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Biomedical subjects

W Rosner

Publications and source records attributed to W Rosner.

At least 73 records · Page 4Linked to original sources
PubMed1983

Radioimmunoassay for human corticosteroid-binding globulin.

We isolated corticosteroid-binding globulin from human plasma and used the isolated protein as the antigen in raising antibodies in rabbits. The resulting antiserum was used to develop an RIA with which we can determine the globulin in as little as 0.007 microL of serum, although we routinely use, after appropriate dilution, 0.02 microL. The mean concentration of corticosteroid-binding globulin in the serum of 40 normal subjects was 32.4 (SD 4.2) mg/L. The correlation (r) between results with this RIA and those by an established binding assay was 0.94.

Female↗
PubMed1982

Synthesis of 16 alpha-[125I] iodo-5 alpha dihydrotestosterone and evaluation of its affinity for the androgen receptor.

Analogs of 5 alpha-dihydrotestosterone, halogenated at carbon 11, were synthesized as potential gamma-emitting ligands for the androgen receptor. These compounds, were chosen for synthesis because estradiol, similarly substituted, is strongly bound to the estrogen receptor, and both androgen and estrogen receptors have generally similar structural requirements for the D-ring. The 16 alpha-halogenated steroids, including 16 alpha-]125I] iodo-5 alpha-dihydrotestosterone were synthesized from 16 beta-bromo-5 alpha-dihydrotestosterone by halogen exchange. The cis-beta-bromohydrin substrate was synthesized from 5 alpha-androstane-3,17-dione by selective ketalization, dibromination at C-16 and stereoselective reduction, 16 alpha-iodo dihydrotestosterone was devoid of androgen activity in vivo at concentrations at which 5 alpha-dihydrotestosterone was fully stimulatory. The 16-alpha-iodo, 16 alpha-bromo, and 16-beta-bromo analogs were allowed to compete with [3H]-dihydrotestosterone for binding to the androgen receptor; the 16 alpha-iodo compound had a relative binding affinity 1/100th and both bromo compounds 1/30th that of dihydrotestosterone. In addition, no specific binding was detected when the 16 alpha-[125I]iodo analog was incubated with prostatic cytosol.

Animals↗
PubMed1982

The effect of corticosteroid-binding globulin on the in vitro incorporation of thymidine into human lymphocytes.

The purpose of these studies was to examine the inhibitory effect of human corticosteroid-binding globulin (CBG) on the mitogenic effect of phytohemagglutinin on cultured human lymphocytes. We found that CBG could inhibit the incorporation of [14C] thymidine into lymphocytes, but the observed inhibition could be accounted for by the cortisol which was bound to the purified CBG, CBG stripped of cortisol or bound to 11-deoxycortisol was inactive in this assay.

Cells, Cultured↗
PubMed1982

Radioimmunoassay for human testosterone-estradiol-binding globulin.

We isolated testosterone-estradiol-binding globulin (TeBG) from human pregnancy plasma and used the isolated protein to obtain a rabbit antiserum to it. Using immunochemical techniques, the antiserum was demonstrated to be both monospecific and to have the ability to remove TeBG-binding activity from human serum. This antiserum was used to develop a RIA for human TeBG. The sensitivity is such that TeBG can be determined in as little as 0.005 microliter serum or plasma, although we routinely use 0.5-1.0 microliter. The correlation between the RIA and an established binding assay, using 63 different specimens, was 0.995.

Animals↗
PubMed1981

Synthesis in vitro of corticosteroid-binding globulin from rat liver messenger ribonucleic acid.

To show that corticosteroid-binding globulin (CBG), a plasma glycoprotein, is synthesized in the liver as well as to develop a tool with which to study the hormonal factors which control its synthesis, we undertook to translate rat CBG mRNA in vitro. Rat liver mRNA was translated in the presence of [35S]methionine in both a rabbit reticulocyte lysate (cell-free) System and a Xenopus laevis oocyte (whole cell) system. A monospecific antibody against highly purified rat CBG was used to precipitate a single 35S-labeled product from the mixture of newly synthesized hepatic proteins in the two systems. In the rabbit reticulocyte lysate system, the protein migrated with an apparent molecular weight of 41,000 on sodium dodecyl sulfate-gel electrophoresis; CBG isolated from rat serum migrated as a doublet with apparent molecular weights of 56,000 and 62,500. Since proteins synthesized by a cell-free system are not glycosylated, the difference in molecular weight can probably be accounted for by the carbohydrate content of plasma CBG. Translation of rat liver mRNA in a Xenopus laevis oocyte system again resulted in immunoprecipitation of a single 35S-labeled protein, but this time with an apparent molecular weight of 53,000 on sodium dodecyl sulfate-gel electrophoresis; this probably represents glycosylated CBG and corresponds to the apparent molecular weight of one of the species isolated from rat plasma.

Animals↗
PubMed1980

Interaction of 16 alpha-[125I]iodo-estradiol with estrogen receptor and other steroid-binding proteins.

This communication describes the synthesis of 16 alpha-[125I]iodo-estradiol (125I-E2) with specific activities greater than 1000 Ci/mmol (1 Ci = 3.7 x 10(10) becquerels). We show that it binds to the same specific estrogen receptor sites as does [3H]estradiol and that it does so with an affinity that is indistinguishable from that for the latter steroid. This is true for receptor obtained from calf uterus and from a pool of human mammary carcinomas. There is no significant binding of 125 I-E2 to the testosterone-estradiol-binding globulin of human plasma. 125I-E2 also binds tightly to anti-estradiol antibodies raised against estradiol derivatized at carbons 3, 6, and 17. Finally, we show that unlabeled I-E2 is an active estrogen in vivo as demonstrated by its ability to increase uterine weight in ovariectomized rats.

Animals↗
PubMed1979

Investigation of a corticosteroid-binding globulin-like molecule in human placenta.

The existence of an endogenous corticosteroid-binding globulin (CBG)-like molecule in human placenta has been reported in studies which depended largely on immunological techniques. The experiments reported herein do not support that contention. Using a well characterized antibody to purified human CBG, we were unable to demonstrate CBG in placental cytosol. Although the immunological methods were too insensitive to detect CBG, the presence of small amounts of it were detected in binding experiments with [3H]cortisol. Simultaneous measurement of serum albumin indicated that the presence of CBG in the placental cytosol could be accounted for by contamination of the preparation with plasma.

Cytosol↗
PubMed1978

An exchange assay for the cytoplasmic glucocorticoid receptor in the liver of the rat.

Cytosolic receptor for glucocorticoids can exist in either the free or bound form; assays now in use measure only the free form. In order to assay the total glucocorticoid receptor content of rat liver, free plus bound, we have developed an exchange assay wherein specifically bound [3H]dexamethasone is shown to be a valid measure of receptor in the presence of high concentrations of corticosterone. The exchange between [3H]dexamethasone and corticosterone is able to proceed because, unter the conditions of the assay, corticosterone is almost completely metabolized.

Adrenalectomy↗
PubMed1978

Enhancement by oleandomycin of the inhibitory effect of methylprednisolone on phytohemagglutinin-stimulated lymphocytes.

Triacetyloleandomycin (TAO) and its parent compound oleandomycin have a "steroid-sparing" effect in asthmatic patients. This study was designed to determine whether oleandomycin would enhance the inhibitory action of glucocorticosteroids in suppressing the blast transformation of phytohemagglutinin-stimulated human lymphocytes. Although oleandomycin could not be shown to inhibit the phytohemagglutinin stimulation of lymphocytes, there was a significant potentiation of steroid suppression at each concentration of methylprednisolone examined. This augmentation of glucocorticosteroid suppression of lymphocyte proliferation in vitro appears to support the clinically observed "steroid-sparing" effect of TAO and therefore its usefulness may not be limited to asthma and bronchitis.

Adult↗
PubMed1978

Effect of protein binding on transfer and metabolism of cortisol in perfused human placenta.

The transfer and metabolism of cortisol and cortisone and the effect of protein binding on these processes have been investigated in vitro in the perfused human placenta. The clearance of cortisol in buffer, expressed as a fraction of the antipyrine transfer rate (clearance index), was 0.50 +/- 0.05 SEM in either direction. Extensive conversion to cortisone (85%) occurred during transfer. Addition of corticosteroid-binding globulin (CBG) in amounts sufficient to bind 50% of the cortisol reduced the clearance (0.40 +/- .026) insignificantly, whereas human serum albumin (HSA) in amounts sufficient to bind 50% of the cortisol reduced the clearance to 0.28 +/- 0.012 (P less than 0.001) even though the association constant for albumin is approximately 1000-fold less. The percent of conversion to cortisone did not change significantly with protein binding. The clearance index of cortisone from a protein-free perfusate was 0.74. With CBG and albumin in the same concentrations as used in the cortisol experiments, the binding of cortisone to CBG was 23% and its clearance was 0.70; with albumin, the binding was 45% and the clearance index was 0.45. The addition of albumin and CBG to the same perfusate resulted in a cortisol clearance equal to that obtained with perfusate containing only albumin. Binding to albumin may be more significant than binding to CBG in controlling the transfer rate of cortisol to the fetus.

Carrier Proteins↗
PubMed1978

Studies of gonadotropin-gonadal dynamics in patients with androgen insensitivity.

Four patients with androgen insensitivity had plasma LH and FSH measured at 20-min intervals for 24 h and at 15- to 30-min intervals for 3 h after the injection of LRH. Twenty-four-hour mean testosterone (T), estradiol, and androstenedione (delta 4) levels were also measured. Patients with androgen insensitivity had significantly elevated LH levels (P less than 0.05) and an increase in the number of LH secretory episodes (P less than 0.001) compared to normal subjects. The amplitude of the LH secretory episodes, expressed as the absolute increment, was significantly higher than normal controls (P less than 0.005). The LH response to LRH (absolute increment) was twice that of normal, but was not significantly different from normal subjects. The 24-h mean FSH levels were normal in three of the patients and elevated in one. This patient had the mildest degree of androgen insensitivity on clinical exam and the greatest degree of testicular atrophy. The 24-h mean T, estradiol, and delta 4 levels were higher than normal, but only the delta 4 was significantly increased (P less than 0.05). To determine if the elevated LH levels were in response to a decrease in the free T level, we measured T-binding capacity (TBG), TBG was higher than normal controls but was not significantly different, suggesting that elevated LH levels were probably in response to a decrease in T action at the hypothalamic-pituitary level. This was further supported by the inability of prolonged dihydrotestosterone administration to affect LH secretion in one of the patients with the Reifenstein syndrome.

Adolescent↗
PubMed1977

Investigation of the binding site of human corticosteroid-binding globulin by affinity labeling. Demonstration of a cysteinyl residue in the binding site.

This communication deals with the investigation of the binding site of purified human corticosteroid-binding globulin (CBG) by the method of affinity labeling. The design of the studies necessitated the rapid removal of unbound ligands, followed by assay of the binding capacity of CBG. We were able to accomplish this by demonstrating that CBG absorbed on DEAE-filter discs bound cortisol as if both molecules were in solution. Using this principle, 6beta-bromoprogesterone was shown to react with CBG in a time-dependent and irreversible fashion with a t1/2 = 15 min at 2 degrees. One mol of 6beta-bromo[3H]progesterone reacted with 1 mol of CBG. After completion of the reaction, one of the two sulfhydrul groups in CBG was no longer titratable with Ellman's reagent. The product of the reaction of 6beta-bromoprogesterone with cysteine is progesterone-6-S-L-cysteine. After acid hydrolysis of CBG, which had been incubated with 6beta-bromoprogesterone, a compound which migrated with the same mobility on thin layer chromatography as the model compound was observed. We conclude that 6beta-bromoprogesterone is an affinity label for CBG and that a cysteinyl residue is present in the binding site.

Affinity Labels↗