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Biomedical subjects

J Hubbard

Publications and source records attributed to J Hubbard.

50 records · Page 3Linked to original sources
PubMed1983

Biochemical characterization of glucocorticoid receptors of rat testis.

Rat testis cytosolic glucocorticoid receptors were characterized by DEAE-cellulose chromatography, Sephadex G-100 columns and sucrose-density gradients. The unactivated [3H]dexamethasone-receptor complex showed two distinct peaks of macromolecular bound radioactivity on DEAE-cellulose chromatography. Peak I eluted just after the column wash, while peak II eluted at 0.28 M KCl. Activation of the complex at 25 degrees C for 45 min resulted in a significant increase in peak I with a concomitant decrease in peak II and the appearance of a third peak at 0.18 M KCl. Both the unactivated and activated [3H]dexamethasone-receptor complex, when analyzed on Sephadex G-100 columns, showed a single macromolecular bound radioactive peak having a Stokes radius of 6.5 nm. Treatment of the [3H]dexamethasone-receptor complex (6.5 nm holo-receptor) with trypsin (0.5 microgram/ml) resulted in the appearance of a smaller (2.0 nm) fragment but no intermediate sized forms of the receptor were observed. The complexes sedimented as 7-8 S (in low salt) and as 4.6 S (in high salt) forms in sucrose gradients in the presence or absence of 10 mM molybdate. Steroid unbound receptors were inactivated at 25 degrees C and 4 degrees C with a T 1/2 of 2 h and 24 h, respectively. Ten mM molybdate slightly protected the unbound testis receptor at 25 degrees C. However, molybdate, dithiothreitol, and molybdate plus dithiothreitol were unable to either enhance or reactivate [3H]dexamethasone binding of unbound receptors at 4 degrees C or 25 degrees C. Activation of testis [3H]dexamethasone-receptor complexes resulted in a 2-3 fold enhancement in subsequent binding to testis nuclei in vitro. In addition, we observed that activated [3H]dexamethasone-receptor complexes were precipitated with 30-35% ammonium sulfate, while unactivated complexes were precipitated with 30-40% ammonium sulfate.

Animals↗
PubMed1983

The effect of calcium on the hepatic glucocorticoid receptor.

The unbound glucocorticoid receptor of rat hepatic cytosol was very unstable at 25 degrees C. This receptor instability was increased by the addition of 1-10 mM Ca2+ such that the unbound glucocorticoid receptor was completely inactivated within 30 min at 25 degrees C in the presence of 5 mM Ca2+. Interestingly, Ca2+ inactivation was blocked approximately 80% by simultaneous addition of either 10 mM molybdate or 10 mM molybdate plus 5 mM dithiothreitol. Ten millimolar leupeptin or 10 mM alpha 2-macroglobulin did not inhibit Ca2+ inactivation of the receptor. In fact, leupeptin alone slightly inactivated the receptor. Prior treatment of cytosol with 10 mM molybdate plus 5 mM DTT at 25 degrees C for 30 min offered a complete protection against the subsequent addition of 5 mM Ca2+. The effects of Ca2+ were found to be irreversible since addition of molybdate or molybdate plus dithiothreitol after 5 mM Ca2+ treatment (20 min for 25 degrees C) did not reactivate partially inactivated receptors but did stabilize the remaining receptor binding at the time of molybdate or molybdate plus dithiothreitol addition. Addition of 1-5 mM Ca2+ to preformed [3H]-dexamethasone--receptor complexes slightly inhibited steroid--receptor complex transformation into a nuclear binding form at 25 degrees C. In addition, 10 mM Ca2+ altered the agarose gel filtration profile of the complex. Control cytosol or cytosol treated with 10 mM EDTA or 10 mM leupeptin showed three distinct macromolecular bound radioactivity peaks having Stokes radii of 65, 40 and 20 A. On the other hand, Ca2+-treated cytosol showed a single large aggregated component which was excluded in the void volume of the column. Trypsin (0.5 microgram/ml) generated a small steroid--receptor fragment of 20 A when added alone, but interestingly when added with 10 mM Ca2+ the [3H]-dexamethasone--receptor complex eluted in the agarose gel exclusion volume. These results show that Ca2+ has a profound effect on rat hepatic glucocorticoid receptors and that molybdate can block Ca2+-dependent receptor inactivation.

Animals↗
PubMed1982

Interaction of sodium thiocyanate with rat hepatic glucocorticoid-receptor complexes.

0.1-0.3 M sodium thiocyanate greatly enhanced the rate of inactivation of unbound rat hepatic glucocorticoid receptors in vitro at 4 degrees C. Prior treatment of the unbound glucocorticoid receptor with 10 mM molybdate (at 25 degrees C for 30 min) protected the receptor from 0.3 M KCl, but not from 0.3 M NaSCN inactivation. When the [3H]dexamethasone-receptor complex was examined on sucrose density gradients containing 0.1 M NaSCN, the receptor sedimented as a 4 S complex rather than the 7 S form observed in 0.1 M KCl gradients. NaSCN was found to be more effective in the extraction of both in vivo and in vitro nuclear-bound [3H]dexamethasone-receptor complexes than KCl. At a concentration of 0.3 M, NaSCN extracted most of the specific nuclear-bound receptor. 50 mM NaSCN significantly blocked the thermal activation of preformed [3H]dexamethasone-receptor complexes. The chaotropic salt, NaSCN, appears therefore to have significant effects on glucocorticoid receptors in vitro. In addition, NaSCN appears to be a useful agent in quantitative extraction of steroid from nuclear-bound steroid-receptor complexes.

Animals↗
PubMed1982

Acquired subglottic stenosis following prolonged endotracheal intubation. A canine model.

We developed a canine animal model of subglottic stenosis following prolonged intubation with modified non-cuffed endotracheal tubes. None of the puppies intubated for seven days had an irreversible stenosing subglottic lesion, whereas all animals intubated for 14 days or more had at least a 40% to 50% reduction of the subglottic lumen secondary to maturing fibrotic stenosis. The model described is more congruent with the known and suspected pathogenesis in those infants and children who require prolonged endotracheal intubation and subsequently acquire subglottic stenosis, and can be used in evaluating the efficacy of medical therapy or surgical therapy, or both, in the prevention or management of this disease.

Animals↗
PubMed1976

Monoamine oxidase activity in the fetal lung and liver.

Monoamine oxidase (MAO) in lung and liver is important in the degradation of circulating 5-hydroxytryptamine. These sites of MAO activity have been investigated histochemically in the human fetus of 12 to 18 weeks gestation. Enzyme activity could be demonstrated in the liver by both tryptamine and adrenaline oxidation. In the lung, MAO activity was present only when adrenaline was used to substrate. It may be, therefore, that in the premature baby the capacity of MAO to metabolize 5-hydroxytryptamine is not fully developed, which could lead to deleterious effects on pulmonary function.

Epinephrine↗