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P Formstecher

Publications and source records attributed to P Formstecher.

At least 73 records · Page 4Linked to original sources
PubMed1987

Microheterogeneity of agonist and antagonist glucocorticoid receptor complexes detected by isoelectric focusing and modifications induced by receptor activation.

Rat-liver glucocorticoid receptor was incubated with either [3H]triamcinolone acetonide or [3H]RU 486, a well known antiglucocorticoid. Once formed, the steroid-receptor complexes were analyzed by isoelectric focusing in agarose gel slabs. A careful slicing of the receptor tracks revealed the presence of three distinct radioactive peaks focused at the following pI values: 5.3 +/- 0.2 (n = 17) and 4.4 +/- 0.1 (n = 17). All these peaks correspond with receptor isoforms as suggested by control experiments. The receptor state was analyzed after focusing by a chromatographic assay on DNA-cellulose, DEAE-trisacryl and hydroxyapatite minicolumns. The peak of pI 4.4 apparently corresponded to the non-transformed receptor and was greatly stabilized in the presence of RU 486, whereas the peaks of pI 4.8 and 5.3 were probably made of transformed receptor and meroreceptor. These results were confirmed by autoradiographic studies after isoelectric focusing of receptor molecules covalently labelled with [3H]dexamethasone mesylate. Thus, the rat-liver glucocorticoid receptor appeared to be a rather acidic protein which became less acidic after transformation by heat, displaying a pI shift which was strongly reduced in case of steroid-receptor complexes formed with the antiglucocorticoid RU 486.

Animals↗
PubMed1987

Approach to the molecular mechanisms of the modulation of growth hormone gene expression by glucocorticoid and thyroid hormones.

Glucocorticoid and thyroid hormones modulate the expression of the growth hormone gene. To investigate this control mechanism, we have determined whether this gene contains sites that bind the human glucocorticoid and thyroid hormone receptors in vitro. To do so, we have designed a novel assay for studying binding of the purified glucocorticoid receptor to cloned fragments of the human growth hormone (hGH) gene, and have adapted a DNA-competition assay for the thyroid receptor in nuclear cell extracts. Two glucocorticoid receptor binding regions were found in the hGH gene, one of high affinity in a fragment of the gene containing the first intron, and one of low affinity located within a 290 bp-fragment of 5'-flanking DNA. In contrast, the thyroid receptor bound with high affinity to the 5'-flanking fragment. Homologous binding regions for the two types of receptor were found in the human placental lactogen (chronic somatomammotropin) gene. DNA binding of the two receptor types appeared to depend on the presence of the hormone, yet antagonist-bound glucocorticoid receptor was still capable of interacting specifically with DNA. There was no evidence for synergism or antagonism of the two receptor types in binding to their respective sites on the hGH gene. The data also make it unlikely that the thyroid receptor negatively controls gene transcription and that the stimulatory effect of thyroid hormone results from a derepression mechanism.

Animals↗
PubMed1987

[Preparation of an anti-idiotypic antibody directed against anti-dexamethasone antibody and demonstration of its ability to acknowledge the receptor of glucocorticoids].

By using a 3-carboxymethyloxime dexamethasone derivative coupled to bovine serum albumin we have prepared specific anti-dexamethasone antibodies in rabbits. These antibodies were then purified by affinity chromatography and administered to a second set of rabbits. One of them produced anti-idiotypic antibodies able to impede the [3H] dexamethasone binding of the initial anti-dexamethasone antibodies and to displace the [3H] dexamethasone-antibodies complexes towards high molecular weight species in gel filtration experiments. Moreover these antibodies we were also able to impede the binding of [3H] dexamethasone to the rat liver glucocorticoid receptor and to recognize the highly purified receptor using Western blot analysis.

Animals↗
PubMed1986

Rapid purification of antisteroid antibodies by high-performance liquid affinity chromatography.

An adsorbent for the high-performance affinity chromatography of antisteroid antibodies was prepared, based on a commercial pre-packed column. The column contained activated microparticulate silica beads bearing epoxide functions, on which the steroid dexamethasone was covalently linked. The column was used successfully for the rapid and complete isolation of several hundred microgram amounts of specific antidexamethasone antibodies from rabbit antisera. The practical aspects of the purification procedure, especially the optimization of the washing and of the elution steps, are detailed. Despite non-biospecific elution with 20% acetonitrile in an acidic buffer, the purification yield was very satisfactory and the biological activity of the purified immunoglobulins appeared excellent.

Antibodies↗
PubMed1986

Physical characterization of the activated and non-activated forms of the glucocorticoid-receptor complex bound to the steroid antagonist [3H]RU 486.

We have compared the physicochemical characteristics of the non-activated (molybdate stabilized) glucocorticoid-receptor complex bound either to [3H]triamcinolone acetonide or to the antagonist [3H]RU 486 with those of the activated (25 degrees C preheated) complexes. The level of activation was measured by a DNA cellulose assay. The physicochemical features of the steroid-receptor complexes were analyzed by various techniques including high-performance size exclusion chromatography, sucrose gradient sedimentation and high-performance DEAE ion exchange chromatography. All the buffers used during the analytical procedures contained sodium molybdate in order to prevent any dissociation of the steroid-receptor complex. The non-activated glucocorticoid receptor bound either to [3H]TA or to [3H]RU 486 sedimented at 9.3S on sucrose gradient, displayed at 70 A Stokes radius in high performance size exclusion chromatography on TSK G4000 SW column, and was eluted at 0.22-0.28 M KCl by anion-exchange chromatography on a DEAE 545 column. After activation the Stokes radius and the sedimentation coefficient declined to 50 A and 4.5S respectively, and the complex was eluted by 0.10-0.12 M KCl on the ion-exchange column. No qualitative difference could be detected between the characteristics of the glucocorticoid-receptor complexes bound either to [3H]TA or to [3H]RU 486. Moreover, the relative distribution of "non-activated" and "activated" forms of the glucocorticoid receptor obtained through physicochemical experiments was highly correlated with the activation level determined by DNA binding experiments. However the activation level of [3H]RU 486-glucocorticoid-receptor complex appeared markedly decreased (15%) when compared with that of the agonist [3H]TA-receptor complex (65%). Experiments done with an antagonist steroid of the 17 beta-carboxamide series of dexamethasone exhibit the same results with an activation level of 12% as compared with triamcinolone acetonide. Thus, two antihormones which have different structures show the same behavior towards the glucocorticoid-receptor complex by strikingly reducing both the activation process and the size reduction of the steroid-receptor complex which is concomitant to activation and which could constitute the first step of this process.

Animals↗
PubMed1985

Characterization of the purified molybdate-stabilized glucocorticoid receptor from rat liver. An in vitro transformable complex.

Rat liver glucocorticoid receptor was purified in the presence of molybdate by a three-step procedure comprising protamine sulfate precipitation, affinity chromatography on a dexamethasone matrix and high-performance size-exclusion chromatography (HPSEC) on a TSK G 3000 SW column. The [3H]triamcinolone-acetonide-receptor complex was obtained in 20% yield with an overall 11 800-fold purification. The dissociation rate constant of this complex was 1.6 X 10(-4) min-1. The purified receptor sedimented at 8.3 S in high-salt and 9.4 S in low-salt sucrose gradients containing molybdate. A 7.0-nm Stokes radius was determined by HPSEC on a TSK G 4000 column in high-salt buffer. The calculated Mr was 278000. Dodecyl sulfate/polyacrylamide gel electrophoresis revealed an almost homogeneous 90 000-Mr band. Three minor bands with Mr of 78 000, 72 000 and 48 000 were also inconstantly seen. An apparent pI = 5.1 was observed for the [3H]steroid complex by isoelectric focusing in agarose gel. Furthermore high-performance ion-exchange chromatography of the purified complex on a DEAE 545 LKB column (DEAE HPLC) yielded a sharp peak eluted at a 315 mM potassium ion concentration. This peak was shown to contain almost all the 90 000-Mr protein. Moreover the purified receptor complex appeared to be transformable to a DNA-binding state after molybdate removal followed by warming 30 min at 25 degrees C in presence of 0.2% bovine serum albumin: 50-78% transformation yield could be demonstrated by DNA-cellulose chromatography. Partial transformation could also be obtained at 0 degrees C in the absence of any added protein and was followed by DEAE HPLC. The transformed complex was eluted by 180 mM potassium.

Animals↗
PubMed1985

Covalent chromatography by thiol-disulfide interchange of the highly-purified non-transformed rat liver glucocorticoid-receptor.

Highly-purified non-transformed rat liver [3H]triamcinolone acetonide-receptor complex was shown to be covalently adsorbed on activated thiol sepharose 4B, a reactive sulfhydryl matrice. Elution by mercaptoethanol in excess and inhibition of binding by previous treatment of the complex with N-ethylmaleimide clearly demonstrated the specificity of the binding by thiol disulfide interchange. The transformed [3H]triamcinolone acetonide-receptor complex, partially purified by DNA-cellulose chromatography, was also retained on activated thiol sepharose 4B. The physicochemical characteristics of both the transformed and non-transformed glucocorticoid receptor complexes eluted from the covalent chromatography column were studied by HP size exclusion chromatography on a TSK G 3000 SW column and were found to be identical to those of the starting complexes. These results provide direct evidence for accessible sulfhydryl groups on the glucocorticoid receptor complex surface, probably distinct from the steroid binding essential sulfhydryl group.

Animals↗
PubMed1985

Nontransformed rabbit liver glucocorticoid receptor: purification, characterization and transformation.

The molybdate-stabilized nontransformed form of the glucocorticoid receptor from rabbit liver has been purified approximately 8,000-fold by a three-step procedure. The first step involved protamine sulfate precipitation which allowed a 5-6-fold purification with 85% yield. The second step, affinity chromatography using a N-(12-dodecyl-amino) 9 alpha-fluoro-16 alpha-methyl-11 beta, 17 alpha-dihydroxy-3-oxo-1,4-androstadiene-17 beta-carboxamide substituted Sepharose gel, purified the receptor 1,500-2,000-fold as calculated by specific radioactivity. The third step involved high performance liquid chromatography resulting in overall purification near 8,000-fold. The final glucocorticoid receptor appeared about 60% pure. The purified nontransformed glucocorticoid receptor had a sedimentation coefficient of 9 S in 0.16 M phosphate containing 5-20% sucrose gradients and the Stokes radius was 6.1-6.3 nm as determined by low pressure gel filtration and HPLC. Binding specificity of the purified receptor was identical to that previously reported in crude rabbit liver cytosol. Isoelectricfocusing and ion-exchange chromatography showed that the purification procedure affected the net charge of the receptor protein. This phenomenon could be related to interactions between the glucocorticoid receptor and cytosolic factors. SDS polyacrylamide gel electrophoresis showed a major Mr = 94,000 protein band which is in good agreement with previously reported values for glucocorticoid receptors. Transformation of the purified receptor was achieved after removal of molybdate by exposure at 25 degrees C to 0.4 M KCl. Characterization of the molecular forms was performed by means of incorporation into isolated nuclei, affinity towards polyanionic exchangers and high pressure size exclusion chromatography. Results show that about 40% of the receptor is in the transformed state.

Adrenalectomy↗
PubMed1985

Binding of the human glucocorticoid receptor to defined regions in the human growth hormone and placental lactogen genes.

An in vitro competition assay was used to investigate whether binding sites for the human glucocorticoid receptor occur in the human genes for growth hormone (hGH) and placental lactogen (chorionic somatomammotropin, hCS). These genes display 95% sequence homology. Two receptor-binding regions were found in the hGH gene, one of which is located within 290 bp upstream, and one within 251 bp downstream from the transcription initiation site. Two binding regions homologous to those in the hGH gene were found in the hCS gene. The receptor-binding DNA fragment from the structural part of the genes, but not that from their promoter area, contained a sequence homologous to a 15-bp consensus sequence proposed earlier for the glucocorticoid receptor binding site. It is unlikely that the putative difference in glucocorticoid sensitivity between the hGH and hCS genes is accounted for by major differences in glucocorticoid receptor binding pattern.

Base Sequence↗
PubMed1984

Inactivation of unbound rat liver glucocorticoid receptor by N-alkylmaleimides at sub-zero temperatures.

A series of N-alkylmaleimides was shown to inactivate effectively the rat liver glucocorticoid receptor at neutral pH. A partial purification of the unbound cytosolic receptor by protamine sulfate precipitation and a careful stabilization of the essential thiol by dithiothreitol and sodium molybdate before the alkylation step appeared essential to obtain pseudo-first-order kinetics. Moreover, performing the experiment at -12 degrees C in buffer containing 40% glycerol as antifreeze agent resulted in increased receptor stabilization and a slowing-down of the inactivation process, which could then be more accurately studied. This process was demonstrated to be dose- and pH-dependent in the case of N-ethyl- and N-nonylmaleimides. Furthermore, comparison of the various N-alkylmaleimides revealed a striking increase of receptor inactivation with increasing chain length of the maleimide derivative. Full protection against inactivation was afforded by previous [3H]dexamethasone binding on the receptor. Long-chain N-alkylmaleimides inactivated by beta-mercaptoethanol were still able to inhibit the [3H]-dexamethasone binding noncovalently. Likewise N-nonylsuccinimide was shown to compete with [3H]dexamethasone for receptor binding. It is suggested that the chain length effect observed in the inactivation process is related to nonpolar interactions in the binding of maleimides to the receptor prior to the irreversible alkylation of sulfhydryl groups. These groups lie in a hydrophobic environment, probably in the steroid binding site itself.

Animals↗
PubMed1982

Progesterone receptor from chick oviduct: purification of molybdate-stabilized form and preliminary characterization.

A molydate-stabilized, 'non-activated' form of the progesterone receptor from the cytosol of oestrogen-stimulated chick oviduct has been purified to homogeneity by a three-step procedure. The first step, affinity chromatography using a N-(12-amino-dodecyl)-3-oxo-4-androsten-17 beta-carboxamide-substituted Sepharose gel, purified the receptor 1500-2700-fold with approximately equal to 50% recovery. In the second step, ion-exchange chromatography through a DEAE-cellulose column, progesterone receptor was eluted as a single peak at 0.1 M KCl. Purification after this step was greater than 6700-fold. The third step was filtration through Ultrogel AcA 34, resulting in overall purification approximately equal to 7400-fold with overall recovery approximately equal to 25% of pure receptor on the basis of 1 binding site/molecule of Mr 85000. The purified molybdate-stabilized receptor had a sedimentation coefficient approximately equal to 7.9S +/- 0.1 (n = 4) in 0.15 M or 0.4 M KCl containing sucrose 5-20% gradient and approximately equal to 8.9S +/- 0.2 (n = 6) in 0.15 M KCl containing glycerol 10-35% gradient, and its Stokes radius was 7.05 +/- 0.10 nm (n = 3) (calculated Mr between 240000 and 280000). Binding specificity of the purified receptor was the same as that found in crude cytosol. SDS-PAGE revealed a single band migrating as a polypeptide of Mr approximately equal to 85000 +/- 2300 (n = 9). PAGE under non-denaturing conditions at total acrylamide concentrations 5%, 7% and 9% showed a single [3H]ORG 2058-protein band (ORG 2058 is a high-affinity analogue more suitable than progesterone for electrophoretic studies). The data suggest that the high molecular weight molybdate-stabilized progesterone receptor purified from oestrogen-primed chick oviduct is composed of only approximately equal to 85000-Mr polypeptide chains.

Animals↗
PubMed1980

Synthesis of steroidal 17 beta-carboxamide derivatives.

Several 17 beta-carboxamide derivatives of natural and fluorinated glucocorticoids have been synthesized. The 17 beta-carboxylic derivatives were obtained by periodic acid oxidation of their side chains. They were then activated by N-hydroxybenzotriazole (HOBT) and coupled to several primary amines. Using this method eleven 17 beta-carboxamide derivatives have been prepared in good yields.

Chemical Phenomena↗
PubMed1977

Carbamyl-phosphate-synthetase deficiency with neonatal onset of symptoms.

The clinical course and biochemical findings in a case of carbamyl-phosphate-synthetase deficiency are described. The patient, a boy, presented 48 h after birth with rapidly developing hypotonia and hypothermia. Pulmonary haemorrhage, melaena and haematemesis ensued and despite ventilatory assistance and peritoneal dialysis the patient died on the fifth day. A virtual absence of carbamyl phosphate synthetase I (N-acetylglutamate dependent) was proved by analysis of tissue samples removed post mortem. Other urea cycle enzymes were normal.

Amino Acids↗
PubMedDate not supplied

Alteration of the glucocorticoid receptor subcellular localization by non steroidal compounds.

The glucocorticoid receptor (GR) engages transient or stable interactions with chaperones (hsp90, hsp70), co-chaperones (p60/hop, hsp40) and several other polypeptides such as immunophilins (Cyp40, FKBP59) and p23 to achieve a high affinity ligand binding state. This complex dissociates in response to hormonal stimuli and holo-GR translocates into the nucleus, where it regulates the activity of glucocorticoid-sensitive genes. GR activity is controlled through its ligand binding domain by steroids displaying either agonistic or antagonistic activity. An alternative approach to modulate GR activity is to target receptor-associated proteins (RAPs), and several non steroidal compounds binding to RAPs affect GR transcriptional activity. We have studied the effect of such drugs on the intracellular localization of a EGFP-GR fusion protein, which has wild type GR pharmacological properties. Agonist and antagonist binding induced nuclear translocation of GR, whereas rifampicin was found to be inactive in our system. Immunosuppressants FK506 and cyclosporin A were able to induce partial nuclear translocation of GR, suggesting that potentiation of glucocorticoid action by these compounds may also proceed through enhanced GR nuclear transfer. Short treatment of cells with the hsp90 inhibitor geldanamycin (GA) did not prevent nuclear translocation of GR. However, longer treatments, in parrallel to the inhibition of GR transcriptional activity, strongly perturbed GR subcellular localization concomitantly to the disruption of the actin network, and caused GR aggregation and down-regulation. The GA-induced transcriptional shutdown was also observed for other nuclear receptors which do not interact stably with hsp90. Thus RAP-binding compounds may exert their effects at least in part through perturbation of the GR cytosol to nucleus partitioning, and identify these proteins as valuable therapeutic targets to control nuclear receptor activity.

Actins↗