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.

Subtractive differential display: a modified differential display technique for isolating differentially expressed genes.

Differential display (DD) is a novel PCR-based technique, very commonly used to study differentially expressed genes at the mRNA level. In this paper we report a modified version of this technique that we have used to study the differences between the mRNA population from brain tissue of adult and old rats. We have modified the technique to enhance reproducibility and reduce false positives and redundancy. It is fast and does not require any expensive or uncommon reagent. We choose to call it as subtractive differential display as it is a differential display performed over subtracted mRNA population. We have used this protocol successfully to clone a number of age-related differentially expressed sequences from rat brain that need to be sequenced to establish the gene identity.

Expression of D2 dopamine receptor in the mouse brain.

The neurotransmitter, dopamine, binds to dopamine receptor (DR), and is involved in several functions of the brain, such as initiation and execution of movement, emotion, prolactin secretion, etc. Of all the five DRs, D2 dopamine receptor has maximal affinity for dopamine. D2 has a short isoform, D2S, and a long isoform D2L. D2L is longer than D2S by 29 amino acid residues. We studied the expression of the gene and protein of D2 receptor in the cerebral and cerebellar cortices of the brain of new born, developing, adult, and old male mice to find out: (i) at what stage of development, expression of the gene peaks and (ii) if it undergoes any changes as the animal ages, which may account for the neurodegenerative changes and symptoms of Parkinson's and other diseases seen in old age. RT-PCR and Western blot studies show that peak expression of D2 gene occurs in the cerebral and cerebellar cortices around 15-day after birth. We speculate that the majority of dopaminergic synapses are established and possibly become functional in the brain around 15-day after birth. The expression of D2 receptor is upregulated in the cerebral cortex in old mice. However, it is down-regulated in the cerebellar cortex.

Expression of muscle creatine kinase gene of mice and interaction of nuclear proteins with MEF-2, E boxes and A/T-rich elements during aging.

The changes in the expression of muscle creatine kinase (MCK) gene in the heart and skeletal muscle of mice during aging were studied. Its expression declines as a function of age in the heart, however, no age-related change is observed in the skeletal muscle. The cis-acting elements, MEF-2, E boxes and A/T rich elements present in the enhancer region of the mouse MCK gene are known to regulate the expression of the gene. Hence, these elements were subcloned and electrophoretic mobility shift assay was carried out to investigate the changes in the binding of the nuclear trans-acting protein factors of the heart with these elements as a function of age. These factors showed specificity for the respective cis-acting elements. Furthermore, the binding of these factors was found to decrease during aging which may contribute to the age-related decline in the expression of the MCK gene and activity of the heart.

Interaction of nuclear proteins with repeat sequences in the 5' flanking region of mouse muscle creatine kinase gene during aging.

The 5' flanking region of the mouse muscle creatine kinase (MCK) gene contains two repeat sequences-a mononucleotide repeat, (A)(22) (-2694 to -2673), and a tetranucleotide repeat, (GTTT)(8) (-2962 to -2931). We show here that these repeats in the mouse MCK gene bind to specific nuclear protein factors. Some of the factors interacting with these sequences are tissue-specific and show age-related decrease in the binding activity. Nonspecific competitor and heterologous DNA probes failed to compete out the complexes showing that the interaction is specific to the repeat sequences. These proteins may have a role in the expression of the gene during aging.

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.

Trans-acting factors that interact with the proximal promoter sequences of ovalbumin gene are tissue-specific and age-related.

Aging or deterioration of functions of various organs occurs after adulthood due to alterations in the expression of genes that maintain adulthood functions. Molecular changes that occur in regulation of genes in higher organisms after adulthood can be understood by using genes whose timing and rate of expression run parallel with specific functions during the life span. One such function is egg formation in birds which is dependent on the expression of egg protein genes, ovalbumin, lysozyme, vitellogenin, etc. Trans-acting factors are known to play a pivotal role in the regulation of transcription of genes. We show here a decrease in the level/binding of nuclear trans-acting factors with specific cis-acting elements of the promoter of the ovalbumin gene of oviduct of old Japanese quail. This parallels the decrease in the expression of the ovalbumin gene in the oviduct after adulthood. These factors are tissue-specific and bind in a cooperative and sequential manner to the ovalbumin gene promoter.

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.

Activation of vitellogenin II gene expression by steroid hormones in the old Japanese quail.

Alterations in the basal transcription rates of eukaryotic genes are believed to involve the binding of trans-acting factor(s) with specific DNA sequences in the promoter. We show here two interrelated events for the VTGII gene of the old, non-egg laying Japanese quail: alterations in the structure of the chromatin encompassing the gene, and binding of trans-acting factors to the promoter of the gene. Estradiol/progesterone alone or together cause alterations in the conformation of the chromatin of the promoter region of the gene. This may allow free access of nuclear protein(s) to the cis-acting elements, ERE, PRE and NF1, in the promoter of the gene and cause activation of transcription.

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 changes in the ovalbumin gene of the Japanese quail.

Northern hybridization studies showed that the level of ovalbumin mRNA decreases in the oviduct of the Japanese quail after adulthood. In order to find out if this is due to changes in the conformation of the chromatin containing the promoter region of the gene, nuclei of the oviduct of young, adult and old birds were digested by DNaseI and micrococcal nuclease (MNase). Southern hybridization with the labelled promoter showed that this region is less sensitive to the two enzymes in the old birds. Both the endonucleases recognized the same hypersensitive sites. The results indicate that the chromatin containing the promoter is present in an open conformation in adult birds. Gel mobility shift assay using a 20-mer dsDNA containing the CAAT-box and nuclear extract of oviduct shows the presence of trans-acting factors that bind to this region. The levels of these factors are lower in old birds. This may be the reason for the lower expression of the gene in old birds.

Interactions of trans-acting factor(s) with the estradiol response element and nuclear factor 1 of the vitellogenin II gene of Japanese quail.

This study was directed at achieving an understanding of the mechanisms by which steroid hormones control the synthesis of vitellogenin (VTG) protein in the liver of the Japanese quail. Northern hybridization shows that administration of estradiol alone or with progesterone stimulates the synthesis of VTG mRNA. Gel mobility shift assay of DNA fragments containing the ERE and NF 1 shows that estradiol alone or with progesterone increases the levels of nuclear proteins that bind to these cis-acting elements of the promoter of the VTG gene. The cooperative effect of the two hormones seen at the level of expression of the VTG gene may be due to protein-protein interactions of trans-acting factors that bind to ERE and NF 1.

Modulation of vitellogenin II gene by estradiol and progesterone in the Japanese quail.

Estrogen and progesterone receptors are reported to functionally cooperate in gene activation if their cognate binding sites are close to one another in the gene. Our studies show that the expression of the vitellogenin (VTG) gene is induced by estradiol alone or along with progesterone. Progesterone alone inhibits the expression completely. Methylation status of the VTG gene remains unaltered by steroid hormones. Gel mobility shift assay shows that qualitative and quantitative changes occur in the trans-acting factor(s) that bind to estradiol and progesterone responsive elements (ERE and PRE) after administration of these steroid hormones. We, therefore, conclude that the interaction of trans-acting factors that bind to ERE and PRE play a role in the regulation of VTG gene expression.

Alterations in histones of the liver and oviduct of Japanese quail during aging.

Age-related changes occur in histones of the liver and oviduct of the female quail. In the liver an extra histone band, named HCX, between H2A and H4, is observed that increases with age. In the oviduct, a large complex of histones is seen which is tissue-specific. This complex declines with increasing age. The changes in the histones of the oviduct of adult and old birds in response to estradiol and progesterone are age-related. In the adult, the histone-complex increases after administration of either one of the hormones. In old birds, however, it is seen only after progesterone administration. Thus, the alterations in histones in the birds are not only tissue- and age-related, but also vary in response to steroid hormones.

Changes in expression and CRE binding proteins of the fibronectin gene during aging of the rat.

The level of plasma fibronectin (pFNT) which is synthesized in the liver and secreted to the plasma has been found to decrease with age. Nuclear run-on transcription, slot-blot, and northern blot analysis also show that the expression of the FNT gene is lower in the liver of old rats. cAMP is known to influence the expression of the gene. Gel mobility shift assay using an oligonucleotide containing the cAMP responsive element (CRE) and nuclear extract of liver shows the presence of trans-acting factors that bind to CRE. These factors change with age. This may be the reason for the lower expression of the gene in the old rat.

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.

DNase I hypersensitive sites of the 5' region of the fibronectin gene of the liver of the rat.

We have mapped the DNase I hypersensitive sites (DH-sites) of the 5' region of the fibronectin (FNT) gene of the liver of the rat. These sites are not detectable in the cerebral hemisphere of the rat. Northern blot hybridization of the gene shows the presence of a 8 kb transcript in the liver which is induced by dexamethasone. Nuclear run on transcription shows that transcription of the FNT gene is much lower than that of the albumin gene in the liver. Gel mobility shift assay using nuclear extract of the liver shows the presence of a trans-acting factor which binds to the cAMP responsive element which overlaps with a DH-site in the region. There is thus good correlation between DH-sites of the FNT gene and its transcription in the liver.

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.

Conformation and expression of the albumin gene of young and old rats.

The conformation and expression of the albumin gene in the liver of young (21-) and old (85-week) rats were studied. Digestion of nuclei with MNase shows no differences in the nucleosomal organization in the coding region of the gene in the two ages. The gene has a DNase I hypersensitive site which is distinctly less sensitive in old rats. Its 5'-CCGG-3' sequences are more methylated in the old in which its rate of transcription is also lower.

Expression of actin and myosin heavy chain genes in skeletal, cardiac and uterine muscles of young and old rats.

The steady-state levels of mRNA and transcription of alpha-skeletal actin (alpha-SKA) and adult myosin heavy chain (MHC) genes were measured in the skeletal, cardiac and uterine muscles of young (22-25 week) and old (123-135 week) female rats. The effects of 10(-8) M 17 beta-estradiol/dexamethasone/T3 alpha on their transcription were also studied. The data show that the alpha-SKA mRNA level is lower in the old skeletal muscle and uterus, but is higher in the old myocardium. The adult MHC mRNA level is not different in the three muscles of both the ages. The transcription of alpha-SKA gene is lower in the skeletal muscle and higher in the uterus of old rats. It is unaltered in the myocardium of old rats. The transcription of adult MHC gene is lower in the old uterus. The effects of hormones on transcription of both the genes are different in the three muscles. We show that the expression of alpha-SKA gene is tissue-specific and age-related. The over-expression of alpha-SKA gene in the old myocardium is possibly due to derepression of the gene caused by hypertrophy of cardiac myocytes, and continuous hemodynamic pressure overload on the old heart.