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Biomedical subjects

B Descomps

Publications and source records attributed to B Descomps.

At least 37 records · Page 2Linked to original sources
PubMed1986

Stimulation of aromatase activity by dihydrotestosterone in human skin fibroblasts.

In order to study the regulation of aromatase activity by androgens in cultured fibroblasts derived from genital skin of normal prepubertal boys, aromatase activity was evaluated in the presence of various concentrations of non-aromatizable androgen DHT(5 alpha-dihydrotestosterone). The estrogen formation was assayed by an enzymatic method, after 24 h incubation of the cells with 10(-6) M androstenedione. Aromatase activity was stimulated 3- to 20-fold by DHT at concentrations 10(-10) and 10(-9) M. It was necessary to preincubate the cells with DHT for 48 h in order to bring about this stimulation. The stimulatory effect was not significant after preincubation for only 24 h. The basal value of aromatase activity was in the range of 8 +/- 1.2 pmol/mg protein/day (mean +/- SEM), while the maximal stimulation 1043 +/- 46 pmol/mg protein/day was obtained at the concentration of 10(-8) M DHT. This stimulation was partially blocked with cyproterone acetate at level of 20 +/- 4 pmol/mg protein/day; stimulation of aromatase activity by DHT could thus be mediated by the androgen receptor. This stimulatory effect was prevented by incubation of the cells with cycloheximide or actinomycin D, suggesting that DHT acts to increase aromatase activity in cultured fibroblasts by inducing the synthesis of new proteinaceous material. In vitro regulation of aromatase activity by androgens could contribute to a new approach to the extraglandular formation of estrogen.

Aromatase↗
PubMed1986

Rapid separation of dehydrogenases by affinity chromatography with new induced specificity phases.

We describe a procedure using immobilized nicotinamide as an affinity chromatographic ligand for the binding of NAD(P)+-dependent dehydrogenases. The procedure involves preparation of nicotinamide N1-(N-(6-aminohexyl)-acetamide)-agarose and modification of the immobilized nicotinamide by the addition of a ketone or an aldehyde to form an adduct. The nicotinamide, which has no affinity for dehydrogenase, becomes a very specific ligand of dehydrogenase, which binds the ketone or the aldehyde as substrate or inhibitor. In tests, the adduct prepared with immobilized nicotinamide and sodium pyruvate bound specifically to lactate dehydrogenase (EC 1.1.1.27), whereas the adduct prepared with alpha-ketoglutarate bound to glutamate dehydrogenase (EC 1.4.1.3). This technique enables the rapid isolation of a given dehydrogenase.

Alcohol Dehydrogenase↗
PubMed1985

Enzymatic androgen assay: some properties of human placental microsomal aromatase.

The androgen content of biological fluids can be determined after their conversion into estrogens using human placental microsomal aromatase (HPMA). The purpose of this paper is to report some physico-chemical properties of HPMA. Using an accurate, specific and sensitive assay for HPMA, Km values for dehydroepiandrosterone (DHEA), androstenedione and testosterone were found to increase with increasing amount of the detergent (Triton X-100) added. Analysis at substrate concentrations 5-10 times above and below the Km values did not indicate any anomalous kinetic behaviour. Triton X-100, used for enzyme solubilization, significantly decreased the rate of aromatization of the three substrates by increasing their Km values. This effect was more important for testosterone than for androstenedione or DHEA. Using a new protocol for the determination of aromatase activity, kinetic properties of aromatase before and after solubilization are described.

3-Hydroxysteroid Dehydrogenases↗
PubMed1985

Testosterone secretion is severely impaired after hepatectomy in rat, dog and man.

In 90%-hepatectomized rats, the plasma testosterone level (0.34 +/- 0.07 ng.ml, mean +/- SEM) is significantly lower (P less than 0.001) than in sham operated male rats (1.7 +/- 0.26 ng.ml, mean +/- SEM). In dogs, after 90% hepatectomy, the mean plasma testosterone concentration fell to 1/10 of the plasma testosterone level measured in sham operated animals either 24 or 72 h after surgery. In hepatectomized men, plasma testosterone is markedly decreased in contrast to what is observed after duodeno-pancreatectomy performed under the same conditions of anesthesia. These results suggest that 90% hepatectomy severely alters the Leydig cell function.

Animals↗
PubMed1985

Sulfonic acid enkephalin: binding specificity to rat brain opiate receptors.

We tested sulfonic-acid enkephalin, a SO3H-Tyr derivative of the Leu-enkephalin, for its binding capacity to rat brain opiate receptors, by competition against several tritiated enkephalins. Using [3H]DADLE as the competitor, we demonstrated that sulfonic-acid enkephalin binds to rat brain opiate receptors, and using [3H]DSLET and [3H]DAGO as the competitors, we demonstrated that sulfonic-acid enkephalin binds preferentially to delta-opiate receptors. The affinity ratio of sulfonic-acid enkephalin for delta-opiate receptors versus mu-opiate receptors, is considerably higher than that of the parent compound, Leu-enkephalin, and of DADLE and is similar to the affinity ratio of DSLET for the same receptors.

Animals↗
PubMed1985

Bromocriptine inhibition of hyperprolactinemia during surgery.

The material included two groups of 10 women undergoing diagnostic laparoscopy. General anesthesia was administered by injection of 0.1 mg fentanyl followed by infusion of propanidide-succinylcholine. The control group received no medication prior to surgery, whereas patients in the experimental group were given 5 mg bromocriptine per os. Blood samples for prolactin determinations were drawn as the patients were placed on the operating table and immediately following surgery. The association of anesthesia and surgery caused prolactin levels to rise from 10.9 +/- 3.5 to 168 +/- 18.7 ng . ml-1 in the control group (p much less than 0.001) and from 3.5 +/- 0.5 to 7.5 +/- 1.1 ng . ml-1 in the test group (p less than 0.001). A significant difference was noted between the two groups for their pre- and postoperative levels and prolactin response (p less than 0.05, p much less than 0.001 and p much less than 0.001, respectively). The proposed protocol successfully suppresses prolactin increase during surgery and constitutes a useful tool for investigating hyperprolactinemia and its consequences during this same time. Possible applications include in vitro fertilization and studies on prolactin receptor-bearing tumors.

Anesthesia, Intravenous↗
PubMed1984

Specific 5 alpha-dihydrotestosterone receptor and 5 alpha-reductase activity in human amniotic fluid cells.

A macromolecular component which specifically binds tritium-labeled-dihydrotestosterone is present in cultured amniotic fluid cells. The androgen-binding complex is characterized by a 3.6S sedimentation coefficient, an apparent dissociation constant of 1 nmol/L, a mean binding capacity of 243 +/- 140 fmol/mg of DNA, and a specificity for testosterone and dihydrotestosterone. Similar properties have been reported for the androgen receptor of the fetal genital skin fibroblast, which suggests that the tritium-labeled-dihydrotestosterone-binding component in amniotic fluid cells is the androgen receptor. Amniotic fluid cell monolayers incubated with serum-free medium containing testosterone are able to transform testosterone into dihydrotestosterone. The 5 alpha-reduced product has been characterized by thin-layer chromatography and capillary column gas-liquid chromatography. Androgen receptors and 5 alpha-reductase activity are expressed in amniotic fluid cells. The prenatal diagnosis of 5 alpha-reductase deficiency or complete androgen insensitivity syndrome (testicular feminization) is thus theoretically possible and obviously prenatal testing would be indicated in the family at high risk.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1984

Physico-chemical properties of the hydroxysterol binding protein of human lymphocyte cytosol. Effects of high salt concentrations and molybdate.

Side chain-hydroxylated derivatives of cholesterol (OH sterol) inhibiting lymphoblastic transformation bind with high affinity and specificity to a hydroxysterol binding protein (OHSBP) in the cytosol of human lymphocytes. These binding properties of OHSBP suggested some analogies with that of steroid hormone receptors. The observation of a nuclear binding of 25-OH[3H]cholesterol prompted us to apply to the cytosolic OH sterol-OHSBP complex the physico-chemical treatments known to 'activate' the steroid hormone receptors. A change of sedimentation coefficient from 8.3 to 4.3 S was observed in hypertonic buffer (0.4 M KCl) but the resulting 4.3 S complex dissociates easily whereas the 'native' 8.3 S form does not. Moreover, molybdate did not prevent the 8.3----4.3 S transformation induced by KCl and neither ammonium sulfate precipitation nor increasing temperature had any effect on the sedimentation coefficient of the 8.3 S complex. Thus, several physico-chemical features differentiate the OH sterol-OHSBP complex from steroid hormone receptors.

Binding, Competitive↗
PubMed1984

Inhibition of androgen metabolism and binding by a liposterolic extract of "Serenoa repens B" in human foreskin fibroblasts.

We previously suggested [Steroids 33, (1979) 3; Steroids 37, (1981) 6] that cultured genital skin fibroblasts should prove useful for screening of potential antiandrogens in human and living target cells. "Serenoa repens" lipidic extract (S.R.E.) was recently reported (Br. J. Pharmacol., in press) to inhibit androgen action in animals. The present investigation was designed to study the antiandrogenicity of this compound in human cells: we therefore analyzed the effects of S.R.E. on the intracellular conversion of testosterone (T) to 5 alpha-reduced derivatives, and we investigated interaction of S.R.E. with the intracellular androgen-receptor complex. Since the chemical structure of the active component of S.R.E. is still unknown, results are expressed in U/ml (one unit is defined as the amount of S.R.E. required to inhibit 50% of the specific binding (IC50) of [3H]1881 to rat prostate cytosol). S.R.E. at different dilutions (5.7 to 28.6 U/ml) is added to culture media containing [3H]T or [3H]dihydrotestosterone (DHT) and incubated at 37 degrees C with cultured fibroblasts. 28.6 U/ml S.R.E. significantly alters the formation of DHT and strongly inhibits 3 ketosteroid reductase mediated conversion of DHT to 5 alpha-androstane-3 alpha, 17 beta-diol, characterized radiochemically by thin-layer chromatography. S.R.E. is a good competitor for the whole cell androgen receptor: 7.1 U/ml S.R.E. gives 50% inhibition of the binding of 2 X 10(-9) M [3H]DHT to its receptor. Competitive binding assays after cell fractionation indicate that S.R.E. is less potent in nuclear than in cytosol receptors. Sucrose gradient centrifugation of the radioactive cell lysate of fibroblasts demonstrates that 28.6 U/ml S.R.E. abolishes 70% of the 3.6 S receptor-complex radioactive peak. The present studies show that S.R.E. inhibits 5 alpha-reductase, 3-ketosteroid reductase and receptor binding of androgens in cultured human foreskin fibroblasts. As the search for the ideal antiandrogen continues, S.R.E. appears to be a new type of antiandrogenic compound as therapeutics for the treatment of benign prostatic hypertrophy, hirsutism and so forth.

Adult↗
PubMed1983

DNA and cholesterol biosynthesis in synchronized embryonic rat fibroblasts. I. Temporal relationships between HMG-CoA reductase activity, sterol biosynthesis and thymidine incorporation into DNA.

Temporal relationships between hydroxymethylglutaryl-CoA reductase activity, biosynthesis of C27 sterols, and [3H]thymidine incorporation into DNA were studied in a rat embryo fibroblast cell line synchronized by double thymidine block and cultured in cholesterol-containing medium. Cyclic variations of HMG-CoA reductase activity and C27 sterols occurred, with two maxima in S and G2M phases; the relative shortness of the G1 phase (3 h) in these cells could be responsible for the shift of sterol synthesis in the S phase. No noticeable variation of the individual C27 sterols was observed during the entire cell cycle. In each experiment, there was a good linear correlation between HMG-CoA reductase activity and C27 sterol synthesis, but from one experiment to another, a given level of enzymatic activity led to varying levels of [2-14C]acetate incorporation into sterols. In our experimental conditions, total HMG-CoA reductase activity is measured, and the preceding observation could be explained by a varying degree of phosphorylation of the enzyme depending on the metabolic state of the cells at the start of the experiment. The cyclic variations of the enzyme activity seem to be due more to increased synthesis at given times of the cycle than to periodic dephosphorylation. We question the existence of a relationship between cell division and cyclic sterol synthesis occurring in cells cultured in cholesterol-containing medium.

Acetates↗
PubMed1983

DNA and cholesterol biosynthesis in synchronized embryonic rat fibroblasts. II. Effects of sterol biosynthesis inhibitors on cell division.

The relationships between cholesterogenesis and cell division were studied by using two inhibitors of hydroxymethylglutaryl-CoA reductase activity--25-hydroxycholesterol and compactin. The effects of both compounds on DNA synthesis were compared in synchronized rat fibroblasts cultured in a cholesterol-containing medium. Compactin did not inhibit DNA synthesis, except after a long time of contact and at high and almost cytotoxic concentrations. 25-Hydroxycholesterol inhibited DNA synthesis (without cytotoxic effects) after only 9-16 h of contact, depending on the phase of the cell cycle at which this compound was added to the culture medium. Sensitivity of cells to 25-hydroxycholesterol was maximal at the end of the S phase/beginning of the G2M phase. The rapid effect of 25-hydroxycholesterol on DNA synthesis appears to be separate from the inhibitory effect on sterol or non-sterol mevalonate-derived compound synthesis. Indeed, under our experimental conditions, the suppression of cholesterol biosynthesis is compensated by the presence of cholesterol in the culture medium, as demonstrated by the lack of effect of compactin on DNA synthesis; moreover, addition of mevalonolactone to the culture medium did not reverse the effect of 25-hydroxycholesterol. 25-Hydroxycholesterol could inhibit DNA synthesis by a direct action on the nucleus, after transfer by the intermediary of a specific hydroxysterol-binding protein.

Acetates↗
PubMed1983

[Collection of mature ovocytes during cycles with spontaneous ovulation for the purpose of in vitro fertilization].

In vitro fertilization was attempted during a spontaneous cycle in 17 out of 97 patients. Preovulatory urinary estrogen and LH maximas were assayed in order to time celioscopy 24 hours after the onset of the triggering urinary LH maxima. In 8 patients, comparison with plasmatic LH shows that the timing of spontaneous ovulation can be precisely predicted from urine assays. However, when the triggering LH release is weak, LH must be above or equal to 25 IU/g of creatinine with a creatininuria above or equal to 0.4 g/l in two urine samples at a three-hour interval.

Estrogens↗
PubMed1983

Bioluminescent assay of femtomole levels of estrone and estradiol.

A rapid and very sensitive enzymatic assay of estrogens was developed using the transhydrogenase reaction of the human estradiol-17 beta dehydrogenase and bioluminescence for the quantification of NADH accumulated during the first reaction. The assay requires two steps: first, the addition of transhydrogenase buffer, and a few minutes later the addition of the bioluminescence reagent. The method can determine 0.1 to 50 pg of estrogens in the assay, in 1 h, with a coefficient of variation of 5%.

17-Hydroxysteroid Dehydrogenases↗
PubMed1983

Enzymatic determination of dehydroepiandrosterone sulfate in biological fluids.

A simple method is described for the determination of androgens, particularly dehydroepiandrosterone sulfate (DHEAS), in human plasma and urine. The method does not involve extraction or chromatography. An internal standard is used as reference. The androgens are enzymatically converted to estrogens and these latter compounds can be measured by the enzymatic method previously described [1]. The range of accuracy was between 99 and 103%. The precision of the method was 2.5%. The sensitivity was 0.1 pM per sample. The method is suitable for routine use, and one worker can easily perform twenty assays in one day.

Aromatase↗
PubMed1983

Active site-directed irreversible inhibition of 3 beta-hydroxysteroid dehydrogenase from human placenta.

To obtain a placental microsome preparation able to convert androstenedione and testosterone specifically to estrogens, 3 beta-hydroxysteroid dehydrogenase must be eliminated. After solubilisation by Triton X-100, the remaining 3 beta-hydroxysteroid dehydrogenase activity is inhibited by 11 alpha-bromoacetoxyprogesterone, an alkylating analogue of progesterone, which behaves as an irreversible active site-directed inhibitor. The enzyme is protected against inactivation and alkylation by steroid and coenzyme substrates. The inhibition is specific to 3 beta-hydroxysteroid dehydrogenase and the estrogen synthetase (aromatase) activity contained in the preparation is not affected by this inhibitor.

3-Hydroxysteroid Dehydrogenases↗
PubMed1983

[Metabolism of dihydrotestosterone in cultured skin fibroblasts: reduction to 5 alpha-androstane-3 alpha, 17 beta-diol].

Reduction of dihydrotestosterone into 5 alpha-androstan-3 alpha, 17 beta diol by 3 alpha hydroxysteroid dehydrogenase was studied in human cultured skin fibroblasts. Characterization of the 3 alpha diol was performed by gas-liquid chromatography coupled with mass spectrometry. Sex skin fibroblasts enzyme has the same Km (9.10 X 10(-6) M) as that of non sex skin fibroblasts (8.75 X 10(-6) M). On the other hand, reduction of DHT is 3 times higher in sex skin fibroblasts (Vmax = 223.9) than in non sex skin fibroblasts (Vmax = 87.9). Thus, human culturel fibroblasts appear again to be an useful tool for the study of a key-enzyme of androgen metabolism in target cells.

Androstane-3,17-diol↗
PubMed1983

Centrifugal analyzer used for enzyme immunoassay of progesterone and choriomammotropin.

Recently we developed an enzyme immunoassay involving the use of steroid delta-isomerase (EC 5.3.3.1) as enzyme label and exclusion-affinity chromatography for rapid separation of free antigen-enzyme conjugate that bound to antibodies (J. Immunol. Methods 35: 267-284, 1980). Here we describe an automated version of this procedure, for immunoassay of progesterone and choriomammotropin (human placental lactogen) in serum with the use of a centrifugal analyzer. After incubation, suitable dilutions of sera or extract plus antiserum, conjugate, and double antibody were filtered on an estradiol affinity gel-filtration column; the enzyme activity of the filtrates was determined with the centrifugal analyzer. Results correlate well with those obtained by radioimmunoassay: r (progesterone) = 0.980, r (choriomammotropin) = 0.940. The within-run and between-run precision, specificity, sensitivity, accuracy, and speed of this system make it a useful tool for immunoassay.

Autoanalysis↗