The binding of 3H-labelled oestradiol- and progesterone-receptor complexes to hypothalamic chromatin of male and female sheep.

Chromatin isolated from hypothalamic nuclei of sexually mature entire male and female sheep was linked to cellulose in u.v. light. The saturation binding of 3H-labelled oestrogen- and progesterone-receptor complexes, prepared by (NH4)2SO4 precipitation from the 105000g supernatant of hypothalamic cytosol, was then measured in vitro in 0.15m-KCl. Saturation binding was also measured after extraction of histones and masking acidic proteins. Salt + urea was observed to be more effective than guanidine hydrochloride in unmasking receptor acceptor sites, and the binding of labelled receptor complexes to dehistonized unmasked chromatin was shown to be largely resistant to 0.4m-KCl extraction. Whereas extents of receptor-complex binding were similar to published values for comparable preparations of hen oviduct chromatin, no sex-related difference was observed. However, binding of progesterone-receptor to chromatin was greater than that of oestradiol-receptor. Binding also increased more after removal of histones and masking acidic proteins, suggesting the presence of a greater number of progesterone-receptor acceptor sites in hypothalamic chromatin than of estradiol-receptor acceptor sites. The failure to demonstrate a sex-related difference in oestradiol-receptor binding to hypothalamic chromatin in vitro is discussed.     

The binding of [3H]oestradiol-receptor complex to hypothalamic chromatin of male and female mice

Histones and masking acidic proteins were removed from hypothalamic chromatin in order to evaluate/measure the number of available acceptor sites for the [3H]oestradiol-receptor complex. This number increases after dehistonizing and unmasking and is lower than published values for comparable preparations. No sex-related difference in [3H]oestradiol-receptor binding to hypothalamic chromatin in vitro was observed. Failure to observe such a difference suggests that sexual differentiation and steroid sensitivity cannot be attributed to marked differences in the degree of chromatin masking.     

Effect of sodium molybdate on the binding of androgen-receptor complexes to germ cell and sertoli cell chromatin

This study explores the effect of molybdate on androgen receptors in relation to chromatin binding. Two fractions of testicular and prostatic cytosol were obtained by ammonium sulfate precipitation at 15–37% (I) and 37–47% (II) saturation. Both fractions from either tissue exhibited specific binding of [³H]-androgens which was thermolabile. With the exception of testicular fraction II, all fractions bound to Sertoli cell and germ cell chromatin.Prostatic [³H]-androgen-receptor complexes isolated from cytosols in the presence of 10 mM sodium molybdate showed a decreased ability (only 20–40%) to bind to either Sertoli cell or germ cell chromatin. Similarly, testicular [³H]-androgen-receptor complexes isolated in the presence of molybdate bound less well (60–70%) to Sertoli cell chromatin. On the other hand, inclusion of molybdate subsequent to ammonium sulfate precipitation did not significantly alter the binding ability of the receptor complexes to either chromatin. Also, the presence of molybdate during the receptor-chromatin interaction did not significantly decrease the ability of either prostatic or testicular androgen-receptor complexes to bind to Sertoli cell chromatin. These results indicate that the addition of molybdate prior to ammonium sulfate precipitation may impede the activation of androgen-receptor complexes by salt, resulting in a decreased ability of the steroid-receptor complexes to bind to chromatin acceptor sites. The data also indicate that molybdate exerts its inhibitory effect directly on the nonactivated receptor complexes and not on the chromatin acceptor sites.     

The binding of [3H]oestradiol-receptor complexes to calf uterine chromatin.

Various aspects of the interaction of oestrogen-receptor complexes with calf uterine chromatin covalently coupled to cellulose were analysed. Partially purified [3H]oestradiol-receptor complexes were bound to intact, or partially deproteinized, chromatin resins. Proteins were removed from the chromatin-cellulose resins by extraction with high molarities of salt, including NaCl/urea, guanidine hydrochloride and guanidine thiocyanate. After extensive washing to remove the salt, [3H]oestradiol-receptor-complex solutions were added to the resins and the degree of binding was determined. The extent of [3H]oestradiol-receptor-complex binding to chromatin was enhanced by extraction of chromosomal proteins. By varying the molarity of the salt, and consequently the extent of protein removal, it was possible to resolve [3H]oestradiol-receptor-complex binding to guanidine thiocyanate-extracted chromatin into two components. Similarly, [3H]oestradiol-receptor-complex binding to guanidine hydrochloride-treated chromatin included three regions of enhanced binding capacity. The [3H]oestradiol-receptor-chromatin interaction was saturable with respect to both intact and salt-extracted resins. Thus uterine chromatin may contain three or more specific classes of acceptors for the oestrogen-receptor complex.     

The effects of sex and neonatal androgenization on neuron dendroarchitectonics in amygdala posterior cortical nucleus

Sex differences in neuron dendroarchitectonics of the amygdala posterior cortical nucleus of adult rats were described for the first time using the Golgi method. Long-axon sparse-branched neurons in male rats possessed a larger number of primary dendrites, while female rats had long-axon dense-branched neurons with longer dendrites. Injection of testosterone propionate at 1250 µg to females on day 5 after birth resulted in a greater number of primary dendrites of long-axon sparse branched neurons in adults, as compared to that in the control. Dendrites of long-axon sparse-branched neurons became much longer, thus enlarging the dendrite area.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 64–67.Original Russian Text Copyright © 2005 by Akhmadeev, Kalimullina.     

Characteristics of the androgenization produced in mice by neonatal exposure to alpha-fetoprotein antibodies

Neonatal male and female mice, ages 1-5 days, were injected intracranially with either anti-alpha-fetoprotein (AFP) IgG, steroids, or various control solutions. The mice were autopsied 60-70 days later and the spleen, thymus, liver and gonads were examined by light microscopy. The antibody to AFP produced a gonadal response which was sexual specific. Histological examination of the tissue sections from female mice treated with either anti-AFP IgG or steroids revealed the presence of polyfollicular ovaries lacking in corpora lutea formation. A comparable antibody-induced specific effect on the testes could not be demonstrated; however, steroidal administration induced aspermatogenesis and delayed maturation in parallel studies. In the anti-AFP IgG-treated groups, there appeared a gross neurological lesion which was termed external hydrocephaly. The physiologic responses in the female gonad were found independent of the presence of this anatomical brain lesion, whereas those in the male gonad were found causally related. The immunological specificity of the gonadal response was demonstrated by the use of IgG devoid of anti-AFP IgG and by various IgG control solutions. Thus, exposure to anti-AFP IgG during the critical period of sexual development in the brain appears to mimic steroidal androgenization of the female mouse.     

Testicular cytosol factors inhibit the binding of steroid-receptor complexes to chromatin

Testicular and prostatic androgen-receptor complexes as well as uterine estradiol-receptor complexes, partially purified by ammonium sulfate precipitation (15-37%), were bound to germ cell chromatin. At equivalent concentrations, less testicular androgen-receptor complexes bound to chromatin than did the other two steroid-receptor complexes. Addition of a partially purified testicular androgen-receptor preparation with prostatic androgen-receptor or uterine estradiol-receptor preparation to the binding interaction mixture reduced the binding of either of the latter two steroid-receptor complexes to chromatin. These data suggest the presence of inhibitory factor(s) in the testicular receptor preparations. Testicular cytosols were fractionated by ammonium sulfate precipitation into fractions A (15-37% saturation), B (37-50%) and C (50-75%). All fractions inhibit binding of these steroid-receptor complexes to chromatin. Fractions A and B appear to be heat labile, while fraction C was more stable. Further fractionation of A and C fractions on DEAE cellulose yielded A1 and C1 (filtrates) as well as A2 and C2 (0.3 M NaCl eluents), respectively. Subfractions A1, A2, and C2 contained inhibitory factors for the binding of steroid-receptor complexes to chromatin while C1 showed no effect. These data demonstrated that testicular cytosol contains a variety of inhibitory factors which affect the binding of both androgen-receptor and estradiol-receptor complexes to chromatin.     

The specific binding of androgens and the subsequent distribution of androgen-receptor complexes within MCF-7 human breast cancer cells

Using a whole cell suspension assay technique, we have examined both the specific binding of testosterone (T) and dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) (DHT) and the subsequent distribution of androgen binding activity within MCF-7 human breast cancer cells. We have observed that both androgens are bound with high affinity to the same single class of receptor which is present at a concentration of approximately 8,000 sites per cell. The incubation of cells with either T or DHT was followed by the nuclear accumulation of specifically bound ligand which increased to maximal values within 1 h and then decreased thereafter. However, only about 30% of the total cellular specific binding activity observed with either androgen was localized within the nuclear compartment at any time during a 4 h incubation. Further examination of the extranuclear binding component suggested that a substantial portion of this activity was localized to the particulate fraction of the cytoplasm. The results of these studies suggest that both T and DHT are capable of exerting biological activity within MCF-7 cells, and raise the interesting possibility that androgen-receptor complexes may participate in the direct regulation of mitochondrial and/or microsormal function in this system.     

The effect of melanotropic peptides on binding of [(3)H]dihydroalprenolol-hydrochloride to hypothalamic membranes

In the present work we studied the interaction of alpha-melanocyte-stimulating hormone (alpha-MSH) and ACTH-(1-24) with beta-adrenergic receptors in hypothalamic membranes from rat brain. Saturation curves for [(3)H]dihydroalprenolol-hydrochloride ([(3)H]DHA) binding in the presence of the peptides revealed a decreased binding capacity (Bmax). The dissociation constant (Kd) was, however, not affected by alpha-MSH or ACTH-(1-24). These data indicate a non competitive interaction between these melanocortin peptides and [(3)H]DHA on beta-adrenergic receptors in hypothalamic membranes.     

The state transitions of normal and mutant androgen-receptor complexes in human genital skin fibroblasts

We have incubated cells from controls and subjects with receptor-defective androgen resistance with 3H-labelled testosterone (T), methyltrienolone (MT), dihydrotestosterone (DHT) or mibolerone (MB) and studied the temperature dependence of the dissociation rate constants of these various androgen-receptor (A-R) complexes both within cells and after they were extracted from them. In control cells, Arrhenius plots for T-, MT-, DHT- and MB-R complexes were linear and formed a hierarchy of dissociation states with energies of state IV greater than III greater than II, greater than I, respectively. Relative to this hierarchy, the dissociation states of the MB-, DHT- and MT-R complexes in mutant cells were displaced to higher, androgen-inappropriate energies in a mutant-distinctive pattern. When extracted from cells control or mutant T- or MT-R complexes, and mutant (but not control) DHT- or MB-R complexes lowered their respective dissociation rates by undergoing state transitions in conformity with the hierarchy. Hence we propose that different A-R complexes reach different dissociative states by undergoing sequential transitions along a common pathway, and that these transitions are co-regulated both by the chemical characteristics of the bound androgen and by other cellular non-receptor factors.     

The effect of phospholipases and proteases on the binding of γ-aminobutyric acid to junctional complexes of rat cerebellum

A preparation enriched in junctional complexes, as judged by marker enzymes and electron microscopy, was prepared from rat cerebellum. The junctional complexes were incubated with γ-amino[¹⁴C]butyric acid at 25°C for 10 min, using [³H]sucrose as a marker for entrapped space. Total binding was determined in the absence of, and non-specific binding in the presence of, an excess of unlabelled γ-aminobutyric acid. The difference between the two binding values, i.e. the specific binding, was saturable and reversible, and showed positive cooperativity with a Hill number of about 2. The specific binding was inhibited by N-methylbicuculline, picrotoxinine and imidazole-4-acetic acid, but not by curare, strychnine or l-2,4-diaminobutyric acid. The above compounds had little effect on the non-specific binding, but addition of ethylenediaminetetraacetic acid decreased non-specific binding by 80%. Trypsin, pronase, phospholipase A₂ (EC 3.1.1.4), lysolecithin and sodium dodecyl sulfate decreased binding. Phospholipase C (EC 3.1.4.3) increased the specific binding by 260%. Phospholipids competed with γ-aminobutyric acid for binding, with phosphatidylethanolamine being more potent than phosphatidylcholine. These results lend support for Watkins' hypothesis that phosphatidylethanolamine competes with γ-aminobutyric acid for binding to the receptor protein.     

The nucleosome binding protein HMGN1 interacts with PCNA and facilitates its binding to chromatin

Proliferating cell nuclear antigen (PCNA) is a ubiquitous protein that interacts with multiple partners and regulates nuclear activities, including chromatin assembly, histone modifications, replication, and DNA damage repair. The role of specific partners in regulating PCNA activities is not fully understood. Here we identify the nucleosome binding protein HMGN1 as a new PCNA-interacting protein that enhances the binding of PCNA to chromatin but not to purified DNA. Two tetrapeptides in the conservative domain of HMGN1 contain amino acids necessary for the binding of HMGN1 to PCNA. Deletion of both tetrapeptides abolishes the HMGN1-PCNA interaction. PCNA preferentially binds to the linker DNA adjacent to an HMGN-containing nucleosome. In living cells, loss of HMGN1 decreases the rate of PCNA recruitment to damaged DNA sites. Our study identifies a new factor that facilitates the interaction of PCNA with chromatin and provides insights into mechanisms whereby nucleosome binding architectural proteins affect the cellular phenotype.     

The binding of ORC2 to chromatin from terminally differentiated cells

Nuclei from terminally differentiated Xenopus erythrocytes lack essential components of the prereplication complex, including the origin recognition complex (ORC) proteins XORC1 and XORC2. In Xenopus egg extract, these proteins are able to bind erythrocyte chromatin from permeable nuclei, but not from intact nuclei, even though they are able to cross an intact nuclear envelope. In this report we use both permeable and intact erythrocyte nuclei to investigate the role of cyclin-dependent kinase activity in modulating the binding of XORC2 to chromatin. We find that elevating the level of cyclin A-dependent kinase in egg extract prevents the binding of XORC2 to chromatin from permeable nuclei and that kinase inhibition reverses this effect. We also observe a nuclear transport-dependent accumulation of H1 kinase activity within intact nuclei incubated in the extract. However, inhibiting this kinase activity does not facilitate the binding of XORC2 to chromatin, suggesting that other molecules and/or mechanisms exist to prevent association of XORC proteins with replication origins within intact nuclei from terminally differentiated cells.     

Sex chromatin frequency in buccal mucosal tissue: The distribution of single,double and triple sex chromatin bodies

Summary A method of recording sex chromatin scores with a reproducibility of ±3% at two standard deviations is used to investigate the incidence of sex chromatin bodies in normal and polysomic-X individuals. The distribution of sex chromatin bodies in nuclei from both groups may be expressed by a single simple distribution function. The form of the function suggests that the condensation of all X chromosomes (normal and abnormal nuclei) is a random process occurring within a limited period of the cell cycle.Supported by a postgraduate research grant from the Science Research Council. This work forms part of a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science, University of Sheffield.     

In vivo binding of retinol to chromatin. The binding is mediated by a lipoprotein

We have previously shown that exposure of responding cells to vitamin A leads to profound modifications of chromatin structure as revealed by an increased susceptibility to DNase I digestion, modified patterns of histone acetylation, and impaired synthesis of a nonhistone chromosomal protein (Ferrari, N., and Vidali, G. (1985) Eur. J. Biochem. 151, 305-310). The present results show that these effects are most probably due to the direct interaction between retinol and chromatin, and analysis of mononucleosomes and higher oligomers obtained from retinol-treated cells shows that retinol is indeed tightly bound to chromatin. Enzymatic digestions of vitamin A containing nucleosomes with proteinase K, phospholipase C, and phospholipase A2 support a model where the final binding of retinol to chromatin is mediated by a lipoprotein: the recognition of the binding sites on DNA being dictated by the proteic component while the hydrophobic retinol is solubilized in the fatty acid moiety.     

Body distribution of 3H-labelled dalargin bound to poly(butyl cyanoacrylate) nanoparticles after i.v. injections to mice

The blood-brain barrier (BBB) limits the penetration of substances into the brain. Because many drugs, particularly peptides, therefore can not be delivered to the brain, carrier systems were developed to overcome this problem. In earlier studies we demonstrated central analgesic effects of a peptide, dalargin (dal), after systemic administration when this substance was bound onto the surface of polybutylcyanoacrylate nanoparticles and coated with polysorbate 80 but not when it was given alone. The aim of the present study was to investigate the body distribution of 3H-labelled dal bound to nanoparticles compared to unbound dal after i.v. injection in mice. The radioactivity in several tissues, including the brain, was separated in subcellular preparations and was measured after a single i.v. injection over time. Dal radioactivity level in brain preparations was 3 times higher when the drug was bound to nanoparticles whereas the first pass pathway in liver was reduced. The results support previous data that nanoparticles can be used to transport peptides across the BBB.     

新生大鼠下丘脑CRF和AVP神经元对急性低氧的应答

目的:观察肾上腺摘除新生大鼠下丘脑促肾上腺皮质激素释放激素(CRF)和精氨酸加压素(AVP)神经元对急性低氧的应答.方法:在低压氧舱中模拟高海拔低氧,用放免法测定AVP和CRP含量.结果:新生大鼠暴露于急性低氧环境下(模拟5 000 m和7 000 m海拔高度,24 h),其下丘脑CRP在3 d和7 d龄大鼠中无明显变化,但14d、21 d和28 d时低于对照;下丘脑AVP在3 d大鼠中亦无变化,但14 d时低于对照,7 d、21 d及28 d时高于对照.两者对低氧的应答模式随日龄而变化.摘除肾上腺后,14 d、21 d及28 d大鼠下丘脑CRF和AVP含量均显著低于同龄完整大鼠,此时暴露于急性低氧环境下,CRF和AVP无进一步的变化.结论:摘除肾上腺抑制下丘脑CRF和AVP的发育,影响它们对低氧应激的正常应答.