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P Robel

Publications and source records attributed to P Robel.

At least 127 records · Page 7Linked to original sources
PubMed1978

Competitive inhibition of specific steroid-protein binding: practical use of relative competition ratios for the derivation of equilibrium inhibition constants.

The relative competition ratio (RCR) is widely used to express the relative affinities of inhibitor(s) and agonist for a binding protein. The RCR is not a constant; it depends on the concentrations of binding sites and of radioactive hormone, and on the presence of nonsaturable binding component(s). According to the assay conditions used, equating the RCR value to the ratio Ka/Ki of the equilibrium association constants of agonist and inhibitor can lead to large errors. In the case of homogeneous non-interacting binding sites, simple correction factors permit one to calculate the ratio Ka/Ki from the measured RCR value. Calculations are given for the eventual contribution of nonsaturable binding components. Corrections can be unnecessary under well defined experimental conditions, where the bound fraction of hormone in absence of competitor is reduced by using a large dilution of binding protein and/or an increased concentration of radioactive hormone.

Binding, Competitive↗
PubMed1978

Cytoplasmic and nuclear estradiol and progesterone receptors in human endometrium.

Estradiol and progesterone receptors have been characterized in normal human endometrial biopsy samples. The cytosol and nuclei were prepared from 150-250-mg samples, either processed immediately or kept in liquid nitrogen. The total concentration of estradiol-and progesterone-binding sites (available or occupied with endogenous hormone) were measured in both fractions. Results were best expressed in femto-moles per mg DNA, or in sites per cell, assuming an even distribution of receptor throughout the endometrial samples. The contribution to total binding of non-saturable binding components and of plasma proteins (transcortin or sex steroid-binding protein) was taken into account. Measurements were obtained in more than 300 patients, among whom 54 had completely normal menstrual cycles on the basis of clinical, hormonal, and histological features. Total estradiol and progesterone receptors were highest in the late proliferative phase (about 8,000 and 12,000 sites/cell, respectively) and were very significantly lower in the late secretory phase. During the proliferative phase, estradiol receptors were increased only in the nuclear fraction, whereas progesterone receptors were increased mainly in the cytoplasm. In the early luteal phase, estradiol and progesterone receptors decreased in the cytosol, whereas they remained high in the nuclei. Both receptors were at their lowest level in cytosol and nuclei in the late secretory phase. The changes of total estradiol and progesterone receptor sites and of their respective subcellular distributions seem to depend upon the plasma levels of both hormones and to follow the same cause and effect relationships as those demonstrated experimentally in laboratory animals.

Cell Nucleus↗
PubMed1977

[Combined effects of testosterone and estradiol on the ventral lobe of the rat postate in organ culture].

After organ culture without hormone, the epithelial gland cells of Rat veantral prostate undergo atrophic changes, whereas the interstitial stroma components tend to increase. Estradiol (1-1,000 nM),added to the culture medium, is ineffective. On the contrary, testosterone (1-100 nM) maintains epithelial cells and prevents the increase of interstitial stroma. When estradiol (1-1,000 nM) is combined with a physiological concentration of testosterone (1-4 nM), the epithelial cells are well maintained, but the inhibitory action of testosterone on the stroma is counteracted so that the glandular epithelium and the interstitial stroma are both stimulated. However, when testosterone is used at supraphysiological (10-100 nM) concentrations, estradiol is completely ineffective and the structure of the prostate is identical to the one given by the androgen alone.

Animals↗
PubMed1977

Simultaneous radioimmunoassay of tesosterone, dihydrotestosterone, 5 alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3beta, 17beta-diol in the plasms of adult male rats.

A single thin layer chromatography and three antibodies were used for the specific radioimmunoassay of four androgens in pooled rat plasma (Sprague-Dawley adult males). The following values were found (pg/ml +/- SD). Testosterone: 3, 138 +/-173; dihydrotestosterone: 374 +/-20; 5alpha-androstane-3alpha, 17beta-diol: 284+/-24; 5alpha-androstane-3beta, 17beta-diol: 223+/-11.

Androstane-3,17-diol↗
PubMed1976

Influence of purified plasma proteins on testosterone uptake and metabolism by normal and hyperplastic human prostate in "constant-flow organ culture".

Surgical samples of human prostate were explanted and submitted to constant-flow organ culture. The medium contained 3H-testosterone 50 nM, and except for controls, increasing concentrations of human serum albumin (HSA) or human sex-steroid-binding plasma protein (SBP). At steady state, the explants were washed and homogenized, and the total radioactivity, radioactive testosterone, androstanolone (17 beta-hydroxyandrostan-3-one), androstane-3 alpha, 17 beta-diol, and androstane-3 beta, 17 beta-diol were determined after the addition of the corresponding internal 14C standards. From these data, testosterone uptake and metabolism were quantitated. The concentration of unbound testosterone in protein-supplemented culture media was measured separately by equilibrium dialysis. In control superfusions without protein, the tissue concentration of total radioactive steroids was equivalent to 182 +/- 18 (mean +/- SEM) pmoles/g of prostate. Androstanolone represented about 2/3, testosterone 1/10, and the two androstanediols together 1/10 of the total radioactivity. No difference was found between "normal" and hyperplastic prostate explants. In experiments with HSA (15-176 muM), is was observed that the uptake of radioactive testosterone in the prostate explants was decreased in direct proportion to the unbound testosterone fraction of the superfusion medium, but the proportions of testosterone metabolities in the superfused explants remained the same. In experiments with SBP (6-135 nM), the concentrations of unbound testosterone in the superfusion medium were reduced to the same levels as in the experiments with HSA. The reduction of tissue radioactivity was somewhat larger than that expected from the reduction of unbound testosterone in the superfusion medium for the concentrations of SBP less than 50 nM, and then remained approximately constant. In addition, SBP altered the metabolism of testosterone: the androstanolone/testosterone ratio in the prostate explants was critically dependent upon the SBP concentration in the superfusion medium. It is therefore suggested that, independent of its effect on the binding of testosterone, SBP has a direct effect on testosterone uptake and metabolism by the human prostate. The underlying mechanism is unknown.

Androstane-3,17-diol↗
PubMed1976

[Androgen receptors. Their value in physiopathological investigations in man].

There is less knowledge of androgen receptors than of oestrogen receptors but, in recent years, important progress has been made. The author thus describes the physico-chemical properties of the androgen receptors. He discusses also the problems raised by their estimation. The exchange techniques are practically unusable as the androgen receptors are easily destroyed even at 15 degrees C. The author proposes and describes another method which isolates the hormone-receptor complex with protamine sulphate. He also considers the prospects of these receptor estimations in prostatic disease, e.g. androgen-dependent adenoma. The absence of androgen receptors in testicular feminisation syndrome finally permits excellent illustration of the importance of the study of receptors in the understanding of cellular insensivity to a hormone.

Chemical Phenomena↗
PubMed1975

Steroid-induced early protein synthesis in rat uterus and prostate.

An early 'induced protein', after exposure of the rat uterus to estradiol, is detected among the soluble proteins with a double-labelling technique and electrophoretic fractionation. Efforts have been directed to establish the subcellular distribution of the induced protein, since such a protein, observable 1 h after hormone administration, may play an important role in the subsequent amplified responses, especially in terms of RNA synthesis. Moreover such an early discrete induced protein was sought in a comparable system responding to another hormone, namely prostate and seminal vesicles under androgens. The induced protein was not found in uterine nuclei of 21-day-old rats after 1 h of estradiol action in vivo and 1 h of tissue incubation with labelled leucine. This negative result summarizes a search among different nuclear protein fractions using various procedures; nor was induced protein observed in mitochondrial and microsomal pellets. Contrary to these negative findings, slight changes of histone labelling were observed under the experimental conditions used to demonstrate induced protein. In addition histone acetylation was increased after 1 h of estradiol action in vivo and 15 min tissue labelling in vitro with radioactive acetate. Furthermore, an increase in total protein synthesis between 0 and 2 h after estradiol action was observed, the relative increase of incorporation of radioactive leucine into protein of estradiol-treated vs non-stimulated uteri being corrected for variations of the acid-soluble radioactive leucine pool. Attempts to obtain an early and discrete induced protein with androgens in prostate and seminal vesicles of immature or castrated rats after different times of exposure to testosterone, androstanolone and estradiol have been unsuccessful. The contribution of both negative and positive findings in steroid-induced early protein synthesis is discussed in the context of the current knowledge of hormone action.

Animals↗
PubMed1975

[Estradiol and progesterone receptors in human endometrium during the menstrual cycle].

The concentrations of estradiol and progesterone receptors are measured in the cytoplasmic and nuclear fractions of endometrial samples obtained by biopsy throughout the menstrual cycle. The standardized micromethod accounts for the sites occupied by endogenous hormones and for interference by androgen receptors and plasma binding proteins. In normal women, the cellular concentration of estradiol receptor increases early in the cycle and decreases steadily after ovulation. The progesterone receptor is maximum between the 10th and 15th day of the cycle and decreases during the luteal phase. The nuclear/cytoplasmic ratios of both receptors follow the variations of circulating hormones. These data constitute a new basis for physiopathological and pharmacological investigations on the hormonal control of the human uterus.

Biopsy↗
PubMed1975

Determination of protein-ligand binding constants at equilibrium in biological samples.

Protein-ligand complexes can be separated functionally into two classes. "Specific" binding is characterized, in relative terms, by a high affinity for the ligand and a low binding capacity. "Non-specific" binding is characterized by a low affinity and a very large capacity. The calculation of equilibrium binding constants for any specific protein-ligand interaction requires the exact determination of the unbound ligand concentration and the specifically bound ligand concentration. These determinations usually require corrections for the contribution of non-specific binding. The use of two correction terms, kn and f, is proposed: kn is the product of the affinity constant k times the number of binding sites n of the non-specific components, while f is the fraction of the non-specific binding included in the experimental estimates of bound ligand. Several theoretical solutions using these terms are proposed for the calculation of specific binding constants. The practical choice of the correction factor may be different when the simultaneous measurement of the affinity constant and maximum number of binding sites, or when only the latter, is desired. In the case of complex binding systesm containing more than one specific component, the individual constants can be determined by non-graphical methods, using computer-aided iterative statistical calculations. A complete solution is given for a system containing two specific plus non-specific interactions and actual experiments are reported for steroid hormone-receptro complexes.

Animals↗
PubMed1975

[The binding of estradiol to human polynuclear eosinophilic leukocytes].

High affinity estradiol binding is found in the fraction of human eosinophil rich, polynuclear leukocytes sedimenting between 800 times 10 g times mn and 24,000 times 20 g times mn. The apparent dissociation constant at equilibrium is approximately 0.6 nM, and the total number of binding sites per blood eosinophil leukocyte is 7,400 sites per cell. Hydrogen peroxyde (1 muM) enhances estradiol binding, a finding similar to that previously observed autoradiographically for the binding of estradiol to the eosinophils of rat uterus.

Binding Sites↗
PubMed1975

Androgen-binding proteins in human benign prostatic hypertrophy.

Prostatic samples were surgically removed from 7 patients suffering from benign prostatic hypertrophy. High-speed supernatants (cytosol) containing 20-25 mg of protein/ml were prepared. Glycerol gradient ultracentrifugations were performed, using cytosol labeled at 0 C with 2-5 nM 3H-17beta-hydroxy-androstan-3-one (androstanolone or dihydrotestosterone) alone, or in the presence of 50-250-fold excess of androstanolone, estradiol, or androstane-3alpha, 17beta-diol (androstanediol). Two high-affinity saturable binding components were observed. One binding component was the androgen receptor. Its sedimentation coefficient was 8 S in low-salt medium. It had a high affinity for androstanolone. The binding of 3H-androstanolone was strongly completed by androstanolone itself, less by estradiol, and not by androstanediol. In one case, endogenous androstanolone found in the 8 S region of glycerol gradients was measured by radioimmunoassay, and it was calculated that more than 90% of the cytosol receptor binding sites might be occupied by this steroid while the total binding capacity of the 8 S receptor was estimated to approximate 2.6 pmol of androstanolone/g of prostate. No testosterone was found in the receptor fraction. The second binding component was attributable, at least in part, to the sex steroid-binding plasma protein (SBP), as indicated by its sedimentation coefficient (congruent to 4 S in low salt medium), its high affinity for androstanolone and androstanediol and its lower affinity for estradiol, and finally, its migration on polyacrylamide gel electrophoresis. In one instance, the concentration of the SBP-like protein in prostate cytosol was measured by equilibrium dialysis, and it was calculated that the binding capacity of the prostate SBP-like component corresponded to 4 pmol of androstanolone/g of prostate, a small (less than 5%) value with regard to SBP concentration in the plasma of the same patient. The blood contamination of the cytosol, as obtained from the measurement of hemoglobin, did not account for the amount of SBP found in the prostate sample. Since SBP-like protein is probably of plasma origin, to determine whether SBP was located in the extracellular space or inside the prostate cells, BPH slices from another patient were incubated in the presence of 3H-testosterone, the cytosol was prepared, and was fractionated by Sephadex G-150 column chromatography. The androstanolone/testosterone ratio in the receptor-containing peak was high (1.7), whereas in the incubation medium it was very low (0.08). In the peak containing the SBP-like protein, the ratio was 0.74, which may suggest that all or part had been exposed to the predominant androstanolone environment inside the prostatic cell.

Aged↗