In vitro activation of rat cardiac glucocorticoid antagonist- versus agonist-receptor complexes.

The synthetic antiglucocorticoid RU 38486 interacts with cardiac cytoplasmic glucocorticoid receptors and competes for in vitro binding with the potent agonist triamcinolone acetonide. In addition to binding to receptors with high affinity, RU 38486 also facilitates the in vitro conformational change in the receptor which is a consequence of the physiologically relevant activation step during which the receptor is converted from a non DNA- to a DNA-binding form. This ability of RU 38486 to promote receptor activation is reflected by both the appropriate shift in the elution profile of [3H]RU 38486-receptor complexes from DEAE-cellulose as well as by an increased binding of these complexes to DNA-cellulose. Although less effective than triamcinolone acetonide, RU 38486 promotes in vitro receptor activation under a variety of experimental conditions, including incubation of labeled cardiac cytosols at 25 degrees C for 30 min or at 15 degrees C for 30 min in the presence of 5 mM pyridoxal 5'-phosphate. Once thermally activated, the cardiac [3H]triamcinolone acetonide and [3H]RU 38486-receptor complexes bind to nonspecific DNA-cellulose with the same relative affinities, as evidenced by the fact that 50% of both activated complexes are eluted at approx. 215-250 mM NaCl. Thus, this pure antiglucocorticoid does promote, at least to some extent, many of the crucial in vitro events including high-affinity binding, activation, and DNA binding which have been shown to be required to elicit a physiological response in vivo.

Immunohistochemical localization of modified C-reactive protein antigen in normal vascular tissue.

BACKGROUND: The prototypic acute phase reactant, C-reactive protein (CRP), is a serum soluble, cyclic pentameric protein, the concentration of which increases markedly within hours of any tissue-damaging, inflammatory event. However, upon dissociation of its pentameric quaternary structure, CRP subunits undergo a spontaneous and irreversible conformational change. The resulting molecule, termed modified CRP or mCRP, has reduced aqueous solubility and a propensity to aggregate into a matrix-like lattice structure. METHODS: Using monoclonal antibodies, normal human tissues were immunohistochemically screened for the presence of CRP as well as mCRP antigens. RESULTS: Significant levels of mCRP were detected in the walls of blood vessels associated with normal human tissues. These data indicate that mCRP is a naturally occurring form of CRP and that it is a tissue-based rather than serum-based molecule. SIGNIFICANCE: This report describes the localization of a stable form of CRP, mCRP, in blood vessels associated with normal human tissues.

[Health problems among the Kaingáng (Xapecó Indigenous Reserve, Santa Catarina) and the health care system]. / Agravos na saúde Kaingáng (Terra Indígena Xapecó, Santa Catarina) e a estrutura dos serviços de atenção biomédica.

The second semester of 1999 was a transition period for the implementation of the Special Indigenous Health District on the Xapecó Indigenous Reserve in western Santa Catarina State. The health clinic in the main village provided treatment with a staff including a general practitioner/obstetrician, pediatrician, dentist, nurse, two nursing assistants, and four nursing technicians. This paper presents the preliminary results of research on the organization of these health care services, their use by the community, and the health/disease profile of the Kaingáng, using patient files as the source of information. In September 1999, a total of 222 Indians were treated (children and adults), 50.5% of whom residing in the main village. Among the Indians ages 0 to 14 years, infectious and parasitic diseases were the most frequent, supporting the idea that the Kaingáng have precarious sanitary and nutritional conditions. Use of the clinic by adults was more varied, since of the 116 who appeared for consultation, 27 were pregnant women (out of a total of 86 women). In addition, prescriptions were written up for children and adults in 85.0% and 81.8% of the consultations, respectively.

Heat shock protein 70 is associated in substoichiometric amounts with the rat hepatic glucocorticoid receptor.

The 70-kDa heat shock protein (hsp70) has been shown to be an important participant in several intracellular events, including protein folding and trafficking. Hsp70 binds to many, if not all, proteins during their translation and maintains its association with some protein complexes as a subunit. We have examined the possibility that hsp70 may be associated with one or more forms of the rat hepatic glucocorticoid receptor (GR). Unliganded GR was immunoprecipitated from cytosol with the anti-GR monoclonal antibody BUGR2 and then subjected to western blotting. Both hsp70 and the 90-kDa heat shock protein (hsp90) were found to be specifically associated with the GR. Hsp70 was also bound to the liganded unactivated and activated (transformed) forms of the GR complex, while as expected, hsp90 was absent from the activated GR. Immunoprecipitation of cytosolic hsp70 with the anti-hsp70 monoclonal antibody N27 resulted in coprecipitation of GR. The components of the immunopurified GR were also analyzed by laser densitometry after polyacrylamide gel electrophoresis and Coomassie blue staining. These experiments revealed that hsp70 is bound to the GR in an approximate 1:5 ratio. Unactivated GR complexes isolated via a ligand affinity purification scheme contained hsp90 and trace amounts of hsp70. Collectively, these experiments demonstrate that hsp70 is specifically associated with several forms of the native rat hepatic GR, although its binding is substoichiometric. This is in direct contrast to hsp90, which binds as a dimeric subunit to the heteromeric unactivated GR complex.

ATP-induced activation of purified rat hepatic glucocorticoid receptors.

We have utilized unactivated rat hepatic glucocorticoid receptor complexes purified to near homogeneity by a three-step scheme which includes affinity chromatography, gel filtration and anion exchange chromatography, to demonstrate for the first time that ATP can interact directly with the receptor protein in stimulating activation. This stimulation is reflected by an increase in DNA-cellulose binding as well as by a shift in the elution profile of the purified receptor complexes from DEAE-cellulose. A concentration of 10 mM Na2MoO4 is able to block both of these effects. ATP stimulates activation in a dose-dependent manner (maximally at 10 mM), and elicits maximal activation within 30 min at 15 degrees C. There appears to be no nucleotide specificity since GTP, CTP and UTP, as well as ADP and GDP also stimulate activation. All of these observations closely parallel data obtained from similar activation experiments performed with crude rat hepatic receptors. ATP does not appear to stimulate activation of receptors (crude or purified) by initiating a phosphorylation reaction since hydrolysis-resistant analogues of ATP are also effective. Pyrophosphate (PPi) is as effective as ATP in promoting receptor activation, since it elicits similar increases in DNA-cellulose binding, shifts in elution patterns from DEAE-cellulose, and dose-response relationships. None of the compounds tested stimulate activation indirectly by pH or ionic strength effects. Despite the fact that high ATP concentrations (3-4-fold higher than those present in vivo) are necessary to stimulate maximal activation, a physiological role of ATP in directly regulating in vivo activation of glucocorticoid receptors cannot be ruled out.

Majority of RNA which co-purifies with unactivated rat hepatic glucocorticoid-receptor complexes is nonspecific and not receptor-associated.

Molybdate-stabilized, unactivated rat hepatic glucocorticoid-receptor complexes were purified by a three-step procedure which includes affinity chromatography, gel filtration and anion exchange chromatography. Following elution of unactivated steroid-receptor complexes from the final DEAE-cellulose column, RNA which remained bound to the anion exchange resin was eluted with 1 M KCl. This RNA was small and heterogeneous in size. Equivalent amounts of RNA were detected after a mock purification which was devoid of receptors, suggesting that the presence of this RNA is not dependent on that of receptors. Both a [32P]DNA complementary to the RNA eluted from DEAE-cellulose and a [32P]DNA probe synthesized from total rat liver RNA gave similar results when hybridized to total rat liver RNA. These data indicated that the RNA which co-purified with unactivated receptors through the first two steps was very similar to total RNA in overall composition. Virtually identical hybridization patterns were also detected when end-labeled probes generated from the DEAE-cellulose eluted RNA or total liver RNA were hybridized to total genomic rat DNA, suggesting that the RNA eluted from the anion exchange resin is not specific or unique. Although these results do not exclude the possibility that there could be specific RNA species associated with the unactivated glucocorticoid receptor, they do indicate that the majority of the RNA eluted from DEAE-cellulose following elution of receptor complexes appears indistinguishable from total rat liver RNA and can be detected in parallel mock purifications.

Effects of bovine pancreatic ribonuclease A, S protein, and S peptide on activation of purified rat hepatic glucocorticoid-receptor complexes.

Bovine pancreatic ribonuclease (RNase) A and S protein (enzymatically inactive proteolytic fragment of RNase A which contains RNA binding site) stimulate the activation, as evidenced by increasing DNA-cellulose binding, of highly purified rat hepatic glucocorticoid-receptor complexes. These effects are dose dependent with maximal stimulation of DNA-cellulose binding being detected at approximately 500 micrograms (50 units of RNase A/mL). RNase A and S protein do not enhance DNA-cellulose binding via their ability to interact directly with DNA or to increase nonspecific binding of receptors to cellulose. Neither S peptide (enzymatically inactive proteolytic fragment which lacks RNA binding site) nor cytochrome c, a nonspecific basic DNA binding protein, mimics these effects. RNase A and S protein do not stimulate the conformational change which is associated with activation and is reflected in a shift in the elution profile of receptor complexes from DEAE-cellulose. In contrast, these two proteins interact with previously heat-activated receptor complexes to further enhance their DNA-cellulose binding capacity and thus mimic the effects of an endogenous heat-stable cytoplasmic protein(s) which also function(s) during step 2 of in vitro activation [Schmidt, T. J., Miller-Diener, A., Webb, M. L., & Litwack, G. (1985) J. Biol. Chem. 260, 16255-16262]. Preadsorption of RNase A and S protein to an RNase affinity resin containing an inhibitory RNA analogue, or trypsin digestion of the RNA binding site within S protein, eliminates the subsequent ability of these two proteins to stimulate DNA-cellulose binding of the purified receptors.(ABSTRACT TRUNCATED AT 250 WORDS)