CORRECTION

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Abstract

Authors

Receptor-mediated Modulation of Human Monocyte, Neutrophil, Lymphocyte, and Platelet Function by Phorbol Diesters

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The tumor promoting phorbol diesters elicit a variety of responses from normal and leukemic blood cells in vitro by apparently interacting with cellular receptors. The biologically active ligand [20-3H] phorbol 12,13-dibutyrate ([3H]PDBu) bound specifically to intact human lymphocytes, monocytes, polymorphonuclear leukocytes (PMN), and platelets, but not to erythrocytes. Binding, which was comparable for all four blood cell types, occurred rapidly at 23° and 37°C, reaching a maximum by 20-30 min usually followed by a 30-40% decrease in cell associated radioactivity over the next 30-60 min. The time course for binding was temperature dependent with equilibrium binding occurring after 120-150 min at 4°C, with no subsequent loss of cell-associated radioactivity at this temperature. Bound [3H]PDBu could be eluted by addition of unlabeled PDBu. Scatchard analysis of data from 4°C binding studies revealed linear plots with high affinity receptors in these cell types with dissociation constants and receptors per cell of 60 nM and 7.8 × 105/cell for lymphocytes, 51 nM and 15.5 × 105/cell for monocytes, 38 nM and 4.0 × 105/cell for PMN, and 19 nM and 2.9 × 104/cell for platelets. Structure-activity studies using unlabeled phorbol-related compounds demonstrated a close correlation between their abilities to inhibit binding of [3H]PDBu to cells and their abilities to induce cellular responses (monocyte and PMN H2O2 secretion, lymphocyte 3HTdR incorporation, and platelet tritiated serotonin release); phorbol and 4-alpha phorbol were inactive while phorbol 12-myristate 13-acetate (PMA), PDBu, mezerein, and phorbol 12,13-diacetate (in decreasing order of potency) inhibited [3H]PDBu binding and elicited the various responses. Thus, these high affinity, specific receptors for the phorbol diesters, present on monocytes, lymphocytes, PMN, and platelets, mediate the pleiotypic effects induced by these ligands.

Authors

Bonnie J. Goodwin

Spectrin beta-chain variant associated with hereditary elliptocytosis.

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An electrophoretically fast-moving variant of the spectrin beta-chain was discovered in the erythrocyte membranes of a woman and her father who both exhibited elliptocytosis and mild hemolytic anemia. This abnormal beta'-subunit (Mr = 214,000) co-existed with a decreased normal beta-chain and represented about half of the total beta-chains in the membrane. In contrast to the spectrin beta-chain, the beta'-chain was phosphorylated neither in the membrane by endogenous protein kinases nor in solution by pure membrane casein kinase whether or not the spectrin was dephosphorylated by erythrocyte cytosolic spectrin phosphatase. The presence of the beta'-chain was associated with a defective self-association of spectrin dimer to form tetramer as manifested by: (a) an excess of spectrin dimer in the 4 degrees C spectrin crude extract, (b) a defective self-association of the spectrin dimer in the 37 degrees C crude spectrin extracts. Gel electrophoretic analysis of the tetramer and dimer species isolated from the proband's 4 degrees C extract showed that the tetramer contained trace amounts of the beta'-chain, whereas in contrast, a large proportion of beta'-chain was present in the dimer. These results demonstrated the responsibility of the beta'-chain for the defective reassociation of spectrin dimer into tetramer. The study of this abnormal spectrin confirms the participation of spectrin beta-chain in dimer-dimer association and strongly suggests that the phosphorylation sites of the normal beta-chain are located at the end of the molecule involved in the dimer-dimer interactions.

Authors

D Dhermy, M C Lecomte, M Garbarz, O Bournier, C Galand, H Gautero, C Feo, N Alloisio, J Delaunay, P Boivin

Changes in Bone Mineral Density of the Proximal Femur and Spine with Aging: DIFFERENCES BETWEEN THE POSTMENOPAUSAL AND SENILE OSTEOPOROSIS SYNDROMES

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We measured bone mineral density (BMD) of the proximal femur, lumbar spine, or both by dual photon absorptiometry in 205 normal volunteers (123 women and 82 men; age range 20 to 92 yr) and in 31 patients with hip fractures (26 women and 5 men; mean age, 78 yr). For normal women, the regression of BMD on age was negative and linear at each site; overall decrease during life was 58% in the femoral neck, 53% in the intertrochanteric region of the femur, and 42% in the lumbar spine. For normal men, the age regression was linear also; the rate of decrease in BMD was two-thirds of that in women for femoral neck and intertrochanteric femur but was only one-fourth of that in women for lumbar spine. This difference may explain why the female/male ratio is 2:1 for hip fractures but 8:1 for vertebral fractures. The standard deviation (Z-score) from the sex-specific age-adjusted normal mean in 26 women with hip fracture averaged −0.31 (P < 0.05) for the femoral neck, −0.53 (P < 0.01) for the intertrochanteric femur, and +0.24 (NS) for the lumbar spine; results were similar for 5 men with hip fractures. By contrast, for 27 additional women, ages 51-65 yr, with only nontraumatic vertebral fractures, the Z-score was −1.92 (P < 0.001) for the lumbar spine. Thus, contrary to the view that osteoporosis is a single age-related entity, our data suggest the existence of two distinct syndromes. One form, “postmenopausal osteoporosis,” is characterized by excessive and disproportionate trabecular bone loss, involves a small subset of women in the early postmenopausal period, and is associated mainly with vertebral fractures. The other form, “senile osteoporosis,” is characterized by proportionate loss of both cortical and trabecular bone, involves essentially the entire population of aging women and, to a lesser extent, aging men, and is associated with hip fractures or vertebral fractures or both.

Authors

B. L. Riggs, H. W. Wahner, E. Seeman, K. P. Offord, W. L. Dunn, R. B. Mazess, K. A. Johnson, L. J. Melton III

Hepatic Uptake of Bile Acids in Man: FASTING AND POSTPRANDIAL CONCENTRATIONS OF INDIVIDUAL BILE ACIDS IN PORTAL VENOUS AND SYSTEMIC BLOOD SERUM

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This investigation was undertaken in order to (a) characterize the postprandial inflow of individual bile acids to the liver and (b) determine if peripheral venous bile acid levels always adequately reflect the portal venous concentration, or if saturation of hepatic bile acid uptake can occur under physiological conditions. In five patients with uncomplicated cholesterol gallstone disease, the umbilical cord was cannulated during cholecystectomy, and a catheter was left in the left portal branch for 5 to 7 d. The serum concentrations of cholic acid, chenodeoxycholic acid, and deoxycholic acid in portal venous and systemic circulation were then determined at intervals of 15 to 30 min before and after a standardized meal. A highly accurate and specific gas chromatographic/mass spectrometric technique was used.

Authors

Bo Angelin, Ingemar Björkhem, Kurt Einarsson, Staffan Ewerth

Mechanisms of proteinuria in human glomerulonephritis.

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We evaluated glomerular barrier function in 28 patients with glomerulonephritis. Neutral dextrans of graded size were used to characterize the size-selective properties of the barrier. Charge selectivity was characterized by electrofocusing excreted urinary proteins. A fractional IgG clearance (relative to freely permeable inulin), smaller or greater than 100 x 10(-5) was used to distinguish patients with minor (group I, n = 13) and major (group II, n = 15) urinary IgG leakage, respectively. Fractional clearances of smaller dextrans (radii 20-50 A) were similar, but those of larger dextrans (radii 52-60 A) were elevated in group II relative to group I patients. A model of solute transport through a bimodal pore size distribution revealed the values for pore radius in the lower mode to approximate 51-55 A in both group I and group II patients. Pore radius in the upper mode, by contrast, was much larger in group II than in group I patients, approximating 87-97 vs. 72-77 A, respectively. Electrofocusing of urinary protein from group I patients revealed mostly albumin (isoelectric point 5.2). In group II patients, however, immunoglobulin excretion was copious. Moreover, the distribution of anionic, neutral, and cationic species (isoelectric points 5.5-8.5) in urinary and plasma eluates of IgG2 and IgG4 was similar. We conclude that when glomerulonephritis is associated with selective albuminuria, as in group I,, there is an isolated reduction of electrostatic retardation of relatively small anionic proteins. Major urinary IgG leakage (group II), however, appears to result from the development in the glomerular membrane of a subpopulation of enlarged pores that are highly permeable towards proteins of large size and varying charge.

Authors

B D Myers, T B Okarma, S Friedman, C Bridges, J Ross, S Asseff, W M Deen

Generation of leukotrienes by purified human lung mast cells.

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Although mediator release from mast cells and basophils plays a central role in the pathogenesis of human allergic disease, biochemical studies have been restricted to rat peritoneal mast cells and basophilic leukemia cells because they could be easily purified. We have used two new techniques of cell separation to purify human lung mast cells to 98% homogeneity. Lung cell suspensions were obtained by dispersion of chopped lung tissue with proteolytic enzymes. Mast cells were then purified from the suspensions by countercurrent centrifugal elutriation and affinity chromatography. The purified mast cells released both histamine and slow-reacting substance of anaphylaxis (SRS-A) (leukotriene C and D) during stimulation with goat anti-human IgE antibody. Moreover, these preparations were able to generate significant quantities of SRS-A (32 +/- 7 x 10(-17) LTD mole-equivalents/mast cell) at all stages of purification, indicating that a secondary cell is not necessary for the antigen-induced release of SRS.

Authors

D W MacGlashan Jr, R P Schleimer, S P Peters, E S Schulman, G K Adams 3rd, H H Newball, L M Lichtenstein

Synthesis of hemoglobin F in adult simian erythroid progenitor-derived colonies.

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The simian hematopoietic system is known to respond to anemic stress with the production of erythrocytes containing large amounts of fetal hemoglobin. To determine the regulatory mechanism responsible for hemoglobin F (HbF) production in stress erythropoiesis, adult simian bone marrow cells were cultured in plasma clots in the presence or absence of erythropoietin and burst-promoting activities, and the HbF content of progenitor-derived colonies was determined by radioimmunoligand assay. Three classes of erythroid progenitors were detected: BFU-E, CFU-E, and a very mature cohort of dense highly erythropoietin-responsive cells (HERC). These classes varied in inverse proportion to their maturity with respect to their potential for HbF accumulation in the colonies to which they give rise. Both erythropoietin and burst-promoting activity stimulated HbF production, particularly in colonies derived from immature progenitors. For example, under conditions of high erythropoietin stimulation, BFU-E colonies contained 13.7-37.7% HbF, CFU-E colonies contained 2.8-13.5% HbF, and HERC colonies 0-1% HbF. These results suggest that under nonstress conditions simian erythrocytes are derived almost entirely from HERC progenitors and proerythroblasts in which gamma chain synthesis is suppressed. During stress erythropoiesis, augmented HbF accumulation could be explained by the rapid entrance into the marrow of proerythroblasts directly derived from immature progenitors. Gamma chain production in these proerythroblasts is additionally regulated by the changes in environmental erythropoietin and burst-promoting activities.

Authors

R M Macklis, J Javid, J M Lipton, M Kudisch, P K Pettis, D G Nathan

Spontaneous activation of latent cytomegalovirus from murine spleen explants. Role of lymphocytes and macrophages in release and replication of virus.

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Cytomegalovirus (CMV) is a major pathogen in the compromised host where many infections result from activation of latent virus. Because latent CMV infection has been difficult to study in humans, murine models have been developed and investigated. Here, we describe the events involved in activation of latent murine CMV (MCMV) from spleen explants in vitro. Infectious virus was no longer detectable in murine organs 4 mo after inoculatioN of 10(5) plaque-forming units of MCMV. 8-10 d after establishment of spleen explants, phagocytic macrophages covered 70-80% of the surface of tissue culture dishes, and lymphocytes were continuously released, reaching titers of 10(6) cells/ml. MCMV was produced spontaneously after 12-18 d from spleen explant cultures of 33 of 34 mice. Virus replicated to titers above 10(4) plaque-forming units/ml, remained at that level for 4-5 wk, and gradually disappeared as macrophages were lysed. Although MCMV was shown to be replicating in macrophages, these cells were never found to be the source of latent virus. Cell separation studies indicated that latent virus was initially released from 70% of lymphocyte cultures and was associated with the B cell enriched fraction. We conclude that MCMV establishes nonreplicating dormant infection in B lymphocytes, activates from these cells in spleen explant cultures, and is augmented in titer by replication in permissive macrophages.

Authors

M C Jordan, V L Mar

Basis of Plasma Iron Exchange in the Rabbit

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Rabbit transferrin in vitro is shown to load ferrous iron at random on its specific binding sites. The release of iron to reticulocytes is shown to be an all-or-none phenomenon. The two monoferric transferrins have similar in vivo plasma iron clearance rates and tissue distribution. Diferric transferrin, while giving a similar tissue distribution of radioiron, has a plasma iron clearance rate approximately twice that of the monoferric transferrins at low plasma iron concentrations. This difference diminishes as the plasma iron concentration increases. These results are consistent with a progressively greater in vivo conversion of mono- to diferric transferrin as transferrin saturation increases. The in vivo plasma iron turnover in the rabbit increases progressively as the plasma iron increases, from a mean value of ∼0.8 mg/dl whole blood per d at a plasma iron concentration of 50 μg/dl to 2.0 at a plasma iron concentration of 300.

Authors

Helmut Huebers, David Uvelli, Antonio Celada, Betty Josephson, Clement Finch

Mechanism of Insulin-resistant Glucose Transport Activity in the Enlarged Adipose Cell of the Aged, Obese Rat: RELATIVE DEPLETION OF INTRACELLULAR GLUCOSE TRANSPORT SYSTEMS

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The effects of increasing cell size on glucose transport activity and metabolism and on the concentrations of glucose transport systems in both the plasma and low density microsomal membranes in isolated adipose cells from the aging rat model of obesity have been examined. Glucose transport activity was assessed by measuring l-arabinose transport and the concentration of glucose transport systems estimated by measuring specific d-glucose-inhibitable cytochalasin B-binding. Basal glucose transport activity increases from 0.3 to 1.4 fmol/cell/min with a 10-fold increase in cell size, but remains constant per unit cellular surface area and is accompanied by a constant 5 pmol of glucose transport systems/mg of membrane protein in the plasma membrane fraction. Maximally insulin-stimulated glucose transport activity, on the other hand, remains constant at 2.3 fmol/cell per min with increasing cell size, but markedly decreases per unit cellular surface area and is accompanied by a decrease from 30 pmol of glucose transport systems/mg of plasma membrane protein to the basal level. These diminished effects of insulin on glucose transport activity and the number of glucose transport systems in the plasma membrane fraction in enlarged cells are paralleled by an 80% decrease in the basal number of glucose transport systems/mg of membrane protein in the low density microsomal membrane fraction, the source of those glucose transport systems appearing in the plasma membrane in response to insulin. The effects of cell size on the metabolism of a low concentration of [1-14C]glucose (0.56 mM) directly parallel those on glucose transport activity and the concentration of glucose transport systems in the plasma membrane fraction, and are not associated with significant alterations in the cell's sensitivity to insulin. Thus, adipose cellular enlargement is accompanied by the development of a marked “insulin resistance” at the glucose transport level, which may be the consequence of a relative depletion of glucose transport systems in the intracellular pool.

Authors

Paul J. Hissin, James E. Foley, Lawrence J. Wardzala, Eddy Karnieli, Ian A. Simpson, Lester B. Salans, Samuel W. Cushman

Characterization of lipase activities in obese Pima indians. Decreases with weight reduction.

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Adipose tissue and muscle lipoprotein lipase and postheparin hepatic and lipoprotein lipase activities have been measured in a group of 21 Pima Indian males over a wide range of body weight to determine the relationship between obesity and these lipase activities. There was a significant positive correlation between adipose tissue lipoprotein lipase and obesity; muscle and postheparin lipoprotein lipase and hepatic lipase were not related to degree of obesity. Fasting insulin levels were not related to any of the measurements of lipase activity. There were racial differences in adipose and postheparin lipoprotein lipase activities; both were significantly lower in the Pimas as compared with a group of weight-matched Caucasian males. Lipase activities were remeasured in eight subjects after a period of weight reduction including several weeks of stabilization at the reduced weights. After the period of weight reduction adipose tissue lipoprotein lipase declined in all subjects. Hepatic lipase also declined in all but two patients. Muscle and postheparin lipolytic activities were not affected by weight loss. The data indicate that (a) there are racial differences in adipose tissue lipoprotein lipase; and (b) the elevated adipose lipoprotein lipase associated with obesity, like many other biochemical variables in the obese state, returns toward normal after weight reduction.

Authors

J S Reitman, F C Kosmakos, B V Howard, M R Taskinen, T Kuusi, E A Nikkila

Mesenchyme-epithelial interactions in human endometrium. Prostaglandin synthesis in separated cell types.

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Glandular epithelium and stromal cells of human endometrium were separated and maintained in monolayer culture. At the time the cells became confluent, cell suspensions were prepared and incubated with [14C]arachidonic acid. Radiolabeled prostaglandin E2 and, to a lesser extent, prostaglandin F2 alpha and metabolites of these prostaglandins, were formed principally in stromal cells. There was considerably less prostaglandin formation in endometrial glands either after maintenance in monolayer culture or in freshly separated glands. In stromal cells of endometrium prostaglandin formation was linear with time of incubation for 2.5 min and with [14C]arachidonic acid concentrations up to 8 microM. When stromal cells and epithelial cells were combined, all prostaglandin formation could be accounted for by that produced in stromal cells. Little or no prostaglandin formation was detected in stromal cells from human adipose tissue or in fibroblasts from human genital or abdominal skin or human fallopian tube.

Authors

D Gal, M L Casey, J M Johnston, P C MacDonald

Human Alveolar Macrophage Growth Factor for Fibroblasts: REGULATION AND PARTIAL CHARACTERIZATION

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The number of fibroblasts composing the alveolar structures in controlled within narrow limits by a strictly modulated rate of fibroblast replication. One possible source of growth-modulating signals for alveolar fibroblasts is the alveolar macrophage, a member of the mononuclear phagocyte family of cells, which collectively are known to be important sources of growth factors for a variety of target cells. To evaluate the role of alveolar macrophages in the control of alveolar fibroblast replication, macrophages from normal individuals obtained by bronchoalveolar lavage were maintained in suspension culture with and without added stimuli, and supernates were evaluated for fibroblast growth-promoting effect. Supernates from unstimulated macrophages contained no growth factor activity. In marked contrast, supernates from macrophages stimulated with particulates and immune complexes contained a growth factor that caused a significant increase in fibroblast replication rate. Maximum growth factor activity was observed 3-4 h after macrophage stimulation, at a concentration of 1-2 × 106 macrophages/ml. The alveolar macrophagederived growth factor eluted from DEAE-cellulose at 0.27 M NaCl at neutral pH had an apparent molecular weight of 18,000, and appeared to be distinct from other characterized growth factors. The alveolar macrophage-derived growth factor stimulated lung fibroblast DNA synthesis within 12 h, with cell division apparent within 48 h. In serum-free culture, the alveolar macrophage-derived growth factor by itself did not promote fibroblast replication, but rather acted as a progression factor causing a synergistic increase in fibroblast replication rate in the presence of competence factors such as fibroblast growth factor or platelet-derived growth factor. These studies suggest that when stimulated, human alveolar macrophages may modulate, in part, the replication rate of alveolar fibroblasts by releasing a growth factor within the alveolar microenvironment.

Authors

Peter B. Bitterman, Stephen I. Rennard, Gary W. Hunninghake, Ronald G. Crystal

Familial Hypercholesterolemia: EVIDENCE FOR A NEWLY RECOGNIZED MUTATION DETERMINING INCREASED FIBROBLAST RECEPTOR AFFINITY BUT DECREASED CAPACITY FOR LOW DENSITY LIPOPROTEIN IN TWO SIBLINGS

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Abstract

Cultured skin fibroblasts were obtained from two siblings with classic clinical features of homozygous familial hypercholesterolemia. Plasma cholesterol values were 970 and 802 mg/100 ml in the siblings, 332 mg/100 ml in the mother, and 426 mg/100 ml in the father. Fibroblast receptor-specific capacity for binding and degradation of 125I-low density lipoprotein (LDL) at 37°C was 11% of normal, consistent with the diagnosis of “homozygous LDL receptor-defective” hypercholesterolemia, a disorder in which LDL binding activity is low but detectable.

Authors

Richard E. Ostlund Jr., Richard A. Levy, Joseph L. Witztum, Gustav Schonfeld

Mechanisms of K⁺ Transport in Isolated Turtle Urinary Bladder: INDUCTION OF ACTIVE K⁺ SECRETION IN A K⁺-ABSORBING EPITHELIUM

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Transepithelial K+ movement was studied in vitro in the short-circuited turtle bladder by increasing luminal K+ permeability and by inhibiting the basolateral Na/K pump. Luminal addition of amphotericin B caused net K+ secretion (180±52 nmol/h) compared with net K+ absorption (42±6 nmol/h) in control bladders. Serosal ouabain and luminal amiloride abolished K+ secretion in amphotericin-treated bladders; ouabain restored net absorption (45±16 nmol/h). The direction and rate of net K+ transport are controlled by the relative K+ permeabilities and the Na/K pump sites at the two cell membranes of the epithelium.

Authors

Russell F. Husted, Philip R. Steinmetz

Tubular Mechanism for the Spontaneous Hypercalciuria in Laboratory Rat

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Recently it has been observed that Ca excretion in laboratory rats does not follow a Gaussian distribution, with ∼10% of them excreting Ca at a rate of 2 SD above the group mean. This phenomenon has been described as spontaneous hypercalciuria (SH). Our studies were designed to define its mechanism. 48 Wistar rats were subjected to metabolic studies to identify SH, prospectively defined as Ca excretion 2 SD above the group mean during 7 d of dietary Ca deprivation (≤0.03% by analysis), in the absence of hypercalcemia, PO4 depletion, or exaggerated natriuresis.

Authors

Kai Lau, Bonnie K. Eby

Proteolysis by Neutrophils: RELATIVE IMPORTANCE OF CELL-SUBSTRATE CONTACT AND OXIDATIVE INACTIVATION OF PROTEINASE INHIBITORS IN VITRO

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Polymorphonuclear leukocytes have been implicated in connective tissue injury in a variety of disease processes. To gain insight into mechanisms by which neutrophils might degrade connective tissue macromolecules in the presence of proteinase inhibitors, we have used a model system that allows neutrophils to be held in vitro under physiologic conditions in close proximity to a very proteinase-sensitive substrate, 125I-labeled fibronectin. We have found: (a) neutrophils spread rapidly on the fibronectin substrate; (b) fibronectin proteolysis by neutrophils is largely attributable to released elastase, and is linearly related to cell number over the range of 2,000 to 30,000 cells per assay; (c) oxidants released from neutrophils stimulated by opsonized zymosan or phorbol myristate acetate do not protect released elastase from inhibition by α1-proteinase inhibitor or α2-macroglobulin; (d) neutrophil myeloperoxidase and enzymatically generated superoxide anion render α1-proteinase inhibitor ineffective against fibronectin proteolysis when neutrophils are added 30 min later; and (e) α1-proteinase inhibitor and α2-macroglobulin incompletely inhibit fibronectin proteolysis by neutrophils (79.8±6.3 and 73.5±12.0%, respectively.) The data suggested that proteolysis due to neutrophils that are in contact with susceptible macromolecules may occur due to partial exclusion of inhibitors from the cell-substrate interface. Although confirming that α1-proteinase inhibitor is ineffective against neutrophil-derived proteolysis after exposure to oxidants, these studies did not support the hypothesis that oxidants released from stimulated neutrophils enhance activity of proteinases they release in the presence of α1-proteinase inhibitor. We anticipate that further studies with this test system will be helpful in defining conditions that modulate inflammatory connective tissue injury in diseases such as pulmonary emphysema and rheumatoid arthritis.

Authors

Edward J. Campbell, Robert M. Senior, John A. McDonald, David L. Cox, Jeanne M. Greco, Jill A. Landis

Treatment of Lactic Acidosis with Dichloroacetate in Dogs

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Lactic acidosis is a clinical condition due to accumulation of H+ ions from lactic acid, characterized by blood lactate levels >5 mM and arterial pH <7.25. In addition to supportive care, treatment usually consists of intravenous NaHCO3, with a resultant mortality >60%. Dichloroacetate (DCA) is a compound that lowers blood lactate levels under various conditions in both man and laboratory animals. It acts to increase pyruvate oxidation by activation of pyruvate dehydrogenase. We evaluated the effects of DCA in the treatment of two different models of type B experimental lactic acidosis in diabetic dogs: hepatectomy-lactic acidosis and phenformin-lactic acidosis. The metabolic and systemic effects examined included arterial blood pH and levels of bicarbonate and lactate; the intracellular pH (pHi) in liver and skeletal muscle; cardiac index, arterial blood pressure and liver blood flow; liver lactate uptake and extrahepatic splanchnic (gut) lactate production; and mortality. Effects of DCA were compared with those of either NaCl or NaHCO3. The infusion of DCA and NaHCO3, delivered equal amounts of volume and sodium, although the quantity of NaHCO3 infused (2.5 meq/kg per h) was insufficient to normalize arterial pH.

Authors

Robert Park, Allen I. Arieff, William Leach, Virginia C. Lazarowitz

Multicompartmental Analysis of Cholesterol Metabolism in Man: QUANTITATIVE KINETIC EVALUATION OF PRECURSOR SOURCES AND TURNOVER OF HIGH DENSITY LIPOPROTEIN CHOLESTEROL ESTERS

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The purpose of this study is to delineate the immediate sources and fractional turnover of high density lipoprotein (HDL) esterified cholesterol in man. Various labeled preparations were administered in 11 experiments to six subjects who had either a complete bile fistula (maximally stimulated cholesterol metabolism) or an intact enterohepatic circulation. The administered tracers included [3H]mevalonic acid; [14C]cholesterol bound to albumin; low density lipoprotein (LDL) free [3H] or [14C]cholesterol; HDL free [3H] or [14C]cholesterol; HDL esterified [3H]cholesterol; and LDL esterified [3H]cholesterol. Blood samples were obtained at frequent intervals for up to 5 d after the administration of tracers. The mass and radioactivity in individual plasma lipoprotein (very low density lipoprotein [VLDL], HDL, and LDL) free and esterified cholesterol were determined.

Authors

Charles C. Schwartz, Mones Berman, Z. Reno Vlahcevic, Leon Swell

Biological Activity, Lipoprotein-binding Behavior, and In Vivo Disposition of Extracted and Native Forms of Salmonella typhimurium Lipopolysaccharides

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Although phenol-extracted gram-negative bacterial lipopolysaccharides (LPS) have been used to study the properties of endotoxins for many years, nothing is known about the behavior of native (unextracted) LPS in vivo. Accordingly, we have compared extracted and native forms of LPS with regard to their biological activity, their ability to bind to plasma high density lipoproteins (HDL), and their fate after intravenous injection into rats. The LPS of Salmonella typhimurium G-30 were labeled with [3H]galactose, and whole bacteria, bacterial outer membranes, outer membrane fragments (harvested from the bacterial culture supernatant), and phenol extracts of the bacteria were prepared. After defining the LPS, phospholipid, and protein composition of these preparations, we compared the activity of the LPS in phenol extracts and membrane fragments in two assays. In both the limulus lysate assay and the rabbit pyrogen test, the LPS in phenol extracts were slightly more potent than the LPS in membrane fragments. We next studied the ability of the LPS in each preparation to bind to rat lipoproteins in vitro, and each preparation was then injected intravenously into rats for measurements of LPS-HDL binding and tissue uptake in vivo. Two patterns of lipoprotein binding were observed. Less than 25% of the LPS in both outer membranes and whole bacteria bound to HDL in vitro. When the outer membranes and whole bacteria were injected into rats, their LPS again bound poorly to HDL and they were rapidly removed from plasma into the liver and spleen. In contrast, >50% of the LPS in both culture supernatant membrane fragments and phenol-water extracts bound to HDL in vitro. When these preparations were injected into rats, ∼50% of the LPS in the membrane fragments and phenol-water extracts bound to HDL and remained in the plasma over the 10-min study period. Moreover, the LPS in these preparations accumulated in the ovary and the adrenal gland, two tissues that use HDL-cholesterol for hormone synthesis. Binding to HDL thus greatly influenced the plasma half-life and tissue uptake of both extracted and native LPS.

Authors

Robert S. Munford, Catherine L. Hall, J. M. Lipton, John M. Dietschy

Presence and formation of cobalamin analogues in multivitamin-mineral pills.

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Because the origin of cobalamin (vitamin B12) analogues in animal chows and animal and human blood and tissues is unknown, we investigated the possibility that multivitamin interactions might convert cobalamin to cobalamin analogues. We homogenized three popular multivitamin-mineral pills in water, incubated them at 37 degrees C for 2 h, and isolated the cobalamin. Using paper chromatography we observed that 20-90% of the cobalamin was present as cobalamin analogues. Studies using CN-[57Co]cobalamin showed that these analogues were formed due to the concerted action of vitamin C, thiamine, and copper on CN-cobalamin. These cobalamin analogues are absorbed from the gastrointestinal tract of mice and either fail to stimulate or actually inhibit cobalamin-dependent enzymes when injected parenterally. We conclude that CN-cobalamin can be converted to potentially harmful cobalamin analogues by multivitamin-mineral interactions and that these interactions may be responsible for the presence of cobalamin analogues in animal chows and animal and human blood and tissues.

Authors

H Kondo, M J Binder, J F Kolhouse, W R Smythe, E R Podell, R H Allen

Rat Anterior Pituitary: DISTINCTION OF AN ∼8S, CORTICOSTERONE-PREFERRING SPECIES FROM DEXAMETHASONE-BINDING GLUCOCORTICOID RECEPTORS

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Abstract

Studies on the feedback inhibition of ACTH release by steroid hormones and on the binding of tritiated steroids by the pituitary have prompted the hypothesis that receptors in addition to or other than classical glucocorticoid receptors may mediate steroid hormone effects in this tissue. Accordingly, we have asked whether more than one glucocorticoid-binding species, distinct from corticosteroid binding globulin, can be found in rat anterior pituitary gland.

Authors

Zygmunt S. Krozowski, John W. Funder

Complement biosynthesis by the human hepatoma-derived cell line HepG2.

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Abstract

The human hepatoma-derived cell line, HepG2, synthesized and secreted functional complement proteins C1r, C1s, C2, C3, C4, C5, factor B, C1 inhibitor, C3b inactivator, a small amount of C6, and trace amounts of C8; but failed to produce detectable C1q, C7, or C9. Immunochemically, C2, C3, C4, C5, and B were isolated from culture medium as proteins with molecular sizes and subunit structures identical to the corresponding components isolated from serum. C2 and factor B from cellular lysates had slightly lower molecular weights than the corresponding proteins in culture medium. C3, C4, and C5 were detected as single chain precursor molecules in cellular lysates. These results demonstrate that human C5, like C3 and C4, is synthesized as a single chain precursor that is converted by limited proteolysis to the native two-chain molecule. It also establishes the precursor-product relationship for human pro-C4 and native C4, pro-C5, and native C5.

Authors

K M Morris, D P Aden, B B Knowles, H R Colten