A DNA study of rat liver oligonucleosomes enriched by transcriptionally active genes during induction due to the administration of an amino acid mixture

A highly active fraction of rat liver oligonucleosome DNA has been isolated and studied by means of thermal denaturation after induction by amino acid mixture or hydrocortisone. A considerable redistribution of DNA content has been shown in sucrose gradient fractions during these forms of induction. The changes are revealed in melting temperature, differential melting profile of DNA, isolated from actively transcribed chromatine fractions. Analysis of melting profiles shows changes of GC content of oligonucleosome DNA, suggesting that there are differences in activation during two studied forms of induction.     

Electrophoretic study of the nuclear membrane ribonucleases of rat liver and hepatoma-27 cells]

Purified cell nuclei from the rat liver and hepatoma-27 cells were used to prepare nuclear membranes from which the enzyme-containing extracts of acid-soluble proteins were prepared. The protein extracts were subjected to disc-electrophoresis in 15% polyacrylamide gel using modified Reisfeld's system. It was found that ribonucleases contained in the acid-soluble proteins of the nuclear membranes of normal liver are represented as several components, and differed by their electrophoretic mobility and also by some other physical and chemical properties from crystalline bovine ribonuclease, as well as from nuclear chromatine ribonucleases.     

Changes in the creatine kinase activity of the brain, heart, liver and plasma of rats subjected to oxygen starvation]

The action of hypoxic and cytotoxic hypoxia on the creatine phosphokinase activity in the blood plasma, cardiac and hepatic tissue, cerebral tissue, as well as on the nuclear and mitochondrial cerebral fractions was investigated. Oxygen deficiency was followed by significant changes of the creatine kinase activity in all the tissues and cell fractions under investigation. The type of changes depended on the isoenzyme tissue perculiarities, as well as on the state of the enzyme oxidative phosphorylation systems and membrane permeability.     

Subcellular localization of UDP-glucuronyltransferase by differential centrifugation. Changes produced by pretreatment of rats with secretin, glucagon, vasoactive intestinal polypeptide and phenobarbitone

Subcellular fractionation of liver homogenates from treated rats was carried out in order to study the mechanism of action of the gastrointestinal polypeptides on glucoronidation. Rats were treated for 90 min with an intravenous infusion of secretin (0.4 cU/h/100 g body weight), glucagon (100 micrograms/h/100 g body weight) and vasoactive intestinal polypeptide (VIP) (300 ng/h/100 g body weight); controls were sham-treated rats. For comparison, another group of animals was treated with a daily injection of phenobarbitone (10 mg/kg), a well-established enzyme inducer. Treatment with the different polypeptides produced minor changes in the subcellular localization of the enzyme. The bulk of activity was always recovered in the microsomal fraction, as identified by both differential centrifugation and the enrichment in specific activity of glucose-6-phosphatase, esterase and NADPH-cytochrome c reductase. Secretin produced a specific increase of bilirubin glucuronidation, more evident in all nuclear fractions. Glucagon increased both bilirubin and p-nitrophenol glucuronidation in all subcellular fractions. VIP had a selective action on p-nitrophenol conjugation of similar extent in nuclear and microsomal fractions. The type of changes observed is suggestive of physicochemical modifications occurring into the cell, perhaps at the membrane environment of different organelles, able to modify the overall conjugation of different substrates by the cell.     

Morphologic changes in cultures of different tissues exposed to the toxins of C1. perfringens types B, C, E and F]

There were revealed morphological peculiarities of the action of C1. perfringens toxins, types B, C, D, E and F on the cultures of fibroblasts of chick embryo, amniotic cells and intestinal tissue. The toxin type B was characterized by a marked vocuolization of the cell cytoplasm; the action of the toxin of type C was expressed in the swelling of the nuclei and the lysis of the chromatine substance, the toxin of type E casued kariorhexis, and the toxin of type F--hyperchromatosis of the nuclei. All the cultures proved to be insensitive to the toxin of type D. Peculiarity of the morphological affection of the cells permitted to differentiate toxin of type B in the cultures of the fibroblasts of chick embryo, whereas the toxins of types C, E and F--in the cultures of the amniotic cells under control of the reaction of neutralization with the homologous antitoxic sera.     

Ultrastructure of hepatocytes after local cooling of the liver

Electron-microscopic and stereometric study of hepatocyte ultrastructure with local liver cooling to -30 degrees C have been performed using the method of vital fixation by rat liver perfusion. Quantitative and qualitative data obtained testify to the presence of destructive changes in hepatocytes immediately after thawing expressed in hyaloplasm lightening, formation of cavities in cytoplasm and injury of mitochondria membrane and endoplasmic reticulum integrity. Changes in nuclear structure of hepatocytes and Kupffer cells (widening of nuclear pores and channels in condensed chromatine, as well as the presence of ribosomal complexes in the nucleus and edge position of the nucleolus) may be connected with the beginning of the reparation process.     

The selective participation of brain-specific non-histone proteins of chromatin Np-3,5 during the reproduction of a defensive habit to food in edible snails]

The role of brain-specific nonhistone proteins of chromatine Np-3.5 in the processes of reproduction of elaborated defensive habit to food was studied in previously learning snails. It was found, that gamma-globulines to Np-3.5 during tens of minutes inhibited behavioural and neuronal reactions elicited by a definite conditioned stimulus--carrot juice, without changing reactions to other conditioned stimulus--apple juice. gamma-globulines to other nonhistone proteins of chromatine did not influence the reproduction of food rejection habits. It was supposed that brain-specific nonhistone proteins of chromatine Np-3.5 were selectively involved in the molecular processes providing for neurophysiological mechanisms of information extraction from the long-term memory.     

Shifts in phospholipid composition of the nuclear matrix in rat liver cells exposed to insulin

It was shown that the total content of phospholipids of rat liver nuclear matrix increased after in vivo action of insulin (2 units per 100 g of animal weight). At the same time the sphyngomyelin content was increased and the content of phosphatydilcholine and phoshpatydilinositol was decreased significantly under the hormone action. The decreasing of monophosphoinositides amount was accompanied by the increasing of triphosphoinositides content which indicates the redistribution among the phosphoinositides fractions under the hormone action and may be the result of insulin initiation of phosphoinositide pathway in nuclear membrances.     

Effect of hydrocortisone on composition of polyphosphoinositides in liver cell nuclear membrane and rat brain

The action of hydrocortisone in vivo was shown to cause changes both in the total amount of phospholipids and polyphosphoinositides of rat liver and brain nuclear membranes. The hormone increased the content of five from seven phospholipid fractions including the fraction of monophosphoinositide when acting in concentration and exposition leading to activation of biosynthetic processes. The increasing of monophosphoinositides amount was accompanied by the decreasing of triphosphoinositides content which indicated the redistribution among the phosphoinositides fractions under the steroid hormone action in both cell types.     

Discrimination of osteoarthritic and rheumatoid human synovial cells in culture by nuclear image analysis.

Rheumatoid arthritic (RA) and osteoarthritic (OA) synovial cells in culture differ in their metabolic and proliferative behaviour. To assess links between these properties and nuclear changes, we used image analysis to study chromatin texture, together with nuclear morphometry and densitometry of OA and RA cells in primary culture. Chromatin pattern at the third day (D3) was heterogeneous and granular with chromatin clumps whereas at the final stage (D11) of culture a homogeneous and finely granular chromatin texture was observed. This evolution indicates global chromatin decondensation. These characteristics were more marked for RA than for OA nuclei. At each culture time, RA nuclei could be discriminated with high confidence from OA ones from parameters evaluating the organization of the chromatine texture. Nuclear image analysis is thus a useful tool for investigating synovial cell biology.     

Effect of protein S-100 on phosphorylation of nuclear proteins of cells of rat brain and liver

The effect of acidic neurospecific protein S-100 on the phosphorylation of brain and liver nuclear proteins with 1 and 10 microM ATP was investigated. It was shown that protein S-100 increases the phosphorylation of brain nuclear proteins, while antigen D, another acidic neurospecific protein half-identical to 14-3-2 protein, inhibits this process. Ca2+ and cAMP at concentration of 10(-6) M do not affect the phosphorylation of brain nuclear proteins. In control assays the tracer 32P is presumably incorporated into high molecular weight nuclear protein fractions (Mr greater than 40000). After addition of protein S-100 the tracer is mainly incorporated into these proteins as well independently of ATP concentration (1 or 10 microM). The phosphorylation of nuclear proteins with molecular weights above 100000 is mostly increased in this case. At ATP concentration of 1 microM protein S-100 decreases histone phosphorylation 2.3 times but does not affect that of non-histone proteins. However, at 10 microM ATP the inhibitory action of this protein on histone phosphorylation is absent. The possible mechanisms of protein S-100 action on nuclear proteins phosphorylation are discussed.     

Differential suppression of progesterone receptors by progesterone in the reproductive tract of female macaques

Ovariectomized cynomolgus macaques were treated with implants of estradiol (E2) for 14 days. Some animals then received an additional implant of progesterone (P) for 7-14 more days. After treatment with either E2 alone or with E2 plus P we removed the reproductive tracts and measured nuclear and cytosolic P receptors by exchange assay. In addition we used steroid radioimmunoassays(RIA) to measure levels of E2 and P in parallel aliquots of the nuclear and cytosolic fractions. P treatment reduced the concentrations of E2 in nuclear and cytosolic fractions in the cervix, endometrium, myometrium and oviduct compared to the amounts present after 14 days of E2; these data are consistent with many reports that P treatment significantly lowers the amount of nuclear and cytosolic estrogen receptors in all of these tissues. In the oviduct, myometrium and cervix both cytosolic and nuclear P receptor levels were lowered during P action. In the endometrium, however, P treatment reduced the amount of P receptor only in the cytosolic but not the nuclear fraction. RIA determinations of the amount of P retained in nuclear fractions of the P-treated animals indicate that P levels were significantly elevated only in the nuclei obtained from endometrium. This specific increase in the retention of P by endometrial nuclei during P action is consistent with the specific retention of P receptor by endometrial nuclei. These results lead to the unexpected conclusion that the stimulatory effects of P as expressed in the maintenance of the progestational state in the primate endometrium may require higher levels of occupied nuclear P receptor than do the suppressive effects of P as expressed in oviductal atrophy, diminished cervical secretion and myometrial quieting.     

PSL, a nuclear cell-cycle associated antigen is increased during retinoic acid-induced differentiation of HL-60 cells

PSL(p55) is a nuclear 55kD antigen present in various mammalian cell systems, which has been first identified by use of human autoimmune antibodies (Barque et al. 1983, EMBO J. 2, 743). It has been shown to be associated with interphase chromatine and to be synthesized in during the S phase of the cell cycle. In this work, we have analysed the status of PSL in promyelocytic HL-60 human cells in exponential or stationary growth, or undergoing granulocytic differentiation in presence of Retinoic acid. By use of 2-dimensional electrophoresis, PSL was found to be composed of two acidic proteins designated p55A and p55B. Unexpectedly, estimated 10-20 fold higher amounts of each species were found in cells treated for 5 days with 10(-6)M Retinoic acid, than in asynchronously growing cells or resting cells. Moreover, the p55A protein was phosphorylated during the process. On the basis of these results, PSL appears to be involved in some steps of the granulocytic differentiation process.     

Effect of novobiocin on degradation and repair of the superhelical structure of nuclear DNA from rat thymocyte fractions

A study was made of the action of novobiocin on degradation and repair events in supercoiled nuclear DNA from three thymocyte fractions obtained by ficoll-paque gradient sedimentation. When added before gamma-irradiation novobiocin (1.9 mg/ml) exerted a radioprotective effect during the "second wave" of supercoiled DNA degradation. It is suggested that this effect may be due to the inhibition of DNA topoisomerase II.     

Chromatin regions,released by endogenous nucleases,are enriched in immunogenic tissue-specific proteins

Localization of immunogenic tissue-specific proteins in chromatin regions, hypersensitive to endogenous nucleases, has been studied using rabbit antibodies against rat thymus chromatin. It is shown that the first 1–2,5% of the chromatin (calculating on DNA), released by Mg²⁺-, Mn²⁺-, and Ca²⁺/Mg²⁺-dependent nuclear endonucleases are drastically enriched in tissue-specific antigenic determinants. The released chromatin fractions are found to contain a heterogeneous set of nonhistone proteins and are deficient in histones. The cleavage of nuclear DNA by endogenous acidic nuclease, independent on bivalent ions, resulted in a significantly less enrichment of the released fractions with immunogenic proteins.