B. Manz

A comparison of cytoplasmic and nuclear estradiol and progesterone receptors in human fallopian tube and endometrial tissue

Quantitative and qualitative aspects of the in vitro binding of 3H-estradiol and 3H-progesterone to receptor components from human endometrium and fallopian tube cytoplasmic and nuclear fractions were studied. The steroid binding macromolecules formed in vitro could be extracted from nuclei by 0.4 M KCl and detected by glycerol gradient centrifugation. Both estradiol- and progesterone-binding compounds formed only one peak (under high ionic strength conditions) with a sedimentation coefficient of about 4-5 S. The number of cytoplasmic and nuclear binding sites for both estradiol and R5020 varied dramatically throughout the menstrual cycle: the estradiol and progesterone receptor concentrations 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 anatomic 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 receptors were seen in the infundibulum and the isthmus.

Gestodene: A novel synthetic progestin — characterization of binding to receptor and serum proteins

Gestodene, 17 alpha-ethinyl-13-ethyl-17 beta-hydroxy-4,15-gonadien-3-one, is a new orally active progestational agent, which is available for clinical use in oral contraceptives. The aim of the present study is to make a broad characterization of gestodene at the receptor level and to discuss the results in comparison to those of established progestogens. Kinetic studies of 3H-gestodene uptake show a rapid increase in the amount of specific binding during the first three hours. After saturation, the amount of specifically bound 3H-gestodene remained almost constant up to 24 hours at 4 degrees C. The dissociation of 3H-gestodene from the cytoplasmic myometrial progestone receptor, measured by displacement of labeled steroid with dextran-coated charcoal treatment at 4 degrees C at various times, showed a biphasic or two-component first order dissociation curve. As anticipated, sucrose gradient centrifugation analysis of the 3H-gestodene-labeled cytosol of human myometrial tissue showed that the gestodene binding components sedimented in the 4S and 8S region. A 200-fold molar excess of nonradioactive gestodene reduced only the 8S binding of 3H-gestodene. 4S binding of 3H-gestodene was not reduced, which indicate the existence of a second high capacity binding component. In biological test systems, such as the Clauberg test or Kaufmann test, gestodene has proved to be a very effective progestogen. Among nortestosterone derivatives it is one of the most potent and resembles progesterone biologically in its progestogenic effects. This biologically identical gestagenic activity of gestodene and progesterone is reflected by a very similar behavior in vitro in terms of binding to progesterone receptors of human uterus cytosol. Furthermore, competitive studies indicated that gestodene like other synthetic progestagens also displays some affinity for androgen and glucocorticoid receptors but no measurable affinity for the estrogen receptor. Remarkable is the high binding affinity of gestodene to the binding sites of the mineralocorticoid receptor of rat kidney with a RBA value of 350% compared to aldosterone.

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.

Steroid side-chain modification and receptor affinity: Binding of synthetic derivatives of corticoids to human spleen tumor and rat liver glucocorticoid receptors

Abstract Human spleen tumors (Hodgkins disease I) contain varying amounts (40–150 fmol/mg protein) of glucocorticoid receptors. The apparent K diss at 0°C is 6.8 ± 2.1 nmol/1. A second glucocorticoid binder, presumably the plasma contaminant human corticosteroid binding globulin (CBG), is shown by a newly developed [ 3 H]-hydrocortisone assay. A series of side-chain modified natural and synthetic corticoids bind with similar affinities to human spleen tumor and rat liver glucocorticoid receptors. 17β-Carboxylic acid derivatives do not bind to both receptors whereas methylation or amidation of the acids partially restore their binding affinities. The one exception, the inactive methyl 17β-carboxester of betamethasone, which bears a 16β-methyl substitution, confirms the hypothesis that receptor binding involves a specific conformation of the side-chain.

One millisecond of light suffices to suppress nighttime pineal melatonin synthesis in rats

The effect of a single high-intensity light pulse with a duration of 1 ms on nighttime pineal activity of male Sprague-Dawley rats was investigated. 10 minutes after light exposure pineal N-actyltransferase activity and melatonin content were significantly reduced. These results show that the rat pineal is capable of responding to very short light flashes of high intensity.

Application ofliquid—liquid partition chromatography in the simultaneous purification of sex-hormone-binding globulin and corticosteroid-binding globulin

Two human serum proteins, corticosteroid-binding globulin (CBG) and sex-hormone-binding globulin (SHBG), were purified to homogeneity by the application of a combination of three different modes of chromatography. Human pregnancy serum was fractionated with ammonium sulphate. SHBG (50% pellet) and CBG (80% pellet) were then purified by affinity chromatography on tresyl-activated Sepharose with 15-aminopentadecanoic acid (for SHBG) and 1,12-diaminododecane (for CBG) as spacers and 17 zeta-aminoethyl-5 alpha-androstan-3 beta,17-diol (for SHBG) and 17 alpha-hydroxy-4-androsten-3-one-17 beta-carboxylic acid (for CBG) as specific ligands for these two proteins. The eluate was injected into a Mono Q anion-exchange column. Fractions containing SHBG or CBG were finally purified by liquid-liquid chromatography on Lipar-Gel 750. This chromatographic sequence clearly separated SHBG and CBG from other proteins, mainly serum albumin, without a loss of protein or binding activity.

Fluorescent deacetylcolchicine: New aspects of its activity and localization in PtK-1 cells

Abstract PtK-1 cells have been used to localize structures which are decorated by deacetylcolchicine conjugated with fluorescein isothiocyanate (FDC). This colchicine derivative competitively inhibits [ 3 H]colchicine binding to soluble tubulin, is able to depolymerize microtubules (MTs) assembled in vitro, and inhibits cell growth by arresting colchicine-sensitive cells in mitosis. Fixed cells were incubated with FDC (10 −5 M), and/or with tetramethyl rhodamine-labelled anti-tubulin antibodies using the indirect immunofluorescence technique. In double label microscopy the cells show corresponding fluorescence of MTs after incubation with anti-tubulin antibodies and FDC. This can be demonstrated most clearly with cells in mitosis which are preincubated with the microtubule-stabilizing agent taxol. FDC binding is decreased, but not completely blocked, by preincubation of fixed cells with unlabelled colchicine (10 −4 M). Trimethyl colchicinic acid (TMCA) conjugated with FITC, a colchicine derivative which does not bind to soluble tubulin, and FITC alone, inactivated by methylamine, do not bind to MTs. These results demonstrate that FDC is able to decorate and to depolymerize sensitive MTs, indicating a direct action of the drug on these structures. Furthermore, the labelling of other cellular structures by FDC, possibly membrane systems as well as chromatin, is compared with recent data.

Radioimmunoassay of Human Serum Serotonin

: A radioimmunoassay for extracted, N-acetylated human serum serotonin (5-hydroxytryptamine) is described. Antisera were raised in rabbits against a conjugate of bovine serum albumin with serotonin hemisuccinamide. Polyethylene glycol in combination with anti-rabbit immunoglobulins was used to separate bound and unbound 125I-Bolton Hunter-serotonin conjugate. Ethanol precipitation of serum proteins was used to extract serotonin, which was subsequently acetylated with acetic anhydride to N-acetyl serotonin. The average recovery was 66%. The minimal detectable concentration of N-acetyl serotonin was 0.012 mumol/l serum (25 fmol per tube). The intra-assay precision (CV) was 6.8% (n = 20) at a level of 0.9 +/- 0.06 mumol/l. The inter-assay CV was 10% at a level of 0.49 +/- 0.049 mumol/l, and 25% (n = 10) at a level of 2.16 +/- 0.53. Analytical recovery of serotonin, corrected for losses during extraction and acetylation, was 99 +/- 13%. The only substance cross-reacting with the antibody was endogenous N-acetyl serotonin. This was detectable when the acetylation step was omitted, and it can be removed by extraction before the acetylation. The observed range for the concentration of serotonin in serum was for 59 women 0.45 - 3.46 (mean +/- SD: 1.37 +/- 0.63 mumol/l) and for 59 men 0.19 - 2.8 (mean +/- SD: 1.18 +/- 0.56 mumol/l). All values are corrected for endogenous N-acetyl serotonin: observed range 0 - 0.18 (mean +/- SD: 0.03 +/- 0.03 mumol/l).

3H-Labelled RU 38486: Characterization of Binding Sites in Human Uterine Cytosol

The behaviour of the antifertilizing synthetic steroid RU 38486 towards human uterine progestin receptor was investigated. RU 38486 competed in the same order of magnitude as progesterone for the [3H]R 5020 binding site of progestin receptor, whereas R 5020 was unable to compete against [3H]RU 38486. This apparent contradiction could be explained by means of HPLC-chromatography. HPLC-chromatography with an anion exchange column (MonoQ, Pharmacia, Uppsala, Sweden) showed that [3H]RU 38486 forms at least two stable complexes with uterine cytosol, on one hand with serum albumin, which presents almost 90% of bound radioactivity, and on the other hand with the two native progestin receptor forms, corresponding to 4 S and 8 S receptor forms in sucrose density gradient analysis. Whether reduced binding of salt-activated RU 38486 receptor complexes to DNA-cellulose is due to reduced activation is still uncertain and remains to be further investigated.

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.