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.

Solubilization of dopamine-D2 receptors from synaptosomal membranes of the bovine caudate nucleus.

Dopamine D2-receptors were solubilized from synaptosomal membranes of the bovine caudate nucleus using different detergents. They were labelled with [3H]-spiperone and assayed by polyethylene glycol precipitation. CHAPS was found to be the best solubilizing agent among all detergents used. Optimal conditions for solubilization were: 0.25% CHAPS, 3.5 mg ml-1 protein, 25 min, 4 degrees C and the yield of D2-receptors was 18.6%. Addition of some sulphobetain detergents increased the extent of solubilization, 125 mM NaCl and 0.25 M sucrose decreased it, while SH-group protecting agents (2 mM dithiothreitol and 6 mM beta-mercaptoethanol), as well as MEGA-9 and MEGA-12 were almost ineffective. -log IC50 values for solubilized dopamine D2-receptors are in linear correlation with the corresponding values for membrane-bound receptors (r = 0.962, slope factor 0.96) and Kd value of solubilized receptors was 3.61 +/- 0.94 nM, while that of membrane-bound receptors was 1.25 +/- 0.10 nM. Specific binding of [3H]-spiperone to the solubilized receptors resolved by linear sucrose density gradient centrifugation shows two maxima, one in the first several fractions from the bottom and the other with an apparent S value of 7.3.

Human immunodeficiency virus V3 peptide-reactive antibodies are present in normal HIV-negative sera.

A structural relation between consensus sequences of the portion of HIV-1 gp120 involving the V3 loop (V3 peptide) and the variable domains of human immunoglobulin members of the VH-III gene family was proposed to trigger an imbalance of the idiotypic network during the course of HIV infection. Thus, the repertoires of immunoglobulins in healthy individuals should contain antigenic determinant(s) complementary to particular V3 loop epitope(s). In this study we investigated the specific binding to the V3 peptide of antibodies present in sera of HIV-positive and of clinically normal HIV-negative subjects. Two groups of HIV-positive sera differing in antibody titers to V3 peptide, arbitrarily referred here as high- and low-reactive HIV-positive sera, were distinguished on the basis of an ELISA. Antibodies were affinity purified on V3 peptide and their titers in both HIV-negative and low-reactive HIV-positive sera were nearly superimposable and much lower than the titers of those from high-reactive HIV-positive sera. Also, the quality of the two groups of antibodies differed: much higher amounts of soluble V3 peptide were needed to partly compete the binding of antibodies from HIV-negative sera to insoluble V3 peptide as compared with those from HIV-positive sera, suggesting that the latter had higher affinity for V3 peptide. All of the affinity-purified antibodies bound poorly to unrelated peptides, even to those sharing sequence similarity with the V3 peptide. The present observations suggest that in HIV infection antigen-driven affinity maturation of preimmune immunoglobulins with idiotypes complementary to V3 epitope(s) participating in physiological autoreactivity might be at work.

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.

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.

Effect of gamma-irradiation on microtubule assembly in vitro.

Microtubular protein was exposed to gamma-radiation from 500 to 1000 Gy, Within that dose range its polymerization ability was decreased by 20-60 per cent when samples were irradiated in assembled state, and by 40-75 per cent when irradiated in unassembled state. Microtubules assembled from irradiated subunits were shorter and of more uniform lengths than control microtubules. For the dose of 1000 Gy the mean length and its standard deviation were reduced to about one-half of the values of the control.

Double and single exponential kinetics of microtubule assembly in vitro.

The kinetics of the microtubule protein assembly were studied in Mes buffer, pH 6.6, at 28 degrees C. The assembly under above conditions follow a kinetic expression containing two exponential terms. The observed two rate constants depend on protein concentration, and are on the order of 10(-2) sec-1 and 10(-3) sec-1. When CaCl2 is added to the system in low concentration, the kinetic expression becomes single exponential. The observed rate constant is independent of protein concentration and its value is 5 X 10(-3) sec-1. It is concluded that the double exponential kinetics correspond to favorable assembly conditions, probably to a high extent of nucleation, whereas the single exponential kinetics correspond to favorable assembly conditions, probably to a high extent of nucleation, whereas the single exponential kinetics is a slower process which occur under hindered assembly conditions.

Dopaminergic activity of some ergot alkaloid derivatives: relationship to their chemical structure.

Several ergot alkaloid derivatives have been tested for their ability to inhibit receptor binding of [3H]spiperone (D2 dopamine receptors) and [3H]dopamine (D3 dopamine receptors) in the bovine caudate nucleus. Dopaminergic activity was correlated to their chemical structure. The potencies of ergot alkaloids for displacing [3H]spiperone binding can be ranked in the following order: lisuride greater than ergosine greater than bromodihydroergosine greater than bromoergosine approximately dihydroergosine approximately ergosinine approximately dihydroergocryptine greater than bromoergocryptine greater than CH-29 717 greater than saccharinoergosinine = saccharinoergosine greater than dihydroergosinine. The potencies of these compounds for displacing [3H]dopamine binding can be ranked in the following order: lisuride greater than ergosinine approximately ergosine greater than dihydroergosine greater than bromodihydroergosine approximately CH-29 717 approximately bromoergocryptine approximately bromoergosine approximately dihydroergocryptine greater than saccharinoergosine, saccharinoergosinine, dihydroergosinine. Displacement of both radioligands was unaffected by GTP. Binding characteristics of ergot alkaloids examined revealed antagonist-like properties of binding to D2 and agonist-like properties of binding to D3 receptors. Introduction of bromine into position 2, selective hydrogenation of 9,10-dihydroderivatives, or isomerization in position 8 of the ergot alkaloid molecule did not drastically change binding parameters of these compounds. However, an alpha-configuration in position 8 combined with hydrogenation of the delta-9,10-double bond of dihydroergosinine, significantly decreased its affinity to both D2 and D3 receptors in comparison with dihydroergosine or ergosinine, suggesting stereoselectivity of dopamine receptors towards the pair dihydroergosine/dihydroergosinine.

Cortisol-binding components of cytosol receptor in normal and adrenalectomized rat liver.

[3H] cortisol was found to be bound to two distinct components of receptor protein in hepatic cytosol of both normal and adrenalectomized adult male rats. In liver cytosol of normal rats, 78% of total bound [3H] cortisol to receptor was associated with the first receptor component, and about 20% to the second one. However, the first component of cytosol receptor in adrenalectomized rats was found to bind 95% of total [3H] cortisol bound to both receptor components, while only 4.5% of the bound hormone was found associated with the second receptor component. These alterations in binding capacity are discussed in relation to possible regulation of nuclear RNA synthesis by cortisol-receptor complexes.

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.

Hyperthermic stress modulates the functions of rat liver glucocorticoid receptor.

A mild whole body hyperthermic stress causes a rapid and reversible reduction of rat liver glucocorticoid receptor (GR) binding capacity and affects the stability of the GR-DNA complexes formed after thermal transformation of the receptor. These changes appear to be physiologically relevant, since they are accompanied by a decrease in dexamethasone induction of hepatic tyrosine aminotransferase (TAT). In spite of the decreased rate of the GR degradation in liver cytosol of hyperthermic as compared to control rats, the total amount of the GR and its proteolytic products recognized by BuGR2 monoclonal antibody was found to be lower in the former cytosol, but higher in the respective nuclei.

Cadmium affects the activity of rat liver tyrosine aminotransferase and its induction by dexamethasone.

The effects of cadmium (Cd) administration to intact rats on hepatic glucocorticoid receptor (GR) steroid binding capacity and DNA-binding ability were examined and correlated with the influence of the metal on rat liver tyrosine aminotransferase (TAT) activity and its induction by dexamethasone. It was found that 24 h after i.p. administration of Cd doses ranging from 0.5 to 4 mg/kg, the GR steroid- and DNA-binding activities were significantly reduced in a dose-dependent manner. The same doses of Cd also affected the basal and dexamethasone-induced level of TAT activity, as well as the concentration of metallothionein in rat liver. The decrease in TAT activity and in its induction by dexamethasone observed in response to low Cd doses was proportional to the alterations of the GR functional properties. Higher doses of Cd, which were more effective in reducing both the GR binding of the hormone and to DNA, however, stimulated TAT activity and potentiated dexamethasone induction of the enzyme. The results led to the conclusion that Cd may alter physiological response of rat liver cells to glucocorticoids interfering with the GR-dependent transcriptional regulation of the TAT gene.

Hyperthermic stress affects glucocorticoid receptor-mediated transcription in rat liver.

Binding capacity of the cytoplasmic and nuclear glucocorticoid receptor (GR) and the activity of tyrosine aminotransferase (TAT) were examined in the liver of intact and adrenalectomized rats exposed to 41 degrees C whole body hyperthermic stress. In glucocorticoid-deprived animals, stress-induced decrease in the cytoplasmic steroid binding was followed by parallel increases in its nuclear binding and TAT activity, suggesting a stimulation of TAT gene transcription by the GR in the absence of the ligand. In intact animals, however, a diminution of the steroid binding in the cytosol, its unchanged nuclear binding and an impairment of TAT activity were observed upon the stress. The results imply that stress could elicit different structural or functional alterations of unliganded vs liganded GR.

Anti-V3 and anti-IgG antibodies of healthy individuals share complementarity structures.

It was recently shown that antibodies reactive with a peptide from the tip of the HIV-1NY5 gp120 V3 loop (V3 peptide) are present not only in sera of HIV-positive patients but also in sera of healthy HIV-negative individuals. In the present study, we show that V3 peptide reactive antibodies are predominantly IgM in sera of HIV negative individuals and that a fraction of the IgG anti-V3 antibodies exhibit features of autoantibodies. These antibodies were purified by chromatography on IgG-sepharose columns from sera as well as from purified IgG anti-V3 antibodies. A higher IgG anti-V3 reactivity was detected in autoantibody preparations from HIV-positive sera as compared with the reactivity of sera and purified antibodies from HIV-negative individuals. This was confirmed by solid phase binding of IgG anti-V3 antibodies both to V3 and to human IgG F(ab')2 antigens. The autoantibodies did not bind to peptides that share sequence similarity with V3 peptide indicating a high epitope specificity. The detection of antibodies against HIV epitopes in HIV-negative individuals may suggest that anti-V3 antibodies after HIV infection represent at least in part a secondary immune response.

Alpha 1-antitrypsin genetic phenotypes in a group of children suffering from pulmonary diseases.

Alpha 1-antitrypsin (AAT), the main protease inhibitor of human sera, was studied in a group of 88 children suffering from different pulmonary diseases, with the hope that some of the potential victims of chronic obstructive lung diseases can be identified in time. AAT genetic phenotypes were determined using acid agarose gel electrophoresis, followed by crossed antigen-antibody electrophoresis in agarose gel. Identification of the banding patterns revealed 10.2% of AAT variants. 4.54% of the patients were MZ, 3.40% were MS and 89.77% were MM. During this study, 1 FF and 1 MV subject were also found. All AAT variants were in the group of younger children, under 6 years of age.

In vitro evidence for modification of rat liver glucocorticoid receptor binding properties and transformation by hyperthermia.

The physico-chemical parameters of the interaction of [3H]triamcinolone acetonide (TA) with the glucocorticoid receptor (GR) and the in vitro activation of glucocorticoid-receptor complexes were studied in liver cytosols of rats exposed to 41 degrees C hyperthermia. A significant reduction in glucocorticoid binding and a slight increase in binding affinity were detected in hyperthermic rats as compared to the controls. The number of binding sites was 0.48 +/- 0.02 and 0.73 +/- 0.03 pmol/mg protein for heat-treated and control rats, respectively. Differences in equilibrium dissociation constants (0.52 +/- 0.08 nM for hyperthermic and 0.94 +/- 0.13 nM for control animals) were reflected in corresponding differences in dissociation rate constants at 25 degrees C (5.1 x 10(-4) and 7.5 x 10(-4) min-1, respectively), whereas association rate constants were similar. The inactivation kinetics of unoccupied GR at 25 degrees C was the same in both groups. Glucocorticoid-receptor complexes in liver cytosol from hyperthermic and control rats were thermally activated to a similar extent, but the activation rate was significantly lower in the former. Mixing experiments with the control and hyperthermic rat liver cytosols suggest that this impairment of glucocorticoid-receptor complexes activation could be at least in part ascribed to heat stress-related changes in the action of activation modulator(s). Sedimentation behaviour of unactivated and activated [3H]TA-receptor complexes of hyperthermic and control rats was identical.

Expression of cyclooxygenase-2 in canine renal cell carcinoma.

Cyclooxygenase-2 (COX-2) has been shown to be the primary enzyme responsible for prostaglandin production during inflammation but is absent in most tissues under normal physiological states. High levels of COX-2 expression have been observed in the macula densa and thick ascending limbs of fetal kidneys; this expression declines to minimal levels during renal maturation. We hypothesized that the neoplastic cells of renal cell carcinoma (RCC) may revert to high expression of COX-2, and we evaluated its expression in three spontaneous cases of canine RCC by using immunohistochemical methods. The neoplastic cells of two of the three cases exhibited moderate to marked COX-2 immunoreactivity. These results suggest that some canine renal cell carcinomas express high levels of COX-2, which may play a role in the modulation of neoplastic cell growth.

In vivo effects of cadmium on rat liver glucocorticoid receptor functional properties.

1. Cadmium (Cd2+) administered in vivo induced a 40% reduction of rat liver glucocorticoid receptor (GR) capacity and inhibition of glucocorticoid-receptor complexes binding to mouse mammary tumor virus (MMTV) DNA fragment containing GR consensus sequence. 2. The effect of Cd2+ on the GR binding activity can be reversed with DTT, suggesting Cd2+ interaction with thiol groups. 3. Cd(2+)-related GR modification seems to be mediated by Cd2+ binding to cytoplasmic components included in the regulation of the receptor function, although the direct binding of the metal to the receptor thiols could not be ruled out.