Effect of sex and bezafibrate on incorporation of blood borne palmitate into lipids of rat liver nuclei

The aim of the present study was to investigate whether lipid metabolism in the nuclei is affected by changes in the metabolism of free fatty acids in the liver. The experiments were carried out on 3 groups of rats: 1 - control-male, 2 - female, and 3 - male, treated with bezafibrate (a peroxisome proliferator). The rats received ¹⁴C-palmitic acid intravenously. Thirty min later liver samples and blood from the abdominal aorta were taken. The liver nuclei were isolated in sucrose gradient. Lipids were extracted from the nuclei and the liver homogenate and subsequently separated into the following fractions: phospholipids, mono-, di- and triacylglycerols, free fatty acids, cholesterol and cholesterol esters. The radioactivity of each fraction was counted. Furthermore, the content of free fatty acids and the fatty acid binding proteins was measured. It was found that radioactivity was present in each lipid fraction obtained from the liver homogenate and from the nuclei. In the female group, the total radioactivity of lipids in the liver homogenate was lower, whereas in the nuclei it was higher in comparison to the male group. The reduction in the radioactivity in the liver was mostly accounted for by decreased radioactivity in the fraction of triacylglycerols and phospholipids. In the nuclei, the radioactivity of the fraction of phospholipids, free fatty acids and diacylglycerols was elevated. Bezafibrate did not affect the total radioactivity of lipids in the liver and reduced it in the nuclei. In the liver, the drug increased radioactivity mostly in the fraction of phospholipids and reduced it mainly in the fraction of triacylglycerols. In the nuclei, the radioactivity of each lipid fraction examined was reduced. The content of the fraction of free fatty acids in the liver and in the nuclei in the female and in the bezafibrate-treated groups did not differ from the respective value in the control group. The content of fatty acid binding proteins in the nuclei of the female and bezafibrate-treated groups increased in parallel to the elevation in their content in the cytosol. It is concluded that the female sex hormones and bezafibrate influence the transport of selected lipids into the nuclei. The effects seem to be a consequence of the action of these factors directly on the nucleus.     

Effect of sex and bezafibrate on incorporation of blood borne palmitate into lipids of rat liver nuclei

The aim of the present study was to investigate whether lipid metabolism in the nuclei is affected by changes in the metabolism of free fatty acids in the liver. The experiments were carried out on 3 groups of rats: 1 - control-male, 2 - female, and 3 - male, treated with bezafibrate (a peroxisome proliferator). The rats received 14C-palmitic acid intravenously. Thirty min later liver samples and blood from the abdominal aorta were taken. The liver nuclei were isolated in sucrose gradient. Lipids were extracted from the nuclei and the liver homogenate and subsequently separated into the following fractions: phospholipids, mono, di- and triacylglycerols, free fatty acids, cholesterol and cholesterol esters. The radioactivity of each fraction was counted. Furthermore, the content of free fatty acids and the fatty acid binding proteins was measured. It was found that radioactivity was present in each lipid fraction obtained from the liver homogenate and from the nuclei. In the female group, the total radioactivity of lipids in the liver homogenate was lower, whereas in the nuclei it was higher in comparison to the male group. The reduction in the radioactivity in the liver was mostly accounted for by decreased radioactivity in the fraction oftriacylglycerols and phospholipids. In the nuclei, the radioactivity of the fraction of phospholipids, free fatty acids and diacylglycerols was elevated. Bezafibrate did not affect the total radioactivity of lipids in the liver and reduced it in the nuclei. In the liver, the drug increased radioactivity mostly in the fraction of phospholipids and reduced it mainly in the fraction of triacylglycerols. In the nuclei, the radioactivity of each lipid fraction examined was reduced. The content of the fraction of free fatty acids in the liver and in the nuclei in the female and in the bezafibrate-treated groups did not differ from the respective value in the control group. The content of fatty acid binding proteins in the nuclei of the female and bezafibrate-treated groups increased in parallel to the elevation in their content in the cytosol. It is concluded that the female sex hormones and bezafibrate influence the transport of selected lipids into the nuclei. The effects seem to be a consequence of the action of these factors directly on the nucleus.     

Studies on the nuclear binding of steroid hormone-receptor complex; characteristics of binding of nuclei from fetal rat liver to 3H-dexamethasone-liver cytoplasmic receptor complex

Binding of 3H-dexamethasone (Dex)-rat liver cytoplasmic receptor complex to nuclei from fetal rat livers in vitro exhibited a high-affinity and saturable nature (Kd=1.5 X 10- M, maximal binding sites=470 fmole/mg DNA), and the binding was inhibited competitively by prior injection of Dex in vivo. While binding of 3H-Dex-receptor complex to nuclei from adult rat liver was in low affinity and unsaturable, and injection of Dex prior to the sacrifice of animals did not influence the nuclear binding to 3H-Dex-receptor complex in vitro. Differential salt-extraction with KCl solution of the nuclear bound 3H-Dex receptor complex revealed the presence of salt-extractable and residual forms of bound receptors. The amount of the fraction extracted with 0.3 M KCl reached its maximum at 10 min after the start of incubation, while the 1.0 M KCl-extractable and residual fractions reached their maximum plateaus after 30 min of the incubation. Scatchard analysis revealed that the binding of the receptor complex to the 0.3M and 1.0M KCl fractions was saturable, while the residual fraction did not show any tendency of saturation under the experimental conditions employed in the present study. The results obtained in this work were compared to those which have been reported by other investigators.     

Stability of receptor complexes in the rat liver bound to glucocorticoids of different biopotencies

To examine the behavior in the receptor-acceptor system of glucocorticoids of different biopotencies, the stability of receptor complexes of dexamethasone (Dex), prednisolone (Pred) and corticosterone (Cort) in cytosols, nuclei and nuclear extracts from the rat liver was compared. Receptor complexes bound to these ligands were relatively stable at 0 degrees C, but at 25 degrees C a rapid liberation of ligands was observed. However, differences in the rate of temperature-dependent decay of these receptor complexes were obvious; the Dex receptor complex was the most stable, the receptor complex bound to Cort liberated the ligand most rapidly and the stability of the Pred-receptor complex was the intermediate of these two. The addition of molybdate and dithiothreitol stabilized the receptor complexes in cytosols but these agents accelerated the liberation of ligands from the complexes in nuclei and nuclear extracts. Among the factors examined, only bovine serum albumin decreased the rate of decay in the nuclear-bound receptor complexes. From these observations, it appears likely that different mechanisms may contribute to the dissociation of ligand from receptor complexes in cytosols, nuclei and nuclear extracts.     

Effect of extraction of histones and their reconstitution on [3H] actinomycin D binding to isolated nuclei of the root of Pinus silvestris.

The purpose of the study presented was to investigate the effect of the extraction of histones on the template activity of DNA, measured by the autoradiographically evaluated intensity of [3H]actinomycin D ([3H]AMD) binding. The study was carried out on nuclei isolated from the root meristem of Pinus silvestris. Histones were removed selectively from them and reconstituted in the nuclei deprived of these proteins. The greatest rise in radioactivity was found after the extraction of the arginine fraction and that of lysine-rich and moderately lysine-rich fractions removed together, whereas the extraction of the lysine-rich fraction does not cause such a considerable increase in radioactivity. The reconstitution of particular histone fractions induced a fall in radioactivity to the level of controls in all the cases examined. No [3H]AMD binding to the nucleolus was found. The extraction of lysine histones results in the decondensation of chromatin and their reconstitution in the formation of complexes of compact chromatin.     

Properties of androphilic proteins in cytosols of human benign prostatic hypertrophy.

Androphilic proteins in the cytosol from the human benign prostatic hypertrophy are separated into two fractions by Sephadex chromatography; void volume fraction and IgG fraction which was eluted near the site of hIgG. In the present study, properties of these two androphilic proteins were compared. Association constants of these proteins were in the order of 10(9) M-1. However, the binding capacity of the former was smaller than that of the latter. These two androphilic proteins well bound to nuclei, and the high-affinity and saturable binding to nuclei was observed in the 3H-dihydrotestosterone-IgG fraction complex, while binding of 3H-dihydrotestosterone-void volume fraction complex to nuclei was low affinity and unsaturable. The binding of the complexes to chromatin seems to be of low affinity and nonsaturable. These androphilic proteins did not bind to calf thymus DNA. Salt extractability of the bound void volume fraction after incubation with nuclei was not different from that of the bound IgG fraction. It was observed that the chromatographic behavior of the androphilic protein in IgG fraction was changed after incubation with nuclei.     

Binding of glucocorticoid-receptor complexes with the acceptor zones of nuclei and effect of glucocorticoids on RNA synthesis in hormone-sensitive and hormone-resistant cell populations

The binding of free radioactive glucocorticoid and the glucocorticoid-receptor complex to rat liver nuclei was studied in vitro. The binding is non-saturated and independent of preliminary injection of the "cold" hormone. In the course of DNA hydrolysis the amount of the radioactive hormone bound to the chromatin moiety in vivo remains practically unchanged relatively to the initial radioactivity of the protein. The liberation of the nuclei into a cell-free medium and the effect of DNAase I on the nuclei are associated with the redistribution of the hormone-receptor complex in the chromatin molecule and with the appearance of new, previously masked acceptor zones of the hormone binding. During the first 1-2 hours following the hormone injection the endogenous RNA-synthesizing activity of the nuclei is decreased. The increase of RNA synthesis in liver nuclei occurs not earlier than 3 hours after the injection. In Zajdela hepatoma nuclei the repression of RNA synthesis persists as long as 3 hours after the injection of dexamethasone. When RNA synthesis is determined in the nuclei in the presence of exogenous RNA-polymerase of E. coli in vitro, the increase in nuclear RNA synthesis can be observed beginning with the 30th min after the hormone injection. It is assumed that this effect is due to conformational changes in the chromatin structure, which are concomitant with the initial steps of association of the hormone-receptor complex.     

Removal of human polymorphonuclear leukocyte surface sialic acid inhibits reexpression (or recycling) of formyl peptide receptors. A possible explanation for its effect on formyl peptide-induced polymorphonuclear leukocyte chemotaxis

Removal of surface sialic acid specifically inhibits human polymorphonuclear leukocyte (PMN) chemotactic responses to N-formyl-methionyl-leucyl-phenylalanine (FMLP). Neuraminidase-treated (NT)-PMN bound and internalized [3H]FMLP (used as receptor marker) as well as normal PMN. NT-PMN, however, retained more [3H]FMLP-associated radioactivity than normal PMN. Subcellular fractionation studies demonstrated that NT-PMN retained more sedimentable (100,000 X G for 180 min) [3H]FMLP-associated radioactivity within light Golgi-containing fractions than normal PMN. Furthermore, NT-PMN exhibited a defect in their ability to reexpress (or recycle) a population of FMLP receptors. Abnormal receptor recycling was associated with inhibition of FMLP-induced PMN chemotaxis. Thus, it appears that recycling of formyl peptide receptors may be necessary for optimal PMN chemotactic responses to FMLP. We postulate that removal of PMN surface sialic acid inhibits FMLP-induced PMN chemotaxis by blocking the reexpression (or recycling) of a population of formyl peptide receptors, perhaps by preventing trafficking of desialated receptors through a light Golgi pathway.     

Differences in the binding of 3-methylcholanthrene and phenobarbital to rat liver cytosolic and nuclear protein fractions

The inducers of cytochrome P-450c and P-450b, 3-methylcholanthrene and phenobarbital, respectively, have been studied in their interaction with subcellular fractions from rat liver. 3-Methylcholanthrene bound to both nuclear and cytoplasmic components as demonstrated by DNA-cellulose chromatography. The binding of 3-methylcholanthrene to cytosolic proteins, on DNA-cellulose, was approximately 27 fmol/mg of applied protein, whereas the binding to nuclear proteins was 250–570 fmol/mg applied protein. Phenobarbital did not bind to proteins of rat serum, rat liver cytosol, or rat liver nuclei which could bind to DNA-cellulose. Further examination of the potential interaction of phenobarbital to rat liver cytosolic proteins was carried out using either DEAE A-50 Sephadex chromatography, charcoal dextran analysis, or sucrose density gradients. No binding of phenobarbital to rat liver cytosolic proteins was observed under these experimental conditions. In contrast, the binding of 3-methylcholanthrene to cytosolic proteins showed four peaks of radioactivity after DEAE A-50 Sephadex chromatography, two peaks by sucrose density gradient analysis, and specific binding (0.13 pmol/mg protein) was observed using the charcoal dextran technique. One of the peaks on sucrose gradients was labile in the presence of salt. The uptake and intranuclear distribution of 3-methylcholanthrene and phenobarbital were markedly different after incubation with whole nuclei: 64% of the available 3-methylcholanthrene but only 3% of the available phenobarbital radioactivity became associated with nuclei. Of this radioactivity, the highest specific activity of the 3-methylcholanthrene radioactivity was associated with the 2 m KCl-resistant nuclear pellet and the highest specific activity of the phenobarbital radioactivity was associated with the nuclear fraction soluble in the absence of salt. These results are interpreted in regard to the induction of cytochrome P-450c.     

Studies on the nuclear binding of steroid hormone-receptor complex; binding of liver and thymus dexamethasone-receptor complex and prostate dihydrotestosterone-receptor complex to nuclei from various tissues.

To examine the binding specificity of steroid hormone-cytoplasmic receptor complexes to nuclei, binding of 3H-dexamethasone (Dex)-liver, 3H-Dex-thymus and 3H-dihydrotestosterone (DHT)-prostate receptor complexes to nuclei from liver, prostate, thymus, spleen and kidney was studied. It was observed that a significant amount of steroid-receptor complexes was bound to any nuclei used in the present study and the extent of the binding of receptor complexes to nuclei from homologous tissues was not always greater than that to nuclei from heterogenous tissues. However, a significant portion of the 3H-Dex-liver and 3H-DHT-prostate receptor complexes was not absorbed by nuclei from kidney, spleem, and thymus, and the unabsorbed complexes were efficiently bound to liver and prostate nuclei. The results obtained indicate that two types of receptor complex with regard to nuclear binding were present in cytosols of liver and prostate; one binds to nuclei from kidney, spleen, thymus, liver and prostate and the other does not bind to nuclei from kidney, spleen and thymus but does bind to nuclei of liver and prostate. The latter type of receptor complex was not observed in the cytosol from the thymus.     

Direct evidence on incorporation of the receptor into the nuclei of rat ventral prostate in the form of the complex with dihydrotestosterone.

Cytosol 9S receptor was prepared from the supernatant fluid at 105,000 X g of rat prostate homogenates by a Sephadex G-100 gel chromatography, and was labeled with 131I. The 131I-labeled 9S receptor retained the activity of forming a complex with 3H-dihydrotestosterone, similarly to the intact cytosol receptor, when examined by a sucrose density gradient centrifugation. When the 131I-labeled cytosol 9S receptor was incubated with isolated prostatic nuclei in the presence of 3H-dihydrotestosterone, it was found that the 131I-labeled receptor was directly incorporated into the nuclei in a form of the complex bound to 3H-dihydrotestosterone. 131I-Labeled receptor-3H-dihydrotestosterone complex which was incorporated into the nuclei was extracted with 0.5 M KCl solution, and the nuclear complex sedimented with a velocity of 5S. The incorporation of 131I-labeled receptor-3H-dihydrotestosterone complex into the nuclei increased along with the raised temperature of incubation, whereas association of the complex with the chromatin reached maximum at 35 degrees C and then decreased gradually beyond this temperature. In the time course study, either the incorporation of the complex into the nuclei or the association with the chromatin reached the maximal levels within 10 min and leveled off up to 60 min. On the other hand, 131I-labeled serum protein was far less efficiently incorporated into the nuclei than the radioiodinated 9S receptor, and association of the serum protein with the chromatin was limited to a very little extent. The 131I-labeled 9S receptor-dihydrotestosterone complex associated with the chromatin was found to be preferably distributed into the non-histone protein as well as the DNA itself of the chromatin. The radioactivity lost by dehistonization of the chromatin was almost negligible. Furthermore, the cytosol 9S receptor fraction bound to 3H-dihydrotestosterone was purified about 100 fold by the two consecutive column chromatographies. The partially purified receptor fraction which was labeled with radioiodine incorporated mostly into non-histone protein and DNA fractions.     

Stability of receptor complexes in the rat ventral prostate and seminal vesicle bound to androgens: thermodynamic study

To examine the behaviour of the receptor-acceptor system of androgen of different biopotencies, we compared the stability of receptor complexes of dihydrotestosterone (DHT), methyltrienolone (R1881) and testosterone (Test) in cytosols, nuclei and nuclear extracts from ventral prostate and seminal vesicle of rats. Liberation of ligand from receptor complexes bound to these ligands followed the first-order kinetics. The rate constant for ligand liberation at 25 degrees C varied with the ligand. The receptor complexes bound to Test were most labile, while the receptor complexes bound to DHT were relatively stable, and intermediate stability was observed in the receptor complexes bound to R1881 under the conditions employed in the present study. Thermodynamic characteristics of the stability of the complexes were also different in these three androgens. The Arrhenius plots of the rate constant for the liberation of ligand from R1881- and DHT-receptor complexes in cytosols and nuclei showed curvilinearities, but the plots for Test-receptor complexes were almost linear. In addition, the stabilizing effect of molybdate on R1881- and DHT-receptor complexes in cytosols was observed in the range of low temperature, while the effect on Test-receptor complexes was significant at the higher temperature. The differences observed in the present study seem to be related to the difference in the biological potency of these androgens.     

Physical and functional association of follitropin receptors with cholera toxin-sensitive guanine nucleotide-binding protein

We have previously reported detergent (Triton X-100) solubilization of a follitropin (FSH) receptor-rich fraction from light membranes of bovine testis that responded to exogenous FSH by activation of adenylate cyclase (Dattatreyamurty, B., Schneyer, A., and Reichert, L. E., Jr. (1986) J. Biol. Chem. 261, 13104-13113). Upon gel filtration of the detergent-extract through Sepharose-6B, two fractions were separated. Each specifically bound [3H]guanosine 5'-imidotriphosphate (Gpp(NH)p) and had guaninetriphosphatase (GTPase) activity. Of these, one fraction (6B-Fraction-1) also bound radioiodinated human follitropin (hFSH), indicating a coelution of the nucleotide-binding protein with receptor. The other fraction (6B-Fraction-2) did not contain detectable FSH receptor activity. Several lines of evidence suggest that 6B-Fraction-1 is a complex consisting of FSH receptor and a guanine nucleotide regulatory protein, probably Ns. 1) The GTP-binding and FSH-binding activities of 6B-Fraction-1 were retained by a GTP-affinity column, and their retention by the affinity matrix could be prevented by simultaneous addition of free Gpp(NH)p. 2) When exogenous GTP was added to 6B-Fraction-1, binding of 125I-hFSH was reduced compared to controls lacking exogenous GTP. This effect of GTP was highly specific and noncompetitive, indicating that GTP did not bind to receptor. In addition, the affinity of receptor for FSH was decreased, and the rate and degree of dissociation of bound labeled FSH from receptor were increased in the presence of exogenous GTP, each in concentration-dependent manner. 3) Exposure of 6B-Fraction-1 to higher concentration of Triton X-100 reduced significantly the receptor-associated GTP-binding activity and also rendered the hormone-binding activity insensitive to GTP. 4) Treatment of highly purified testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminated the ability of GTP to modulate the 125I-hFSH binding to receptor. 5) After cholera toxin-induced [32P]ADP-ribosylation of testis membranes, a major peak of radioactivity (presumably Ns) was coeluted with FSH receptor activity from the Sepharose-6B column. These results and the observation that the effect of GTP is noncompetitive at FSH receptor level suggest that FSH binding inhibition and the increased rate of hormone dissociation from receptor were the result of GTP interaction with a guanine nucleotide regulatory protein, probably Ns, which itself was functionally associated with the FSH receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of estrogen receptor in complex with a discrete nuclear subfraction from hen oviduct.

A nuclear subfraction containing bound estrogen receptor in presumed complex with its nuclear acceptor site has been partially purified from hen oviduct. Sucrose density gradient ultracentrifugation was used to separate mechanically sheared chromatin (i.e. lysed nuclei) into several fractions which differed in protein to DNA ratio as well as in vitro template activity. Gradient fractions were then examined for the presence of bound estrogen receptors. Care was taken to use physiological ionic strength buffers when preparing nuclei since the number of estrogen receptors per nucleus decreased from 5600 to 1600 when nuclei prepared in low ionic strength (mu = 0.013 M) were compared with nuclei prepared in physiological ionic strength (mu = 0.2 M). [3H]Estradiol was introduced into nuclear estrogen receptors by exposing minced oviduct to labeled hormone in tissue culture or by exchanging nuclear estrogen receptor complexes formed in vivo with labeled hormone. In all cases, receptor was found in a fast sedimenting nuclear subfraction of low in vitro template activity. Sodium dodecyl sulfate-gel electrophoresis revealed no differences between proteins from receptor-containing and slower sedimenting fractions. Hybrdization experiments using a cDNA probe made from ovalbumin mRNA indicated no enrichment of this gene in DNA from receptor-containing nuclear material. Salt-extracted nuclear estrogen receptor was shown to partially aggregate to fast sedimenting species of heterogeneous size when sedimented in gradients containing low salt concentrations. Bound receptors were distinguished from such receptor aggregates using a novel electrophoresis technique. In addition, receptor aggregates could be disrupted in high salt, while bound receptors were resistant to this treatment. The number of exchangeable nuclear estrogen receptors in immature chicks given secondary estrogen stimulation was compared with birds that had been withdrawn from hormone. The number of receptors per nucleus was shown to be higher in animals given secondary stimulation, and these receptors were associated exclusively with fast sedimenting nuclear material.     

Transfer of retinoic acid from its complex with cellular retinoic acid-binding protein to the nucleus

Cellular retinoic acid-binding protein (CRABP), a potential mediator of retinoic acid action, enables retinoic acid to bind in a specific manner to nuclei and chromatin isolated from testes of control and vitamin A-deficient rats. The binding of retinoic acid was followed after complexing [3H]retinoic acid with CRABP purified from rat testes. The binding was specific, saturable, and temperature dependent. If CRABP charged with nonlabeled retinoic acid was included in the incubation, binding of radioactivity was diminished, whereas inclusion of free retinoic acid, or the complex of retinol with cellular retinol binding protein (CRBP) or serum retinol binding protein had no effect. Approximately 4.0 X 10(4) specific binding sites for retinoic acid were detected per nucleus from deficient animals. The number of binding sites observed was influenced by vitamin A status. Refeeding vitamin A-deficient rats (4 h) with retinoic acid lowered the amount of detectable binding sites in the nucleus. CRABP itself did not remain bound to these sites, indicating a transfer of retinoic acid from its complex with CRABP to the nuclear sites. Further, CRBP, the putative mediator of retinol action, was found to enable retinol to be bound to testicular nuclei, in an interaction similar to the binding of retinol to liver nuclei described previously.     

Binding of benzo(a)pyrene to rat liver nuclear matrix

Binding of benzo(a)pyrene (B(a)P) to nuclei isolated from rat liver was investigated. After incubation with ¹⁴C-B(a)P, the nuclei were subfractionated into an envelope fraction, two chromatin fractions and a matrix fraction. About 50% of the B(a)P that entered the nuclei was associated with the matrix fraction. Covalently bound B(a)P in the matrix fraction also exceeded that in the chromatin fractions. The radioactivity of ¹⁴C-B(a)P attained by the matrix DNA was 3–5 times higher than that attained by the chromatin DNAs. These findings suggest that the nuclear matrix is a major intranuclear binding site of B(a)P.     

Binding of glucocorticoids to liver nuclei and chromatin of fetal,immature and adult rats

The interaction of dexamethasone with nuclei and chromatin was investigated following incubation of liver slices from fetal, immature (6-day-old) and adult rats with the labeled steroid at 37°. The number of specific binding sites for dexamethasone in purified liver nuclei increases with the age of the animal in a manner similar to that previously reported for the cytoplasmic receptor. The high affinity nuclear binding approaches saturation at 40 and 500 nM dexamethasone in fetal and adult liver, respectively. In comparison with dexamethasone, the relative efficiency of corticosterone to accumulate in the nucleus is 9 percent in fetal liver and only 1 percent in adult liver.Specifically bound dexamethasone in adult nuclei exists in at least three forms; a Tris-soluble, a KCl-soluble, and a residual (non-extractable with KC1 or DNase) form. Part of the Tris-soluble steroid is associated with macromolecules sedimenting at about 4 S both in the presence and absence of 0.4 M KCl. This form of the receptor was not detected in fetal liver nuclei. In liver chromatin, bound dexamethasone exists in a KCl-soluble and a residual form, the latter comprising the major fraction of steroid associated with chromatin from both fetal and adult tissue (60 and 75 percent, respectively). Treatment with Triton X-100 releases about 20 percent of the radioactivity in adult liver nuclei, but has no effect on fetal liver nuclei.In contrast with the above observations in the intact tissue, the major fraction of steroid bound to chromatin in cell-free systems is KCl- and DNase-soluble, only 30 percent remaining in the residual pellet.     

Recycling of tumor necrosis factor-alpha receptor in MCF-7 cells

Kinetics of regulation of membrane receptors for tumor necrosis factor-alpha (TNF) in human breast adenocarcinoma MCF-7 cells was investigated. When MCF-7 cells were incubated with radioiodinated human recombinant TNF, they bound TNF specifically and accumulated it intracellularly. Preincubation of cells with native TNF up to 1 x 10(-9) M for 12 h stimulated specific binding of TNF, indicating that concentrations of membrane receptors for TNF were regulated by the ligand. Accumulation of radioactivity in cells incubated with [125I]TNF proceeded at a constant rate for up to 24 h. Kinetics of binding and internalization of TNF were similar in the presence and absence of protein synthesis for at least 1 h, suggesting that the TNF receptor was either replenished from an intracellular pool of receptors or was recycled (reutilized) during the course of TNF internalization. Data were analyzed kinetically by fitting equations of compartmental models of ligand-cell interactions with and without the term for receptor recycling. Fits were obtained only for the model with receptor recycling; absence of the term for receptor recycling resulted in physically impossible best-fit parameter values. Analysis of the best-fit parameters indicated that both internalization and recycling of the receptor were stimulated by the ligand.     

The in vivo disposition of aflatoxin B1 in rat liver

The disposition of a non-toxic i.p. dose of [3H]-aflatoxin B1 (0.70 micrograms/kg) in the blood, plasma, and liver was studied in male Wistar rats. Uptake into the blood, plasma, and liver was biphasic; there was an initial rapid rise (0-2 hr) followed by a second phase (2-12 hr) of a gradual increase. Most of the radioactivity in the blood was bound noncovalently to albumin. Distribution of radioactivity in the subcellular fractions of liver showed that the microsomes exhibited the highest labeling which increased over the time course; labeling of the cytosol reached a maximum at 2 hr then decreased to a new steady state, whereas the mitochondria and nuclei reached a plateau. When the content of aflatoxin B1 in the nuclear subfractions was examined, greater than 92% of the total radioactivity was found in the deoxyribonucleoprotein fraction, and 84% of this was bound noncovalently. These results suggest that aflatoxin B1 is transported from the site of injection through the blood to the liver and its subcellular and subnuclear fractions primarily in a noncovalent form.     

Covalent binding of benzo[a]pyrene metabolites to DNA, RNA and chromatin proteins in the AKR mouse embryo cell-line

A specific fraction from the nuclei of the AKR mouse embryo cell-line (fraction I) displayed a much greater localization of radioactivity compared to fraction II and III when the chemical carcinogen, [3H]benzo[a]pyrene (B[a]P) was incubated with the cells for 24 h. The radioactivity in fraction I consisted of both covalently and non-covalently bound metabolites. Isolation of the DNA, RNA and protein of fraction I revealed that 94% of the covalently bound radioactivity was to protein, 5% to RNA and 1% to DNA. Analysis of the fraction I proteins by SDS gel electrophoresis revealed that there was more radioactivity covalently bound to the larger proteins than to smaller proteins. Isoelectric focusing (IEF) of the purified proteins displayed two peaks of radioactivity, one at a pH of 5 and the other at 11. The former proteins bound more radioactivity per mass of protein than the latter proteins. Analysis of fraction I histones on acid urea polyacrylamide gels showed that the radioactivity coincided with histones H3 and H2B and low levels of radioactivity associated with histones H1, H2A and H4. Two significant peaks of radioactivity closely migrated near but did not co-migrate with histone H1. The distribution of the bound radioactivity is probably a reflection of the availability of the proteins to the reactive carcinogen metabolites. The possible binding of B[a]P metabolites to phosphorylated histones and to the high mobility of group (HMG) proteins 1 and 2 is discussed.