Modifications of rat liver glucocorticoid receptor by insulin-induced hypoglycemia.

Stability-, equilibrium- and kinetic binding parameters, transformation rate and sedimentation properties of liver cytosol glucocorticoid receptor from insulin-treated rats were studied. 40% elevation of cytosolic glucocorticoid binding and a lower affinity of the receptor for ligand were observed in hypoglycemic rats as compared to the controls. A small but significant decrease of [3H]triamcinolone acetonide-receptor complexes association rate and an increase of dissociation rate were also found. The rate and the extent of activation of the complexes from insulin-treated rats were somewhat higher compared to the controls, and the complexes from both groups showed higher affinity for the nuclei isolated from insulin-treated animals. Mixing experiments suggested that insulin treatment lead to alterations at the level of both the receptor protein and the nuclear binding sites. Sedimentation properties of transformed and untransformed receptor remained unchanged upon insulin treatment. The physiological relevance of the data was confirmed by hypoglycemia-related stimulation of tyrosine aminotransferase induction by dexamethasone.

Cardiac Nitric Oxide Synthases and Na⁺/K⁺-ATPase in the Rat Model of Polycystic Ovary Syndrome Induced by Dihydrotestosterone.

Nitric oxide synthases (NOSs) and Na(+)/K(+)-ATPase are enzymes essential for regular functioning of the heart. Since both enzymes are under insulin and androgen regulation and since insulin action and androgen level were disturbed in polycystic ovary syndrome (PCOS), we hypothesized that cardiac nitric oxide (NO) production and sodium/potassium transport would be deteriorated in PCOS. To test our hypothesis we introduced animal model of PCOS based on dihydrotestosterone (DHT) treatment of female Wistar rats and analyzed protein expression, phosphorylation or subcellular localization of endothelial NOS (eNOS), inducible NOS (iNOS) and alpha subunits of Na(+)/K(+)-ATPase in the heart. Obtained results indicate that DHT treatment significantly decreased cardiac eNOS protein level and activating phosphorylation at serine 1,177, while inhibitory phosphorylation at threonine 495 was increased. In contrast to expression of eNOS, iNOS protein level in the heart of DHT-treated rats was significantly elevated. Furthermore, cardiac protein level of alpha 1 subunit of the ATPase, as well as its plasma membrane content, were decreased in rats with PCOS. In line with this, alpha 2 subunit protein level in fraction of plasma membranes was also significantly below control level. In conclusion, DHT treatment impaired effectiveness of NOSs and Na(+)/K(+)-ATPase in the female rat heart. Regarding the importance of NO production and sodium/potassium transport in the cardiac contraction and blood flow regulation, it implicates strong consequences of PCOS for heart functioning.

Association of the rat liver glucocorticoid receptor with Hsp90 and Hsp70 upon whole body hyperthermic stress.

The influence of whole body hyperthermic stress (41 degrees C, 15 min) on association of the glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70 was followed in rat liver cytosol during a 24 h period after the stress. Total cytosolic concentration of the GR, Hsp90 and Hsp70 and the amounts of Hsp90 and Hsp70 co-immunopurified with the GR were determined by a quantitative Western blotting using appropriate monoclonal antibodies. A significant decrease in the cytosolic GR level in response to the stress was noticed. The ratio of the amount of the GR to Hsp90 recruited by the GR was found to be unaltered by hyperthermia, in spite of the stress-induced increase in the total Hsp90 concentration in the cytosol. Hsp70 was also found in association with the GR and its 2.5-fold induction by the stress was accompanied by about 3-fold increase in its relative amount that co-immunopurified with the GR. The results suggest that heat stress influences the interaction of the GR with Hsp70 through the mechanisms controlling the untransformed rat liver GR heterocomplexes assembly process.

DNA-binding and non-DNA-binding forms of the transformed glucocorticoid receptor.

In this work we have probed the mechanism responsible for two non-DNA-binding states of the mouse glucocorticoid receptor. In the first case, transformed receptors were treated with hydrogen peroxide. It is known that oxidizing agents promote the formation of disulfide bonds in the glucocorticoid receptor, but it has not been determined what domains are involved in any disulfide bond formation that leads to inactivation of DNA-binding activity. We show here that hydrogen peroxide inhibits DNA-binding by the 15-kDa tryptic fragment containing the DNA-binding fingers with the same concentration dependency as it inhibits DNA-binding by the uncleaved receptor. This suggests that all of the effect of peroxide is on sulfhydryl groups within the zinc fingers. After dissociation (transformation) of cytosolic heteromeric glucocorticoid receptor complexes, only a portion (40-60%) of the dissociated receptors can bind to DNA-cellulose. We show that the 15-kDa tryptic fragment derived from the portion of transformed receptors that do not bind to DNA is itself competent at DNA-binding.

Release of microparticles in LDL apheresis.

Particle contamination of blood always takes place in extracorporeal systems and few studies have been conducted to evaluate potential risks. Particle concentration was measured in the efferent blood line on original equipment for two established LDL elimination procedures (DALI) (Fresenius) and Liposorber (Kaneka). Acquired data were compared with standards for infusion solutions from European (EP) and American (USP) Pharmacopoeia. All values were well below the given limits. Even in extreme situations (>20 pump stops) particle concentration did not exceed the standards. Considering an average treated blood volume of 7.31 for the DALI-System and 17.01 for Liposorber (long term clinical studies) the absolute amount of particles infused per treatment was 167,000 (DALI) and 465,000 (Liposorber) particles > or = 2 microm.

Accidental selenium poisoning of growing pigs.

Chronic selenium (Se) toxicosis was diagnosed in two groups of growing pigs. Emaciation, loss of hair, necrotic areas in the skin, lesions of the coronary band and hooves, postnecrotic atrophic cirrhosis of liver, and lumbal poliomyelomalacia were the principal findings. High Se concentrations were detected in blood plasma. Addition of the calculated amounts of sodium selenite directly to the feedstuff instead to mineral premix was the cause of this intoxication.

Hypothalamic-pituitary-adrenocortical axis hypersensitivity and glucocorticoid receptor expression and function in women with polycystic ovary syndrome.

INTRODUCTION: Molecular mechanisms underlying pathophysiology of polycystic ovary syndrome (PCOS), especially those related to cortisol signaling, are poorly understood. We hypothesized that modulation of glucocorticoid receptor (GR) expression and function, may underlie possible PCOS-related impairment of feedback inhibition of hypothalamic-pituitary-adrenocortical (HPA) axis activity and thus contribute to increased adrenal androgen production in women with PCOS. MATERIALS AND METHODS: 24 normal-weight and 31 obese women with PCOS were compared to 25 normal-weight controls. Fasting blood samples were collected for measurements of serum concentrations of dehydroepiandrosterone sulfate, testosterone, sex hormone-binding globulin, insulin, basal cortisol and cortisol after oral administration of 0.5 mg dexamethasone. Concentrations of GR mRNA, GR protein, mineralocorticoid receptor (MR) protein and heat shock proteins (Hsps), as well as the number of GR per cell (B(max)) and its equilibrium dissociation constant (K(D)) were measured in isolated peripheral blood mononuclear cells. RESULTS: An increase in HPA axis sensitivity to dexamethasone, an elevation of the GR protein concentration, and unaltered receptor functional status were found in both normal-weight and obese women with PCOS vs. healthy controls. Lymphocyte MR, Hsp90 and Hsp70 concentrations, and MR/GR ratio were similar in all groups. Correlation between B(max) and K(D) was weaker in the group of obese women with PCOS than in the other 2 groups. CONCLUSIONS: The results did not confirm the initial hypothesis, but imply that PCOS is associated with increased GR protein concentration and HPA axis sensitivity to dexamethasone.

Long-term imipramine treatment affects rat brain and pituitary corticosteroid receptors and heat shock proteins levels in a gender-specific manner.

Gender-related differences in the effects of imipramine, on the protein levels of glucocorticoid receptor (GR), and heat shock proteins Hsp90 and Hsp70, as well as on dexamethasone binding to corticosteroid receptors (CRs) in the pituitary, hypothalamus, hippocampus and brain cortex of non-depressed rats were studied. Differences between female and male animals in the GR protein level in the tissues of untreated animals were not noticed. However, imipramine led to opposite changes in the cellular level of GR protein in the brain of female and male rats, as well as to gender- and tissue-specific changes in in vitro dexamethasone binding to GR and mineralocorticoid receptor (MR) in the hippocampus and brain cortex. Gender-related differences in the expression of Hsp90 and Hsp70 were noticed mainly in the hippocampus, only after imipramine treatment. The observed changes in the response of GR to imipramine suggest that this antidepressant may affect both the level of the receptor protein and the mechanisms regulating its binding ability in a gender-related manner.

The effect of alkaline phosphatase on the activation of glucocorticoid-receptor complexes in rat liver cytosol.

Calf intestinal alkaline phosphatase was found to stimulate the rate of in vitro activation of rat liver glucocorticoid-receptor complexes. This effect was registered both at 0 and 25 degrees C and could be prevented by sodium molybdate. The resulting change in sedimentation behaviour (shift of sedimentation coefficient from 9.6 S to 4.8 S for molybdate-stabilized and alkaline phosphatase-treated complexes, respectively) was similar to that observed after heat activation.

Glucocorticoid receptor interaction with Hsp90 remains unaltered after whole body hyperthermia.

The influence of 41 degrees C whole body hyperthermic stress on glucocorticoid receptor (GR) association with 90-kDa heat shock protein (Hsp90) in the rat liver cytosol was examined. Total cytosolic GR and Hsp90 concentrations, as well as the amount of Hsp90 co-immunoprecipitated with the GR were determined by quantitative Western blotting using BuGR2 as anti-GR and AC88 as anti-Hsp90 monoclonal antibody. After exposure of the animals to the heat stress, the level of cytosolic Hsp90 increased, while its ratio to apo-receptor within non-activated GR heterooligomeric complexes remained unaltered. Therefore, the Hsp90 recruitment by the GR was not dependent on Hsp90 total cytosolic concentration.

Background and indications for protein A-based extracorporeal immunoadsorption.

Protein A (SPA), a major cell wall component of Staphylococcus aureus, has occupied numerous investigators from its discovery in the late fifties. Its availability and avid binding to human immunoglobulins have led to extensive usage for diagnostic and research purposes. Today, SPA-based extracorporeal immunoadsorption relies on two rather different systems, namely, SPA-silica (Prosorba), and SPA-Sepharose (Immunosorba). Both systems are approved by the Food and Drug Administration for the core indications of rheumatoid arthritis and idiopathic thrombocytopenic purpura (SPA-silica) or hemophilia with inhibitors (SPA-Sepharose). Off label indications include immune disorders with a conceivable connection between autoantibody titers and disease activity, like forms of glomerulonephritis, systemic lupus erythematodes, myasthenia, and the Guillain-Barré syndrome as well as alloantibody formation in the context of e.g., transplantation. This review summarizes historical developments and important properties of SPA. Indications for extracorporeal therapy are discussed on the basis of available information and personal experience.

Cadmium affects the redox state of rat liver glucocorticoid receptor.

It has previously been documented that cadmium displays high affinity for protein thiol groups and induces an impairment of glucocorticoid receptor (GR) cellular functions. The present study examined the possibility that cadmium exerts these effects on GR activity by disturbing the receptor's redox equillibrium. To that end, the influence of cadmium on the rat liver GR potential to form intramolecular and intermolecular disulfide bonds under nonreducing conditions and under oxidizing conditions produced by the addition of hydrogen peroxide (H2O2) to the cytosol was examined by nonreducing SDS-PAGE and immunoblotting. The results show that cadmium inhibits formation of disulfide bonds within the GR both in the absence and in the presence of H2O2. The creation of intermolecular disulfide linkages between the apo-GR and associated heat shock proteins Hsp90 and Hsp70, which was evident in the presence of H2O2, was also significantly impaired after cadmium administration. These observations are consistent with the assumption that cadmium affects the redox state of the receptor, possibly by binding to its sulfhydryl groups.

The influence of hyperthermic stress on the redox state of glucocorticoid receptor.

Glucocorticoid receptor (GR) is hormone-dependent transcription factor which participates in intracellular signal transduction. The reduced state of the receptor sulfhydryl groups is considered a necessary prerequisite for its normal functioning under the homeostatic conditions. The aim of the work presented in this paper was to examine the influence of non-homeostatic conditions - whole body hyperthermic stresses at 41 degrees C and 42 degrees C, on GR redox state. Non-reducing SDS-PAGE and immunoblot analysis were used to trace alterations of the receptor's redox state. The steroid binding assay was performed in order to examine direct influence of the whole body heat stresses on the receptor thiols. The results obtained show that the 41 degrees C stress leads to formation of intermolecular disulfide bonds between apo-GR and associated heat shock proteins (Hsp90, Hsp70). Apart from intermolecular GR-Hsp90 and GR-Hsp70 disulfide linkages, 42 degrees C hyperthermic stress also caused creation of intramolecular ones within GR. The results imply malfunctioning of intracellular redox control mechanisms under the hyperthermic conditions.

Mercury inhibits rat liver and kidney glucocorticoid receptor hormone binding activity.

The present study was focused on the influence of mercury on the rat liver and kidney glucocorticoid receptor (GR) binding properties. The time-course and dose-dependence of mercury effects, as well as possible involvement of thiol groups were examined after in vivo and in vitro administration of the metal in the form of HgCl2. Mercury led to reduction of the liver and kidney GR hormone binding capacity. In both examined tissues maximal reduction was noticed 4 h after administration of the metal at 2 and 3 mg Hg/kg bw, but the effect was more prominent in kidney as compared to liver. On the other hand, binding affinity in the two tissues was similar. The complete reversal of mercury effects on GR binding capacity by 10 mmol/L DTT was achieved in liver and partially in kidney. The reversal by DTT suggested that mercury caused the decrease of GR binding activity by interacting with thiol groups. The difference in the response of the two tissues reflected the fact that kidney contained a higher mercury concentration and a lower thiol content in comparison to liver. The implicated thiols probably belong to GR, since when applied in vitro at 0 degrees C, mercury produced reduction of the receptor binding activity similar to that observed in vivo. GR protein level examined by quantitative Western blot was either unchanged, when determined by polyclonal antibody, or reduced, when determined by BuGR2 antibody, suggesting that Hg might affect BuGR epitope availability.

Flow cytometric analysis of reticulated platelets.

Flow Cytometric Analysis of Reticulated Platelets (G. Matic, G. Rothe, and G. Schmitz, University of Regensburg, Regensburg, Germany). In the last several years, flow cytometry has emerged as one of the tools of choice in evaluation of platelets. In particular, the distinction between reticulated platelets and mature platelets based on RNA fluorescence has proved to be a vital tool in the clinical hematology laboratory. Further, as is now well understood, it is important to evaluate platelets in whole blood rather than in isolated populations. Platelet quantification in a dual-color whole-blood method is of interest for the characterization of thrombocytopoiesis in (immune)-thrombocytopenia or in the regenerating bone marrow.

Heat stress affects the glucocorticoid receptor interaction with heat shock protein Hsp70 in the rat liver.

The association of glucocorticoid hormones receptor (GR) with heat shock protein Hsp70 in the liver cytosol of rats exposed to 41 degrees C whole body hyperthermic stress was examined by quantitative immunoblotting of the two proteins within immunopurified untransformed GR multiprotein complexes. The presence of Hsp70 in the rat liver GR heterocomplexes was confirmed, and 2-fold increase in the Hsp70 relative to the steroid binding protein content within the complexes was recorded 2 and 12 h after the stress. This increase exceeded the stress-induced elevation in the total cytoplasmic Hsp70 level, but could not be seen 24 h after the stress, when cytoplasmic Hsp70 returned to basal level. The results suggest that hyperthermic stress alters the composition of the rat liver untransformed GR heterocomplexes increasing the Hsp70 share.

The level and phosphorylation of Hsp70 in the rat liver cytosol after adrenalectomy and hyperthermia.

Hepatic heat shock protein Hsp70 synthesis and in vitro phosphorylation were studied in the liver cytosol of intact, adrenalectomized and dexamethasone-administered adrenalectomized rats after 41 degrees C whole body hyperthermic stress. Hsp70 was detected by immunoblotting with N27F3-4 monoclonal antibody recognizing both constitutive and inducible forms of the protein. A comparison between basal and heat stress-induced levels of the protein in the liver cytosol of the three groups of animals suggested that glucocorticoid hormones stimulate the basal synthesis of Hsp70 and inhibit its induction by stress. In both unstressed and hyperthermia-exposed animals, hepatic Hsp70 was detected as a phosphoprotein. The extent of its in vitro phosphorylation was found to be significantly reduced by heat stress or adrenalectomy, but dexamethasone failed to restore it to the original level.

Some disturbances related to iodination and utilisation of thyroglobulin and 27 S iodoprotein in non-toxic multinodular goitre.

The relationships among the thyroid iodoproteins, their biosynthesis in vitro in thyroid slices and the histological structure of a multinodular, sporadic goitre was examined after strumectomy from a euthyroid 51-year-old woman. Using sedimentation methods 27S, thyroglobulin (TG), 12S and 3--8S proteins were found. Besides these, 4 to 5 other proteins with molecular weights between 165,000 and 36,000 daltons were detected by polyacrylamide gel electrophoresis. The concentration of soluble proteins was very low (3 mg/100 mg wet tissue), particularly TG (0.69 MG/100 MG). The sedimentation constant (18.3S) of goitrous TG was lower than mature 19S-TG and it was poorly iodinated (0.06% w/w). 27S iodoprotein was present in the goitre extract. Thyroid slices of goitre tissue incorporated 14C-leucine into proteins and synthesized TG and its subunits. Newly synthesized proteins were rapidly released from the microsomes. Pathohistologically, struma colloido-microfolliculare with dystrophic-proliferative changes was noted. In the large follicles which were filled with colloid, dystrophic alterations in thyrocytes and desquamation of the follicular epithelium were observed. In conclusion, it is suggested that the presence of poorly iodinated, immature TG in nontoxic multinodular, sporadic goitre, in the absence of iodine deficiency, probably results in disturbances in the utilization of TG from the follicular lumen. In the follicular lumen under these conditions it is possible that 27S protein is formed from pre-existing poorly iodinated TG. These findings suggest that inability to resorb colloid may be a cause of nodule formation.