Characterization of Serum Resistance of Neisseria gonorrhoeae that Disseminate: ROLES OF BLOCKING ANTIBODY AND GONOCOCCAL OUTER MEMBRANE PROTEINS

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Abstract

Neisseria gonorrhoeae isolated from patients with disseminated infection (DGI) often resist complement (C′)-dependent killing by normal human serum (NHS) and less commonly by convalescent DGI serum. 7 of 10 NHS specimens completely inhibited killing of serum-resistant (serr) gonococci by convalescent or immune DGI serum. Immunoglobulin G (IgG) purified from NHS was shown to be the blocking agent. In addition, IgM (plus C′) purified from NHS was shown to be fivefold more effective (wt/wt) in killing serum-sensitive (sers) gonococci than equivalent amounts of IgM tested in the presence of IgG (whole serum). Although inhibition of NHS killing of sers gonococci required a 640% excess of IgG, only a 40% excess was required to block immune serum killing of serr gonococci. F(ab′)2 prepared from IgG also blocked killing of serr gonococci by immune serum indicating antigenic specificity of blocking IgG.

Authors

Peter A. Rice, Dennis L. Kasper

Metabolic Requirement for Inorganic Phosphate by the Rabbit Proximal Tubule: EVIDENCE FOR A CRABTREE EFFECT

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Abstract

These studies examine the effects of acute changes in the availability of inorganic phosphate on the function of isolated proximal renal tubules from rabbit kidney. We removed phosphate from the extracellular fluids and measured fluid absorption rates in isolated perfused tubules and oxygen consumption rates in suspensions of cortical tubules. In proximal convoluted tubules, the selective removal of phosphate from the luminal fluid reduced fluid absorption rates from 1.11±0.12 to −0.01±0.08 nl/mm · min. This effect on fluid absorption was dependent on the presence of glucose transport and metabolism. The addition of phlorizin to the phosphate-free luminal fluid preserved fluid absorption rates (1.12±0.12 nl/mm · min) as did the substitution of nonmetabolized α-methyl d-glucopyranoside for glucose (1.05±0.21 nl/mm · min) or the addition of 2-deoxyglucose, an inhibitor of glycolysis, to the bathing medium (1.01±0.15 nl/mm · min). There was no effect on fluid absorption if phosphate was removed from the bath only. Additionally, removal of phosphate from the luminal fluid of proximal straight rather than convoluted tubules had no effect on fluid absorption rates. Oxygen consumption rates in suspensions of cortical tubules were reduced from 18.9±0.6 to 10.6±0.6 nmol O2/mg tubular protein · min by the removal of phosphate from the medium. This inhibition was prevented by the substitution of α-methyl d-glucopyranoside for glucose in the phosphate-free medium. The data indicate that under certain conditions, proximal convoluted tubules require the presence of phosphate in the luminal fluid to preserve tubular function. In the absence of intraluminal phosphate, glucose metabolism causes a reduction in both oxidative metabolism and fluid absorption. This response is analogous to the Crabtree effect and suggests limitations on the intracellular availability of inorganic phosphate.

Authors

Peter C. Brazy, Steven R. Gullans, Lazaro J. Mandel, Vincent W. Dennis

Elevation of Plasma Neurotensinlike Immunoreactivity after a Meal: CHARACTERIZATION OF THE ELEVATED COMPONENTS

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Abstract

The detection of an elevation in neurotensinlike immunoreactivity in peripheral plasma for several hours after a meal has been confirmed and shown to be primarily due to the presence of aminoterminal fragments of neurotensin (NT) rather than to NT itself. We have developed a procedure to separate and characterize these N-terminal cross-reacting substances, and to estimate the contributions of these constitutents to plasma neurotensinlike immunoreactivity. Gel chromatography of pooled plasma extracts on Sephadex G-25 followed by reverse-phase high pressure liquid chromatography indicated that peptides coeluting with NT and its N-terminal partial sequences NT(1-8) and NT(1-11) were present in plasma. Comparison of plasmas collected before and 1 h after a defined meal, in five experiments, demonstrated no change in C-terminal immunoreactivity and an 8- to 10-fold rise in N-terminal immunoreactivity. Chromatographic analysis of pooled pre- and postmeal plasma in four experiments showed that essentially all of this elevation in neurotensinlike immunoreactivity measured with an N-terminal directed antiserum was due to increases in NT(1-8) and NT(1-11), while NT itself, measured using a C-terminal directed antiserum, did not increase appreciably in peripheral plasma 1 h after the meal. Generation of tritiated substances with the same elution times as NT(1-8) and NT(1-11) did occur after incubation of [3H]NT with whole blood in vitro, providing supporting evidence that these fragments are metabolites of NT. The marked elevation in the circulating levels of these fragments reflects that an increased secretion of NT occurred in response to the test meal. The secreted NT may have acted as a hormone before it was metabolized, or it may only have had a local (paracrine) effect.

Authors

Robert A. Hammer, Robert E. Carraway, Susan E. Leeman

Release of Somatostatin-like Immunoreactivity from Incubated Rat Hypothalamus and Cerebral Cortex: EFFECTS OF GLUCOSE AND GLUCOREGULATORY HORMONES

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Abstract

Somatostatin (SRIF) is localized in the hypothalamus, extrahypothalamic brain, and throughout the gastrointestinal tract. Release of gastrointestinal SRIF-like immunoreactivity (SRIF-LI) is under nutrient regulation but the effect of nutrients on neural SRIF-LI is unknown. The present studies examined the effects of glucose uptake and metabolism and hormones influencing glucose disposition on SRIF-LI release from medial basal hypothalamus (MBH) and cerebral cortex (Cx) incubated in Krebs-Ringer bicarbonate containing bacitracin. After a preincubation to achieve stable secretion, tissues were incubated for 20 min in 14 mM glucose (basal) and then, for 20 min in fresh medium with test materials. MBH SRIF-LI release was inversely related to medium glucose concentration with release in the absence of glucose (235±42 pg/MBH per 20 min) more than five times that in the presence of 25 mM glucose (46±4 pg/20 min). In the presence of 14 mM glucose MBH SRIF-LI release was stimulated above basal by agents interfering with glucose uptake including 3-O-methyl-d-glucose (42 mM; 70±5 vs. 42±3 pg/20 min, P < 0.05), phlorizin (50 mM; 351±63 vs. 29±2 pg/20 min, P < 0.001) or cytochalasin B (20 μM; 110±7 vs. 22±2 pg/20 min, P < 0.001). Inhibition of glucose metabolism by 2-deoxy-d-glucose resulted in dose-related stimulation of MBH SRIF-LI release (maximal at 28 mM; 201±28 pg/20 min vs. 32±4 pg/20 min, P < 0.001). Viability of MBH was unimpaired by incubation in the absence of glucose or following exposure to 2-deoxy-d-glucose as determined by retention of SRIF-LI responsiveness to stimulation by potassium (60 mM) or neurotensin (5 μM). In contrast, Cx SRIF-LI release was slightly inhibited by decreases in medium glucose and unaffected by inhibition of glucose uptake or metabolism. These results provide evidence for nutrient regulation of MBH but not Cx SRIF-LI release and may explain inhibition of growth hormone seen in the rat in response to hypoglycemia. Insulin (10 nM-1 μM) stimulated MBH but not Cx SRIF-LI release while glucagon was without effect. Our previous demonstration that MBH SRIF-LI release was stimulated by somatomedin-C, but not insulin at physiologic concentrations, is consistent with an action of insulin through the somatomedin-C receptor at the doses studied. Our studies indicate a regional specificity for the control of SRIF secretion within the brain and suggests the possibility of a role for hypothalamic SRIF in metabolic regulation.

Authors

Michael Berelowitz, Daniel Dudlak, Lawrence A. Frohman

Insulin Infusion in Conscious Dogs: EFFECTS ON SYSTEMIC AND CORONARY HEMODYNAMICS, REGIONAL BLOOD FLOWS, AND PLASMA CATECHOLAMINES

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Abstract

Cardiovascular actions of insulin were studied by intravenous infusions of insulin (4 and 8 mU/kg per min) in normal conscious dogs. This resulted in increases in cardiac output, heart rate, and left ventricular derivative of pressure with respect to time (dP/dt) and dP/dt/P, as blood glucose was reduced. The inotropic and chronotropic effects of insulin were not related to hypoglycemia, as they persisted even when blood glucose was restored to control values or when it was prevented from falling by a simultaneous infusion of glucose. These cardiac effects were accompanied by increases in plasma catecholamines, and were abolished by propranolol pretreatment. Both plasma epinephrine and norepinephrine increased during insulin hypoglycemia, but only norepinephrine increased during insulin infusion when euglycemia was maintained.

Authors

Chang-Seng Liang, John U. Doherty, Robert Faillace, Kishio Maekawa, Stephanie Arnold, Haralambos Gavras, William B. Hood Jr.

Human Skin Collagenase in Recessive Dystrophic Epidermolysis Bullosa: PURIFICATION OF A MUTANT ENZYME FROM FIBROBLAST CULTURES

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Abstract

Recessive dystrophic epidermolysis bullosa, a genodermatosis characterized by dermolytic blister formation in response to minor trauma, is characterized by an incresaed collagenase synthesis by skin fibroblasts in culture. Since preliminary studies of partially purified recessive dystrophic epidermolysis bullosa collagenase suggested that the protein itself was aberrant, efforts were made to purify this enzyme to homogeneity, so that detailed biochemical and immunologic comparisons could be made with normal human skin fibroblast collagenase. Recessive dystrophic epidermolysis bullosa skin fibroblasts obtained from a patient documented to have increased synthesis of the enzyme were grown in large scale tissue culture and both serum-free and serum-containing medium collected as a source of collagenase. The recessive dystrophic epidermolysis bullosa collagenase was purified to electrophoretic homogeneity using a combination of salt precipitation, ion-exchange, and gel-filtration chromatography. In contrast to the normal enzyme, the recessive dystrophic epidermolysis bullosa collagenase bound to carboxymethyl-cellulose at Ca2+ concentrations at least 10 times higher than those used with the normal enzyme. Additionally, this enzyme was significantly more labile to chromatographic manipulations, particularly when serum-free medium was used. However, rapid purification from serum-containing medium yielded a preparation enzymatically equivalent to normal human skin collagenase. Like the normal enzyme, the recessive dystrophic epidermolysis bullosa collagenase was secreted as a set of two closely related zymogens of ∼60,000 and ∼55,000 daltons that could be activated by trypsin to form enzymically active species of ∼50,000 and ∼45,000 daltons, respectively. Amino acid analysis suggested slight variations between the normal and recessive dystrophic epidermolysis bullosa collagenases. Cyanogen bromide digests demonstrated peptides unique to the enzyme from each source. The recessive dystrophic epidermolysis bullosa proenzyme was significantly more thermolabile at 60°C than the normal, a finding that correlated with an approximate fourfold decrease in the affinity of the mutant enzyme for Ca2+, a known activator and stabilizer of human skin collagenase. Aside from the altered affinity for this metal cofactor, kinetic analysis of the structurally altered recessive dystrophic epidermolysis bullosa collagenase revealed that its reaction rates and substrate specificity for human collagen types I-V were identical to those for the normal enzyme. Likewise, enzymes from both sources displayed identical energies of activation and deuterium isotope effects. Antisera were raised to the normal and putatively mutant procollagenases respectively, and, although they displayed a reaction of identity in double diffusion analysis, immunologic differences were present in enzyme inhibition and quantitative precipitation studies. These studies indicate that recessive dystrophic epidermolysis bullosa is characterized by the increased synthesis of an enzymically normal, but structurally aberrant, collagenase.

Authors

George P. Stricklin, Howard G. Welgus, Eugene A. Bauer

Dopamine Stimulation of Active Na and Cl Absorption in Rabbit Ileum: INTERACTION WITH α₂-ADRENERGIC AND SPECIFIC DOPAMINE RECEPTORS

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Abstract

The effects of dopamine on active intestinal ion transport have been evaluated. An epithelial sheet preparation of rabbit ileum was used in vitro with the Ussing chamber-voltage clamp technique. Dopamine, in the presence of 1 mM ascorbic acid, added to the serosal bathing solution caused a dose-dependent decrease in short-circuit current, with a half-maximal effect at 1.2 μM and maximal effect of −50 μA/cm2 at 50 μM; dopamine decreased the potential difference, and increased the conductance and net Na and net Cl absorption. There was no effect on the residual ion flux. Dopamine did not alter the change in short-circuit current caused by mucosal glucose (10 mM) or serosal theophylline (10 mM). Mucosal dopamine had no effect. The effect of dopamine on short-circuit current was inhibited by the dopamine antagonists haloperidol and domperidone and the α2-adrenergic antagonist yohimbine; there was no effect of the α1-antagonist prazosin and the β-antagonist propranolol. In addition, the α2-adrenergic agonist clonidine, but not the α1-agonist methoxamine caused a dose-dependent decrease in short-circuit current. The ileal effects of dopamine did not occur via conversion into norepinephrine or release of norepinephrine from the peripheral nerves since “peripheral sympathectomy” with 6-hydroxydopamine did not alter the dopamine-induced change in ileal short-circuit current. The dopamine effects were not associated with a change in basal ileal cyclic AMP content but were associated with a decrease in total ileal calcium content as measured by atomic absorption spectrometry and as estimated by 45Ca++ uptake. The decrease in calcium content could be attributed to a dopamine-induced decrease in 45Ca++ influx from the serosal surface. Because of the presence of dopamine in ileal mucosa and these effects on ileal electrolyte transport, it is possible that dopamine may be involved in the physiologic regulation of active intestinal electrolyte absorption.

Authors

Mark Donowitz, Sheila Cusolito, Laurie Battisti, Ronald Fogel, Geoffrey W. G. Sharp

Muscle Glucose Metabolism following Exercise in the Rat: INCREASED SENSITIVITY TO INSULIN

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Abstract

Muscle glycogen stores are depleted during exercise and are rapidly repleted during the recovery period. To investigate the mechanism for this phenomenon, untrained male rats were run for 45 min on a motor-driven treadmill and the ability of their muscles to utilize glucose was then assessed during perfusion of their isolated hindquarters. Glucose utilization by the hindquarter was the same in exercised and control rats perfused in the absence of added insulin; however, when insulin (30-40,000 μU/ml) was added to the perfusate, glucose utilization was greater after exercise. Prior exercise lowered both, the concentration of insulin that half-maximally stimulated glucose utilization (exercise, 150 μU/ml; control, 480 μU/ml) and modestly increased its maximum effect. The increase in insulin sensitivity persisted for 4 h following exercise, but was not present after 24 h. The rate-limiting step in glucose utilization enhanced by prior exercise appeared to be glucose transport across the cell membrane, as in neither control nor exercised rats did free glucose accumulate in the muscle cell.

Authors

Erik A. Richter, Lawrence P. Garetto, Michael N. Goodman, Neil B. Ruderman

Inactivation of Factor XIa by Plasma Protease Inhibitors: PREDOMINANT ROLE OF α₁-PROTEASE INHIBITOR AND PROTECTIVE EFFECT OF HIGH MOLECULAR WEIGHT KININOGEN

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Abstract

Factor XIa is a plasma protease that, by activating Factor IX, plays an important role in the early phase of the intrinsic pathway of blood coagulation. Four plasma protease inhibitors, α1-protease inhibitor, antithrombin III, C1-inhibitor, and α2-plasmin inhibitor, have been reported to inactivate human Factor XIa, but their quantitative contribution to the inactivation of Factor XIa in plasma has not been fully assessed. Using purified systems, we observed that the second-order rate constants for the reaction of Factor XIa with α1-protease inhibitor, antithrombin III, and CI-inhibitor were 4.08, 10, and 14.6 M−1 min−1 × 103, respectively. The pseudo-first-order rate constants, at plasma concentration of the inhibitors, were 1.86 × 10−1, 4.68 × 10−2, and 2.4 × 10−2 min−1, respectively. These kinetic data predict that α1-protease inhibitor should account for 68%, antithrombin III for 16%, and C1-inhibitor and the equipotent α2-plasmin inhibitor each for 8% of the total inhibitory activity of plasma against Factor XIa. The rate of inactivation of Factor XIa in various plasma samples specifically deficient in inhibitors was consistent with these predictions.

Authors

Cheryl F. Scott, Marc Schapira, Harold L. James, Allen B. Cohen, Robert W. Colman

Studies of the Transferrin Receptor on both Human Reticulocytes and Nucleated Human Cells in Culture: COMPARISON OF FACTORS REGULATING RECEPTOR DENSITY

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Abstract

The transferrin receptor, present on reticulocytes and nucleated cells in tissue culture, has been measured with both immunoassay techniques and transferrin binding studies. The total cellular immunoreactive receptor is rapidly lost from erythrocytes during the process of reticulocyte maturation (from as many as 400,000 molecules to <20,000 molecules/reticulocyte). This event parallels the loss of cell surface transferrin binding sites and RNA content, and correlates with previous studies that have measured the decline in hemoglobin synthesis.

Authors

Janet L. Frazier, Jennifer H. Caskey, Mark Yoffe, Paul A. Seligman