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D Bellabarba

Publications and source records attributed to D Bellabarba.

At least 55 records · Page 3Linked to original sources
PubMed1985

Characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase in human adrenal cortex.

Our study compares the properties of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from a human metastatic virilizing carcinoma and that from normal adrenal glands obtained from kidney donors. Optimal conditions for enzyme assay were obtained when a 50-mM imidazole buffer (pH 7.2) containing 5 mM EDTA, 250 mM NaCl, 1 mM phenyl-methylsulfonylfluoride, 0.1 mM leupeptin, and 5.5 mM dithiothreitol (DTT) was used. A 30-min preincubation period preceding addition of substrates enhanced reductase activity by 1.75-fold. In crude microsomal preparations, Km values were similar for both tumor and normal tissues and varied between 4 and 5 microM (S)HMG-CoA. The presence of NaF in homogenization and incubation media decreased the maximum velocity, but not the Km. A partially purified rat liver phosphorylase phosphatase preparation or a similar preparation from the carcinoma restored to maximal levels the reductase activity of microsomes prepared in the presence of NaF. A Km of 96 microM NADP was found for the carcinoma microsomal preparation. Preincubation of microsomes in the presence of monothioglycerol or DTT resulted in an increased reductase activity, suggesting a possible inactive enzyme precursor(s) consisting of disulfide-linked units. Reactions of the DTT-activated enzyme incubated in the presence of increasing amounts of NADPH showed sigmoidal kinetics. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed the presence of a 92.5K mol wt protein band that reacted with a rat antireductase antibody. Reductase activity in different regions of the carcinoma varied from 679-1763 pmol/mg protein, with an average of 1146 pmol. In three normal adrenal glands we found values of 23.4, 48.1, and 36 pmol. We concluded that the expression of HMG-CoA reductase activity was elevated in human adrenal carcinoma.

Adrenal Cortex↗
PubMed1984

Corticosteroid receptors in the kidney of chick embryo. III. Nature, properties, and ontogeny of aldosterone receptor.

An aldosterone receptor in the cytosol from kidney of chick embryos which had a sedimentation coefficient of 8.2 S and a molecular weight higher than 100,000 was identified. Kinetic analysis at 4 degrees revealed a rapid association of the hormone to the receptor that followed second-order reaction kinetics and a dissociation of pseudo-first-order reaction kinetics. The association (ka) and dissociation (kd) rate constants were, respectively, 4.94 X 10(5) M-1 sec-1 and 8.33 X 10(-6) sec-1. From their ratio a KA value of 5.9 X 10(10) M-1 was calculated. In a series of experiments performed with kidneys of 17-day-old embryos, the KA at equilibrium, obtained from the Scatchard plot, was 3.1 +/- 1.2 X 10(8) M-1, whereas the Nmax was 172 +/- 14 fmol/mg protein. Competition studies with various steroids demonstrated that corticosterone had an affinity for the receptor close to that of aldosterone, thus suggesting a degree of resemblance of the mineralo- and glucocorticoid receptors in the chick embryo. However, the profiles of the binding affinities and capacities during the embryogenesis showed that the aldosterone-binding sites had a pattern completely different from that of the glucocorticoid receptor, indicating that the two receptors are most likely separate entities.

Aldosterone↗
PubMed1984

Increased 3-hydroxy-3-methyl-glutaryl coenzyme A reductase activity in a virilizing adrenal carcinoma.

HMG-CoA reductase activity was determined on microsomal preparations of an adrenal carcinoma and on a control adrenal obtained from palliative surgery for breast carcinoma. In both tissues we also measured [14C]pyruvate incorporation to study the formation of sterols. The endogenous adrenal content of cholesterol and its esters was quantitated. The content of various steroids was also determined in tissues and media before and after incubations in Krebs-Ringer. The carcinoma had a HMG-CoA reductase activity of 972.0 pmol/mg protein/min vs 13.8 for the control adrenal. The tumor incorporated 4.6 pmol of [14C]pyruvate per mg protein per 90 min into digitonin precipitable sterols compared to 0.5 pmol found for the control gland. Free cholesterol and cholesterol esters in tumoral tissue were 0.09/100 mg and 0.02/100 mg tissue respectively, compared to 0.18 and 2.56 in control tissue. The output of corticosteroids and androgens was very high when calculated for the whole tumor. These results suggest that the carcinoma had acquired a high capacity for de novo synthesis of cholesterol which could have served as substrate for the observed high plasma androgen level.

Adrenal Cortex Hormones↗
PubMed1984

Triiodothyronine nuclear receptors in liver, brain and lung of neonatal rats. Effect of hypothyroidism and thyroid replacement therapy.

In this study we have examined the effect of neonatal hypothyroidism and triiodothyronine (T3) replacement therapy on the maximal binding capacity (MBC) and the affinity of T3 receptors prepared from liver, brain and lungs. Rats were radiothyroidectomized at birth and administered T3 or its placebo starting at 8 days of age. The thyroid state in normal, hypothyroid and hypothyroid T3-treated rats was assessed by serum determination of thyroxine, T3 and by the measurement of the hepatic alpha-glycerophosphate dehydrogenase. The animals were sacrificed at 8, 16 or 24 days of age and the T3 binding was estimated in isolated nuclei and in salt nuclear extracts. The MBC of the T3 receptors was higher in the hypothyroid rats at all ages when it was determined in isolated nuclei, but not when was measured in nuclear extracts. At 8 days, the MBC had risen twofold or more in the receptors from brain (1.0 +/- 0.37 vs. 0.4 +/- 0.1 ng T3/mg DNA in controls) and from lungs (0.6 +/- 0.25 vs. 0.3 +/- 0.15 ng T3/mg DNA in controls), but was only slightly elevated in the hepatic receptor (0.62 +/- 0.08 vs. 0.48 +/- 0.15 ng T3/mg DNA in controls). At 16-24 days, the highest value of MBC was observed in the hepatic receptor (0.83 +/- 0.04 vs. 0.41 +/- 0.1 ng T3/mg DNA in controls) followed by the brain receptor (0.65 +/- 0.03 vs. 0.35 +/- 0.02 ng T3/mg DNA in controls), and that of lung (0.39 +/- 0.07 vs. 0.20 +/- 0.03 ng T3/mg DNA in controls).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Luteinizing hormone-releasing hormone binds to enriched human placental membranes and stimulates in vitro the synthesis of bioactive human chorionic gonadotropin.

Partially purified cell membranes were prepared from midterm and term placentas after sedimentation on a sucrose density gradient. Biochemical characterization showed that the sucrose density pellet was enriched 8-fold in alkaline phosphatase activity and also contained the majority of [125I]LHRH binding sites. This enrichment was also confirmed by electron microscopy. Specific binding of LHRH was then determined by incubating iodinated LHRH or two of its superanalogs with increasing doses of the corresponding radioinert ligand. Scatchard representation of the data showed curvilinear plots whose first component revealed, for both stages of pregnancy, saturable binding of [125I]LHRH and its agonists with similar association constants (Ka) that ranged between 5.5 X 10(5) M-1 and 1.1 X 10(7) M-1. When standardized per milligram of DNA content, the number of binding sites ranged between 225 and 310 X 10(-12) M. Specificity was evidenced by the inability of a biologically active LHRH antagonist, oxytocin, and TRH to inhibit [125I]LHRH binding. Short term placental cultures incubated with 1.5 X 10(-6)M LHRH had increased production rates of both immunoassayable and bioassayable hCG, and this effect was 4-fold higher in midterm placental cultures. Placental incubations with either buffer or equimolar concentrations of oxytocin or TRH had no effect on hCG production. These observations expand information on extrapituitary binding sites of LHRH and suggest a role for this peptide in the physiology of the human placenta.

Cell Membrane↗
PubMed1983

Corticosteroid receptors in the kidney of chick embryo. I. Nature and properties of corticosterone receptor.

Cytosol from kidney of chick embryo (age 16-18 days) contained a corticosterone-binding site with the features of a putative receptor. This receptor was a thermolabile protein, readily digested by proteolytic enzymes, with a sedimentation coefficient of 7-8 S and with an apparent molecular weight greater than 100,000. Simultaneous studies with transcortin (CBG) revealed several differences between the renal- and serum-binding protein pertaining to the effect of temperature, the sedimentation coefficient, the charcoal "stripping" and, finally, the binding and competition of various steroids for the two proteins. Kinetic analysis showed a rapid association (10 min), which followed second-order reaction kinetics, and a dissociation of pseudo-first-order reaction kinetics with a t1/2 of 168 min at 0 degrees. The analysis of the Scatchard plot showed the presence of a single class of binding sites with an association constant (KA) of 1.3 X 10(8)M-1 and a binding capacity (nmax) of 500-700 fmol/mg protein. We obtained similar results when we used dexamethasone as a ligand. The association (ka) and dissociation (kd) rate constants were respectively 2.9 X 10(6)M-1 sec-1 and 6.86 X 10(-5) sec-1. From their ratio a KA value of 4.2 X 10(10) M-1 was obtained. Studies with various steroids demonstrated that only dexamethasone and, to a lesser degree, progesterone competed for the binding site. These data showed that the kidney of chick embryo possessed one type of receptor for the glucocorticoids, which was similar to the type II described in rat kidney.

Animals↗
PubMed1983

Corticosteroid receptors in the kidney of chick embryo. II. Ontogeny of corticosterone receptor and cellular development.

In this study the ontogeny of cytosol receptors for corticosterone in the chick embryo kidney was examined and then this ontogenic profile was compared with that of an index of cellular development, i.e., the enzyme ornithine decarboxylase (ODC). The corticosterone receptor concentration (nmax) increased by sevenfold from Day 12 to Day 15 of embryogenesis and then declined to its lowest level by the time of hatching (Day 21). Similar results were obtained when dexamethasone was used as ligand, except that the baseline values at Day 9 and 21 were higher than those found with corticosterone. An identical ontogenic profile was obtained when the results were corrected for the endogenous glucocorticoids present in the cytosol. This increase of the corticosterone receptor occurred simultaneously with the enhanced adrenal corticoid synthesis. The ODC also showed a marked increase and a rapid fall during chick embryogenesis, but the enzyme activity was at its maximum when the corticosterone receptor number was still low (Days 12 and 13) and quickly decreased by the time the receptors had reached their highest levels (Days 14 and 15). The lowest level of ODC was observed immediately before hatching. These results indicate that during chick embryogenesis adrenal corticoids may induce the development of the corticosteroid receptor and that such development may cause a suppression of ODC activity. This suppressive effect of glucocorticoids could represent a mechanism of hormonal action on the kidney.

Age Factors↗
PubMed1983

Properties of triiodothyronine-binding proteins in liver cytosol of rat.

The electrophoretic mobility and the sedimentation coefficient were determined in partially purified preparations of both rat liver cytosol and serum triiodothyronine (T3)-binding proteins. Crude cytosol and serum, each labeled with [125I]T3, were filtered through a Sephadex G-100 column. The cytosol yielded a single T3-binding peak, whereas three binding components were recognized in the serum. Protamine sulfate precipitated the cytosol T3-binding protein, but had no effect on the serum T3-binders. The cytosol protein and the three binding proteins from serum were analyzed by polyacrylamide gel electrophoresis and sucrose density gradient centrifugation. The cytosol binder migrated as a single peak on gel electrophoresis with an Rf of 0.53, whereas the serum proteins had RfS between 0.27-0.33. The sedimentation coefficient of the cytosol protein was 6.3 S, whereas it was 4.1 S for the major binding protein of the serum. These data indicate that: (i) preliminary purification by gel chromatography is a useful step for better characterization of the T3-binding proteins of the cytosol and serum; (ii) the cytosol binder is an acidic protein with completely different properties from those of the serum T3-binding proteins.

Animals↗
PubMed1982

Exchange assays using sodium thiocyanate to measure total nuclear content of estrogen receptors in the hypothalamic pituitary axis of mice.

We studied the effect of sodium thiocyanate (NaSCN) in the determination of specific nuclear estrogen receptors from the hypothalamic-pituitary axis (HPA) of mice. We compared the results with those of the exchange assay using either suspensions of whole nuclei or KC1-nuclear extracts. Our findings demonstrated that 0.5 M NaSCN was more efficient than 0.5 M KC1 in extracting [3H] E2 nuclear content from HPA (91% vs 79.3%). Nuclear fractions extracted with 0.5 M NaSCN revealed the presence of a single class of low-capacity-high affinity binding sites that sedimented in the 4.0 S area. Nuclear binding was T degrees dependent and reached maximum levels of 450 +/- 156 (S.E.) fmol/mg DNA after an overnight incubation at 4 degrees C. Such levels were comparable to those observed in whole nuclei suspensions after 1 hour incubation at 37 degrees C (618 +/- 71 fmol/mg DNA, p greater than 0.05) but two-fold higher (p less than 0.01) than the concentration of binding sites measured in KC1-extracted nuclear fractions under similar experimental conditions. We conclude that NaSCN extracted the total content of nuclear estrogen receptors in HPA of mature mice.

Animals↗
PubMed1981

Relationship between hepatic malate dehydrogenase and thyroid hormones in the chicken during embryogenesis and the neonatal period.

The synthesis of hepatic cytosol malate dehydrogenase (MDH) can be stimulated by thyroid hormones under various conditions, and consequently, this enzyme has been considered as a marker of the expression of thyroid hormone action. Since the concentrations of serum thyroid hormones increase considerably during the late phase of chick embryogenesis, we have studied the relationship between this phenomenon and liver MDH activity. A delay of 5 days was observed between the increase in circulating T4 and T3 concentrations and the enhancement of MDH activity. However, treatment with pharmacological doses of T4 (60 microgram/day) resulted in increased MDH activity 48 h after administration of the hormone, while hypothyroidism induced by methimazole decreased the level of the enzyme. Sex-linked differences in MDH activity were found in newborn chickens between 5-9 days of age. No such differences were observed for serum T3 and T4. These findings suggest that under physiological conditions MDH activity is not directly modulated by thyroid hormones in the chick embryo. However, these hormones may well play a permissive role with this enzyme.

Animals↗