethasone-receptor complexes with rat liver nuclei and DNAs]

The role of DNAs in the nuclear binding of dexamethasone-receptor complexes (DRC) was studied. The cytosolic receptors from rat liver have a sedimentation coefficient of about 7S, the Stock's radius--of about 50 A and possess a high affinity to dexamethasone (Kas = 2,6 X 10(8) M-1). Their capacity is 3 X 10(-13) and 5.5--7.0 X 10(-12) mole of dexamethasone per mg cytosolic protein and mg DNA, respectively. DRC has the ability to bind to the nuclei of rat liver. DRC binding to nuclei is increased approximately 3-fold by temperature activation of cytosol. The nuclear acceptor sites are saturated at the level of 16.2 pmoles of bound DRC per mg nuclear DNA. Free DNA has the ability to compete with nuclei for binding with DRC. Temperature-activated DRC can bind both with homo- and heterologous DNAs. Secondary DRC-DNA complexes were isolated by means of gel filtration on Sepharose 4B. Thermal denaturation of DNA decreases its ability to bind DRC approximately 2-fold. DNAs of a similar nucleotide composition, i.e. DNA from rat liver (GC = 43 mole%) and DNA from Photobacterium belozerskii (GC = 44 mole%), have a close DRC-binding ability. At the same time, these DNAs bind about 1.5-fold less DRC, as compared to DNA from Pseudomonas aeruginosa (GC = 67 mole%) and about 1.5-fold more, than does DNA from T2 phage (GC = 35 mole%). Thus the positive correlation between the GC composition of DNA and its DRC-binding ability was established. Unique sequences (Cot greater than 600) bind several times less DRC than the reiterated sequences (also denaturated) (Cot = O--600) of rate liver DNA. Thus, DNA can be considered as a nuclear acceptor of DRC. It is assumed, that DRC is able to recognise in DNA certain short GC-rich sequences, distributed in the rate genome in a non-random fashion.