Neutral lipids of nuclear membranes and chromatin in rat liver after hydrocortisone administration

It is shown that over 80% of nuclear neutral lipids are found in the nuclear membrane preparations, whereas only 7%--in chromatin. The injection of hydrocortisone to animals significantly decreases the neutral lipids content in both fractions. These results and the data obtained previously by the authors indicate the possible participation of nuclear membrane and chromatin lipids in the process of hormonal regulation of the genetic activity.     

Changes in rat liver chromatin after administration of a mixture of amino acids

It was shown that injections of an amino acid mixture essentially increase the number of nuclease-sensitive regions of chromatin and its active fraction (Mg2+-soluble fraction), while hydrocortisone increases the amount of the latter, which is less sensitive to the effect of DNAase II. Genome activation during nonhormonal induction after administration of the amino acid mixture or during hydrocortisone injection reflects in different ways on the parameters of melting of chromatin active fractions and on the relative content of its protein fractions.     

Conformational changes in rat liver chromatin after liver regeneration.

N-Pyrenemaleimide, a fluorescent probe that specifically labels histone H3 of rat liver chromatin in situ, was used to monitor the accessibility of histone H3 in chromatin isolated from rat liver at different times during degeneration. At times of maximum DNA synthesis (18--24 h after hepatectomy), the accessibility of the probe was found to be markedly (40--50%) increased. This increase is abolished, however, by treatment of the chromatin fibres with high salt (2 M-NaCl) or detergent. Tryptophan fluorescence was also enhanced at points of maximum DNA synthesis, suggesting that some non-histone tryptophan-containing protein was being synthesized. The polarization of the labelled histone H3 is not markedly altered, suggesting that fibre aggregation or dissociation does not occur. Mononucleosomes extracted from sham-operated and hepatectomized animals did not exhibit any difference in binding to the probe. Also, analysis of the chromatin protein by electrophoresis on detergent- and acid/urea/ Triton-X-100-containing polyacrylamide gels showed no detectable difference in histone H3 : 1, H3 : 2 or H3 : 3 subclasses.     

Isolation of chromatin fragments rich in non-histone protein after endonuclease digestion of rat liver nuclei

Detergent-washed rat liver nuclei, prepared in the presence of a protease inhibitor, were incubated for up to 60 min at 37 °C. The action of endonucleases produced chromatin fragments which could be removed from the nuclei by extraction with 8 M urea 50 mM phosphate, pH 7.6, 15% of the total nuclear DNA being extracted. No DNA could be detected in this extract after incubation of nuclei at 4 °C. The chromatin fragments were sedimented by centrifugation at 90000 g or by chromatography on Sepharose 4B. The DNA fragment sizes were similar to those found in nucleosome particles but the protein/DNA ratio was approx. 5.3:1. The nuclei were prelabelled with [³H]tryptophan and 60% of the label present in the 8 M urea extract was found to sediment with the chromatin fraction. SDS polyacrylamide gel electrophoresis of the latter showed the presence, in addition to histones, of at least 25 polypeptide species of tightly bound non-histone proteins with molecular weights in excess of 30000.     

Ribonuclease activity of rat liver perchloric acid-soluble protein, a potent inhibitor of protein synthesis.

Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not known. Here we show that the protein is a unique ribonuclease and that this activity is responsible for the inhibition of translation. The addition of perchloric acid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 microM led to an almost complete inhibition of protein synthesis. The kinetics are unlike those of hemin-controlled inhibitor, a protein that acts at the initiation step. The inhibition appears to be due to an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80 S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by thin-layer chromatography of [alpha-32P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleoside 3'-monophosphates Ap, Gp, Up, and Cp, providing direct evidence that the protein is an endoribonuclease. When either 5'- or 3'-32P-labeled 5 S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the single-stranded regions of the molecule.     

Effect of triiodothyronine on rat liver chromatin protein kinase.

1. Injection of triiodothyronine to rats stimulates protein kinase activity in liver chromatin nonhistone proteins. A significant increase was found after two daily injections. A 4-fold increase was observed with the purified enzyme after eight daily injections of the hormone. No variations were observed in cytosol protein kinase activity. Electrophoretic pattern, effect of heat denaturation, effect of p-hydroxymercuribenzoate seem to indicate that the enzyme present in treated rats is not identical to the enzyme in control animals, which suggests that thyroid hormone has induced nuclear protein kinase. Diiodothyronine, 3, 3', 5'-triiodothyronine have no effect on protein kinase. 2. Chromatin non-histone proteins isolated from rats injected with triiodothyronine incorporated more 32P when incubated with [gamma-32P]ATP than the chromatin proteins from untreated rats. Thyroidectomy reduced the in vitro 32P incorporation. It is suggested that some of the biological activity of thyroid hormone could be mediated through its effect on chromatin non-histone proteins.     

Reverse sphingomyelin-synthase in rat liver chromatin

The chromatin phospholipid fraction is enriched in sphingomyelin content which changes during cell maturation and proliferation. Recently, we have demonstrated that the sphingomyelin variations can be due to chromatin neutral sphingomyelinase and sphingomyelin-synthase activities which differ in pH and K(m) optima from those present in nuclear membranes. The sphingomyelin can be used also as a source of phosphorylcholine for phosphatidylcholine synthesis by reverse sphingomyelin-synthase. In the present work we have studied the possible existence of reverse sphingomyelin-synthase activity in nuclear membrane and chromatin. A very low activity was detected in the homogenate, cytosol and nuclear membrane (0.93+/-0.14, 2.61+/-0.33 and 0.87+/-0.13 pmol/mg protein/min, respectively), whereas the activity present in chromatin was 37.09+/-2.05 pmol/mg protein/min. The reverse sphingomyelin-synthase decreases the intranuclear diacylglycerol pool and increases the intranuclear ceramide pool, whereas sphingomyelin-synthase has an opposite effect. The possible correlation between these enzymes is discussed.     

Effects of cortisone on regenerating rat liver

The effects of continuous administration of cortisone on the metabolism of regenerating rat liver have been studied. Whereas the restoration of the weight of the liver after partial hepatectomy was not markedly affected by cortisone, the multiplication of cells was reduced to a significant degree after the first 2 days of regeneration. Liver restoration in terms of nucleic acids was similarly inhibited by cortisone. The results are consistent with the interpretation that the inhibition of cell multiplication in this system is dependent on and keeps pace with the inhibition of nucleic acid synthesis by this drug. At almost any time after hepatectomy, the nucleic acid content of the liver cells was the same in treated and in untreated animals. In ancillary studies, it was shown that cortisone caused the cells of regenerating liver to be increased in size and weight through the increased infiltration of lipids. Changes in water, protein, and carbohydrate content of the liver cells did not contribute to this increase in the weight of the cells. Since all animals were treated with cortisone for 5 days before hepatectomy, data were also obtained on the effect of this agent on the resting liver. This course of treatment brought about a significant decrease in the number of cells per unit wet weight and in the water content of the livers. The nucleic acid content of the cells at hepatectomy, on the other hand, was unchanged.     

Properties of activated chromatin from rat liver shortly after partial hepatectomy

The properties of rat liver chromatin 1, 2 and 6 hours after partial hepatectomy have been studied by means of cytochemical and biochemical methods. An increase in the accessibility of DNA to low molecular weight ligands, RNA--polymerase and RNAse I and also of the distances between nucleosomes and their heterogeneity in length on electron -- microscopic photographs has been found. Analysis of the isotherms of adsorption has revealed an increase in the number of binding sites for ethidium bromide on DNA and accordingly a decrease in the extent of the filling of the template with protein in activated chromatin. Two hours after partial hepatectomy rat liver chromatin does not differ in all parameters studied from control chromatin. Limited digestion of chromatin with DNAse I almost fully eliminates the difference between the fractions of activated and control chromatin in the number of binding sites for the ligands to the fractions resistent in these conditions to nuclease. A suggestion that the changes in the properties of chromatin upon activation are due to the change in the character of chromatin proteins interaction with DNA are discussed.