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J R Hubbard

Publications and source records attributed to J R Hubbard.

33 records · Page 2Linked to original sources
PubMed1985

Rapid, high temperature exchange assay for the hepatic glucocorticoid receptor.

Liver cytosol from adrenalectomized rats was prebound for 2 hr at 4 degrees C with unlabeled 10(-5) M corticosterone. After treatment of cytosol with dextran-coated charcoal to remove free steroid, samples were incubated at 15-25 degrees C in the presence of 10 mM molybdate plus 5 mM dithiothreitol (followed by a 60 min incubation at 4 degrees C). Essentially, complete exchange of [3H] dexamethasone for receptor-bound unlabeled steroid was observed after 120 min at 15 degrees C, and near complete (80-95%) exchange occurred within 60 min at 25 degrees C using these conditions. However in control, 5 mM dithiothreitol (alone) and 10 mM molybdate (alone) treated samples, less than 50% exchange was found. Using a similar protocol, only partial exchange was found in brain and kidney cytosols, suggesting at least partial specificity for the hepatic system. We have used this rapid, high temperature exchange assay to study the regulation of hepatic cytoplasmic glucocorticoid receptors under some experimental conditions.

Adrenalectomy↗
PubMed1985

Interaction of the hepatic glucocorticoid-receptor complex with Affigel blue.

Unlike the unactivated glucocorticoid-receptor complex, the thermally activated glucocorticoid-receptor complex was able to bind to Affigel blue (a matrix previously shown to bind proteins containing a dinucleotide fold region) under low ionic conditions (0.05 M K C1). Glucocorticoid-receptor complex binding capacity to Affigel blue was enhanced by increasing salt concentration. Optimal binding was obtained at 0.15 M K C1 and remained at a plateau level up to 0.4 M K C1. In contrast to Affigel blue binding, glucocorticoid-receptor complex binding to nuclei was optimum at low ionic strength buffer, declined at 0.15 M K C1 and became negligible at 0.4 M K C1. Interestingly, at physiological ionic strength (0.15 M K C1) both nuclei and Affigel blue bound to the glucocorticoid-receptor complex with almost identical capacity. Glucocorticoid-receptor complexes incubated 45 min at 25 degrees C (activation conditions) in the presence of 10 mM molybdate were unable to bind to Affigel blue (or isolated nuclei) as expected. The results obtained suggest that Affigel blue mimics isolated nuclei in the binding of activated glucocorticoid-receptor complexes under physiological (0.15 M K C1) conditions. In addition, Affigel blue may provide a rapid and easy method to study glucocorticoid-receptor complex activation and interaction with nuclear acceptor sites.

Animals↗
PubMed1984

Influence of proteinase inhibitors on glucocorticoid receptor binding.

The influence of several proteinase inhibitors on rat liver cytosolic glucocorticoid receptor binding to [3H]triamcinolone acetonide has been investigated. E-64 (36 microM), an active-site directed cysteine proteinase inhibitor, significantly (about 40%) inhibited receptor binding. Tos-Lys-CH2Cl (1-2 mM) and Tos-Phe-CH2Cl (1-2 mM) also depressed receptor binding (20-67%). Interestingly, 5 mM dithiothreitol was able to prevent Tos-Lys-CH2Cl and Tos-Phe-CH2Cl effects, but had no apparent influence on E-64 action. A degree of proteinase inhibitor specificity was indicated by the lack of effect of several other proteinase inhibitors such as diisopropylfluorophosphate (1 mM), phenylmethylsulfonyl fluoride (1-2 mM), soybean trypsin inhibitor (1-2 mg/ml), tissue inhibitor of metalloproteinase (3.8 U/ml), cystatin (4-8 microM), and phosphoramidon (20-40 microM). These results suggest that thiol reactive proteinase inhibitors block glucocorticoid receptor binding, and that E-64 may prove to be a useful chemical probe in studying glucocorticoid receptor interaction.

Animals↗
PubMed1984

Tosyl-lysyl chloromethane alters glucocorticoid-receptor complex nuclear binding and physical properties.

We have investigated the effect of tosyl-lysyl chloromethane (a serine proteinase inhibitor) on rat liver cytosolic glucocorticoid-receptor complex binding to isolated nuclei. Tosyl-lysyl chloromethane (1-2 mM) significantly blocked nuclear binding when added before (but not after) thermal activation. Fifty percent inhibition occurred at about 1 mM tosyl-lysyl chloromethane. Interestingly, several other serine proteinase inhibitors (tosyl-phenylalanyl chloromethane, phenylmethyl sulphonylfluoride, and diisopropyl fluorophosphate) also depressed glucocorticoid-receptor complex nuclear binding. Tosyl-lysyl chloromethane, like molybdate, inhibited dilution-induced nuclear binding at low temperature, altered the DEAE-cellulose binding characteristics of heat-treated glucocorticoid-receptor complexes, and caused glucocorticoid-receptor complexes to sediment at about 9-10S (control complexes sedimented at 7-8S) in low salt-sucrose density gradients. Overall, these results suggest that tosyl-lysyl chloromethane modulates several properties of the glucocorticoid-receptor complex, that tosyl-lysyl chloromethane effects resemble those of molybdate, and that a serine proteinase(s) could be involved in the mechanism of glucocorticoid-receptor complex activation into a nuclear binding form.

Amino Acid Chloromethyl Ketones↗
PubMed1983

Chloroquine stabilizes hepatic glucocorticoid receptors.

Chloroquine (an antiarthritic, antimalarial, lysosomotropic amine) was found to significantly stabilize rat unbound hepatic glucocorticoid receptors in vitro for 2 h at 25 degrees C. Chloroquine stabilization was concentration dependent with statistically significant protection at 0.3 mM concentration and optimal effectiveness at approximately 3 mM. KC1 (0.3 M) induced unbound receptor inactivation at low temperature was also markedly reduced in the presence of 3 mM chloroquine. In addition, steroid prebound complexes were significantly stabilized at 4 degrees C and 25 degrees C by 3 mM chloroquine. Unlike molybdate (perhaps the most commonly used glucocorticoid receptor stabilizing reagent), chloroquine did not alter the sedimentation of glucocorticoid-receptor complexes in sucrose-density gradients. These results suggest that chloroquine may have useful application in glucocorticoid receptor quantitation, characterization and purification and may have interesting implications into the biological and pharmacological effects of chloroquine.

Animals↗
PubMed1983

Inactivation of hepatic glucocorticoid receptors by heparin.

Heparin dramatically enhanced the rate of unbound glucocorticoid receptor inactivation in vitro in a concentration, time and temperature-dependent manner. Control specific binding decreased only about 25% after incubation for 6 h at 4 degrees C. However in the presence of heparin (40 micrograms per ml cytosol) receptor binding decreased about 75%. At 25 degrees C liver receptor specific binding was found to have a half-life of about 60 min in control cytosol. However, in the presence of heparin (40 micrograms per ml cytosol) the glucocorticoid receptor had a half-life of only 15 min at 25 degrees C. Interestingly, 10 mM molybdate (with or without 5 mM dithiothreitol) greatly inhibited heparin-dependent receptor inactivation at 4 degrees C. Dithiothreitol (alone) significantly stabilized receptor binding in control samples at 4 degrees C, but provided no protection from heparin-dependent receptor inactivation. Heparin had no apparent inactivating effect on prebound glucocorticoid receptor complexes at 4 degrees C. Interestingly however, heparin altered the sedimentation coefficient of prebound hepatic glucocorticoid-receptor complexes in low salt gradients from 7-8 S to about 3-4 S. When molybdate plus dithiothreitol were added with heparin, the sedimentation coefficient was found to be approx. 6-7 S. These results demonstrate that heparin, which is often used pharmacologically and which occurs naturally in animal tissues, has significant effects on liver glucocorticoid receptors in vitro.

Animals↗
PubMed1983

Differential effects of chelating agents on cytosolic glucocorticoid receptor stability and nuclear binding in vitro.

The effect of several metal chelators (EDTA, EGTA, and 1,10 phenanthroline) on rat liver glucocorticoid receptor properties in vitro was investigated. At 4 degrees C 10 mM EDTA (unlike 10 mM EGTA and 10 mM 1,10 phenanthroline) had a significant stabilizing effect on unbound hepatic glucocorticoid receptors. At higher temperature (25 degrees C) 10 mM EGTA appeared to act as a chemical stabilizer of unbound receptors. 1,10 Phenanthroline had no stabilizing effect at either temperature. Scatchard analysis indicated that the alteration in receptor binding after incubation at 4 and 25 degrees C in the presence and absence of chelating agents was due to a change in the number of steroid binding sites rather than perturbation of receptor affinity. Unlike results obtained with unbound receptors, all three chelating agents appeared to enhance prebound glucocorticoid-receptor complex inactivation. Interestingly these chelating reagents also significantly altered glucocorticoid-receptor complex binding to isolated nuclei.

Animals↗
PubMed1983

Development of an exchange assay for cytosolic glucocorticoid receptors using the synergistic effects of molybdate plus dithiothreitol.

A glucocorticoid receptor exchange assay has been developed for the accurate quantification of both free and steroid-bound receptors in rat liver cytosol. Hepatic cytosol from adrenalectomized rats was saturated in vitro with unlabeled corticosterone. Cytosol was subsequently treated with [3H]dexamethasone (with and without 1000-fold cold dexamethasone) for 2-28 h at 4 C in the presence of 10 mM molybdate plus 5 mM dithiothreitol (DTT). Complete exchange occurred between 16-28 h in the presence of molybdate plus DTT. In control and 10 mM molybdate (alone) treated samples only about 50% exchange was achieved. In the presence of 5 mM DTT (alone) approximately 60-70% exchange was observed. The exchange assay (utilizing molybdate plus DTT) was also applied to hepatic cytosol of adrenalectomized rats injected with corticosterone in vivo and to samples prebound with unlabeled dexamethasone.

Adrenalectomy↗
PubMed1982

Regulation of cartilage acid hydrolases by growth hormone.

The in vivo regulation of three acid hydrolases, namely cathepsin D, cathepsin B, and acid phosphatase, by GH was investigated. The costal cartilage cathepsin D and acid phosphatase activities of hypophysectomized rats were reduced relative to those found in normal controls. Treatment of hypophysectomized animals with GH enhanced rat growth rate and increased these two enzyme activities toward normal levels. Results of pepstatin experiments suggested that the elevated cartilage cathepsin D activity corresponded to an increase in enzyme concentration. A degree of specificity in this regulation was apparent because cartilage cathepsin B, unlike cathepsin D and acid phosphatase, was refractory to hypophysectomy and GH treatment. In contradistinction to cartilage, none of these hepatic enzymes responded to GH, and only cathepsin B activity was diminished by hypophysectomy. Centrifugational and detergent studies indicated that changes in enzyme activities induced by GH treatment were not due to the differential release of acid hydrolases from subcellular compartments. Overall, our results suggest that costal cartilage cathepsin D and acid phosphatase activities are GH dependent and may be related to cartilage growth. These observations may provide insight into the mechanism of GH action and, derivatively, skeletal growth.

Acid Phosphatase↗
PubMed1981

Enhancement of cartilage protease activity during age and growth hormone-dependent growth.

We wish to report an intriguing relationship between cartilage protease activity and rat growth rate. This was demonstrated by comparing protease activities of rats having different growth rates, i.e., normal rats of different ages, hypophysectomized and growth hormone-treated hypophysectomized rats. Protease activity, assessed by hydrolysis of a gelatin film by cartilage microtome slices, at pH 4.0, was time and temperature dependent. Preincubation of cartilage tissue at various temperatures resulted in an increase of protease activity from 4 degrees C to 37 degrees C and a decrease in activity from 37 degrees C to 100 degrees C. The activity of younger (4 week old) more rapidly growing rats, was greater than that of older, less rapidly growing animals. Hypophysectomy reduced protease activity to approximately one-third normal levels. However, injection of bovine growth hormone into hypophysectomized rats restored the activity. These results suggest that a positive correlation exists between cartilage protease activity and growth rate. Our results support the novel hypothesis that cartilage growth could be mediated, at least in part, via growth hormone-dependent proteolytic activity.

Aging↗
PubMed1980

Somatomedin-like bioactivities of a growth hormone fragment on embryonic chick cartilage and cultured human fibroblasts.

Somatomedins are growth hormone-dependent peptides which appear to mediate many of the effects of growth hormone in vivo. These peptides are commonly assayed by their enhancement of proteoglycan sulfation in cartilage (or fibroblasts). We now report that fragment A-II (bovine growth hormone, 96--133) stimulates sulfation in chick embryonic cartilage and cultured fibroblasts. Enhancement of fibroblast proteoglycan sulfation by fragment A-II was log-dose dependent with maximal stimulation at 10(-8) M. The 25--100% maximal enhancement by fragment A-II was similar to that reported with a preparation of somatomedin A (Wasteson, A., Uthne, K., Westermark, B. (1973) Biochem. J. 136, 1069--1074). Sulfation of chick cartilage, in the presence of both serum (hypopituitary human) and fragment A-II was greater than the sum of the effects of each substance tested separately. Fragment A-II was tested between 10(-12) and 10(-8) M; maximal stimulation occurred at 5 . 10(-11) M. To our knowledge, no other growth hormone fragment has yet been shown to possess these somatomedin-like bioactivities. Our results suggest that fragment A-II may be very useful as a model peptide to study the actions and mechanism of naturally occurring sometomedins.

Animals↗
PubMed1976

The bonded alumina crown. 2. Construction using the twin foil technique.

A technique has been developed for making aluminous procelain crowns bonded to thin tin oxide coatings on platinum foil. These crowns require only very thin metal linings of less than 0.05 mm, thus reducing metal costs and considerably improving aesthetics. In addition, by using the "twin foil" technique, porcelain butt fits may be obtained without loss of fit.

Aluminum↗
PubMedDate not supplied

The influence of stress intrusion on immunodepression in generalized anxiety disorder patients and controls.

Previous work from our group has examined the relationship between stress and immunodepression in medical students taking National Boards, Part I, and has described a relationship between stress intrusion scores (SIS) and immunodepression. We have also shown that a high proportion of individuals with generalized anxiety disorders (GAD) and panic disorders (PD) exhibit enhanced stress intrusion (SI) and are more prone to upper respiratory infections (URI). In the present preliminary study, we sought to establish a model to evaluate further the role of SI level on the extent of immunodepression. This would serve to assess in further studies the mechanism(s) of stress-induced immunodepression, its relationship to morbidity, and the role of therapeutic interventions. In 14 GAD patients and 14 controls, we correlated the expression of interleukin-2 receptors (CD25) on T lymphocytes stimulated with anti-CD3 in short term cultures and the frequency of URI and the SIS to assess the relationships among these parameters. A decreased expression of CD25 correlates linearly with increasing SIS and with a higher number of sick days with URI. These results support our previous observations that GAD patients are more susceptible to URI. Moreover, they suggest that there may be a direct relationship between immunodepression and morbidity and between SIS and immunodepression.

Anxiety Disorders↗