The structural changes in chromatin at different levels of its organization during aging]

The data about changes in the molecular organization of the cell nucleus during aging are reviewed. Changes in DNA primary and secondary structure include deletions of some sequences, changes in base methylation pattern and the increasing number of DNA breaks. Conditions underlying poor reproducibility of the results obtained in corresponding experiments are discussed. Changes observed in the nucleosomal and supranucleosomal chromatin structure reflect either its increasing compactization or the loss the nucleosomal structure during aging. The data about the increased DNA superhelicity and topoisomerase activity in aging organisms contradict the accepted views about age-related decrease in chromatin activity. It is suggested that the gene activity in aging organisms is specifically altered rather than generally decreased. The data are presented about the ways of preventing or reverting the age-related changes in chromatin structure.     

Live-cell imaging microscopy and quantitative analysis of Ca2+-dependent effects of neurotransmitters on DNA in snail neurons

The effects of serotonin and glutamate on DNA spatial fluorescent pattern after preliminary application of nifedipine and verapamil (inhibitors of L-type of potential-regulated Ca²⁺ channels) or caffeine (ryanodine receptors agonist) or BAPTA AM (membrane-permeable Ca²⁺ buffer) were in vivo studied in L-RPl1 neurons in isolated snail CNS preparation stained with SYTO16 AM. It was found out that preliminary applied BAPTA AM, caffeine, nifedipine or verapamil suppressed the neurotransmitter-induced changes in the number and size of spots in the neuronal nucleus of edible snail. At the same time by 30–120 min after an application of neurotransmitters in the presence of nifedipine, verapamil or BAPTA AM but not caffeine a pronounced increase in the number of large spots is produced, that probably reflects an activation of DNA compactization and chromatin condensation for this period. Obtained data suggest the presence of two consequent phases of DNA transformation evoked by neurotransmitters: (1) — extracellular Ca²⁺ dependent phase of chromatin decondensation and DNA decompactization (duration: about 1–2 h) and (2) subsequent (in 30 min after application of transmitters) extracellular Ca²⁺ independent caffeine-susceptible phase within which DNA compactization, chromatin condensation and the restoration of the initial DNA state occur.     

Age-related changes in chromatin of liver cell nuclei of different ploidity.

A comparison of the Feulgen hydrolysis curves and the chromatin compactness of the liver cell nuclei of young and old rats was made. It was found that the rate of DNA depurination and chromatin compactness are higher in the liver cell nuclei of old rats, both in di-and tetraploidal cells. The effect of fixation upon the course of the hydrolysis curves is discussed.     

Age-related changes in chromatin of liver cell nuclei of different ploidity

Summary A comparsion of the Feulgen hydrolysis curves and the chromatin compactness of the liver cell nuclei of young and old rats was made. It was found that the rate of DNA depurination and chromatin compactness are higher in the liver cell nuclei of old rats, both in di-and tetraploidal cells. The effect of fixation upon the course of the hydrolysis curves is discussed.This investigation was supported by grant 474/VI Committee of Cell Pathology, Polish Academy of Sciences     

Incorporation of [2-14C orotic] acid into chromatin RNA of tissues of animals of various ages

The structure and biological activity (the level of the labelled precursor incorporation into RNA) of active and repressed chromatin of the liver and small intestine mucosa were studied in adult (6-8 months) and old (24-26 months) rats. The content of repressed chromatin fraction in both tissues is found to increase with age. In the liver of old rats the level of [14C[ orotic acid incorporation into RNA of chromatin fractions decreases, radioactivity of the acid-soluble fraction being unchanged. In the small intestine mucosa a high leve of [14C] orotic acid incorporation into chromatin RNA with ageing is due to an increase in permeability of the mucosa cells.     

Kinetic properties of 5'-nucleotidase in the adipose tissue, liver and blood of rats of different ages

The kinetic characteristics (Vmax and Km) of membrane-bound and serum 5'-nucleotidases from rats of different age groups are studied. The age-related increase of Km parameter in plasma membranes of fatty tissue and liver demonstrated a lower enzyme affinity to the substrate in ageing membranes. The specific activity of 5'-nucleotidase in blood serum of aged animals was below that in young ones, while Km value remained stable. Tissue differentiation of the catalytic properties of the enzyme and their aged-related characteristics may be to a considerable extent determined by the physicochemical state of cell membranes.     

Fine structural and cytochemical analysis of the processes of cell death of oocytes in atretic follicles in new born and prepubertal rats

The process of cell death of oocytes was studied in atretic ovarian follicles of rats aged from 1 to 28 days using light and electron microscope and cytochemical methods. These methods were TUNEL procedure for DNA breaks, active caspase-3 and lysosome-associated membrane protein 1 (LAMP-1) immunolocalizations. The structural features of the process of oocyte death are mainly characterized by the presence of abundant clear vacuoles and autophagosomes, as well as by the absence of large clumps of compact chromatin associated to the nuclear envelope and apoptotic bodies. These features are common to oocytes in all types of follicles studied. Cytochemical features consisting in positive reactions to TUNEL method, active caspase-3 and LAMP-1 immunolocalizations, are common to the cell death of oocytes in all types of follicles. Particular features of the process of cell death of oocytes are found in different types of follicles. Two morphological patterns of cell death occur in pre-follicular oocytes of the new born and in primordial follicles in 1 to 5 days old rats. One is distinguished by clear nucleoli and moderate compaction of chromatin in clumps frequently resembling meiotic bivalents. The second pattern is characterized by nucleolar condensation and by the absence of compact chromatin. The process of cell death of oocytes in antral follicles is characterized by ribonucleoprotein ribbon-like cytoplasmic structures, pseudo-segmentation, and loss of contact with granulosa cells.     

The content of various active and inactive genes in chromatin fractions with different accessibilities of DNA to polycyclic aromatic hydrocarbons

Nuclear chromatin was separated into four fractions according to solubility in low (0.2 mM MgCl2, 10 mM Tris-HCl, pH 7.6) and high (2 M NaCl) ionic strengths after digestion of nuclear DNA with nucleases. In nuclear matrix DNA the ratio of active to inactive genes was always higher than that in the original total DNA, i.e., 25 times greater in rat liver nuclei. In DNA released from the nuclei into a low ionic strength buffer the active to inactive gene ratio was lower than in total DNA (3.7 times as low in case of rat liver nuclei). The amount of carcinogens in matrix DNA exceeded that of DNA soluble in a low ionic strength buffer (3-4 times in case of rat liver nuclei and 16 times in case of hamster embryo cells). The two other fractions occupied an intermediate position between the above said fractions of DNA. The experimental results suggest that the level of carcinogen-induced modifications may be increased in active genes, including transcribed protooncogenes.     

Distribution of estradiol receptor and vitellogenin gene in chick liver chromatin fractions.

The distribution of estradiol receptor and vitellogenin gene was studied in estradiol stimulated chick liver chromatin fractions prepared by limited DNAse II digestion and MgCl2 precipitation. The receptor was found in all fractions, undigested chromatin (P1), Mg2+ insoluble chromatin (P2) and Mg2+ soluble chromatin (S2). This last fraction was rich in acidic proteins, had a high protein:DNA ratio (7.0 w/w), contained 28% of rapidly labelled RNA, 20% of the receptor, 3-5% of chromatin DNA and showed a 2 fold enrichment of vitellogenin DNA sequences over unfractionated chromatin as well as P1 and P2 DNA. On isopycnic metrizamide gradients, all chromatin fractions showed a receptor peak banding at 1.23 g/cm3, the density of nucleoproteins. Hybridization experiments showed that the DNA banding at this density in fraction S2 was enriched 4 fold in vitellogenin DNA sequences over unfractionated chromatin as well as P1 and P2 DNA. These results suggest an association of hormone receptor complex with nucleoprotein structures of an apparently active chromatin fraction.     

Action of hydrocortisone on the lipid composition of active and inactive chromatin fractions in the rat liver

A composition of phospholipids and neutral lipids of rat liver chromatin and its active and inactive fractions has been investigated. It is shown that the hydrocortisone action results in marked increase in phospholipid/neutral lipid ratio of both chromatin and its active fraction. The changes in lipid content is clearly expressed in active chromatin fraction, the lipid content of inactive fraction is not changed. It is concluded that the increase of content in certain phospholipids and simultaneous decrease of neutral lipids in chromatin promotes the hormonal activation of genome.     

Effect of emotional-pain stress on RNA and protein synthesis in rats of different age]

The intensity of RNA and protein biosynthesis is studied in different tissues as well as in active and low-active fractions of liver chromatin, when adult and old rats are subjected to emotional-painful stress during 3 days. Significant stimulation of RNA and protein biosynthesis in chromatin fractions in liver and total RNA and protein in adrenals and hypothalamus is observed.     

Species differences in the toxicity of p-chloro-o-toluidine to rats and mice. Covalent binding to hepatic macromolecules and hepatic non-parenchymal cell DNA and an investigation of effects upon the incorporation of [3H] thymidine into capillary endothelial cells

The interaction of p-[14C] chloro-o-toluidine with hepatic macromolecules of rats and mice has been investigated. At all time points after single administration the extent of binding decreased in the order protein greater than RNA greater than DNA in both species. The level of binding to mouse liver DNA was greater than that to rat liver DNA after both single and repeated administration. In vitro studies showed that mouse liver fractions catalysed the binding of p-chloro-o-toluidine to calf thymus DNA more readily than rat liver fractions. Conversely, binding to protein and RNA was more marked in the rat than in the mouse. Species differences in DNA repair rates were not observed. The results failed to demonstrate a preferential persistence of binding to mouse liver nonparenchymal cell DNA. Autoradiographic determinations did not demonstrate any effect of p-chloro-o-toluidine upon the incorporation of [3H] thymidine into subcutaneous capillary endothelial cells. The results suggest that different reactive metabolites are responsible for binding to DNA and protein, and that the pattern of reactive metabolites formed from p-chloro-o-toluidine in the mouse differs from that formed in rats.     

Effect of ethanol on the DNA-polymerase activity in the liver subcellular fractions of adult and old rats

The effect of 5% ethanol on DNA polymerase activity in nuclei, mitochondria, microsomes and cytosol of intact and regenerating liver of adult and old rats has been studied. No changes in DNA polymerase activity were detected in subcellular fractions of adult rat liver. On the contrary, the increased activity of intact liver nuclei and decreased activity of regenerating liver microsomes was observed with ageing. These age-dependent peculiarities of DNA polymerase activity in response to 5% ethanol may be related to changes in the enzyme molecules or microenvironment associated with ageing.     

DNA-protein interactions and chromosome banding

Pancreatic DNase I was used as a probe to study DNA-protein interactions in condensed and extended chromatin fractions isolated from Chinese hamster liver, and in human lymphocyte and mouse L cell metaphase chromosomes in situ. By studying the rate of digestion of chromatin DNA by DNase, we have previously shown that DNA in extended chromatin is more sensitive to DNase digestion than that in condensed chromatin. In the current investigation, we have examined whether this differential sensitivity of the chromatin fractions to DNase is due to differences in protein binding to DNA or differences in the degree of chromatin condensation. By “decondensing” the condensed chromatin and comparing its rate of digestion to that of untreated condensed and extended chromatin, it was found that differences in the degree of binding of proteins to DNA rather than the degree of condensation of the chromatin primarily determines the sensitivity of each fraction to DNase. Extraction of the various classes of chromosomal proteins, followed by DNase digestion of the residual chromatin revealed that both the histone and non-histone proteins protect the DNA in the chromatin fractions from DNase attack; however, the more tightly associated non-histones appear to be specifically responsible for the differential sensitivity of the chromatin fractions to DNase digestion. These non-histones may be more tightly associated with the DNA in condensed than in extended chromatin, thereby protecting the DNA in condensed chromatin against DNase attack to a greater extent than that in extended chromatin. When metaphase chromosomes were briefly digested with DNase in situ and subsequently stained with Feulgen reagent, incontrovertible C-banding and some G-banding was obtained. This DNaseinduced banding demonstrates that the DNA in C-band and possibly G-band regions is less accessible to DNase than that in the interband regions, and our biochemical data suggest that this differential accessibility is caused by differential DNA-protein binding such that the non-histones are more tightly coupled to the DNA in the G- and C-band regions than they are in the interbands. Differences in the binding of non-histones to DNA in different segments of the metaphase chromosome may be involved in the mechanism of G- and C-banding.