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Y A Lefebvre

Publications and source records attributed to Y A Lefebvre.

At least 37 records · Page 2Linked to original sources
PubMed1989

Characterization of high affinity and low affinity dexamethasone binding sites on male rat liver nuclear envelopes.

Steroids must traverse the nuclear envelope before exerting their action at the chromatin. However, few studies have been done to elucidate the mechanism by which steroids traverse this membrane barrier. As first steps towards investigating the mechanism, we have characterized the binding sites for dexamethasone on male rat liver nuclear envelopes. The nuclear envelopes, prepared in the presence of dithiothreitol, were isolated from purified nuclei after treatment with DNase 1 at high pH. Binding of dexamethasone to the nuclear envelopes was measured after 16 h of incubation at 0-4 degrees C. At pH 7.4, only a single high capacity, low affinity binding site for dexamethasone was identified. However, at pH 8.6, two sites were identified; a low capacity, high affinity site and a high capacity, low affinity site. Adrenalectomy of the animal before preparation of the membranes caused loss of the high affinity site and reduction in the number of the lower affinity sites. Acute dexamethasone treatment of adrenalectomized rats resulted in the reappearance of the high affinity site but long term treatment with dexamethasone was required for complete restoration of the high affinity sites and reappearance of any of the low affinity sites. The steroid specificity of these nuclear envelope binding sites was different from that of the cytosolic glucocorticoid receptor, generally showing broader specificity. However, triamcinolone acetonide, which is a potent competitor for binding to the glucocorticoid receptor, did not complete effectively. The binding sites were sensitive to protease treatment and salt extraction studies revealed that the dexamethasone binding sites do not represent proteins non-specifically bound to the nuclear envelope. The affinity and the hormone responsiveness of the high affinity site are similar to those of the nuclear glucocorticoid receptor. Therefore, the nuclear envelope may be a site of action of glucocorticoids.

Animals↗
PubMed1988

A histone 1-like antigen is a component of the nuclear envelope.

Rabbit antibodies have been raised against rat liver nuclear envelopes. An enzyme-linked immunosorbent assay (ELISA) demonstrated high titer antiserum specific for the nuclear envelope preparation. Immunocytochemical studies showed that the antiserum stained the nuclear envelopes, but not intra-nuclear components of HEp-2 (human malignant epithelial) cells. When electrophoretically separated peptides were tested by immunoblotting techniques, the rabbit antiserum specifically stained proteins with molecular masses of 26 and 28 kD. These peptides had similar mobilities to purified histone 1 (H1). Indeed purified calf thymus H1 recognized the antiserum. The antigens are not loosely bound to the nuclear envelope, as they could not be extracted with low salt. Therefore, we have established that the 26 and 28 kD nuclear envelope peptides are not contaminants of the nuclear envelope preparation and that they express determinants that are immunologically cross-reactive with purified H1, but not with intra-nuclear H1.

Animals↗
PubMed1987

Chemical demonstration of nuclear androgen receptor following affinity chromatography with immobilized ligands.

With increasing purification of the androgen receptor from nuclei of rat ventral prostate, a receptor-like protein could be demonstrated by chemical staining with silver nitrate. After sonication and digestion of nuclei with micrococcal nuclease, the solubilized receptor was applied to a column of Matrex Gel Green A and eluted with a linear gradient of 0-2 M NaCl. Characterized by specific binding of dihydrotestosterone, this form of the receptor was also androgen dependent and yielded an apparent Mr of 33,000 when analyzed by polyacrylamide gel electrophoresis and silver nitrate staining. To facilitate recovery following chromatography, the receptor was precipitated with 0-40% ammonium sulfate. Analysis of the 15-fold enriched fraction by sucrose density-gradient centrifugation confirmed the presence of a 3S androgen-binding protein. About 200 ng of the precipitated protein was applied to a column of dihydrotestosterone-17 beta-succinyl agarose (ligand concentration, 0.25 mumol/ml). The fractions eluted with 50 microM dihydrotestosterone were electrophoresed and stained as before; again, the presence of a 33,000 Mr protein sensitive to castration was demonstrated. Alternatively, when the precipitated protein was fractionated by fast protein liquid chromatography utilizing a Superose 12 HR 10/30 column, the receptor coeluted with nuclear proteins in the 29,000-36,000 Mr range as determined both by retention time and electrophoresis. In combination, the above methods may be used to obtain a receptor protein purified to near homogeneity with a yield of 5-10%. The amount of receptor afforded by the purification sequence is small but nevertheless sufficient for chemical detection. We anticipate that with modification, the procedures may prove suitable for the recovery of nuclear androgen receptor on a preparative scale.

Animals↗
PubMed1986

Diet fat alters the structure and function of the nuclear envelope: modulation of membrane fatty acid composition, NTPase activity and binding of triiodothyronine.

Mice were fed a diet either high or low in P/S ratio to determine the effect of altering dietary lipid on the fatty acid composition of liver nuclear envelopes and thereby on functions of the nuclear envelope. Mice fed the high P/S diet exhibited higher levels of C18:2 omega 6 and unsaturates in liver nuclear envelopes, higher specific activity of NTPase and specific binding for L-triiodothyronine at 15 degrees C and 22 degrees C compared with the low P/S diet fed group. These observations indicate that diet-induced differences in the fatty acid composition of nuclear envelope lipid affects functions of the nuclear envelope.

Animals↗
PubMed1986

Interaction of steroids with the nuclear envelope.

Three approaches have been taken to determine the molecular mechanism by which steroid hormones traverse the nuclear envelope on their way to the genome. The first approach involved characterization of steroid binding to nuclear envelope preparations. We have characterized androgen binding to nuclear envelopes isolated from the rat ventral prostate, the rat liver, and androgen-responsive and androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma and glucocorticoid binding to rat liver. Relatively high affinity binding sites for steroids have been identified on nuclear envelopes. Importantly, the number and specificity of the sites correlates with the responsiveness of the tissue to the steroid. In the second approach, we have undertaken to identify the steroid binding site directly. As the characteristics of the rat ventral prostate site resembled those of the nuclear androgen receptor, we have begun purifying that receptor and have found fast protein liquid chromatography to be very effective. By affinity labelling studies, the dexamethasone binding site on the rat liver nuclear envelope has been identified as a peptide of molecular weight of approximately 90,000. The third approach we have used is to identify androgen-dependent peptides in nuclear envelope preparations. In both the rat ventral prostate and an androgen-responsive cell line of the Shionogi mouse mammary carcinoma, we have identified abundant androgen-dependent peptides. The relationship of these peptides to the binding sites identified by the first two approaches and their role in steroid transport is being investigated.

Animals↗
PubMed1985

Multiple thyroid hormone binding sites on rat liver nuclear envelopes.

Nuclear envelopes relatively free of plasma membrane contamination were isolated from the male rat liver. Equilibrium binding of T3 to nuclear envelopes occurred after incubation for 3 h at 20 degrees C. Scatchard analysis revealed two classes of binding sites; a high affinity site having a KD of 1.8 nM with a maximum binding capacity of 14.5 pmol/mg protein and a low affinity site having a KD of 152.1 nM with a maximum binding capacity of 346.8 pmol/mg protein. No degradation of the radioligand occurred during incubation with the nuclear envelope. T4, rT3 and Triac competed effectively for the binding of T3 to the high affinity site whereas only T4 competed well for binding to the lower affinity site. The binding site was protease sensitive but not salt extractable. Multiple T3 binding sites having similar affinities have been reported on plasma membranes. An intriguing possibility is that membrane binding sites may be involved in translocation of thyroid hormone across membrane barriers.

5'-Nucleotidase↗
PubMed1985

Correlation of androgen-responsiveness of Shionogi mouse mammary carcinoma cell lines with binding of dihydrotestosterone to nuclear envelopes.

Purified nuclear envelopes have been isolated from an androgen-responsive and two androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma. The binding of dihydrotestosterone to nuclear envelope fractions isolated from the three variant cell lines is correlated with the androgen-responsiveness of the cell line. Nuclear envelopes prepared from the two androgen-unresponsive cell lines did not bind dihydrotestosterone specifically following incubation with radioactive dihydrotestosterone from 2.5 to 45.0 nM at 20 degrees C for 18 h. Under the same binding conditions, nuclear envelopes prepared from the androgen-responsive cell line demonstrated saturable, specific binding of dihydrotestosterone. Scatchard analysis revealed a class of binding sites with an apparent Kd of 14.2 nM and a maximum binding capacity of 28.7 fmol/mg protein. Proteinase and heat treatments resulted in the complete loss of androgen-binding activity, whereas DNAase treatment resulted in the loss of 38% of the binding activity. The binding sites were specific for dihydrotestosterone. Testosterone was only a weak competitor and estradiol did not compete. Extraction with concentrations of KCl up to 1.0 M did not result in loss of androgen binding.

Animals↗
PubMed1985

Androgen interactions with intact nuclear envelopes from the rat ventral prostate.

Intact nuclear envelopes containing nuclear pore complexes have been prepared from the rat ventral prostate. The polypeptide profile of the nuclear envelopes from the rat prostate resembled that of nuclear envelopes prepared from the male rat liver. Isolation of the nuclear envelopes after incubation of purified nuclei with radioactive dihydrotestosterone results in labelling of the membrane. More dihydrotestosterone is bound after incubations at 22 degrees C for 18 h than at 2 degrees C for 18 h or 22 degrees C for 2 h. Scatchard analysis revealed a class of binding sites with an apparent Kd of 46 nM. Dihydrotestosterone, testosterone, cyproterone acetate and methyltrienolone were effective as competitors of labelled dihydrotestosterone binding to the nuclear envelopes, while estradiol did not compete. Castration of the rats 24, 48 and 96 h prior to preparation of nuclei resulted in loss of androgen binding to the membranes. Extraction with 0.6 M NaCl resulted in the loss of 72% of the androgen binding.

Androgens↗
PubMed1985

Isolation and characterization of nuclear envelopes from three variant cell lines of the Shionogi mouse mammary carcinoma: identification of androgen-dependent peptides.

We have isolated and purified, with good yields, nuclear envelopes from an androgen-responsive and from two androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma after subjecting purified nuclei to DNase at high pH and characterized them morphologically, chemically, and enzymatically. Phase-contrast microscopy revealed the nuclei to be free of cytoplasmic tags and that the nuclear envelopes were isolated as membrane "ghosts." Electron micrographs clearly showed the double-membrane system with nuclear pore complexes which illustrates that the nuclear envelopes were ultrastructurally intact. The nuclear envelopes contained little DNA, low levels of arylesterase or acid phosphatase activity, and undetectable levels of succinate dehydrogenase and 5'-nucleotidase activity. Coomassie blue staining of the nuclear envelope fractions on sodium dodecyl sulfate-polyacrylamide gels for all three cell lines revealed that most of the polypeptides were similar. However, we have identified androgen-dependent peptides of molecular weights 29 000, 32 000, and 34 000 in nuclear envelopes of the androgen-responsive cell line peptide profiles by comparing the nuclear envelopes prepared from the androgen-responsive cell line grown in intact mice, in castrated mice, and in mice which had been injected with testosterone after castration. Further investigation of the androgen regulation of these nuclear envelope peptides may help us understand the molecular mechanisms involved during morphological changes of the nucleus which occur in response to different hormonal environments.

Acid Phosphatase↗
PubMed1984

Androgen-dependent peptides of the rat ventral prostate nuclear envelope.

Nuclear envelope peptides of the rat ventral prostate in the molecular weight region 18,400 to 19,400 show marked androgen dependence. After castration these peptides disappear. Re-administration of testosterone restores them. They were not extracted by 0.1 M NaCl or 5% HClO4 but were partially extracted by 0.35 M NaCl and 1% Triton. These peptides were not present in rat liver nuclear envelopes. As these androgen-dependent non-histone peptides have similar characteristics to androgen-dependent peptides identified in the prostate nucleus, we conclude that the nuclear peptides are at least partially localized to the nuclear envelope.

Animals↗
PubMed1984

Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver.

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3',5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3',5'-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.

Animals↗
PubMed1982

Binding of dihydrotestosterone to a nuclear-envelope fraction from the male rat liver.

Intact nuclear 'ghosts' containing small amounts of DNA were obtained from rat liver. Incubation of radiolabelled dihydrotestosterone with isolated nuclear-envelope fraction from male rat liver resulted in specific binding of the dihydrotestosterone to the membranes. Optimal binding occurred at 20 degrees C after 20h incubation. Storage for 2 weeks at -80 degrees C resulted in little loss of specific binding. Scatchard analysis revealed a class of binding sites with a KD of 23.2 nM. Pronase and heat treatment destroyed the binding site. Androgens and glucocorticoids competed for labelled dihydrotestosterone binding to the ghosts, whereas oestrogens did not compete. Castration 24h before preparation of ghosts did not alter the binding site, and a similar class of binding sites was identified on female rat liver nuclear envelopes.

Animals↗
PubMed1982

Characterization of androgen uptake by purified nuclei from an androgen-dependent and two androgen-independent cell lines of the Shionogi mouse mammary carcinoma.

Properties of androgen uptake by nuclei prepared from an androgen-dependent tumour cell line occurred after 2 h incubation at 20 degrees C; 2. Nuclei from three tumour cell lines displayed similar affinity for DHT but the two androgen-independent cell lines had less than one-quarter the number of uptake sites; 3. Loss of label from nuclei which had been pre-incubated with [3H]-DHT for 18 h at 20 degrees C was greater from the AD cell line nuclei than from the androgen-independent cell lines; 4. Whole cell contamination of the nuclear preparations did not contribute to specific DHT uptake.

Androgens↗
PubMed1980

Binding of androgens to a nuclear-envelope fraction from the rat ventral prostate.

A nuclear-envelope fraction was isolated from the rat ventral prostate which is virtually free of DNA and contains little RNA or plasma membrane. Isolation of this nuclear-envelope fraction after incubation of purified nuclei with radioactive dihydrotestosterone results in labelling of the membrane. More binding of dihydrotestosterone is observed after incubations at 22 degrees C for 17 h than at 4 degrees C for 17 h or at 22 degrees C for 60 min. Scatchard analysis revealed a class of binding sites with KD 8.4 nM. Dihydrotesterone and testosterone were almost equally effective as competitors of labelled dihydrotestosterone binding on the purified nuclear-envelope fraction, whereas diethylstilboestrol was less effective and dexamethasone did not compete well. When the outer membrane of the nuclei was removed with Triton X-100, a 24% decrease in specific binding of androgens was observed. Castration 24 h before preparation of nuclei resulted in loss of the androgen binding to the membrane.

Animals↗
PubMed1976

Diphosphoinositide metabolism in bovine adrenal medulla.

(1) A phosphatidylinositol kinase (EC 2.7.1.67) of a chromaffin vesicle membrane preparation isolated from bovine adrenal medulla was characterized. Its activity towards endogenous and exogenous phosphatidylinositol was very similar to the kinase activity of the microsomal fraction prepared from the same tissue. (2) Phosphomonoesterase (EC 3.1.3.36) and diesterase activity hydrolysing membrane bound phosphatidylinositol 4-phosphate was located mainly in the microsomal fraction. No hydrolytic activity was present in the vesicle membrane. (3) Phosphorylation of chromaffin vesicle membrane phosphatidylinositol did not increase calcium-binding by the membranes.

Adrenal Medulla↗