Age-dependent expression of S100beta in the brain of mice.

S100beta is a soluble calcium binding protein released by glial cells. It has been reported as a neurotrophic factor that promotes neurite maturation and outgrowth during development. This protein also plays a role in axonal stability and in long term potentiation in the adult brain. The ability of S100beta to modulate neuronal morphology raises the important question whether there is an age-related difference in the expression of S100beta in the cerebral and cerebellar cortices of AKR strain mice and is this change is region specific. Our RT-PCR and Western blotting experiments show that the expression of S100beta gene in the cerebral and cerebellar cortices starts from 0 day, peaks at about 45 days. However, in 70-week old mice its expression is significantly up-regulated as compared to that of 20-week old mice. S100beta follows the same age-related pattern in both cerebral and cerebellar cortices. These results suggest that S100beta is important for brain development and establishment of proper brain functions. Up-regulation of S100beta in old age may have some role in development of age-related pathological systems in the brain.

Methylation of repetitive DNA sequences in the brain during aging of the rat.

Methylation of repetitive DNA sequences (RDS) of the genomic DNA of the brain of 15- and 88-week old rats was analysed by digestion with HpaII, MspI, EcoRI + HpaII and EcoRI + MspI followed by end-labelling. mCpG doublets are present in two RDS of approximately 5 and 0.4 kb, and are also randomly distributed throughout the genome. Hemimethylated mCpC doublets also occur. Both mCpG and mCpC doublets are found more in the old than in the young. This age-related increase in DNA methylation occurs both at CCGG sites of the RDS and in the entire genome. Such increase in DNA methylation may alter chromatin conformation and gene expression in the brain as the rat ages.

Alterations in tyrosine aminotransferase isoenzymes of liver of rats as a function of age.

The specific activities (units/mg protein) of tyrosine aminotransferase (TAT) and its isoenzymes, cytoplasmic (c-), mitochondrial (m-) and nuclear (n-TAT), of the liver of 6-, 35- and 76-week old male albino rats were determined. The activities of all the isoenzymes are highest at 6 weeks and are significantly lower at 35 weeks. Total TAT and c-TAT remain unchanged, m-TAT decreases and n-TAT increases significantly after 35 weeks. c-TAT was partially purified from the liver of rats of the three ages to study its kinetic and molecular properties. These properties apparently do not change as a function of age.

Comparative studies on myosin ATPase of a flying and nonflying bird.

Myosin was purified from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird. Ki (ADP) of myosin ATPase of pigeon is higher, but the Km (ATP) is lower than that of fowl. The specific activity (mumole of Pi liberated/min/mg protein) is higher for the fowl. A0.5 (CaCl2) of myosin of both pigeon and fowl is similar. However, the two proteins differ in their interactions with ADP, ATP and p-chloromercuribenzoate. The two proteins have the same tyrosine, tryptophan and sulfhydryl contents. The electrophoretic patterns of the two myosins on SDS-polyacrylamide gels are different. These studies show significant molecular differences in the myosin derived from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird.

Activity and modulation of ornithine decarboxylase and concentrations of polyamines in various tissues of rats as a function of age.

The activities of ornithine decarboxylase (ODC) on the soluble and nuclear fractions of the cerebral cortex, heart and lungs of 4- (young), 38- (adult) and 85-week (old) male rats were studied. Also, the effects of aminophylline, histamine and estradiol on the activity of soluble ODC have been determined in vitro using slices of these tissues. The activity of ODC is significantly higher in the soluble fraction of all the tissues in comparison to that of nuclear fraction. Its activity in both the fractions is highest in the immature and decreases with increasing age in all the tissues except in the nuclear fraction of the lungs in which it increases with age. The ODC of the heart, lungs and cerebral cortex appear to be different as seen from the differences in their sensitivities to aminophylline, histamine and estradiol. In general, there is a decrease in its sensitivity to the three effectors with increasing age. This may be due to a decrease in the receptors and a concomitant decrease in ODC activity. A direct relationship between ODC activity and polyamine levels of the brain exists at various ages of the rat.

Molecular biology of ageing.

The process of deterioration or ageing of functions that occurs in all organisms after the attainment of reproductive ability is the sum total of the decline in activity of various organs. The functions of different organs begin to deteriorate at different times of the life span and at different rates. It is believed that different genes are involved in the ageing of different organs. Studies on isoenzyme patterns of enzymes show that the genes responsible for coding of different subunits of the enzymes are sequentially expressed during the life span. Also, the decrease in the levels of enzymes seen after adulthood is reversible and can be raised to adult level by inducing their genes by steroid hormones. Another factor that contributes to the decrease in the levels of enzymes is increasing compaction of the chromatin that houses the genes as seen from digestion of chromatin by DNase I and MNase. This decreases the rate of transcription of genes. The expression of many genes declines after adulthood which is due to the decrease in trans-acting nuclear proteins that bind to specific cis-acting sequences in the promoter regions of genes. These proteins are inducible by steroid hormones. Hence the deterioration of functions that occurs after adulthood can be delayed, and the adulthood period can by prolonged by manipulation of the expression of genes.

Age-related and steroid induced changes in the histones of the quail liver.

Histones were extracted from the liver of young, adult and old Japanese quails. Both linker and core histones were found to vary with age. An extra band, H3X, was found between H2A and H4. Its level is higher in young and old birds in comparison to that of the adult. H3X shows interesting changes under steroid induction. Its level is higher in progesterone administered young birds. In adult and old birds, it is higher in progesterone treated (P) and progesterone primed estradiol administered (P+E) birds, and lower in all estradiol (E) and estradiol primed progesterone administered (E+P) birds. The relative levels of H3X in the steroid administered birds also vary with age. Such changes may influence chromatin conformation and gene expression.

Polyamines modulate phosphorylation and acetylation of non-histone chromosomal proteins of the cerebral cortex of rats of various ages.

In vitro phosphorylation and acetylation of NHC (non-histone chromosomal) proteins and their modulation by spermine and spermidine were studied using slices of cerebral cortex of female albino rats of various ages. The total DNA, histone and NHC proteins do not change significantly with age. Phosphorylation and acetylation of total and individual NHC proteins decreases with increasing age of the rat. Spermine and spermidine stimulate phosphorylation and acetylation of specific NHC proteins in immature rats. This effect decreases with increasing age. It is suggested that such modulatory effects of polyamines may cause alterations in the expression of specific genes during aging.

Age-related and sex-related alterations in beta-adrenergic receptors in different regions of rat brain.

The binding of (-)[3H]dihydroalprenolol, an antagonist of norepinephrine, to beta-adrenergic receptors in different regions of the brain of male and female rats of various ages was measured. The binding to the synaptosomal fraction of corpus striatum, hypothalamus, cerebral cortex, cerebellum and the brainstems shows a significant decrease in the binding in old rats of both sexes. Only in the female corpus striatal region, the binding in the adult and the old is the same. In the case of females, the highest binding is seen in the young. In the male, an increase in binding occurs up to adulthood, after which it declines, suggesting a definite sex-related difference in the beta-adrenergic receptor.

Effect of 17beta estradiol and progesterone on the conformation of the chromatin of the liver of female Japanese quail during aging.

Plasma levels of 17beta estradiol and progesterone were measured by radioimmunoassay in immature, adult and old female Japanese quails. The levels of progesterone and the progesterone/estradiol ratio were maximum in adult, egg laying birds. Conformation of the chromatin of the liver of birds of various ages before and after administration of steroid hormones was studied by digesting the nuclei with micrococcal nuclease (MNase) and pancreatic deoxyribonuclease I (DNase I) followed by electrophoretic resolution of the DNA fragments. The pattern of bands shows that the chromatin of the adult is more sensitive to DNase I and MNase than that of young and old birds. Administration of 17beta estradiol and progesterone enhances the digestion of the chromatin by DNase I and MNase in old birds. Such enhancement does not occur in adult birds as the chromatin is already in relaxed and open conformation due to the high levels of the hormones already present at this age.

Methylation of DNA of the brain and liver of young and old rats.

In vitro methylation of purified DNA and chromatin-DNA in nuclei of the liver and brain of young (18 week) and old (120 week) female rats has been studied using 3H-SAM as the -CH3 group donor. Incorporation of -CH3 group is higher in old liver and brain, but it is far higher in the latter. 5 mC is 11% lower in the old brain, but there is no difference in the liver. Methylation by Hpa II methylase does not show any difference in the incorporation of -CH3 group into DNA of the liver of the two ages. However, its incorporation is lower in the old brain. Methylation by Msp I methylase causes slightly higher incorporation of -CH3 groups in the old brain. This shows a higher percentage of unmethylated external cytosines in the 5'-CCGG-3' sequences. On the contrary, methylation by Eco RI methylase is considerably higher in the old brain. These studies show alterations in the methylation status of the DNA during ageing which may cause changes in the expression of genes.

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.