Metabolic Consequences of Oral Administration of 24,25-Dihydroxycholecalciferol to Uremic Dogs

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Abstract

24,25-dihydroxycholecalciferol [24,25-(OH)2D3], once considered a relatively inert metabolite of vitamin D3, has been recently recognized as a metabolically active product in some species. In previous studies, we have shown that infusion of 24,25(OH)2D3 into the thyroid artery of normal dogs results in prompt and complete suppression of parathyroid hormone (PTH) secretion. In this study, we have examined the metabolic consequences of oral administration of this metabolite in dogs with experimentally induced renal hyperparathyroidism. Dogs with comparable degrees of renal insufficiency (glomerular filtration rate, 10-15 ml/min) were treated for 3 wk with daily doses of either 2 μg of 24,25(OH)2D3 or 50% ethanol, the vehicle in which the metabolite was suspended. After a 6-wk recovery period, treatments were reversed: dogs who had previously served as controls received the metabolite while dogs previously treated with metabolite received the vehicle. Administration of 24,25(OH)2D3 resulted in a 40-60% decrease of immunoreactive PTH. This was associated with a small (0.1-0.2 mg/dl) but unequivocal decrease of serum ionized calcium. Calcium balance, which was slightly negative under control conditions, became slightly but definitively positive on treatment with 24,25(OH)2D3. All other parameters measured, including total serum calcium, magnesium, phosphorus, creatinine, electrolytes, phosphorus excretion, and phosphorus balance, remained unchanged. The data support the hypothesis that 24,25(OH)2D3 not only decreases PTH secretion but also functions as an anabolic hormone in bone under the conditions of this experiment.

Authors

Janet M. Canterbury, George Gavellas, Jacques J. Bourgoignie, Eric Reiss

Beta Adrenergic Receptors of Polymorphonuclear Particulates in Bronchial Asthma

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We have tested the beta adrenergic receptor theory of bronchial asthma by determining the number and affinity of binding sites of the beta adrenergic radioligand [3H]dihydroalprenolol (DHA) and the activity of adenylate cyclase in broken cell preparations of polymorphonuclear leukocytes (PMN). We studied 31 control subjects (group 1), 30 asthmatics receiving no systemic adrenergic medication (group 2), and 17 asthmatics receiving adrenergic agonists systemically (group 3). Control subjects and asthmatics taking no adrenergic drugs bound similar amounts of DHA at 0.5 nM and 30 nM DHA and had about 900 binding sites per PMN. In contrast, asthmatics receiving adrenergic agonists had a >70% decrease in their number of DHA binding sites per PMN (254±57). In a subset of our three groups of subjects (eight from group 1, six from group 2, and five from group 3) we measured DHA binding at several DHA concentrations and found similar values (0.4-0.7 nM) for the dissociation constant of DHA among these subjects.

Authors

Stanley P. Galant, Lakshmi Duriseti, Sharon Underwood, Sandra Allred, Paul A. Insel

Binding of Retinoids to Human Breast Cancer Cell Lines and their Effects on Cell Growth

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Vitamin A and its analogues (retinoids) regulate the differentiation of epithelial tissues. Retinoids inhibit the induction of rat mammary cancers by carcinogens in vivo, and cellular binding proteins for retinoids have been demonstrated in some human breast cancer samples. In this study, we examined the model system of human breast cancer cell lines in long-term tissue culture for effects of retinoids on growth and for the presence of cellular retinoid binding proteins. Retinoic acid and retinol inhibit the growth of of MCF-7, Hs578T, and ZR-75-B cell lines. Retinoic acid is more potent than retinol in this regard: 50% growth inhibition is achieved by 6 nM retinoic acid in ZR-75-B and by 700 nM in MCF-7 and Hs578T, whereas 5-8 μM retinol is required in all three cell lines. The time to onset of growth inhibition varies markedly between cell lines and is not related to cell density or doubling time. Retinoic acid increases the doubling time of MCF-7 and ZR-75-B by two- to threefold, but causes cell death in Hs578T. The growth inhibition is reversible in every cell line by removal of retinoic acid. Specific and distinct binding of [3H]retinoic acid and [3H]retinol is present in cytosols of MCF-7 and Hs578T cells as assessed by sucrose density gradient centrifugation. In ZR-75-B, [3H]retinoic acid binding was present, but no binding of [3H]retinol was detectable. This study reveals that retinoids may play an important role in the regulation and treatment of human breast cancer and that human breast cancer cell lines represent a useful model to study this role.

Authors

Andre Lacroix, Marc E. Lippman

Linkage Analysis between the Major Histocompatibility System and Insulin-dependent Diabetes in Families with Patients in Two Consecutive Generations

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We have histocompatibility (HLA) genotyped 28 families with insulin-dependent diabetics in two or more consecutive generations, usually parent and child. This strategy of ascertainment was used to maximize the likelihood of obtaining a homogeneous type of disease within a family, and an autosomal dominant mode of inheritance. 76 diabetics and 169 nondiabetics were studied in these families.

Authors

Jose Barbosa, M. Myra Chern, V. Elving Anderson, Harriet Noreen, Sandra Johnson, Nancy Reinsmoen, Ronald McCarty, Richard King, Leonard Greenberg

Preservation of androgen secretion during estrogen suppression with aminoglutethimide in the treatment of metastatic breast carcinoma.

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Abstract

We evaluated the comparative effects of aminoglutethimide (AG) on androgen and estrogen levels estrone ([E1], estradiol [E2], plasma dehydroepiandrosterone-sulfate [DHEA-S], testosterone [T], dihydrotestosterone [DHT], delta 4-androstenedione [delta 4-A]), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin in postmenopausal patients with breast cancer randomly allocated to either AG treatment or bilateral surgical adrenalectomy as a control group. In response to either treatment, the plasma levels of E1 fell 62-75% (P less than 0.001) and urine E1 85.7-88.7% (P less than 0.001) in all study days over a 12-wk period. Similarly, the concentrations of E2 in plasma and urine fell 40-72% without statistically significant differences between the two treatment modalities. The relatively weak androgen, DHEA-S, was reduced by 92% (877.3 +/- 184.6 to 71.8 +/- 14.5 ng/ml) at 12 wk in women treated with AG, but suppressed nearly 99% (1,151 +/- 262 to 5.8 +/- 3.3 ng/ml) in adrenalectomized women. At all time points after treatment, the DHEA-S levels were significantly higher in patients receiving AG. Plasma concentrations of the potent androgens, T and DHT, were also relatively preserved during AG treatment. T levels were never significantly reduced by AG, and DHT concentrations were decreased only at the 4th wk to a maximum of 20%. delta 4-A levels fell 56% in response to this drug only on the 12th wk of therapy (basal, 0.79 +/- 0.09 ng/ml; 12 wk, 0.35 +/- 0.07 ng/ml). In marked contrast, all androgens fell significantly at each time period in response to surgical adrenalectomy, with an 81% maximum suppression of T, 73% of DHT, and 97% of delta 4-A. In response to estrogen suppression, plasma levels of FSH, LH, and prolactin did not change significantly throughout the treatment period in either therapy group. To examine possible contributions of the postmenopausal ovary to hormone levels during therapy, data from surgically castrate and spontaneously menopausal women were evaluated separately. No significant differences between the two groups were observed for E1, E2, T, DHT, DHEA-S, delta 4-A, LH, FSH, and prolactin. We conclude that equivalent and highly significant estrogen suppression occurs with either AG or surgical adrenalectomy although androgen secretion is preserved during AG treatment but not after surgical adrenalectomy. The combined effects of estrogen deprivation associated with androgen preservation might be significant in the therapeutic action of AG in hormone-responsive neoplasms.

Authors

E Samojlik, J D Veldhuis, S A Wells, R J Santen

Volumetric and functional heterogeneity of human monocytes.

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Volume analysis of purified human blood monocytes revealed distinct populations of large and small cells. Computer curve fitting suggested a third, intermediate-sized population. These monocytes were designated M1, M2, and M3 in order of increasing size, and their approximate volumes were 150, 250, and 480 micron3, respectively. The three subpopulations were present in all 30 normal individuals tested. Two new techniques were developed that separate monocytes into M1 + M2 and M3 fractions; one used preferential incorporation of carbonyl iron particles by M3 cells and the other used the selective aggregation of M3 cells by thrombin in the presence of platelets. The chemotactic response to zymosan-activated human serum by total monocytes, M1 + M2 monocytes, and M3 monocytes was determined by the agarose plate method. In all experiments M3 monocytes were 10-fold more responsive than M1 + M2 monocytes and were significantly more so than total monocytes. These findings suggest that M3 cells are the major subpopulation capable of directional migration. This investigation establishes the existence of volumetrically definable subpopulations of human monocytes that are functionally distinct.

Authors

E B Arenson Jr, M B Epstein, R C Seeger

Resistance of Gram-negative Bacteria to Purified Bactericidal Leukocyte Proteins: RELATION TO BINDING AND BACTERIAL LIPOPOLYSACCHARIDE STRUCTURE

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The sensitivity or resistance of gram-negative bacteria to antibacterial systems appears to be related to the length of the saccharide chain of the bacterial envelope lipopolysaccharides (LPS). To explore this relationship further, we made use of two bactericidal, membrane-active cationic proteins, recently purified to near homogeneity, one from human and one from rabbit polymorphonuclear leukocytes (PMN). We have studied the effects of these two closely similar proteins on strains of Salmonella typhimurium and Escherichia coli, each separate strain differing in the saccharide chain length of its outer membrane LPS. Binding of these proteins to the bacterial outer membrane is required for killing, and is accompanied by an almost immediate increase in outer membrane permeability to normally impermeant actinomycin D. Sensitivity to the bactericidal and permeability-increasing activities of the human and rabbit proteins increases with decreasing LPS-saccharide chain length (chemotype: [S < Ra < Rb3 < Rc < Rd1]). S. typhimurium G-30 and E. coli J5, mutant strains lacking UDP-galactose-4-epimerase, synthesize incomplete LPS (chemotype Rc) when grown without galactose, and are then as sensitive to both PMN proteins as the S. typhimurium strains 395 R10 (Rd1) and R5 (Rb3). However, when these mutants are grown with galactose, they synthesize complete LPS (chemotype S) and exhibit nearly the same relative insensitivity as the smooth strains S. typhimurium 395 MS and E. coli 0111:B4.

Authors

Jerrold Weiss, Susan Beckerdite-Quagliata, Peter Elsbach

Familial rheumatoid arthritis: linkage of HLA to disease susceptibility locus in four families where proband presented with juvenile rheumatoid arthritis.

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The occurrence of a chronic seronegative polyarthritis has been studied in four families in which the proband presented with some form of juvenile rheumatoid arthritis. In these families, histocompatibility testing suggested that susceptibility to arthritis was controlled by a dominant allele with variable penetrance and expressivity at the rheumatoid-like arthritis, first locus (RLA-1). The combined lod scores for the four families (2.70) indicated that the odds in favor of genetic linkage between the major histocompatibility complex and the postulated disease susceptibility gene, RLA-1, were 500:1. In one family, a recombinant event permitted localization of RLA-1 centromeric to HLA-D. Of major interest was the fact that there was significant pleomorphism in the clinical manifestations of arthritis in affected individuals. In some, symptoms first occurred in childhood and in others, in adult life. Even among those with childhood-onset arthritis, different types of juvenile rheumatoid arthritis were observed within the same family.

Authors

R D Rossen, E J Brewer, R M Sharp, J Ott, J W Templeton

Prevention of Collagen Deposition Following Pulmonary Oxygen Toxicity in the Rat by Cis-4-Hydroxy-l-Proline

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Exposure of rats to high oxygen tensions causes increased collagen content of lungs and alveolar enlargement in 3-6 wk. We tested whether cis-hydroxyproline, a proline analogue that inhibits collagen synthesis, could prevent the collagen accumulation and alveolar enlargement. Rats were exposed to hyperoxia for 60 h and then to room air and hyperoxia for alternate 24-h periods for 11.5 d. Treated oxygen-exposed rats received 200 mg/kg cis-hydroxyproline twice daily over the 14-d exposure period. Control rats breathed room air. Examination of lungs on day 14 showed collagen content of oxygen-exposed lungs to be 48% greater than control (P < 0.05). The collagen content of the treated oxygen-exposed lungs was −12% of control (NS). Total lung volume was 16% greater than control in oxygen-exposed rats (P < 0.05) and 8% greater than control in treated oxygen-exposed rats (NS). Morphometric studies showed alveolar size was greater than control in oxygen-exposed rats (188±11 [SE] vs. 143±6 μμl [P < 0.05]). Oxygen-exposed, treated rats had a mean alveolar volume of 150±7 μμl. Lung pressure-volume curves were significantly shifted to the left of control in the oxygen-exposed rats, whereas the curves of the oxygen-exposed, treated group were identical to control. These data suggest that cis-hydroxyproline prevented the accumulation of collagen in the lungs in pulmonary oxygen toxicity. In addition, there was apparent protection from airspace dilatation and decreased lung elasticity, suggesting that alveolar enlargement after oxygen toxicity is linked to the deposition in lung tissue of new connective tissue fibers.

Authors

David J. Riley, Richard A. Berg, Norman H. Edelman, Darwin J. Prockop

Effect of apoproteins on hepatic uptake of triglyceride emulsions in the rat.

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The addition of apoprotein E isolated from human very low density lipoproteins to both rat lymph chylomicrons and a triglyceride emulsion significantly increased the hepatic uptake of these particles in a nonrecycling isolated rat liver perfusion system. The cleared triglyceride was removed without apparent hydrolysis by the hepatocyte. When lymph chylomicrons were loaded with both Apo E and Apo C proteins by exposure to rat plasma, no increment in hepatic clearance was observed. Sequential evalutions of the influence of the C apoproteins on the hepatic clearance of both emulsions and chylomicrons revealed that the CIII (CIII-1) protein had a pronounced inhibitory effect on hepatic removal. The inhibition was observed for both Apo E-enriched chylomicrons and those containing little of this apoprotein.

Authors

F Shelburne, J Hanks, W Meyers, S Quarfordt

Evaluation of Role Played by Mediators of Immediate Hypersensitivity in Exercise-induced Asthma

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To determine whether mediators of immediate hypersensitivity played a role in the pathogenesis of exercise-induced asthma, we measured the concentration of histamine and neutrophil-chemotactic activity present in systemic arterial blood during thermal challenges in five asymptomatic asthmatics. Because exercise-induced asthma has been shown to be a result of respiratory heat loss and because respiratory heat loss during isocapnic hyperventilation has been shown to give identical responses, we chose the latter provocational method in order to minimize increases in cardiac output that might interfere with the interpretation of mediator concentrations in arterial blood. Multiple aspects of pulmonary mechanics were also recorded before and after provocation. The results of these studies were then compared with the effects observed when the same subjects inhaled aerosols of specific antigens on the same day. Each challenge produced identical alterations in lung function, and neither was associated with consistent changes in arterial histamine. However, antigen provocation evoked a sustained and prolonged release of neutrophil chemotactic activity in each subject, whereas isocapnic hyperventilation with cold air was without effect. These data strongly suggest that mast-cell derived mediators are not involved in the development or maintenance of the bronchial obstruction that follows exercise in asthmatics.

Authors

E. Chandler Deal Jr., Stephen I. Wasserman, Nicholas A. Soter, R. H. Ingram Jr., E. R. McFadden Jr.

Circulating heparan sulfate proteoglycan anticoagulant from a patient with a plasma cell disorder.

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A woman, aged 68, with multiple myeloma (immunoglobulin[Ig]A kappa type) developed an anticoagulant with properties suggestive of heparin. The anticoagulant prolonged the thrombin time but not the reptilase time and was resistant to boiling, proteolytic enzyme digestion, and trichloracetic acid precipitation. The thrombin time was corrected by the addition (in vitro) of protamine sulfate or the addition of purified platelet Factor 4 (PF4) to the plasma. The anticoagulant was isolated by PF4-Sepharose affinity chromatography and analyzed in terms of its molecular weight, uronic acid, and amino acid composition. The proteoglycan isolated had a mol wt of 116,000 and appears to consist of two 38,000 dalton polysaccharide units interconnected by peptide material totaling 39,000 daltons. Electrophoretic analysis of the pronase digested peptidoglycan using the lithium acetate-agarose technique suggested the material was of the heparan sulfate type. The peptidoglycan had about one-tenth the specific activity of commercially available heparin on a weight basis. The isolated proteoglycan was indistinguishable from commercial heparin when analyzed in terms of its ability to act as a cofactor in the antithrombin III inhibition of thrombin.

Authors

M S Khoory, M E Nesheim, E J Bowie, K G Mann

Direct Radioimmunoassay of Nuclear 3,5,3′ Triiodothyronine in Rat Anterior Pituitary

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Previous tracer studies have suggested that 5′-monodeiodination of l-thyroxine (T4) in anterior pituitary may contribute a substantial portion of specifically bound nuclear 3,5,3′ l-triiodothyronine (T3) in this tissue in rats. To evaluate this possibility, a radioimmunoassay for nuclear T3 in individual anterior pituitaries was developed. Animals received [125I]T3 60 min before removal of the anterior pituitary and isolation of the nuclei by differential centrifugation. This allowed calculation of the nuclear:serum T3 ratio and comparison of expected with measured T3. T3 was extracted in ethanol, dried, and reconstituted in assay buffer. In untreated hypothyroid rats, anterior pituitary nuclear T3 was 0.18 ± 0.06 pg/μg DNA which was 0.13 pg/μg DNA greater than expected from the serum T3 concentration and the pituitary nuclear:serum [125I]T3 ratio. In 10 hypothyroid rats given a single bolus of 400 ng T3/100 g body wt., the nuclear T3 by radioimmunoassay was 1.0 ± 0.06 pg/μg DNA, whereas that expected from the T3 specific activity calculations was 0.85 pg/μg DNA (P < 0.025). Serum T4 concentrations in these rats were < 0.25 μg/dl but the nuclear T3 derived from as little as 0.2 μg/dl T4 could explain a large portion of these small discrepancies between observed and measured nuclear T3. In 29 normal rats, anterior pituitary nuclear T3 was 0.63±0.04 pg/μg DNA, whereas that expected from the serum T3 concentration (55±2 ng/dl) was 0.23±0.02 pg/μg DNA (P < 0.001). Total pituitary T3 based on this measurement was 92±6 pg. Because the maximal nuclear binding capacity for T3 in rat anterior pituitary is 0.77 pg/μg DNA, these results suggest there is 82% occupancy of these nuclear receptors. The requirement for normal serum concentrations of both T4 and T3 to achieve normal nuclear T3 saturation in anterior pituitary is in marked contrast to the situation in liver, kidney, and heart muscle which appear to require only a normal serum T3. As a consequence, the anterior pituitary can monitor both serum T4 and T3 and respond appropriately to changes in their concentrations.

Authors

P. R. Larsen, S. Z. Bavli, M. Castonguay, R. Jove

Adrenergic Mechanisms for the Effects of Epinephrine on Glucose Production and Clearance in Man

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The present studies were undertaken to assess the adrenergic mechanisms by which epinephrine stimulates glucose production and suppresses glucose clearance in man: epinephrine (50 ng/kg per min) was infused for 180 min alone and during either alpha (phentolamine) or beta (propranolol)-adrenergic blockade in normal subjects under conditions in which plasma insulin, glucagon, and glucose were maintained at comparable levels by infusion of somatostatin (100 μg/h), insulin (0.2 mU/kg per min), and variable amounts of glucose. In additional experiments, to control for the effects of the hyperglycemia caused by epinephrine, variable amounts of glucose without epinephrine were infused along with somatostatin and insulin to produce hyperglycemia comparable with that observed during infusion of epinephrine. This glucose infusion suppressed glucose production from basal rates of 1.8±0.1 to 0.0±0.1 mg/kg per min (P < 0.01), but did not alter glucose clearance. During infusion of epinephrine, glucose production increased transiently from a basal rate of 1.8±0.1 to a maximum of 3.0±0.2 mg/kg per min (P < 0.01) at min 30, and returned to near basal rates at min 180 (1.9±0.1 mg/kg per min). Glucose clearance decreased from a basal rate of 2.0±0.1 to 1.5±0.2 ml/kg per min at the end of the epinephrine infusion (P < 0.01). Infusion of phentolamine did not alter these effects of epinephrine on glucose production and clearance. In contrast, infusion of propranolol completely prevented the suppression of glucose clearance by epinephrine, and inhibited the stimulation of glucose production by epinephrine by 80±6% (P < 0.001). These results indicate that, under conditions in which plasma glucose, insulin, and glucagon are maintained constant, epinephrine stimulates glucose production and inhibits glucose clearance in man predominantly by beta adrenergic mechanisms.

Authors

R. A. Rizza, P. E. Cryer, M. W. Haymond, J. E. Gerich

Inherited Methylmalonyl CoA Mutase Apoenzyme Deficiency in Human Fibroblasts: EVIDENCE FOR ALLELIC HETEROGENEITY, GENETIC COMPOUNDS, AND CODOMINANT EXPRESSION

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We have measured and characterized methylmalonyl coenzyme A (CoA) mutase activity in extracts of cultured human fibroblasts from 23 patients with inherited deficiency of the mutase apoenzyme and from eight obligate heterozygotes for this defect. The mutant cell lines fall into two categories. Those without detectable residual mutase activity in cell extracts (>0.1% of control), and whose ability to utilize propionate in intact cells is refractory to supplementation of the culture medium with hydroxocobalamin, are designated mut° mutants. Those with detectable residual activity in cell extracts (∼0.5-50% of control), and whose ability to utilize propionate in intact cells in markedly increased by hydroxocobalamin supplementation, are designated mut− mutants. The mutant enzyme in the mut− mutants exhibits a 50- to 5,000-fold elevated Michaelis constant (Km) for adenosylcobalamin in vitro, a normal Km for methylmalonyl CoA, and a strikingly reduced thermal stability at 45°C relative to control. Mutase from one mut− mutant turns over at a rate three to four times that of control enzyme when cells are grown in hydroxocobalamin-supplemented medium.

Authors

Huntington F. Willard, Leon E. Rosenberg

Estrogen Dependence of a Gonadotropin-induced Steroidogenic Lesion in Rat Testicular Leydig Cells

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Leydig cells isolated from the testes of rats treated with intravenous exogenous gonadotropin (hCG) or subcutaneous gonadotropin-releasing hormone (GnRH) show markedly decreased luteinizing hormone (LH) receptors and a partial block in testicular 17,20 desmolase activity. In contrast, Leydig cells from animals with equivalent degrees of LH receptor loss induced by subcutaneous hCG treatment show no change in 17,20 desmolase activity. These findings indicated that the acuteness of gonadotrophic stimulation, rather than the extent of LH receptor loss, was responsible for the steroidogenic lesion. A role of estradiol in the enzymatic block produced in vivo by acute elevation of circulating gonadotropin (intravenous hCG or GnRH-stimulated endogenous LH) was suggested by rapid elevations of testicular 17β-estradiol within 30 min after intravenous hCG, whereas more gradual increases in estradiol occurred 4-8 h after subcutaneous hCG. The inhibitory effect of endogenous estrogen on testicular steroidogenesis was confirmed by the ability of an estrogen antagonist (Tamoxifen) to prevent the reduction of testosterone responses caused by intravenous hCG and subcutaneous GnRH. In addition, Tamoxifen significantly increased the number of LH receptors in Leydig cells from both control and gonadotropin-desensitized animals. These findings indicate that the acute elevations of intratesticular estrogen produced by treatment with hCG or GnRH are responsible for the steroidogenic lesion seen in gonadotropin-desensitized Leydig cells. These results also suggest that locally produced estrogens contribute to the regulation of testicular LH receptors and 17,20 desmolase activity.

Authors

S. B. Cicorraga, S. Sorrell, J. Bator, K. J. Catt, M. L. Dufau

Glucagon deficiency and hyperaminoacidemia after total pancreatectomy.

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The first goal of this study was to investigate whether totally pancreatectomized patients are glucagon deficient and if so, to what degree. Immunoreactive glucagon (IRG) concentrations in peripheral plasma of nine pancreatectomized patients were not significantly different from those of 10 normal controls as measured by two antisera (30-K and RCS-5) both detecting the COOH-terminal portion of the molecule and one (RCS-5) postulated to be specific for pancreatic glucagon. Plasma from six of nine pancreatectomized patients were fractionated over Sephadex G-50 and IRG was measured with both antisera in the column eluates. Using 30-K, 80.8 +/- 9% of the IRG eluted within the void volume. This material was rechromatographed on Sephadex G-200 and found to have an apparent mol wt of approximately 200,000. Only 18.3 +/- 9% eluted in the IRG3500 region. IRG3500 was significantly reduced in pancreatectomized patients as compared to normal controls (49 +/- 9 vs. 18 +/- 9 pg/ml, P less than 0.05). Using RCS-5, all IRG (corresponding to 20 +/- 6 pg/ml of plasma) eluted in the IRG3500 region. The second goal of this study was to investigate the effects of chronic glucagon deficiency on plasma amino acids. In the nine pancreatectomized patients studied, postabsorptive plasma concentrations of serine, alanine, arginine, glycine, threonine, citrulline, alpha-aminobutyrate, and tryosine were significantly elevated compared to values obtained from 20 normal controls. Physiological glucagon increments produced in two pancreatectomized patients by infusion of glucagon (6.25 and 8.0 microgram/h, respectively) resulted in normalization of the hyperaminoacidemia within 22 h. We conclude (a) that pancreatectomized patients are partially glucagon deficient because of diminished basal as well as diminished stimulated glucagon secretion; (b) that fasting concentrations of certain glucogenic amino acids are elevated in pancreatectomized patients probably as result of reduce; hepatic gluconeogenesis; and (c) that the RCS-5 antiserum is not "pancreatic glucagon" specific.

Authors

G Boden, R W Master, I Rezvani, J P Palmer, T E Lobe, O E Owen

Epinephrine-induced Insulin Resistance in Man

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Endogenous release of epinephrine after stress as well as exogenous epinephrine infusion are known to result in impaired glucose tolerance. Previous studies of man and animals have demonstrated that this effect of epinephrine results from inhibition of insulin secretion and augmentation of hepatic glucose production. However, the effect of epinephrine on tissue sensitivity to insulin, and the relative contributions of peripheral vs. hepatic resistance to impaired insulin action, have not been defined. Nine young normal-weight subjects were studied with the insulin clamp technique. Plasma insulin was raised by ∼100 μU/ml while plasma glucose concentration was maintained at basal levels by a variable glucose infusion. Under these conditions of euglycemia, the amount of glucose metabolized equals the glucose infusion rate and is a measure of tissue sensitivity to insulin. Subjects received four studies: (a) insulin (42.6 mU/m2·min), (b) insulin plus epinephrine (0.05 μg/kg·min), (c) insulin plus epinephrine plus propranolol (1.43 μg/kg·min), and (d) insulin plus propranolol. During insulin administration alone, glucose metabolism averaged 5.49±0.58 mg/kg·min. When epinephrine was infused with insulin, glucose metabolism fell by 41% to 3.26 mg/kg·min (P < 0.001). After insulin alone, hepatic glucose production declined by 92% to 0.16±0.08 mg/kg·min. Addition of epinephrine was associated with a delayed and incomplete suppression of glucose production (P < 0.01) despite plasma insulin levels >100 μU/ml. When propranolol was administered with epinephrine, total glucose metabolism was restored to control values and hepatic glucose production suppressed normally. Propranolol alone had no effect on insulin-mediated glucose metabolism. These results indicate that epinephrine, acting primarily through a β-adrenergic receptor, markedly impairs tissue sensitivity to an increase in plasma insulin levels, and that this effect results from both peripheral and hepatic resistance to the action of insulin.

Authors

David C. Deibert, Ralph A. Defronzo

Immunochemical Evidence for Protein Abnormalities in Platelets from Patients with Glanzmann's Thrombasthenia and Bernard-Soulier Syndrome

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Crossed immunoelectrophoresis of Triton X-100 solubilized proteins from normal and abnormal platelets was performed with rabbit antibodies raised against normal platelets. In Bernard-Soulier platelets protein 13 was not detected, and neither the amphiphilic (probably GP Ib) nor the hydrophilic (glycocalicin) glycocalicin-related proteins were seen when monospecific antiglycocalicin antiserum was used. The most prominent precipitate, 16, and platelet fibrinogen, 24 were not detected in platelets of two patients with type I thrombasthenia, whereas in one patient with type II thrombasthenia fibrinogen was clearly detected, but the amount of protein 16 remained severely reduced. Protein 16 was heavily labeled after lactoperoxidase-catalyzed 125I iodination of normal platelets, and was precipitated by IgG-L, an alloantibody from a polytransfused thrombasthenic patient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or protein 16 cut out from immunoplates showed two 125I-labeled glycoprotein bands, which migrate as GP IIb and GP IIIa. SDS-PAGE of 125I-labeled type I thrombasthenic platelets showed no periodic acid-Schiff bands or peaks of radioactivity in the GP IIb and GP IIIa regions, whereas in the GP I region both the periodic acid-Schiff band intensity and the radiolabeling were within the normal range. Autoradiography after crossed immunoelectrophoresis of iodinated thrombasthenic platelets showed that the bulk of radioactivity was bound to protein 17. This glycoprotein, which was also present in normal and Bernard-Soulier platelets, migrates in the GP I region on SDS-PAGE. Thus, the bulk of radioactivity observed in the GP I region after SDS-PAGE is associated with protein 17 and not with glycocalicin.

Authors

Inger Hagen, Alan Nurden, Ole Jannik Bjerrum, Nils Olav Solum, Jacques Caen

Lack of influence of fetal hemoglobin levels or erythrocyte indices on the severity of sickle cell anemia.

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Persons with sickle cell anemia who have elevated fetal hemoglobin or lowered erythrocyte mean corpuscular volume are reputed to have less severe clinical manifestations and a greater probability of survival. This study examines the relationship between seven clinical indicators of morbidity in sickle cell anemia and seven hematological parameters that were collected from 214 patients. Risks of sickle cell crisis, acute chest syndrome, hospital admissions, cerebrovascular accident, aseptic necrosis, meningitis/septicemia, and death were used as indicators of morbidity. The hematological parameters included percent fetal hemoglobin, absolute fetal hemoglobin, percent hemoglobin A2, hemoglobin concentration, packed cell volume, mean corpuscular volume, and mean corpuscular hemoglobin concentration. Statistical analyses of the data showed no relationship between the hematological parameters and six of the seven clinical indicators of the severity of sickle cell anemia. The only significant finding was an increased risk of stroke in those patients with lower levels of fetal hemoglobin. Therefore, with this exception, there is no predictable relationship between morbidity and mortality in sickle cell anemia and levels of fetal hemoglobin or erythrocyte indices. Thus, the general belief that there is an association between severity of sickle cell anemia and the levels of fetal hemoglobin has not been established.

Authors

D R Powars, W A Schroeder, J N Weiss, L S Chan, S P Azen

Amino acid modulation of renal phosphatidylcholine biosynthesis in the rat.

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The hypothesis that amino acids act as modifiers of phospholipid biosynthesis was tested in renal cortical cells from normal rats. The rate of [14C]-choline incorporation into phospholipid in cortical slices was enhanced by the addition of lysine or arginine to the incubation medium, and reduced by phenylalanine, aspartic acid, or four other amino acids. Lysine and aspartic acid appeared to modify the cholinephosphotransferase reaction in which cytidine 5'-diphosphocholine (CDP-choline) and 1,2-diacylglycerol react to form phosphatidylcholine, the major phospholipid of renal membranes. Since this enzymatic reaction takes place in the endoplasmic reticulum, the effect of single amino acids on microsomal preparations was examined. Lysine increased CDP-choline:1,2-diacylglycerol cholinephosphotransferase activity by 95%, whereas aspartic acid reduced activity by 65%, in a concentration-dependent manner. For both substrates in the reaction, amino acids modulated enzyme activity by altering the maximum velocity without changing the apparent Km. These observations in intact renal cells and in microsomal preparations indicate that changes in cellular amino acid concentrations could modify the biosynthetic rate of phosphatidylcholine, and suggest a mechanism that could coordinate the biosynthesis of phospholipid and protein.

Authors

L J Havener, F G Toback

Stereoselective Interaction of Phenylbutazone with [¹²C/¹³C]Warfarin Pseudoracemates in Man

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Abstract

To evaluate the interaction of phenylbutazone with racemic warfarin or R,S-(±)-warfarin in man, S-(−)-warfarin or levowarfarin was synthesized with 13C label in the 2-position of the coumarin nucleus and added to [12C]R(+)-warfarin or dextrowarfarin to form a [12C/13C]pseudoracemate of warfarin. In six normal human subjects, a single oral dose of this “cold labeled” pseudoracemate, 1.5 mg/kg body weight, was administered with and without a daily dosage of phenylbutazone, 300 mg orally, beginning 3 d before the warfarin dose and continuing throughout the hypoprothrombinemia. Plasma samples were obtained daily and analyzed for warfarin content and for one-stage prothrombin activity. Unchanged warfarin in the plasma was fractionated by normal-phase, high-pressure liquid chromatography, and the enantiomorphic ratios were determined by chemical-ionization mass spectrometry with pentadeuteriowarfarin as the internal standard. A highly significant augmentation of the hypoprothrombinemia of the pseudoracemate occurred during the phenylbutazone regimen (P < 0.001) compared with pseudoracemic warfarin administered alone. There was a highly significant increase in the plasma clearance of dextrowarfarin (P < 0.01) and a significant decrease in the plasma clearance of levowarfarin (P < 0.05) during the phenylbutazone regimen compared with administration of warfarin alone. It was concluded that phenylbutazone augmented the hypoprothrombinemia of pseudoracemic warfarin stereoselectively by inhibiting the metabolic disposition of the more hypoprothrombinemic levowarfarin, yet reduced the plasma levels of pseudoracemic warfarin by greatly augmenting the metabolic disposition of dextrowarfarin.

Authors

Robert A. O'Reilly, William F. Trager, Catherine H. Motley, William Howald

Contraction of Cultured Rat Glomerular Cells of Apparent Mesangial Origin after Stimulation with Angiotensin II and Arginine Vasopressin

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Abstract

Studies to identify the physiological role of glomerular mesangial cells were undertaken using homogeneous cultures of rat glomerular cells of apparent mesangial origin (MS). Cultured MS cells were treated with arginine vasopressin (AVP), angiotensin II (AGII), prostaglandin E2, and parathyroid hormone. AVP (0.1 nM) and AGII (1 nM) stimulated contraction of MS cells in vitro that was complete by 2 min at 37°C or 10 min at 23°C as observed by phase contrast and electron microscopy. Relaxation recurred 15 min after hormonal addition at 23°C. Similar experiments in cloned rat glomerular epithelial cells or “renin”-producing cells did not demonstrate a contractile response. The contraction of MS cells was independent of cyclic AMP (cAMP) and cyclic 3′,5′-guanosine monophosphate (cGMP) production, even when cyclic nucleotides were measured as early as 30 s after hormonal stimulation. To demonstrate that contraction was a function of hormone-receptor interaction, binding of [3H](8-lysine)vasopressin was studied. Specific binding for 1.6 and 5 nM hormone was both time- and dose-dependent. The estimated apparent affinity was 10 nM. In late MS cell passages (>16th) that no longer demonstrated hormone-stimulated contraction, no specific binding of [3H](8-lysine)vasopressin was observed.

Authors

Dennis A. Ausiello, Jeffrey I. Kreisberg, Christian Roy, Morris J. Karnovsky

In vitro idiotypic suppression of chronic lymphocytic leukemia lymphocytes secreting monoclonal immunoglobulin M anti-sheep erythrocyte antibody.

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Abstract

A patient with chronic lymphocytic leukemia was found to have B cells with surface immunoglobulin (Ig)M manifesting anti-sheep erythrocyte (SRBC) specificity together with a high titer serum monoclonal anti-SRBC IgM antibody. By immunizing a sheep with the monoclonal IgM antibody, followed by multiple absorptions against normal human IgM, an anti-idiotype (Id) antibody was obtained. The serum IgM anti-SRBC antibody was then demonstrated to share the same idiotypic determinants with the surface IgM with anti-SRBC specificity on the patient's B cells. The anti-Id antibody suppressed the spontaneous secretion of anti-SRBC antibody as well as the pokeweed mitogen-induced anti-SRBC antibody production as measured by a hemolysis-in-gel plaque-forming cell assay. In contrast, pokeweed mitogen-induced anti-SRBC plaque-forming cell responses of normal individuals were not suppressed by the anti-Id antibody. Thus, this study demonstrates in vitro suppression of human B-cell function by anti-Id antibody.

Authors

C A Bona, A S Fauci

Plasma deoxyadenosine, adenosine, and erythrocyte deoxyATP are elevated at birth in an adenosine deaminase-deficient child.

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Abstract

We have determined concentrations of adenosine, deoxyadenosine, and deoxyATP (dATP) in cord blood from an infant prenatally diagnosed as ADA deficient. Plasma deoxyadenosine and adenosine were already elevated in cord blood (0.7 and 0.5 microM vs. normal of less than 0.07 microM). Elevation of plasma deoxyadenosine has not previously been documented in these children. Erythrocyte dATP content was also elevated at birth (215 nmol/ml packed erythrocytes vs. normal of 2.9). These elevated concentrations of adenosine, deoxyadenosine, and dATP are similar to those we observed in another older adenosine deaminase-deficient patient and may explain the impaired immune function and lymphopenia seen at birth.

Authors

R Hirschhorn, V Roegner, A Rubinstein, P Papageorgiou

Common pattern of two distinct types of colony-stimulating factor in human tissues and cultured cells.

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Abstract

Conditioned media prepared from human lung, placenta, peripheral leukocytes, cultured human pancreatic carcinoma cells, and cultured cervical carcinoma cells exhibit a common pattern of two distinct colony-stimulating factors (CSF) separable by isoelectrofocusing. Type I CSF appears polydisperse on isoelectrofocusing, showing multiple peaks with an isoelectric point of 3.6-4.7 and mol wt 50,000. It has a much higher activity in mouse than in human bone marrow, and stimulates the formation of predominately macrophage colonies. Type II CSF has an isoelectric point of 5.7 and mol wt 27,000 and exhibits higher activity in human than in mouse marrow, yielding predominately granulocytic colonies.

Authors

M C Wu, A A Yunis