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.

scholarly journals Characterization of a monoclonal antibody that probes the functional domains of the glucocorticoid receptor

1987 ◽  
Vol 246 (1) ◽  
pp. 55-65 ◽  
Author(s):  
N M Robertson ◽  
W F Kusmik ◽  
B F Grove ◽  
A Miller-Diener ◽  
M L Webb ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Functional Domain ◽  
Cytoplasmic Staining ◽  
Receptor Complexes ◽  
Specificity And Sensitivity

Monoclonal antibodies to the rat hepatic glucocorticoid receptor (GR) were produced by using 4000-fold-purified unactivated rat hepatic GR as the immunogen in an immunization in vitro. Hybridomas were screened for anti-GR antibody production by using an enzyme-linked immunosorbent assay. The antibody, 3A6, described here, is an IgM (lambda). The interaction of 3A6 with the purified GR was explored by sedimentation analysis, where a shift of the 9 S GR to a form with a higher s20,w value was demonstrated. Binding specificity and sensitivity were demonstrated by protein immunoblotting. 3A6 cross-reacted with all rat tissue glucocorticoid receptors (GRs) examined, except those of the brain. Species cross-reactivity was observed with other mammalian GRs (from human CEM-C7 cells and from pig and mouse liver). Immunocytochemical localization of the GR was assessed by indirect immunofluorescence in intact fixed cells, which demonstrated intense cytoplasmic staining in the absence of pretreatment with glucocorticoids and nuclear localization when cells were pretreated with glucocorticoids. This monoclonal antibody significantly inhibited steroid binding to unoccupied receptor and DNA binding of activated steroid-receptor complexes. Furthermore, preincubation of the purified activated GR complex with 3A6 prevented phosphorylation of the GR in vitro. Thus 3A6 differs from previous monoclonal antibodies to the GR in its capacity to cross-react with the human GR and by its specificity for an epitope on or near a functional domain of the GR.

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.

Glucocorticoid-receptor complexes are associated with small RNA in vitro

1989 ◽  
Vol 32 (5) ◽  
pp. 633-642 ◽  
Author(s):  
Gian Paolo Rossini ◽  
Ann-Charlotte Wikström ◽  
Jan-Åke Gustafsson
Keyword(s):  
Glucocorticoid Receptor ◽  
Small Rna ◽  
Receptor Complexes

Artificial maturation, fertilization, and early development of the American eel (Anguilla rostrata)

2010 ◽  
Vol 88 (11) ◽  
pp. 1121-1128 ◽  
Author(s):  
K. Oliveira ◽  
W. E. Hable
Keyword(s):  
Early Development ◽  
Anguilla Rostrata ◽  
Sargasso Sea ◽  
American Eel ◽  
Important Species ◽  
Vitro Fertilization ◽  
Commercially Important ◽  
Elusive Species ◽  
American Eels

Spawning for the American eel ( Anguilla rostrata (Le Sueur, 1817)) takes place in secretive locations within the Sargasso Sea, which has thus far prevented investigations of gametogenesis and early development in this ecologically and commercially important species. Attempts to induce maturation and reproduction in this species have been few and have produced limited results, with a single report of the production of gastrula-stage embryos. Here we report the successful maturation of female American eels. Maturation occurred within 13 weeks and ovulation was induced with a single injection of 17α,20β-dihydroxy-4-pregnen-3-one (DHP). Following in vitro fertilization, embryogenesis through hatching was observed and larvae were maintained for up to 6 days. We show that a crucial factor for successful fertilization is the stage of the oocyte at the time of induced ovulation. Oocytes that had not reached the migratory nucleus stage, or had passed this stage, were not successfully fertilized. These findings demonstrate that American eel can reproduce in the laboratory and previously untestable hypotheses pertaining to the developmental biology of this elusive species can now be explored.

Steroid hormone stimulation of Na+ transport in A6 cells is mediated via glucocorticoid receptors

1993 ◽  
Vol 264 (4) ◽  
pp. C875-C884 ◽  
Author(s):  
T. J. Schmidt ◽  
R. F. Husted ◽  
J. B. Stokes
Keyword(s):  
Cell Line ◽  
Glucocorticoid Receptors ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Mineralocorticoid Receptors ◽  
Na Transport ◽  
Receptor Complexes ◽  
Binding Forms ◽  
Mineralocorticoid Antagonists

The A6 cell line derived from the toad kidney forms polarized, highly differentiated epithelial monolayers in culture and has been utilized as an experimental model for studying regulation of transepithelial Na+ transport by aldosterone. In the present study we evaluated the specific role(s) of glucocorticoid and mineralocorticoid receptors in mediating this enhanced electrogenic Na+ transport, which was measured experimentally as an increase in short-circuit current (Isc). Our data demonstrate that specific glucocorticoid agonists (100 nM), including RU 28362 and RU 26988, elicit “mineralocorticoid-like” increases in Isc that are blocked by the glucocorticoid antagonist RU 38486 but are unaffected by mineralocorticoid antagonists including RU 28318 and RU 26752. The stimulatory effects of aldosterone (100 nM) were also blocked by RU 38486 and not by mineralocorticoid antagonists. These data extend earlier studies suggesting that in this cell line aldosterone mediates its physiological effects via binding with relatively low affinity (dissociation constant Kd congruent to 25-50 nM) to glucocorticoid receptors, despite the presence of apparently normal mineralocorticoid receptors. Our in vitro biochemical studies also demonstrate that A6 glucocorticoid receptor complexes can be thermally activated or transformed to DNA binding forms which exhibitaltered elution profiles from anion-exchange resins. Thus, based on several criteria, these amphibian glucocorticoid receptors appear very similar to classical mammalian receptors and are capable of mediating all of the stimulatory effects of aldosterone on net Na+ transport.

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.

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

Description and first occurrence of Myxidium zealandicum (Protozoa: Myxosporidia) in the North American eel Anguilla rostrata LeSueur

1977 ◽  
Vol 55 (1) ◽  
pp. 52-59 ◽  
Author(s):  
M. P. Komourdjian ◽  
W. C. Hulbert ◽  
J. C. Fenwick ◽  
T. W. Moon
Keyword(s):  
North American ◽  
Kidney Tissue ◽  
Quebec City ◽  
Anguilla Rostrata ◽  
Fast Green ◽  
American Eel ◽  
The North ◽  
First Occurrence ◽  
Scanning Electron ◽  
St Lawrence River

Myxidium zealandicum Hine, 1975 is described from gills and kidney of the North American eel Anguilla rostrata collected from the St. Lawrence River near Quebec City and Cornwall. Cysts of M. zealandicum on gills measured up to 1 by 2 mm and in kidneys up to 15 by 20 mm. In addition to single spherical cysts, several polymorphous forms were also observed on the gills. Polymorphous cysts were not found in the kidney. Different stages of spore development were evident in gill cysts and were differentiated by means of a lead hematoxylin – fast green stain. Number and pattern of spore striations were examined by scanning electron microscopy and were highly variable. The invasion of the parasite into kidney tissue appeared to result in less physiological damage to the host than did gill invasion. The existence of this parasite, previously found in eel species in New Zealand, in a North American eel species is discussed.

Sign in / Sign up

Export Citation Format

Share Document