Methylation, acetylation and phosphorylation of the bases of DNA of young and old rats

The possibility of methylation, acetylation and phosphorylation of the bases of DNA has been studied in vitro by incubating nuclei of the liver and cerebral hemisphere of young (18 wk) and old (120 wk) rats with radioactive donors, [3H]S approximately adenosyl methylmethionine, [3H]-acetyl approximately CoA and [32P]-gamma-ATP for methylation, acetylation and phosphorylation of the bases, respectively. Nuclei were also incubated with S approximately adenosyl homocysteine to inhibit methylation with sodium butyrate to stimulate acetylation and with alkaline phosphatase to remove phosphate groups incorporated into the bases. DNA was then extensively purified and incorporation of each type of label was estimated. The data show that both methylation and acetylation of DNA of old rats were significantly higher than those of young rats, and phosphorylation is lower in old rats. Such modifications may prevent base pairing between the two strands of DNA, alter its conformation and binding of trans-acting factors at specific sites, and thereby alter gene expression.     

PCR assay of DNA damage and repair at the gene level in brain and spleen of gamma-irradiated young and old rats.

The PCR amplification of fragments of transcribed (beta-actin, p53) and nontranscribed (IgE, heavy chain) genes in brain and spleen DNA from gamma-irradiated and unirradiated 2- and 28-month-old rats was studied. The amplification levels of fragments of these genes in DNA from old rats were substantially lower than those from young rats, which suggested that these gene fragments in old-rat DNA contained lesions blocking thermostable polymerase in PCR. The beta-actin and IgE gene fragments of spleen DNA from old rats exhibited a significantly higher level of lesions inhibiting Tth polymerase compared to analogous fragments of brain DNA from the same animals. DNA from the tissues of gamma-irradiated rats showed the amount of damage inhibiting amplification to be dependent on animal age and the postirradiation time before DNA isolation. As judged from the changes in the amplification level of gene fragments, there was no preferential fast repair of lesions in the actively transcribed gene beta-actin compared to the nontranscribed gene IgE (heavy chain) in the brain and spleen of gamma-irradiated young and old rats. The amplification results suggest that equal amounts of DNA lesions were repaired in the brain of both old and young rats during the first 0.5 h of the postirradiation time (fast-repair phase), whereas in the subsequent postirradiation period over 5 h (slow-repair phase), the efficiency of damage elimination in the brain DNA of old rats was markedly lower. As for the spleen tissue, the elimination of lesions blocking Tth polymerase was much lower in old gamma-irradiated animals for both of the repair phases.     

Energy metabolism in the liver of young and old rats during starvation

The concentration of key intermediate products of energy metabolism is determined in the liver of young and old rats under normal conditions and 24h after fasting. A decrease in the stationary concentrations of glucose, glucose-6-phosphate, ATP, ADP and AMP and an increase in the concentrations of lactate, glutamate, alpha-glycerophosphate and Pi were found in the liver of rats in ageing. The carbohydrate metabolism response to fasting is also disturbed. The total content of adenine nucleotides it the rat liver during ageing is 25-30% lower. The deficit of adenine nucleotides is not associated with the activation of AMP-desaminase; it may result from both physiological and functional disturbances in the ageing liver.     

Analysis of chromatin of the brain of young and old rats by nick-translation

Chromatin of the brain of young (22-23 week) and old (118-119 week) rats has been analysed by nick-translation reaction following its digestion by DNaseI, EcoRI, MspI and HpaII. The incorporation of (3H)-dTMP in the old is only about 50 percent of that of the young. The difference in the incorporation following digestion of nuclei by MspI and HpaII that quantitate the degree of methylation of internal cytosines in the 5' CCGG 3' sequences, is nearly two-fold higher in the old. These data indicate that the chromatin undergoes increasing condensation as a function of age. One of the contributory factors may be increasing methylation of DNA. This may decrease the active fraction of chromatin.     

Regeneration and transplantation of muscles in old rats and between young and old rats.

In order to compare the regenerative ability of skeletal muscle between young (5 month) and old (26 month) rats, sliced or intact extensor digitorum longus muscles were freely autografted into young and old rats and also reciprocally grafted from young to old inbred animals and vice versa. Sixty days after grafting, the transplants were analyzed for contractile and histochemical properties. There was a relative similarity between the contraction times of both normal control muscles and of all groups of transplants, although the contraction time tended to be prolonged and histochemical fiber pattern was more often found to be uniform in grafts of senescent animals. All groups of transplants possessed histochemically heterogeneous fiber types at 60 days. The experiments demonstrate that skeletal muscle in old rats possesses a substantial degree of regenerative ability and that the free tranpllantation of entire muscles in old animals is feasible.     

Distribution of kappa opioid receptors in the brain of young and old male rats

The experiments to be described have been designed in order to: (a) provide new information on the concentrations of opioid kappa receptors in different regions of the brain of the male rats; and (b) to analyze whether the density of brain kappa receptors might be modified by the process of aging. The concentration of kappa receptors was investigated in the hypothalamus, amygdala, mesencephalon, corpus striatum, hippocampus, thalamus, frontal poles, anterior and posterior cortex collected from male rats of 2 and 19 months of age. 3H-bremazocine (BRZ) was used as the ligand of kappa receptors, after protection of mu and delta receptors respectively with dihydromorphine and d-ala-d-leu-enkephalin. The results obtained show that: (1) in young male rats, the number of kappa opioid receptors is different in the various brain areas examined: the hypothalamus and the striatum have a concentration of kappa binding sites which is significantly higher than that found in the mesencephalon and in the amygdala; much lower concentrations of kappa binding sites have been found in the thalamus, the frontal poles, the hippocampus, the anterior and posterior cerebral cortex. (2) Aging exerts little influence on the number of kappa receptors in the majority of the brain structures considered. However in the amygdala and in the thalamus the number of kappa receptors was increased in old animals. To the authors' knowledge, the data here presented are the first ones which suggest that age may increase rather than decrease the number of neurotransmitter receptors in the brain.     

Incorporation of 14C-thymidine into liver and brain DNA of protein-deficient rats.

The authors studied the effect of a protein-free diet on 14C-thymidine incorporation into rat liver DNA in vivo and found, after 2-3 weeks, a marked decrease in uptake of the radioactive base into the liver DNA, followed by a decrease in the proportion of DNA in liver cell homogenates. The total nuclear count/mg liver tissue displayed an increase during protein depletion, except for the 5th week, when a decrease was recorded. The incorporation of 14C thymidine into the brain DNA likewise displayed no great differences, although a significant drop was observed during the 2nd to 4th week of depletion. In the 5th week we recorded an increase in uptake of the radioactive base by brain DNA, exceeding the incorporation values in the controls.     

Analysis of chromatin of the brain of young and old rats by micrococcal nuclease and DNase I

Micrococcal nuclease (MCN) and DNase I were used to study the conformational changes in chromatin of the brain of rats of different ages. Purified nuclei and chromatin were digested separately by MCN and DNase I. Kinetics of digestion of chromatin by MCN are similar for young, adult and old rats. Also agarose gel electrophoresis of DNA fragments do not show any differences. The kinetics of digestion with DNase I, on the other hand, are greater and faster for 20-week old rats than for 90-week old rats. High performance denaturing polyacrylamide gel electrophoresis reveals that a greater amount of smaller fragments of DNA are produced in the 20-week old rats than in the 90-week. These conformational changes occur in the chromatin during aging.     

Oxidative phosphorylation by in situ synaptosomal mitochondria from whole brain of young and old rats

Synaptosomes, isolated from the whole brain of young (3 months) and old (24 months) rats were used to study the major bioenergetic systems of neuronal mitochondria in situ, within the synaptosome. Approximately 85% of the resting oxygen consumption of synaptosomes from both young and old rats was a result of proton leak (and possibly other ion cycling) across the mitochondrial inner membrane. There were no significant differences between synaptosomes from the young and old rats in the kinetic responses of the substrate oxidation system, the mitochondrial proton leak and the phosphorylation system to changes in the proton electrochemical gradient. Flux control coefficients of 0.71, 0.27 and 0.02 were calculated for substrate oxidation system, phosphorylation system and the proton leak, respectively, at maximal ATP producing capacity in synaptosomes from young animals. The corresponding values calculated for synaptosomes from old animals were 0.53, 0.43 and 0.05. Thus substrate oxidation had greatest control over oxygen consumption at maximal phosphorylating capacity for synaptosomes from whole brain, with proton leak, having little control under maximal ATP producing capacity. The uncoupled rate of oxygen consumption, in the presence of the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), was significantly lower (p = 0.0124) in synaptosomes from old rats (6.08 +/- 0.42, n = 11) when compared with those from the young rats (7.87 +/- 0.48, n = 8). Thus, there is an impaired flux through the substrate oxidation system is synaptosomes from old rats, as compared to synaptosomes from the young animals. These in situ results may have important implications for the interpretation of theories that age-dependent impairment of mitochondrial energy production may result in increased susceptibility to neurodegeneration.     

Testosterone uptake by prostatic tissue from young and old rats.

The uptake of [3H]-testosterone in vitro by the ventral lobe of the prostate of rats more than 11 months old was significantly less than that of rats 4-5 weeks old. There were significant decreases between young and old rats in the RNA and DNA content of the prostate but not in the activity of acid or alkaline phosphatases. Alkaline phosphatase activity was higher than that of acid phosphatase. Testosterone uptake by the prostate was higher in culture medium TC199 than in Krebs-Ringer buffer solution.     

The specific activity of aldolase in the livers of old and young rats.

The catalytic activity of liver fructose-biphosphate aldolase (EC 4.1.2.13) of young and old rats in units per manomole sequence has been calculated. No evidence of the accumulation with age of altered enzyme molecules of low catalytic activity was obtained. This is contrary to results obtained using mice and rabbits and indicates that the accumulation of altered enzymes may not always be associated with aging. The possibility that altered proteins are formed, but do not accumulate, cannot be ruled out.     

Effect of adrenaline and triamcinolone on cytochrome oxidase activity in the brain and liver of young and adult rats.

The effect of adrenaline (0.15 i.p./kg b.w.) and of the synthetic glucocorticoid triamcinolone (40 mg i.p./kg b.w.) on cytochrome oxidase activity, the terminal enzyme of the cytochrome system, was studied in homogenates of the cerebral cortex, subcortical formations (including the basal ganglia, the thalamus and the hypothalamus), the medulla oblongata and the liver of 5-day-old and adult rats. Activity in the above mentioned homogenates was measured polarographically 15 and 30 min after administering adrenaline or 48 h after administering triamcinolone. Fifteen minutes after its injection, adrenaline caused a statistically significant drop in cytochrome oxidase activity in the cerebral cortex, subcortical formations and liver of 5-day-old rats. The decrease still persisted 30 min after administration of the hormone, but was intensified only in the liver. In adult rats, on the other hand, a significant increase in activity was observed in the cerebral cortex and liver after adrenaline. Triamcinolone had no effect on cytochrome oxidase activity in any of the given parts of the brain in either young or adult rats. It significantly stimulated cytochrome oxidase activity in the liver of 5-day-old rats, but severely inhibited it in the liver of adult rats.