Properties and ontogeny of the glucocorticoid receptor in the placenta and fetal lung of the sheep

1984 ◽  
Vol 103 (1) ◽  
pp. 31-42 ◽  
Author(s):  
A. P. F. Flint ◽  
R. D. Burton
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Gel Filtration ◽  
Fetal Lung ◽  
Maternal Circulation ◽  
Whole Cell ◽  
Cytosolic Receptor

ABSTRACT The cytosolic glucocorticoid receptor of ovine placental zona intima has been characterized and measured between day 51 of pregnancy and term, and levels compared with those in fetal lung. By ion-exchange and gel-filtration chromatography the molybdate-stabilized receptor was found to be an acidic molecule with Stokes radius approximately 8 nm; these physicochemical characteristics of the ovine placental receptor are comparable to those of receptors in glucocorticoid target tissues from non-ruminants. Concentrations of cytosolic receptor in placenta (mean, 139 fmol/mg protein) were lower than those in fetal lung (627 fmol/mg) at all stages of gestation investigated. To some extent this difference was accounted for by a twofold higher concentration of protein in placental cytosols compared with those from fetal lung. In both tissues, cytosolic receptor concentrations were maximal between days 91 and 130, when fetal adrenal steroid secretion is low; receptor concentrations decreased before term. Fetal hypophysectomy, which resulted in prolonged gestation, raised receptor concentrations in placenta, but not in fetal lung. In both tissues, apparent dissociation constants for [3H]dexamethasone binding to glucocorticoid receptors were in the range 0·5–7·1 nmol/l; these dissociation constants did not change consistently between day 100 and term. In whole-cell preparations of placenta and fetal lung incubated in vitro there was time-dependent specific binding of [3H]dexamethasone by nuclei, and binding of labelled cytosolic receptor to isolated nuclei occurred at all stages of gestation investigated. Binding of [3H]dexamethasone by cytosolic receptor from placenta and fetal lung was inhibited by progesterone and 17α-hydroxyprogesterone, as well as by cortisol, cortisone, 11-deoxycorticosterone and 11β-hydroxyprogesterone; 20α-hydroxyprogesterone and 17α,20α-dihydroxypregn-4-en-3-one were less effective. In experiments to evaluate the possible antagonistic action of progesterone in whole-cell preparations, uptake of [3H]dexamethasone by nuclei was increased up to twofold in placental minces incubated with aminoglutethimide or epostane, when progesterone synthesis was reduced by 98 and 92 per cent respectively. Nuclear uptake in minces of fetal lung was blocked by concentrations of progesterone found in placenta. The existence of a placental glucocorticoid receptor confirms that fetal cortisol may act directly on the placenta to induce the enzymatic changes controlling the onset of labour. Its availability early in pregnancy is consistent with the ability of administered glucocorticoid to induce labour at any time after day 90 of gestation. Progesterone in the placenta may act as a glucocorticoid antagonist, protecting the fetus against inappropriate induction of preterm labour resulting from high levels of glucocorticoids in the maternal circulation. J. Endocr. (1984) 103, 31–42

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

scholarly journals Clearance and binding of native and defucosylated lactoferrin

1983 ◽  
Vol 212 (2) ◽  
pp. 249-257 ◽  
Author(s):  
M J Imber ◽  
S V Pizzo
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Specific Binding ◽  
Gel Filtration ◽  
Peritoneal Macrophages ◽  
Mononuclear Phagocyte ◽  
Stable Complex

These studies explore the role of carbohydrate recognition systems and the direct involvement of terminal alpha 1-3-linked fucose in the clearance of lactoferrin from the murine circulation and in the specific binding of lactoferrin to receptors on murine peritoneal macrophages. As previously reported, radiolabelled lactoferrin cleared very rapidly (t1/2 less than 1 min) after intravenous injection into mice. However, competing levels of ligands specific for the hepatic galactose receptor (asialo-orosomucoid), the hepatic fucose receptor (fucosyl-bovine serum albumin), and the mononuclear-phagocyte system pathway recognizing mannose, N-acetylglucosamine and fucose (mannosyl-, N-acetylglucosaminyl- and fucosyl-bovine serum albumin) did not block radiolabelled lactoferrin clearance in vivo or binding to mouse peritoneal macrophage monolayers in vitro. Almond emulsin alpha 1-3-fucosidase was used to prepare defucosylated lactoferrin in which 88% of the alpha 1-3-linked fucose was hydrolysed. No difference in clearance or receptor binding was observed between radiolabelled native and defucosylated lactoferrin. Fucoidin, a fucose-rich algal polysaccharide, completely inhibits the clearance in vivo and macrophage binding in vitro of lactoferrin. This effect, however, is probably not the result of competition for binding to the fucose receptor, since gel-filtration studies demonstrated formation of a stable complex between lactoferrin and fucoidin. The present results indicate that the lactoferrin-clearance pathway is distinct from several pathways mediating glycoprotein clearance through recognition of terminal galactose, fucose, N-acetylglucosamine or mannose. Furthermore, alpha 1-3-linked fucose on lactoferrin is not essential for lactoferrin clearance in vivo or specific binding to macrophage receptors in vitro.

The glucocorticoid receptor binds to a sequence overlapping the TATA box of the human osteocalcin promoter: a potential mechanism for negative regulation

1991 ◽  
Vol 11 (6) ◽  
pp. 3379-3383
Author(s):  
P E Strömstedt ◽  
L Poellinger ◽  
J A Gustafsson ◽  
J Carlstedt-Duke
Keyword(s):  
Glucocorticoid Receptor ◽  
Specific Binding ◽  
Tata Box ◽  
Receptor Interaction ◽  
Receptor Binding Site ◽  
Osteocalcin Gene ◽  
Close Proximity ◽  
Human Osteocalcin

Expression of the human osteocalcin promoter is negatively regulated by glucocorticoids in vivo. In vitro DNase I and exonuclease III footprinting analysis showed binding of purified glucocorticoid receptor in close proximity to and overlapping with the TATA box of the osteocalcin gene. These results imply competition or interference with binding of the TATA box-binding transcription factor IID as a mechanism of repression of this gene by glucocorticoids. In support of this notion, point mutation analysis of the receptor binding site indicated that flanking nucleotides and not the TATA box motif per se were important for receptor interaction. Moreover, DNA binding competition assays showed specific binding of the receptor only to the TATA box region of the osteocalcin gene and not to the corresponding region of an immunoglobulin heavy-chain promoter.

scholarly journals Clinical implications of glucocorticoid receptor studies in childhood acute lymphoblastic leukemia

Blood ◽  
1980 ◽  
Vol 56 (6) ◽  
pp. 1036-1040 ◽  
Author(s):  
R Mastrangelo ◽  
R Malandrino ◽  
R Riccardi ◽  
P Longo ◽  
FO Ranelletti ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Lymphoblastic Leukemia ◽  
Poor Response ◽  
The Other ◽  
Clinical Implications ◽  
Short Term

Abstract We have performed in parallel, in 19 children with acute lymphoblastic leukemia, a quantitative determination of glucocorticoid levels, in vitro steroid induced inhibition of nucleic acid precursors, and a short-term clinical trial of corticosteroids alone, before the treatment was given, which included corticosteroids and other drugs. From our results it appears that high glucocorticoid receptor levels in acute lymphoblastic leukemia of children do not guarantee a clinical response to corticosteroids. On the other hand, glucocorticoid receptors may turn out to be of value in predicting a poor response to corticosteroids only if their levels are considerably low.

scholarly journals Clinical implications of glucocorticoid receptor studies in childhood acute lymphoblastic leukemia

Blood ◽  
1980 ◽  
Vol 56 (6) ◽  
pp. 1036-1040 ◽  
Author(s):  
R Mastrangelo ◽  
R Malandrino ◽  
R Riccardi ◽  
P Longo ◽  
FO Ranelletti ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Lymphoblastic Leukemia ◽  
Poor Response ◽  
The Other ◽  
Clinical Implications ◽  
Short Term

We have performed in parallel, in 19 children with acute lymphoblastic leukemia, a quantitative determination of glucocorticoid levels, in vitro steroid induced inhibition of nucleic acid precursors, and a short-term clinical trial of corticosteroids alone, before the treatment was given, which included corticosteroids and other drugs. From our results it appears that high glucocorticoid receptor levels in acute lymphoblastic leukemia of children do not guarantee a clinical response to corticosteroids. On the other hand, glucocorticoid receptors may turn out to be of value in predicting a poor response to corticosteroids only if their levels are considerably low.

Interaction of β2-Glycoprotein-I with Human Blood Platelets: Influence Upon the ADP-Induced Aggregation

1985 ◽  
Vol 54 (02) ◽  
pp. 397-401 ◽  
Author(s):  
Johannes Nimpf ◽  
Helmut Wurm ◽  
Gerhard M Kostner
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Human Platelets ◽  
Glycoprotein I ◽  
Unspecific Binding ◽  
Induced Aggregation ◽  
Human Blood Platelets

SummaryThe interaction of β2-glycoprotein-I (β2-G-I), a plasma constituent of unknown function, with blood platelets was studied. The following results were obtained: 1) β2-G-I binds to washed human platelets isolated by centrifugation (WP) at one kind of specific, saturable binding sites. The dissociation constant was found to be approx. 1 × 10−6M.2) In the presence of physiological concentrations of Ca++ (2.5 mM), this specific binding is markedly reduced. Unspecific binding of β2-G-I to platelets, however, is not influenced by Ca++.3) Platelets prepared by gel filtration (GFP), differing in their in vitro aggregability from WP, exhibit no specific binding of β2-G-I. Binding to GFP is also not induced by activation with thrombin, collagen or ADP.4) β2-G-I causes significant alteration of the ADP-induced aggregation of GFP. Aggregation induced by thrombin, collagen, arachidonic acid or PAF-acether, however is not altered by β2G-I.It is suggested, that pelleting during centrifugation causes irreversible rearrangements in the membrane of platelets.

scholarly journals Partial purification of a protein from maize (Zea mays) coleoptile membranes binding the Ca2+-channel antagonist verapamil

1989 ◽  
Vol 257 (1) ◽  
pp. 95-100 ◽  
Author(s):  
H J Harvey ◽  
M A Venis ◽  
A J Trewavas
Keyword(s):  
Zea Mays ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Electrophoretic Analysis ◽  
Partial Purification ◽  
Single Class ◽  
Maize Coleoptile ◽  
Final Purification Step

A protein that binds the calcium-channel antagonist verapamil has been partially purified from maize (Zea mays) coleoptile membranes. The protein was solubilized with the detergent CHAPS ([ 3-(3-cholamidopropyl)dimethylammonio]propane-1-sulphonate) and purified by a combination of ion-exchange, gel-filtration and hydrophobic-interaction chromatography. This resulted in a 120-fold purification. SDS/polyacrylamide-gel-electrophoretic analysis of the polypeptides from the final purification step indicated that the verapamil-binding protein may have a major component of Mr 169,000. The dissociation constants for specific binding of [3H]verapamil to crude and CHAPS-solubilized maize coleoptile membrane fractions are 72 nM and 158 nM respectively, with respective binding-site concentrations of 135 pmol/mg of protein and 78 pmol/mg of protein. In both cases the Scatchard plots are linear, indicating a single class of binding sites. [3H]Verapamil binding to crude maize coleoptile membrane fractions could not be displaced by unlabelled desmethoxyverapamil or by nifedipine, but could be displaced by unlabelled methoxyverapamil.

scholarly journals Epididymal glucocorticoid receptors promote intergenerational transmission of paternal stress

2018 ◽  
Author(s):  
Jennifer C Chan ◽  
Bridget M Nugent ◽  
Kathleen E Morrison ◽  
Eldin Jašarević ◽  
Natarajan V Bhanu ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Intergenerational Transmission ◽  
Noncoding Rna ◽  
Glucocorticoid Receptors ◽  
Drugs Of Abuse ◽  
Disease Risk ◽  
Glucocorticoid Treatment ◽  
Paternal Stress

AbstractPaternal preconception exposures and insults, including stress, dietary challenge and drugs of abuse, can shape offspring health and disease risk outcomes, as evidenced from retrospective human studies and more recent animal models1–16. Mechanistic examination has implicated small noncoding RNA populations in sperm, including microRNA (miRs), as carriers of paternal environmental information that consequently influence offspring development15,17–21. However, the cellular mechanisms by which these paternal signals are relayed to sperm and how they may persist remain unknown. Here, using our previously established paternal stress mouse model we identify caput epididymal epithelial glucocorticoid receptors as crucial upstream mediators of long-lasting germ cell programming. We show that glucocorticoid treatment of caput epididymal epithelial cells results in increased glucocorticoid receptor levels and enduring changes to the miR content of secreted extracellular vesicles (EVs), or epididymosomes, known to interact with sperm and alter their RNA content22,23. Further, significant changes were detected in the caput epididymal histone code long after stress ended, both in vitro and in vivo, as a potential mechanism whereby stress programmed enduring changes to EV miRs. Genetic targeting to reduce caput epididymal epithelial-specific glucocorticoid receptors reversed stress-induced chromatin remodeling and promoted cellular resilience to paternal stress, ultimately rescuing transmission of a stress dysregulated offspring phenotype. Taken together, these studies identify glucocorticoid receptor regulation of EV miRs in the caput epididymis as a key contributor in the intergenerational transmission of paternal environmental stress experiences.

scholarly journals The glucocorticoid receptor binds to a sequence overlapping the TATA box of the human osteocalcin promoter: a potential mechanism for negative regulation.

1991 ◽  
Vol 11 (6) ◽  
pp. 3379-3383 ◽  
Author(s):  
P E Strömstedt ◽  
L Poellinger ◽  
J A Gustafsson ◽  
J Carlstedt-Duke
Keyword(s):  
Glucocorticoid Receptor ◽  
Specific Binding ◽  
Tata Box ◽  
Receptor Interaction ◽  
Receptor Binding Site ◽  
Osteocalcin Gene ◽  
Close Proximity ◽  
Human Osteocalcin

Expression of the human osteocalcin promoter is negatively regulated by glucocorticoids in vivo. In vitro DNase I and exonuclease III footprinting analysis showed binding of purified glucocorticoid receptor in close proximity to and overlapping with the TATA box of the osteocalcin gene. These results imply competition or interference with binding of the TATA box-binding transcription factor IID as a mechanism of repression of this gene by glucocorticoids. In support of this notion, point mutation analysis of the receptor binding site indicated that flanking nucleotides and not the TATA box motif per se were important for receptor interaction. Moreover, DNA binding competition assays showed specific binding of the receptor only to the TATA box region of the osteocalcin gene and not to the corresponding region of an immunoglobulin heavy-chain promoter.

A detailed investigation of the cytoplasmic cortisol-binding receptor of North American eel (Anguilla rostrata) tissues

1984 ◽  
Vol 62 (10) ◽  
pp. 991-997 ◽  
Author(s):  
John A. DiBattista ◽  
A. Z. Mehdi ◽  
Thomas Sandor
Keyword(s):  
Glucocorticoid Receptor ◽  
North American ◽  
Glucocorticoid Receptors ◽  
Thermodynamic Characteristics ◽  
Relative Mass ◽  
Anguilla Rostrata ◽  
Hydroxyl Groups ◽  
American Eel ◽  
Receptor Complexes

The in vitro binding of tritiated cortisol to ammonium sulfate precipitate (35% saturation) prepared from the gill and gut mucosal cytosol of the North American eel (Anguilla rostrata) was investigated. The sodium molybdate stabilized cytoplasmic preparations bound tritiated cortisol with the following parameters: gill, equilibrium dissociation constant (KD) = 3.7 ± 0.4 nM, (± SEM; n = 4), the maximum concentration of binding sites (Nmax) = 294 ± 26 fmol/mg protein; gut, KD = 5.2 ± 0.4 nM, Nmax = 1085 ± 288 fmol/mg protein. The [3H]cortisol–receptor complexes sedimented on linear (16–41% w/v) glycerol density gradients in single peaks at 6.7S–7.0S or 3.0S–3.6S in hypo- or hyper-tonic (± 0.4 M KCl) gradients, respectively. Sephacryl S-300 column chromatography of the hormone–receptor complex yielded the following hydrodynamic parameters: gill, relative mass (Mr) = 292 000 daltons, Stokes radius (Rs) = 78.7 Å(1 Å = 0.1 nm), frictional ratio (f/f0) = 1.79; gut, Mr = 242 000 daltons, Rs = 68.8 Å, f/f0 = 1.66. Competition studies revealed the following competitive hierarchies of radioinert steroids vis-à-vis the inhibition of [3H]cortisol binding to the receptor with both tissues: cortisol > 11-deoxycortisol > 21-deoxycortisol > 17α-hydroxyprogesterone [Formula: see text] corticosterone [Formula: see text] 11-deoxycorticosterone > 11β-hydroxyprogesterone. Aldosterone, cortisone, progesterone, or promegestone (R5020) hardly competed. These findings underline the importance of the C-17, C-21, and C-11 hydroxyl groups in receptor binding. Hydroxylation of progesterone in positions C-17, C-21, and C-11 contributed free energy changes (ΔG) of −5.8 to −6.2, −3.1 to −3.9, and −1.3 kJ/mol, respectively, to the binding of steroids to the eel glucocorticoid receptor. From these data we conclude that the piscine cortisol receptor is different from other vertebrate glucocorticoid receptors because of its physical and thermodynamic characteristics and its function in mediating electrolyte homeostatic action of a typical glucocorticoid in the transport epithelia. It is conceivable that the fish glucocorticoid receptor is an ancestral form of the glucocorticoid and (or) mineralocorticoid receptors of vertebrates.

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