PubMed Health⌕ Search

Biomedical subjects

B Manz

Publications and source records attributed to B Manz.

At least 55 records · Page 3Linked to original sources
PubMed1984

High-performance and ion-exchange chromatography and chromatofocusing of the human uterine progesterone receptor: its application to the identification of 21-[3H]dehydro Org 2058-labelled receptor.

Two independent lines of evidence were used to identify the human uterine progesterone receptor. First, three differently tritiated progestogens (Org 2058, R 5020, progesterone) were used for reversible labelling of the receptor. Secondly, the highly potent affinity label 21-[3H]dehydro Org 2058 was used to label covalently the steroid-specific binding site of the receptor. The labelled cytosols were chromatographed on a Mono Q high-performance anion-exchange column in the absence or presence of a high molar excess of the respective unlabelled competitor steroids. In the case of 21-[3H]dehydro Org 2058, Org 2058 was used as the unlabelled competitor. After elution with a NaCl gradient, the radioactivity was determined in each fraction and the elution profiles (absorption, A at 280 nm; radioactivity, dpm) were superimposed. Free steroid was eluted with the washing buffer. When the NaCl gradient was performed, two peaks of radioactivity were located. The specifically protein-bound radioactivity was eluted at 0.08 M NaCl. Two non-specific steroid-binding entities were eluted at 0.1 and 0.22 M NaCl, the second of which was identified as albumin. The elution profiles of tritiated progesterone, R 5020, Org 2058 and the affinity label 21-dehydro Org 2058 were identical. In a second set of experiments, Org 2058- and 21-dehydro Org 2058-labelled cytosols were subjected to high-performance liquid chromatography on a Mono P high-performance chromatofocusing column in the absence or presence of a high molar excess of unlabelled Org 2058. After elution with Polybuffer 74, only one specifically labelled protein (pH 6.4) was detected. When the Mono P-purified receptor was submitted to sodium dodecyl sulphate polyacrylamide gel electrophoresis, two labelled polypeptides with Mr = 45,000 and 27,000 were detectable.

Binding, Competitive↗
PubMed1984

Characterization of human spleen tumor glucocorticoid receptors using [3H]cortisol as ligand.

The present report describes an assay system allowing the quantification and characterization of [3H]cortisol binding to glucocorticoid receptors in bloodrich human tissue. The essence of this assay lies in the selective binding of 17 beta-carboxylic acids of natural corticoids to corticosteroid binding globulin (CBG). In the presence of 1240 nmol/l 11 beta-hydroxy-3-oxo-4-androstene-17 beta-carboxylic acid only glucocorticoid receptors were detectable with the expected properties: high affinity for synthetic and natural glucocorticoids, but failure to bind to the respective 17 beta-carboxylic acids, apparent Kd's at 0-4 degrees C for [3H]cortisol of approx 30 nmol/l, Nmax similar to those determined with [3H]dexamethasone and the typical sequence of relative binding affinities (dexamethasone greater than cortisol greater than progesterone greater than 17 beta-methyl-testosterone, estradiol).

Binding, Competitive↗
PubMed1984

[A new LH-RIA rapid test for determining peak levels of serum LH--a methodological contribution to in-vitro fertilization].

A highly sensitive, easy to operate LH-RIA rapid test was developed by changing the incubation period and temperature and by reducing the standard curve to three measurement data, using commercially available reagents. The results obtained by means of the LH-RIA rapid test correlate most loosely with those of a conventional 5-hour LH-RIA. The new LH-RIA rapid test is ideally suited for LH peak search at short intervals for exact determination of the time of ovulation as part of in-vitro fertilisation or insemination.

Adult↗
PubMed1984

Criteria for the establishment of a double-labeling assay for simultaneous determination of estrogen and progesterone receptors.

The availability of [125I]-16 alpha-iodo-3,17 beta-estradiol ( [125I]-E2) with binding characteristics similar to estrogen receptor (ER) enabled us to establish a double-labeling assay for the simultaneous determination of ER and progesterone receptors (PgR) using 125I-E2 and [3H]-R5020. The criteria for the establishment of such a double-labeling assay are described. 150 human mammary tumor cytosols have been investigated with the standard routine receptor assay for ER as well as PgR and the results were compared to those obtained by the double-labeling assay. ER: a coefficient of correlation of 0.691 was obtained, the parameters of the regression line were Y = 1.025 X X-0.036. When referring to the standard assay, 3 determinations were false-positive and 4 false-negative. PgR: a correlation coefficient of 0.984 was found, the parameters of the regression line were Y = 0.960 X X + 12.16. One value was false-negative and 1 false-positive. An equivalence of the two methods could be demonstrated. This new assay reduces by half the amount of tissue necessary for a valid 4- to 6-point saturation analysis, the time required for performing the assay and its cost.

Binding, Competitive↗
PubMed1984

17 beta-carboxamide steroids: highly effective inhibitors of the phytohaemagglutinin mediated blastogenesis of normal human peripheral lymphocytes.

Several novel 17 beta-carboxamide analogues of dexamethasone were synthesized. The common precursor, 9-fluoro-16 alpha-methyl-11 beta,17-dihydroxy-3-oxo-1,4-androstadiene-17 beta-carboxylic acid, did not bind to the glucocorticoid receptors of rat liver and human spleen tumours. In addition, no inhibition of the mitogen-induced blastogenesis of cultured human peripheral lymphocytes was observed. The 17 beta-carboxamide analogues, however, bound with similar affinities to the glucocorticoid receptors of both tissues. They inhibited the mitogen-induced blastogenesis of peripheral lymphocytes, showing the same potency and same order of binding affinity as the natural glucocorticoids.

Animals↗
PubMed1983

3H-estradiol and 3H-R5020 binding in cytosols of normal and neoplastic human ovarian tissue.

High-affinity cytoplasmic estrogen and progesterone receptors in normal and abnormal ovarian tissues were studied. Estradiol receptor was detectable in 65% and progesterone receptor in 36% of the malignant tumors; 39% of all malignant ovarian tissues were estradiol- as well as progesterone-receptor-positive. Tumors were said to be receptor-positive when the receptors bound greater than 5 fM steroid/mg cytosol protein. No correlations were found between receptor status and histopathological diagnosis. In normal ovarian tissues collected at various phases of the menstrual cycle no changes in [3H]-estradiol and [3H]-R5020 binding could be detected. Analysis of the receptor concentration for both steroid hormones with regard to the menopausal status demonstrated highest levels in postmenopausal women. No significant difference could be found when two groups of patients with advanced ovarian carcinoma associated with the cytosol estrogen receptor status were compared in terms of two different therapeutic schemes (cytosolic chemotherapy with and without tamoxifen).

Cytosol↗
PubMed1983

Synthesis of biotin-labelled dexamethasone derivatives. Novel hormone-affinity probes.

A new, general methodology for 'sandwich' affinity chromatography of steroid hormone receptors is proposed, the part purification of the human spleen tumor glucocorticoid receptor is quoted as an illustration. 9-Fluoro-16 alpha-methyl-11 beta, 17-dihydroxy-1,4-androstadiene-3-one-17 beta-carboxylic acid was coupled to biotin using pentamethylenediamine (BioDex 1) as a spacer. The bifunctional derivative binds to glucocorticoid receptors and avidin-Sepharose and efficiently protects the glucocorticoid receptor against inactivation when previously added during homogenisation. We have standardized the capacity and optimum conditions for elution of receptor-BioDex-1 complexes which are bound to avidin-Sepharose. Receptor purification of several thousand fold can be obtained with good yield.

Affinity Labels↗
PubMed1983

16 alpha-iodo-3,17 beta-estradiol: a stable ligand for estrogen receptor determinations in tissues with high 17 beta-hydroxysteroid dehydrogenase activity.

Recently, the successful synthesis of radioiodinated 16 alpha-iodo-3,17 beta-estradiol-[125I] [125I]E2 was reported [1]. This new ligand has similar binding characteristics to the estrogen receptor (ER) [2-5] as the currently used tritium labeled estradiol [3H]E2. However, it offers several advantageous features: (a) high specific activity (theoretically 2,000 Ci/mmol) [1]; (b) minor problems with radioactive waste due to its short half life and (c) the possibility of simultaneous determination of ER and progesterone receptors (PgR) by double labeling with [125I]E2 and [3H]R5020 [6, 7]. As we are presently trying to determine ER and PgR in human placental cytosols we were interested in the stability of different labeled estrogens under the conditions of ER-assay. Placental cytosols [8] as well as cytosols of other tissues such as endometrium [9, 10], ovary [11] or mammary carcinomas [12] have been reported to contain significant amounts of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity. Conversion of labeled estradiol to estrone during incubation for ER-quantification would diminish the amount of labeled estradiol thus leading to errors in ER-concentrations, as estrone has only about 10% of estradiol's binding activity [13].

17-Hydroxysteroid Dehydrogenases↗
PubMed1983

Methyl 17 beta-carboxyester derivatives of natural and synthetic glucocorticoids: correlation between receptor binding and inhibition of in vitro phytohaemagglutinin-induced lymphocyte blastogenesis.

Several methyl 17 beta-carboxyester derivatives of natural and fluorinated glucocorticoids were synthesized in order to compare their potency to compete for [3H]dexamethasone binding sites in human spleen tumour cytosols (as a source of large quantities of white blood cells) with their potency to inhibit phytohaemagglutinin-induced blastogenesis of normal human peripheral lymphocytes. The 17 beta-carboxylic acids neither show binding activity nor inhibition of blastogenesis. Methylation partially restores the binding capacity and the intensity of this effect depends on the kind of ring substitutions. The sequence of binding potency is identical compared to that of parent steroids and was found to be in the following order: desoxymethasone greater than dexamethasone greater than corticosterone greater than cortisol greater than progesterone greater than 17-hydroxyprogesterone. The phytohaemagglutinin-induced stimulation of [3H]thymidine incorporation resembles the order of binding potency. The methyl 17 beta-carboxyester derivatives of progesterone, 17-hydroxyprogesterone and betamethasone are inactive. The N-benzyl 17 beta-carboxamide analogs of dexamethasone and betamethasone behave like their corresponding carboxyesters, suggesting an important influence of the side chain conformation of 17 beta-carboxyl derivatives on glucocorticoid receptor binding.

Binding, Competitive↗
PubMed1983

Comparison of [3H]oestradiol and [125I]oestradiol as ligands for oestrogen receptor determination.

The newly available ligand [16 alpha-125I]3,17 beta-oestradiol was investigated for its suitability for routine oestrogen receptor determinations. Unlabeled 16 alpha-iodo-oestradiol was synthesized. No difference in the relative binding affinity to human uterine oestrogen receptor could be demonstrated, but unlabeled as well as labeled 16 alpha-iodo-oestradiol do not bind to serum sex hormone binding globulin. At 0--4 degrees C [3H]oestradiol reaches, when incubated with oestrogen receptor its equilibrium after 3 h, [125I]oestradiol after 9 h. In 100 human mammary tumour cytosols oestrogen receptor concentrations were determined using either [3H]oestradiol or [125I]oestradiol. The mean Kd-values obtained were 0.5 +/- 0.3 nmol/l using [3H]oestradiol and 0.42 +/- 0.25 nmol/l for [125I]oestradiol. For the linear regression (independent variable [3H]oestradiol, dependent variable [125I]oestradiol) the following sample estimates were obtained: n = 100, r = 0.982, yi = 3.04 + 1.00035 x. For a 0.95 probability of confidence it can be stated that the sample estimates are indeed comparable according to the criteria of the two ligands.

Binding, Competitive↗
PubMed1982

Steroid hormone receptors in human melanoma.

Human melanomas were investigated for the presence of high-affinity estrogen-, gestagen-, and glucocorticoid-binding proteins. A statistically significant difference was found for mean estrogen receptor (ER) concentrations in melanomas of male versus female origin: female origin 37.6 (0-107) fmol/mg protein, male origin 3.9 (0-8.3) fmol/mg protein. No significant difference between sexes was found for gestragen receptors: 41.5 (0-194) fmol/mg protein for melanomas of female origin versus 99 (0-362) fmol/mg protein for male. Sucrose density gradient analyses revealed specific binding for both receptor types in the 4-5 S region as well as in the 8 S region. The binding affinities were in the same order of magnitude as reported for receptors found in typical steroid target organs. No significant difference in receptor values depending on sex was found for the glucocorticoid receptor: 19.2 (0-43) fmol/mg protein.

Centrifugation, Density Gradient↗
PubMed1982

Estradiol and progesterone binding components in the cytosol of normal human fallopian tubes.

The in vitro binding of labeled estradiol and R5020 to receptor proteins from human fallopian tube cytoplasmic fractions has been studied. Both compounds give rise to two separate peaks in density gradient analysis experiments under low ionic strength conditions: a "8 S" steroid-hormone-specific peak and a "4 S" peak accounted for by non-specific binding. The receptor molecules were further characterized by a competition studies, electrophoretic technique and gel chromatography. The receptor content for both steroid hormones varied throughout the menstrual cycle. The estradiol and progesterone receptor concentration were highest during the proliferative phase and were very significantly lower in the second half of the menstrual cycle. Furthermore measurement of both receptors in the cytosol revealed differences among the anatomical segments of the fallopian tube. The highest estradiol and progesterone binding could be detected in the ampullary region, significantly lower levels of estradiol and progesterone receptor were seen in the infundibulum and the isthmus.

Binding, Competitive↗
PubMed1982

Synthesis of a new disulfide affinity adsorbent for purification of human uterine progesterone receptor.

For purification of the human uterine progesterone receptor, an affinity adsorbent was synthesized in which the specific ligand (16 alpha-ethyl-3-oxo-19nor-androst-4-ene 17 beta-carboxylic acid) was bound to derivatized celulose using a disulfide-group-containing spacer. The purified receptor protein, isolated by reductive cleavage of the disulfide bond, bound the synthetic gestagen R5020 with high affinity (Kd 12.2 nmol/l). The affinity gel was highly efficient. A 24000-fold purification of progesterone receptor with a recovery of 40% could be achieved in a single step within 6 h. By means of dodecyl sulphate/polyacrylamide gel electrophoresis two main polypeptides with molecular weights of about 43000 and 108000 could be demonstrated.

Adsorption↗