Characterization of cytosolic glucocorticoid receptor of fetal rat epiphyseal chondrocytes.

The cytosolic glucocorticoid receptor of 21st gestational day rat epiphyseal chondrocytes has been evaluated. The receptor, a single class of glucocorticoid binding component approached saturation, utilizing [3H]triamcinolone acetonide ([3H]TA) as the radiolabeled ligand, at approximately 1.8-2.0 x 10(-8) M. The dissociation constant (Kd) reflected high-affinity binding, equaling 4.0 +/- 1.43 x 10(-9) M (n = 7) for [3H]TA. The concentration of receptor estimated from Scatchard analysis was approximately 250 fmol/mg cytosolic protein and when calculated on a sites/cell basis equalled 5800 sites/cell. The relative binding affinities of steroid for receptor were found to be triamcinolone acetonide greater than corticosterone greater than hydrocortisone greater than progesterone greater than medroxyprogesterone acetate much greater than 17 alpha-hydroxyprogesterone much greater than testosterone greater than 17 beta-estradiol. Cytosolic preparations activated in vitro by warming (25 degrees C for 20 min) were shown to exhibit an increased affinity for DNA-cellulose. 46% of the total specifically bound activated ligand-receptor complex was bound to DNA-cellulose. Cytosol maintained at 0-4 degrees C in the presence of 10 mM molybdate or activated in vitro in the presence of molybdate, bound to DNA-cellulose at 8 and 10% respectively. DEAE-Sephadex elution profiles of the nonactivated receptor were indicative of a single binding moiety which eluted from the columns at 0.4 M KCl. Elution profiles of activated receptor were suggestive of an activation induced receptor lability. The 0.4 M KCl peak was diminished, while a concomitant increase in the 0.2 M KCl peak was only modestly discernible. Evaluation of endogenous proteolytic activity in chondrocyte cytosol using [methyl-14C]casein as substrate show a temperature-dependent proteolytic activity with a pH optimum of 5.9-6.65. The proteolytic activity was susceptible to heat inactivation and was inhibitable, by 20 mM EDTA. The sedimentation coefficient of the nonactivated receptor was 9.3s (n = 6) on sucrose density gradients and exhibited steroid specificity and a resistance to activation induced molecular alterations when incubated in the presence of 10 mM molybdate. Receptor activation in vitro, in the absence of molybdate induced an increased receptor susceptibility to proteolytic attack and/or enhanced ligand receptor dissociation as evidenced by a diminution of the 9.3s binding form without a concomitant increase in 5s or 3s receptor fragments.

Influence of oestradiol on protein expression and methionine utilization during morphogenesis of Paracoccidioides brasiliensis.

The temporal sequence of cytosolic protein expression during phase transition of Paracoccidioides brasiliensis was examined. Electrophoretic analysis of cytosol proteins by one-dimensional SDS-PAGE revealed numerous differences between the mycelial and yeast forms as well as alterations induced by 17 beta-oestradiol. Using either protein staining or fluorography of [35S]methionine-labelled proteins 30 phase-specific bands were detected, 12 mycelial-associated bands (range 30 to 140 kDa) and 18 yeast-associated bands (range 22 to 127 kDa). In cells undergoing mycelial to yeast transition after a shift from 25 degrees C to 37 degrees C, the protein patterns showed a temporal progression toward the yeast profile with the accumulation of yeast bands prior to observable morphogenesis. Five novel protein bands (range 23 to 50 kDa) were detected by silver staining during transition. Treatment of temperature-shifted mycelial cultures with 2.6 x 10(-7) M-oestradiol altered observed profiles; 4 of 12 mycelial-associated bands were maintained whereas the appearance of the 5 novel transition bands and 9 of 18 yeast-associated bands was blocked or delayed. Analysis of [35S]methionine-labelled proteins revealed that oestradiol induced label uptake by mycelial cells, blocked the synthesis of a 92 kDa yeast-specific band 72 h into transition, and diminished label incorporation 120 h into transition. In conjunction with these steroid-induced alterations of protein expression, little or no morphological transformation occurred. These results support our hypothesis that, analogous to mammalian steroid receptor action, the functional responses of P. brasiliensis to oestradiol are related to regulation of protein expression, presumably mediated via a specific binding protein-ligand complex.

Methoxyflurane enhances allyl alcohol hepatotoxicity in rats. Possible involvement of increased acrolein formation.

The effect of methoxyflurane anesthesia on allyl alcohol-induced hepatotoxicity and the metabolism of allyl alcohol was studied in male rats. Hepatotoxicity was assessed by the measurement of serum alanine aminotransferase activity and histopathological examination. Allyl alcohol-induced hepatotoxicity was enhanced when allyl alcohol (32 mg/kg) was administered 4 hr before or up to 8 days after a single 10-min exposure to methoxyflurane vapors. The possibility that methoxyflurane increases alcohol dehydrogenase-dependent oxidation of allyl alcohol to acrolein, the proposed toxic metabolite, was evaluated by measuring the rate of acrolein formation in the presence of allyl alcohol and liver cytosol. The effect of methoxyflurane on alcohol dehydrogenase activity in liver cytosol was also assessed by measuring the rate of NAD+ utilization in the presence of ethyl alcohol or allyl alcohol. Alcohol dehydrogenase activity and rate of acrolein formation were elevated in methoxyflurane-pretreated rats. The results suggest that a modest increase in alcohol dehydrogenase activity and rate of acrolein formation markedly enhances allyl alcohol-induced hepatotoxicity.

Presence of benzodiazepine-like molecules in mammalian brain and milk.

From bovine cerebral cortex extracts, used to isolate n-butyl beta carboline-3-carboxylate (8), fractions active in displacing [3H] flunitrazepam binding were purified and shown to contain benzodiazepine-like molecules. These were recognized by UV spectra, retention time in HPLC, and interaction with a specific monoclonal antibody. Such molecules were localized in synaptic vesicles and cytosol of synaptosomes. Similar molecules were also found in cow milk. The possible dietary origin of these benzodiazepine-like molecules is discussed.

Binding of steroids in nuclear extracts and cytosol of rat pituitary and estrogen-induced pituitary tumors.

We have determined binding sites for estrogen, progestin, androgen and glucocorticoid in anterior pituitaries from Sprague-Dawley rats, a strain with low estrogen sensitivity, and in diethylstilbestrol-induced pituitary tumors in Fischer 344 rats, a strain with high estrogen sensitivity. Binding sites differ in their quantity and subcellular distribution. Cytosolic sites for [3H]estradiol in normal pituitaries from untreated rats were high prevailing over sites for other hormones, but they were depleted in the tumors due to their retention in nuclei under the influence of estrogen. Unoccupied nuclear sites for estrogen in normal glands also prevailed over sites for other steroids, and were similar to those in tumors. Second, the progestin site labeled with [3H]R 5020 was concentrated 5.7-fold in cytosol and 8.5-fold in nuclei of the tumors over the values found in glands from normal males estrogenized for 3 days. Third, glucocorticoid receptors labeled with [3H]dexamethasone were predominantly cytosolic in normal glands, but very low in cytosol and more evident in nuclear extracts from the tumors, the reverse of the profile found in normal pituitaries. Last, limited and comparable amounts of androgen receptors were measured in the subcellular fractions of both tissues. It is suggested that the subcellular distribution of some steroid receptors may be controlled in part by the cell population of the tissue and its degree of genetic activity.

Variations in soluble and particulate ABP of rat testis during sexual development.

Androgen binding activity (ABP) was determined in two different fractions of developing rat testicular homogenates: in cytosol (cABP) and in a particulate fraction isolated by differential centrifugation (pABP). Homogenates were prepared under stabilization conditions by adding 350 nM testosterone to the homogenization buffer. cABP and pABP concentrations were maximal in 22- to 32-day-old animals, to decrease thereafter during sexual maturation. However, both cABP and pABP increased with age when results were expressed on a per organ basis. pABP could be solubilized under conditions in which it could retain its binding activity. It was then photoaffinity labeled and chromatographed on a Sephadex G 200 column using cytosolic epididymal ABP as a control. Similarities between cABP and pABP include not only the same androgen binding characteristics but also the same exclusion volume after Sephadex G 200 chromatography. Since pABP is only present in Sertoli cells, it might represent ABP before being secreted. Because of its intracellular localization, it could play a role in the compartmentalization of androgens within the testis.

Stearic acid desaturation in rat liver microsomes: stimulation by fatty acid binding protein.

A soluble protein (Mr = 12,000) showing the characteristics of fatty acid binding protein is partially purified from rat liver cytosol (15-fold on the basis of its affinity for oleic acid) using ammonium sulphate precipitation. More oleate than stearate is removed from liver microsomes incubated with similar amounts of both fatty acids and the protein, indicating that it has a higher affinity for oleic than for stearic acid. When added to microsomes, a fraction enriched in this protein stimulates stearic acid desaturation. Such an effect is abolished if the protein is pre-saturated with oleic acid. It is suggested that the stimulation of stearic acid desaturation by fatty acid binding protein may involve a selective removal of the product, oleic acid, from the microsomal membranes.

Stearic acid desaturation in rat liver microsomes: stimulation by fatty acid biding protein

Una proteína soluble (Mr=12 000) similar a FABP es parcialmente purificada a partir de citosol de hígado de rata (15 veces, considerando su afinidad por ácido oleico) por precipitación con sulfato de amonio. Durante la incubación de microsomas de hígado de rata conteniendo cantidades similares de los ácidos esteátrico y oleico, con esta proteína, hubo una disociación selectiva del ácido oleico desde la membrana microsomal. Cuando se agrega a microsomas una fracción enriquecida en esta proteína se estimula la desaturación del ácido esteárico. Este efecto es eliminado si la proteína es presaturada con ácido oleico. Se sugiere que esta proteína estimula la desaturación del ácido esteárico por un mecanismo que involucra la remoción del producto de la reacción

Stearic acid desaturation in rat liver microsomes: stimulation by fatty acid biding protein

Una proteína soluble (Mr=12 000) similar a FABP es parcialmente purificada a partir de citosol de hígado de rata (15 veces, considerando su afinidad por ácido oleico) por precipitación con sulfato de amonio. Durante la incubación de microsomas de hígado de rata conteniendo cantidades similares de los ácidos esteátrico y oleico, con esta proteína, hubo una disociación selectiva del ácido oleico desde la membrana microsomal. Cuando se agrega a microsomas una fracción enriquecida en esta proteína se estimula la desaturación del ácido esteárico. Este efecto es eliminado si la proteína es presaturada con ácido oleico. Se sugiere que esta proteína estimula la desaturación del ácido esteárico por un mecanismo que involucra la remoción del producto de la reacción (AU)