Biomedical subjects

Z Krozowski

Publications and source records attributed to Z Krozowski.

53 records · Page 3Linked to original sources

PubMed1989

Generation of polyclonal antibodies against purified rat whey acidic proteins and the synthesis of a tracer fusion protein suitable for use in radioimmunoassays.

Rodent milk consists mainly of caseins and whey proteins. A major component of the latter group is the whey acidic proteins (WAP) the gene for which has been cloned recently and shown to contain several potential glucocorticoid receptor binding sites. Studies on the regulation of this gene by glucocorticoids would be greatly enhanced by the availability of a radioimmunoassay for WAP. Rat milk was obtained from lactating Sprague-Dawley rats and the WAP purified to homogeneity by ammonium sulphate fractionation, gel filtration and ion exchange chromatography. Rabbit polyclonal antibodies were raised against the purified WAP and used to probe a Western blot of whey proteins. The major band recognized by the antibody corresponded in molecular weight to purified WAP. Problems associated with radiolabelling the tyrosine-free WAP molecule necessitated the fusion of a tyrosine containing protein with the rat milk protein. A rat WAP cDNA clone was ligated to the glutathione transferase gene, the fusion protein expressed in Escherichia coli and purified by a one-step procedure on a glutathione affinity column. Purified WAP readily displaced the radiolabelled recombinant tracer in a radioimmunoassay.

Animals↗

PubMed1989

Human chorionic gonadotrophin-induced suppression of serum inhibin involves reduction in ovarian inhibin alpha-subunit messenger RNA levels in the pregnant mare serum gonadotrophin-primed female rat.

We have investigated the effect of administration of human chorionic gonadotrophin (hCG) to immature female rats pretreated with pregnant mare serum gonadotrophin (PMSG) on ovarian inhibin alpha-subunit mRNA, serum inhibin and circulating gonadotrophin levels. PMSG stimulation alone caused a 5-fold increase in relative inhibin alpha-subunit mRNA levels and a 12-fold increase in serum inhibin by 48 h. However, injection of hCG at 40 h suppressed PMSG-stimulated ovarian inhibin alpha-subunit mRNA and serum inhibin to levels at 48 h not statistically significantly different from controls. Serum follicle-stimulating hormone (FSH) fell after PMSG treatment, but rose after combined PMSG-hCG treatment. Serum luteinizing hormone (LH) was unchanged after PMSG alone but also rose after combined PMSG and hCG therapy. In conclusion, hCG suppresses ovarian inhibin synthesis in PMSG-stimulated immature female rats with preservation of the reciprocal relationship between inhibin and FSH. This immature female rat model therefore provides further insight into the effects of LH or hCG on granulosa cell inhibin production just prior to ovulation.

Animals↗

PubMed1988

Analysis of renal and hippocampal type I and type II receptors by fast protein liquid chromatography.

Type I and Type II adrenal steroid receptors from rat renal and hippocampal cytosols were studied by the technique of Fast Protein Liquid Chromatography. Type I receptors were labelled with [3H]aldosterone plus excess RU26988, and Type II receptors with [3H]dexamethasone. On a Mono Q anion exchange column the molybdate-stabilized renal and hippocampal Type I receptors both eluted as single symmetrical peaks at 0.27 M NaCl, with a recovery of approximately 90% and 60-fold purification (renal) and 10-15-fold (hippocampal). Molybdate-stabilized Type II binding sites from both hippocampal and renal cytosols co-eluted with the Type I sites. On Superose gel filtration renal Type I receptor-steroid complexes consistently eluted two fractions later than hippocampal Type I complexes, suggesting that the renal complexes are smaller; Type II receptor-steroid complexes from both cytosols co-eluted, consistently one fraction behind hippocampal Type I sites. Sequential gel filtration and anion exchange chromatography achieved a 1000-fold purification of renal Type I binding sites, with an overall recovery of 10%.

Androstanols↗

PubMed1988

Pregnant mare's serum gonadotropin stimulates inhibin subunit gene expression in the immature rat ovary: dose response characteristics and relationships to serum gonadotropins, inhibin, and ovarian steroid content.

We have investigated the temporal changes and dose responses of ovarian inhibin gene expression, circulating inhibin, and ovarian estradiol content in PMSG-primed immature female rats. The relative levels of ovarian inhibin alpha- and beta A-subunit messenger RNA (mRNA), serum inhibin, and ovarian steroid content were measured after stimulation of immature female rats with 20 IU PMSG. Each of these variables rose in parallel and peaked at 48 h (relative ovarian inhibin alpha- and beta A-subunit mRNA increased 3.8-fold and 2.8-fold, respectively, serum inhibin 269 U/ml, vs. control 31, P less than 0.01, ovarian estradiol content 114 pmol/pair of ovaries vs. controls 4.7 P less than 0.001), corresponding to the time of maximal follicular development in this animal model. Subsequently each fell to reach a nadir at 96 h. The data are consistent with the hypothesis that PMSG stimulation of inhibin secretion occurs by a mechanism involving inhibin gene transcription. The increase in ovarian inhibin and estradiol synthesis during folliculogenesis and their simultaneous decline before ovulation suggest that 1) the circulating levels of inhibin and estradiol reflect follicular maturation and 2) these two hormones are regulated via a common mechanism during follicular development in the rat.

Animals↗

PubMed1988

The effect of hypophysectomy on inhibin production by adult rat ovaries: changes in ovarian inhibin gene expression and serum inhibin.

We have investigated the effect of hypophysectomy on adult rat ovarian inhibin alpha- and beta A-subunit mRNA, serum inhibin, and circulating gonadotrophin levels. Immediate declines in ovarian inhibin gene expression and serum inhibin were observed following hypophysectomy, with a nadir on day 4. However, inhibin synthesis determined by these parameters was still apparent at 16 days post-hypophysectomy. Gonadotropin levels were suppressed following hypophysectomy and the fall in inhibin production was highly correlated with disappearance of FSH from the circulation. This study demonstrates that removal of pituitary gonadotropin stimulation results in diminished ovarian inhibin synthesis and provides further evidence indicating that FSH stimulates granulosa cell inhibin synthesis by enhancing inhibin gene transcription.

Animals↗

PubMed1987

The chicken progesterone receptor: sequence, expression and functional analysis.

The complete mRNA sequence of the chicken progesterone receptor (cPR) has been determined. Expression of the cloned cDNA both in vivo and in vitro produces a protein that has the same apparent mol. wt on SDS--polyacrylamide gels as the 'natural' cPR form B (109 kd) as determined by immunoblotting and photoaffinity labelling. When expressed in HeLa or in Cos-1 cells the 'cloned' cPR displays hormone binding characteristics indistinguishable from the 'natural' receptor and, in the presence of progestins, exhibits 'tight nuclear binding'. A protein corresponding in size to the cPR form A (79 kd) could be detected by expressing in vivo and in vitro an N-terminally truncated cPR starting at methionine 128. A protein of the same apparent mol. wt results from internal initiation during in vitro translation. In contrast, such a protein was barely detectable after in vivo expression of the cPR cDNA in Cos-1 cells. These results suggest that form A is generated by an oviduct cell specific process involving either internal initiation of translation and/or proteolysis in the vicinity of methionine-128. The cPR contains two highly conserved regions C and E, a characteristic of the steroid/thyroid hormone receptor supergene family. By expression of a series of cPR deletion mutants, region E could be defined as the hormone binding domain whereas region C is indispensable for the tight nuclear association of the progestin-receptor complex. In the presence of progestins, the cloned cPR efficiently trans-activates transcription from the long terminal repeat region (LTR) of the mouse mammary tumor virus (MMTV). Deletion of the entire N-terminal region A/B or of the hormone binding domain E results in a 100-fold reduction of transcriptional activation. No stimulation of transcription can be detected when the C-terminal deletion extends into region C, indicating that this region is involved in the recognition of the hormone responsive element (HRE) of the MMTV LTR.

Amino Acid Sequence↗

PubMed1987

Inhibin gene expression in the human corpus luteum.

We report inhibin alpha- and beta A-subunit gene expression in the human corpus luteum and placenta using human alpha-subunit and bovine beta A-subunit nucleic acid probes. In addition, we have demonstrated the presence of immunoreactive and bioactive inhibin in human corpora lutea. Our findings suggest that this tissue is a significant source of inhibin during the luteal phase of the normal human menstrual cycle.

Corpus Luteum↗

PubMed1986

Inhibin A-subunit gene expression in the ovaries of immature female rats is stimulated by pregnant mare serum gonadotrophin.

We have used a bovine cDNA probe for the A-subunit of inhibin to demonstrate the presence of inhibin in the ovaries of immature female rats: the levels of mRNA were increased following treatment of the rats with pregnant mare serum gonadotrophin (PMSG), suggesting modulation by PMSG of ovarian inhibin synthesis at least at the level of transcription. Furthermore, we report the unexpected finding of significant levels of inhibin mRNA in the corpora lutea of mature female rats. No inhibin mRNA was demonstrable in the uterus or placenta.

Age Factors↗

PubMed1986

Cloning of the chicken progesterone receptor.

Monospecific antibodies directed against the chicken progesterone receptor (PR) form B were used to screen a randomly primed phage lambda gt11 cDNA expression library prepared from size-fractionated chicken oviduct mRNA. Two independent immunoreactive clones, lambda cPR1 and lambda cPR2, were isolated. Antibodies selected from anti-PR form B antiserum on matrices of lambda cPR1 and lambda cPR2 fusion proteins detected two proteins on electrophoretic immunoblots of crude and purified PR preparations. These proteins had the same apparent molecular weights as did PR forms A and B crosslinked with the tritiated progestin R 5020. Thus, lambda cPR1 and lambda cPR2 fusion proteins contain epitopes present in both PR forms A and B. A cDNA clone, lambda cPR3, containing the inserts of both lambda cPR1 and lambda cPR2, was isolated from a randomly primed lambda gt10 oviduct cDNA library, indicating that both cDNA inserts were derived from the same oviduct mRNA. Additional evidence that these cDNAs correspond to PR mRNA was provided by sequencing the lambda cPR3 cDNA insert, since it was found to encode the sequence of three tryptic peptides prepared from purified PR form B. A fourth and a fifth cDNA clone, lambda cPR4 and lambda cPR5, were sequentially isolated from the same lambda gt10 cDNA library beginning with a probe derived from the 3' end of the lambda cPR3 insert. Partial DNA sequencing of lambda cPR4 and lambda cPR5 revealed the presence of a sequence coding for a cysteine-rich domain that is strikingly homologous to the amino acid sequences present in the putative DNA-binding domain of the human and chicken estrogen receptors, human glucocorticoid receptor, and v-erbA gene product of the avian erythroblastosis virus.

Amino Acid Sequence↗

PubMed1984

Extravascular CBG-like sites in rat kidney and mineralocorticoid receptor specificity.

To determine the intrarenal distribution of extravascular corticosteroid-binding globulin- (CBG) like sites, we measured the specific binding of [3H]corticosterone to supernatants prepared from papilla-inner medulla, outer medulla, and cortex from adrenalectomized rats. Renal papilla-inner medulla of exhaustively perfused kidneys contained 8-20 times higher concentrations of such sites than cortex or outer medulla. The ontogeny and steroid specificities of such sites were identical to those of plasma CBG; their molar concentration in the inner medulla-papilla was 4 times that of plasma CBG, consistent with local accumulation of CBG. Cytosols from all three zones bound [3H]aldosterone in the presence of RU-26988, a highly specific synthetic glucocorticoid, with high affinity (Kd 4 degrees C, 0.3-0.4 X 10(-9) M) and limited capacity (papilla-inner medulla, 23-34; outer medulla, 15-16; cortex, 12-17 fmol/mg). In papilla-inner medulla the apparent specificity of such [3H]aldosterone binding sites was aldosterone greater than corticosterone greater than dexamethasone, in contrast to aldosterone = corticosterone greater than dexamethasone in outer medulla and cortex. We propose that extravascular CBG confers mineralocorticoid specificity on the papilla-inner medulla [3H]aldosterone binding sites. In addition, given the higher levels of extravascular than intravascular CBG and the recurrent vascular architecture of the renal medulla-papilla, a countercurrent exchange model is proposed for renewable sequestration of corticosterone in the region.

Aldosterone↗

PubMed1983

Reliability of steroid hormone receptor assays: an international study.

The reproducibility of oestrogen and progestin receptor assays performed by laboratories participating in an international breast cancer treatment trial has been assessed. Three tissue reference powders containing low, medium and high oestrogen receptor levels (22 +/- 4, 88 +/- 7, 227 +/- 13 fmol/mg cytosol protein respectively) were prepared in Louisville, KY, U.S.A., assayed repeatedly and multiple samples of each shipped on solid CO2 to the coordination-distribution centre in Berne, Switzerland. Samples were dispatched from Berne to Cantons within Switzerland, to Yugoslavia, South Africa, Australia, New Zealand and also back to the United States for oestrogen and progestin receptor assays. Results were returned to Berne, Switzerland. There was a decrease in the levels of oestrogen and progestin receptors during the time of storage and transit. However, the ability to assign a powder to either the low, medium or high level of oestrogen receptor was not affected. Laboratories also determined progestin receptor. All laboratories clearly identified the powder containing the low level of progestin receptor, but there was poor quantitation with the other two assay standards. It is recommended that clinical hormone receptor laboratories, especially those participating in clinical trials, establish regular quality control procedures for both daily evaluation internally and periodic outside monitoring of interlaboratory variation.

Animals↗

PubMed1982

The mechanism of mineralocorticoid action of carbenoxolone.

The principal side effects of the drug carbenoxolone (Biogastrone; 18 beta-glycyrrhetinic acid sodium hemisuccinate) are sodium retention, hypokalemic alkalosis, suppressed plasma renin, and hypertension. In previous animal studies, carbenoxolone appeared not to have intrinsic mineralocorticoid activity but, rather, to enhance aldosterone action by displacing it from nonspecific binding sites. We here report studies showing that carbenoxolone has demonstrable affinity for rat kidney mineralocorticoid receptors, intrinsic mineralocorticoid activity in the adrenalectomized rat at doses consistent with its receptor affinity, and, in addition, a powerful action of amplifying the electrolyte effects of near-maximal doses of aldosterone.

Adrenalectomy↗

PubMed1981

Mineralocorticoid receptors in rat anterior pituitary: toward a redefinition of "mineralocorticoid hormone".

There is increasing evidence that a pituitary factor(s) in addition to ACTH can influence aldosterone secretion. By analogy with glucocorticoids and ACTH, such a factor(s) may be under mineralocorticoid negative feedback control, presumably via specific mineralocorticoid receptors. We here report studies in which mineralocorticoid receptors in the anterior pituitary are described and clearly distinguished from glucocorticoid receptors. Pituitary mineralocorticoid receptors closely resemble those previously reported in rat kidney and salivary gland in terms of their affinity for aldosterone, the hierarchy of affinities they display for other steroids, and their sedimentation values on sucrose density gradients.

Aldosterone↗

PubMed1981

Mineralocorticoid receptors in the rat lung.

In lung cytosol from adrenalectomized rats, tritiated aldosterone binds to two classes of limited capacity sites with affinities (Kd at 37 C) of 0.7 and 80 nM. The predominant binding species sediments at approximately 9S on sucrose density gradients in low ionic strength, molybdate-containing buffer. Binding of tritiated aldosterone (5 nM) is displaced by a 4-fold excess of nonradioactive hormone in the following hierarchy: 9 alpha-fluorocortisol greater than aldosterone greater than deoxycorticosterone much greater than corticosterone greater than dexamethasone. Thus, pulmonary tissue contains steroid-binding sites with affinity and specificity characteristics equivalent to those previously shown for mineralocorticoid receptors in classic target tissues. The pulmonary cell type(s) in which these putative mineralocorticoid receptors are located and possible physiological roles for mineralocorticoids in adult and fetal lung remain to be explored.

Adrenalectomy↗

PubMed1980

Cytosol oestradiol and progesterone receptors in endometrial hyperplasia and adenocarcinoma: determination by single saturating dose exchange assays.

Available oestradiol and progesterone binding site concentrations in endometrial cytosols were determined by simplified single saturating dose assay in 16 normal women with proliferative endometrium, in 10 patients with endometrial hyperplasia, and in 20 patients with endometrial adenocarcinoma. The mean oestrogen receptor level tended to be higher in endometrial hyperplasia than in either proliferative or carcinomatous endometria. Mean progesterone receptor levels were comparable in proliferative and hyperplastic endometria, but were sequentially lower in highly differentiated carcinoma, moderately differentiated carcinoma, and undifferentiated carcinoma. Correlation between results obtained by single saturating dose assays and by conventional Scatchard analysis in those endometria subjected to both procedures was high for both oestrogen (r = 0.998) and progesterone (r = 0.949) and indicates that with the simplified assays, large scale clinical studies on hormone sensitivity of endometrial adenocarcinomas are feasible.

Adenocarcinoma↗

PubMedDate not supplied

Expression of the 11beta-hydroxysteroid dehydrogenase type II enzyme in breast tumors and modulation of activity and cell growth in PMC42 cells.

Manipulating the metabolism of glucocorticoids may serve as a useful adjunct in the treatment of breast cancer. The 11beta-hydroxysteroid dehydrogenase type 2 enzyme (11betaHSD2) potently inactivates glucocorticoids thereby protecting the non-selective mineralocorticoid receptor (MR) in fluid transporting tissues. In the present study, Western blot analysis showed the presence of 11betaHSD2 in 66% of the breast tumor samples. The 11betaHSD2 and MR are also present in the breast tumor cell line PMC42. Glycyrrhetinic acid abolished glucocorticoid metabolism and inhibited cell growth by 40%, the latter at concentrations consistent with glucocorticoid receptor (GR) and MR binding studies. Metabolism was increased by glucocorticoids, the anti-glucocorticoid RU 38486 and anti-mineralocorticoid spironolactone, while aldosterone had no effect. Neither cortisol nor aldosterone affected cell proliferation, but both RU 38486 and spironolactone caused a significant decrease in cell number. The effects of RU 38486 were only observed at micromolar concentrations and are inconsistent with an action via GR or progesterone receptor (PR). This study shows that 11betaHSD2 activity and cell proliferation of PMC42 cells can be modulated via steroid receptors.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMedDate not supplied

The type I and type II 11beta-hydroxysteroid dehydrogenase enzymes.

Local tissue concentrations of glucocorticoids are modulated by the enzyme 11beta-hydroxysteroid dehydrogenase which interconverts cortisol and the inactive glucocorticoid cortisone in man, and corticosterone and 11-dehydrocorticosterone in rodents. The type I isoform (11beta-HSD1) is a bidirectional enzyme but acts predominantly as a oxidoreductase to form the active glucocorticoids cortisol or corticosterone, while the type II enzyme (11beta-HSD2) acts unidirectionally producing inactive 11-keto metabolites. There are no known clinical conditions associated with 11beta-HSD1 deficiency, but gene deletion experiments in the mouse indicate that this enzyme is important both for the maintenance of normal serum glucocorticoid levels, and in the activation of key hepatic gluconeogenic enzymes. Other important sites of action include omental fat, the ovary, brain and vasculature. Congenital defects in the 11beta-HSD2 enzyme have been shown to account for the syndrome of apparent mineralocorticoid excess (AME), a low renin severe form of hypertension resulting from the overstimulation of the non-selective mineralocorticoid receptor by cortisol in the distal tubule of the kidney. Inactivation of the 11beta-HSD2 gene in mice results in a phenotype with similar features to AME. In addition, these mice show high neonatal mortality associated with marked colonic distention, and remarkable hypertrophy and hyperplasia of the distal tubule epithelia. 11Beta-HSD2 also plays an important role in decreasing the exposure of the fetus to the high levels of maternal glucocorticoids. Recent work suggests a role for 11beta-HSD2 in non-mineralocorticoid target tissues where it would modulate glucocorticoid access to the glucocorticoid receptor, in invasive breast cancer and as a mechanism providing ligand for the putative 11-dehydrocorticosterone receptor. While previous homologies between members of the SCAD superfamily have been of the order of 20-30% phylogenetic analysis of a new branch of retinol dehydrogenases indicates identities of > 60% and overlapping substrate specificities. The availability of crystal structures of family members has allowed the mapping of conserved 11beta-HSD domains A-D to a cleft in the protein structure (cofactor binding domain), two parallel beta-sheets, and an alpha-helix (active site), respectively.

11-beta-Hydroxysteroid Dehydrogenases↗