Effects of Long-Acting Thyroid Stimulator on Thyrotropin Stimulation of Adenyl Cyclase Activity in Thyroid Plasma Membranes

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Abstract

Both thyroid-stimulating hormone (TSH) and long-acting thyroid stimulator (LATS) stimulated adenyl cyclase activity in plasma membranes obtained from bovine thyroid glands. The stimulation induced by LATS was much less than that obtained with maximal amounts of TSH. LATS inhibited TSH stimulation of adenyl cyclase activity while an equivalent amount of normal human γ-globulin did not influence basal or TSH-stimulated activity. The inhibition by LATS appeared to be noncompetitive and was greatest when the plasma membranes were initially exposed to LATS for 30 min at 0°C before being incubated with TSH for 10 min at 37°C. Inhibition could still be demonstrated when the plasma membranes were incubated for 30 min at 0°C with TSH before the addition of LATS. Prolonging the period of incubation of plasma membranes with LATS from 30 to 60 min did not augment the stimulation of adenyl cyclase or increase the inhibition of the effect of TSH. Papain digests of LATS also increased adenyl cyclase activity of thyroid plasma membrane and inhibited the stimulation induced by TSH. The inhibitory effect of LATS was not completely specific for TSH and thyroid plasma membranes since glucagon stimulation of adenyl cyclase in hepatic plasma membranes was also inhibited, but to a lesser extent. In contrast to the results obtained with thyroid plasma membranes, LATS did not influence basal adenyl cyclase activity in hepatic plasma membranes. Furthermore equivalent amounts of normal human γ-globulin also decreased glucagon stimulation of adenyl cyclase activity in plasma membranes obtained from liver. The present data suggest that LATS stimulation of adenyl cyclase in thyroid plasma membranes might be due to a change in the membrane configuration rather than binding to a specific receptor site. Such modification of the membrane structure could interfere with the binding of TSH to specific receptors or to the subsequent stimulation of adenyl cyclase. However, the results do not exclude the possibility that some component in the preparation other than LATS might be responsible for the inhibition of the stimulation by TSH.

Authors

Kamejiro Yamashita, James B. Field

Simultaneous Measurement of Thyroxine and Triiodothyronine Peripheral Turnover Kinetics in Man

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Abstract

Serum triiodothyronine (T3) kinetics in man have been difficult to define presumably due to the interference of iodoproteins generated during the peripheral metabolism of T3. The use, in the present study, of an anion-column chromatographic method for separation of serum T3 as well as thyroxine (T4) from these iodoproteins has overcome this technical handicap. Simultaneous measurement of serum 125I-T3 and 131I-T4 kinetics were performed in 31 subjects from the clinical categories of euthyroid, primary hypothyroid, thyrotoxic and posttreatment hypothyroid Graves' disease, factitial thyrotoxic, and idiopathically high and low thyroxinebinding globulin states. The normal mean T3 fractional turnover rate (kT3) was 0.68 (half-life = 1.0 days), increased in toxic Graves' disease patients to 1.10 (half-life = 0.63 days), and decreased in primary hypothyroid patients to 0.50 (half-life = 1.38 days). The mean T3 equilibration time averaged 22 hr except in hypothyroid and high thyroxine-binding globulin (TBG) patients where the equilibration period was delayed by 10 hr. The mean T3 distribution space in normal subjects was 38.4 liters. This was reduced in subjects with high TBG levels (26 liters) and increased in patients with low TBG and in all hyperthyroid states (53-55 liters). The normal serum T3 concentration was estimated by radioimmunoassay to be 0.106 μg/100 ml. Combined with the mean T3 clearance value of 26.1 liters/day, the calculated T3 production rate was 27.6 μg/day. The mean T3 production rate increased to 201 μg/day in thyrotoxic Graves' disease patients and was reduced to 7.6 μg/day in primary hypothyroid subjects. T3 production rate was normal in subjects with altered TBG states. The ratio of T3 to T4 production rate in normal subjects was 0.31 and was unchanged in patients with altered TBG values. This ratio was increased in all Graves' disease patients with the highest value being 0.81 in the posttreatment hypothyroid Graves' disease group. This apparent preferential production of T3 may have been responsible for the retention of rapid turnover kinetics for T3 and T4 observed in treated Graves' disease patients. The finding that factitial thyrotoxic patients also displayed similar rapid T3 and T4 turnover kinetics indicates that these alterations are not a unique feature of Graves' disease per se. When comparing the peripheral turnover values for T3 and T4 in man, it is apparent that alterations in metabolic status and serum TBG concentration influence both hormones in a parallel manner; however, changes in metabolic status seem to have a greater influence on T3 kinetics while alterations in TBG concentrations have a greater effect on T4. These observations probably relate to the differences in TBG binding affinity and peripheral tissue distribution of these two hormones.

Authors

John T. Nicoloff, James C. Low, Jean H. Dussault, Delbert A. Fisher

Renal Tubular Permeability during Increased Intrarenal Pressure

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Abstract

Renal tubular permeability was studied by microinjection techniques during increased intrarenal pressure in anesthetized diuretic rats. Intrarenal pressure, as evidenced by intratubular pressure (ITP), was increased by elevation of ureteral pressure, partial renal venous constriction, or massive saline diuresis. Various combinations of radioactive inulin, creatinine, mannitol, sucrose, and iothalamate in isotonic saline were microinjected into superficial proximal and distal convolutions, and recovery of the isotopes was measured in the urine.

Authors

William B. Lorentz Jr., William E. Lassiter, Carl W. Gottschalk

Succinyl-CoA: 3-Ketoacid CoA-Transferase Deficiency. A CAUSE FOR KETOACIDOSIS IN INFANCY

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Abstract

To explain the cause of a unique form of severe and intermittent ketoacidosis in an infant who expired after 6 months of life, tissue culture fibroblasts and post mortem tissue were examined for enzyme activities that catalyze glucose and ketoacid oxidation. No measurable succinyl-CoA: 3-ketoacid CoA-transferase (CoA-transferase) activity could be detected in homogenates of the post mortem brain, muscle and kidney tissue, or in the cultured skin fibroblasts. Since seven other enzyme activities involving both glycolysis and ketone body oxidation were present in these same tissues, it was reasonable to conclude that the observed absence of CoA-transferase activity was not an artifact of homogenate preparation. It was concluded that the absence of CoA-transferase activity resulted in a loss of intracellular homeostasis leading to ketoacidosis. In addition, the absence of this enzyme appears to be a reasonable explanation for the alteration in glucose metabolism that was previously reported in fibroblasts from this patient.

Authors

J. Tyson Tildon, Marvin Cornblath

Renal Tubular Acidosis in Infants: the Several Kinds, Including Bicarbonate-Wasting, Classic Renal Tubular Acidosis

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Abstract

In four infants with renal tubular acidosis (RTA), including three with apparently classic RTA and one with Fanconi syndrome (FS), the physiologic character of the renal acidification defect was investigated. In two of the infants with apparently classic RTA, the acidification defect was physiologically separable from that described in both adult patients and children with classic RTA (type 1 RTA) in the following ways. (a) The fractional excretion of filtered bicarbonate (CHCO3̄/Cln) was not trivial but substantial (6-9%), as well as relatively fixed, over a broad range of plasma bicarbonate concentrations (15-26 mmoles/liter). (b) This value of CHCO3̄/Cln, combined with a normal or near normal glomerular filtration rate, translated to renal bicarbonate wasting (RBW). (c) RBW at normal plasma bicarbonate concentrations was the major cause of acidosis, and its magnitude was the major determinant of corrective alkali therapy (5-9 mEq/kg per day), just as in the patient with FS, who was found to have type 2 (“proximal”) RTA. (d) Persistence of RBW at substantially reduced plasma bicarbonate concentrations, which did not occur in FS, accounted for the spontaneous occurrence of severe acidosis and its rapid recurrence after reduction in alkali therapy. (e) During severe acidosis the urinary pH was >7, a finding reported frequently in infants with apparently classic RTA and “alkali-resistant” acidosis but rarely in adult patients with classic RTA. Continued supplements of potassium were required to maintain normokalemia during sustained correction of acidosis with alkali therapy. Yet, in at least two of the three infants with apparently classic RTA, but in distinction from the patient with FS and other patients with type 2 RTA, fractional excretion of filtered potassium decreased when plasma bicarbonate was experimentally increased to normal values. In one of the two infants with apparently classic RTA and RBW, CHCO3̄/Cln and the therapeutic alkali requirement decreased concomitantly and progressively over 2 yr, but RBW continued. Renal tubular acidosis has persisted in all four patients for at least 3 yr, and in three for 4 years.

Authors

Elisabeth McSherry, Anthony Sebastian, R. Curtis Morris Jr.

Thyroid Hormone Binding by Human Serum Prealbumin (TBPA). ELECTROPHORETIC STUDIES OF TRIIODOTHYRONINE-TBPA INTERACTION

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Abstract

Thyroxine-binding prealbumin (TBPA) in normal human serum has been shown in a polyacrylamide gel electrophoresis system to bind 7-9% of tracer level purified [125I]triiodothyronine (T3), and more than 30% of T3 in serum deficient in thyroxinebinding globulin (TBG). The T3-TBPA interaction has been confirmed at pH 9.0 and pH 7.4 in this electrophoretic demonstration of TBPA binding of T3 in serum. Purified human TBPA has also been shown to bind T3. Progressive additions of unlabeled thyroxine (T4) to serum containing tracer [125I]T3 displace T3 from TBG, its principal carrier, to TBPA and albumin; however, T4 loading does not lead to significant T3 displacement from TBPA even at T4 levels known to saturate TBPA. Loading of serum with unlabeled T3 results in displacement of more than 50% of [125I]T3 from TBPA, as well as from TBG, to albumin. Studies carried out with serum containing diphenylhydantoin (DPH) or MK-185, known inhibitors of T4 binding by TBG, also showed T3 displacement from TBG to TBPA and albumin. Although salicylate and tetraiodothyroacetic acid (TETRAC) displace T4 from sites on TBPA, they have only minimal effects on T3-TBPA interaction.

Authors

Paul J. Davis, Barry S. Handwerger, Robert I. Gregerman

The Genesis of Lower Esophageal Sphincter Pressure: Its Identification through the Use of Gastrin Antiserum

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Abstract

The purpose of this study was to evaluate the role of gastrin in the genesis of lower esophageal sphincter (LES) pressure by the use of a high titer gastrin antiserum. Intravenous infusions of increasing amounts of rabbit gastrin antiserum, but not control antiserum, produced graded reductions in the resting LES pressure in anesthetized opossums. A maximal inhibition in LES pressure of 80.0±3.1% (mean ±SE) was achieved when gastrin antiserum was administered in an amount estimated to bind almost all endogenous circulating gastrin in the opossum. Gastrin antiserum also inhibited the LES response to endogenous gastrin release (gastric deacidification) and to exogenous intravenous administration of gastrin I. The inhibition of the LES response to exogenous gastrin I by gastrin antiserum could be eliminated by giving excess gastrin I. Studies performed in vitro showed that gastrin antiserum inhibited the contractile response of LES circular muscle to gastrin I, but not to acetylcholine. These studies indicate that gastrin antiserum: (a) specifically antagonized the response of LES circular muscle to gastrin, in vitro; (b) diminished the LES response to the endogenous release and to the exogenous administration of gastrin; and (c) markedly reduced the resting level of LES pressure. We conclude that endogenous gastrin is the major determinant of resting LES pressure.

Authors

William Lipshutz, William Hughes, Sidney Cohen

Metabolic Regulation of Heme Catabolism and Bilirubin Production. I. HORMONAL CONTROL OF HEPATIC HEME OXYGENASE ACTIVITY

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Abstract

Heme oxygenase (HO), the enzyme system catalyzing the conversion of heme to bilirubin, was studied in the liver and spleen of fed, fasted, and refed rats. Fasting up to 72 hr resulted in a threefold increase in hepatic HO activity, while starvation beyond this period led to a gradual decline in enzyme activity. Refeeding of rats fasted for 48 hr depressed hepatic HO activity to basal values within 24 hr. Splenic HO was unaffected by fasting and refeeding.

Authors

Arne F. Bakken, M. Michael Thaler, Rudi Schmid

Lipogenesis in Human Adipose Tissue: Some Effects of Nibbling and Gorging

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Abstract

Six grossly obese patients were fed 5000 calorie diets for 4 wk. During one period of 2 wk, the calories were consumed over 4 hr (gorging) and during the other 2 wk, the dietary intake was spread over 20 hr (nibbling). Each of these periods followed a low caloric intake which lasted at least 10 days. Three male patients (group I) were studied at or near their maximal weight and three females (group II) after a weight loss of 50-70 kg. The patients in group II gained more weight than those in group I. Lipogenesis from pyruvate was greater in group II than in group I. Rapid ingestion of food (gorging) was accompanied by a significant increase in glyceride-glycerol-14C and fatty acids-14C from pyruvate-14C. The enzymatic activity of sn-glycerol 3-phosphate dehydrogenase and mitochondrial glycerophosphate oxidase paralleled the rate of formation of glyceride-glycerol. Lipogenesis from pyruvate was significantly lower when the bicarbonate concentration was reduced from 25 to 10 mM. Citrate and acetate were also converted to fatty acids but there was no difference between gorging and nibbling. An inhibitor of carbonic anhydrase significantly reduced the conversion of pyruvate into CO2, glyceride-glycerol, and fatty acids. These data on gorging and nibbling have been related to other studies suggesting that the frequency of food intake may be inversely related to obesity.

Authors

George A. Bray

Characterization of Human Platelet Vascular Permeability-Enhancing Activity

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Abstract

Human platelet acid extract obtained from both whole platelets and from isolated subcellular granules was partially purified by DEAE-cellulose chromatography and Sephadex gel filtration. The heat-stable, nondialyzable cationic protein fraction with a mol wt of approximately 30,000 produced a biphasic increase in vascular permeability in rabbit skin and also had antiheparin activity. The acute (15 min) increase in vascular permeability was blocked by prior treatment of the animal with antihistamine and was characterized histologically by edema of perivascular tissues and dilation of capillaries and veinules. The delayed (3 hr) permeability effect was not blocked by antihistamine and was characterized histologically by leukocytic infiltration into the skin. The experiments described suggest that human platelet lysosomal release of cationic proteins may increase vascular permeability by several mechanisms including endogenous histamine release as well as delayed chemotaxis.

Authors

Ralph L. Nachman, Babette Weksler, Barbara Ferris

Metabolism of Glutamine by the Intact Functioning Kidney of the Dog STUDIES IN METABOLIC ACIDOSIS AND ALKALOSIS

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Abstract

The renal conversion of glutamine to glucose and its oxidation to CO2 were compared in dogs in chronic metabolic acidosis and alkalosis. These studies were performed at normal endogenous levels of glutamine utilizing glutamine-34C (uniformly labeled) as a tracer. It was observed in five experiments in acidosis that mean renal extraction of glutamine by one kidney amounted to 27.7 μmoles/min. Of this quantity, 5.34 μmoles/min was converted to glucose, and 17.5 μmoles/min was oxidized to CO2. Acidotic animals excreted an average of 41 μmoles/min of ammonia in the urine formed by one kidney. In contrast, in five experiments in alkalosis, mean renal extraction of glutamine amounted to 8.04 μmoles/min. Of this quantity, 0.92 μmole/min was converted to glucose, and 4.99 μmoles/min was oxidized to CO2. Alkalotic animals excreted an average of 3.23 μmoles/min of ammonia in the urine. We conclude that renal gluconeogenesis is not rate limiting for the production and excretion of ammonia in either acidosis or alkalosis. Since 40% of total CO2 production is derived from oxidation of glutamine by the acidotic kidney and 14% by the alkalotic kidney, it is apparent that renal energy sources change with acid-base state and that glutamine constitutes a major metabolic fuel in acidosis.

Authors

R. F. Pitts, L. A. Pilkington, M. B. MacLeod, E. Leal-Pinto

The Effects of Cyanate In Vitro on Red Blood Cell Metabolism and Function in Sickle Cell Anemia

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Abstract

Cyanate, which is in equilibrium with urea, combines with the α-amino group of the aminoterminal valine of hemoglobin in an irreversible, specific carbamylation reaction. Partial carbamylation (0.72 residues/hemoglobin tetramer) as determined by cyanate-14C incorporation or hydantoin analysis diminishes the in vitro sickling phenomenon. Since cyanate may react not only with hemoglobin but also with functional groups of other red blood cell proteins, the in vitro effect of cyanate was studied on sickle cells. Cells were incubated with 10 mM KCl (control) or 10 mM KNCO (carbamylated) for 1 hr, washed, and resuspended in autologous plasma. Glycolysis, ATP and 2,3-diphosphoglyceric acid (DPG) stability, autohemolysis, and osmotic fragility were not affected by carbamylation. Potassium loss in carbamylated cells (2.8 mmol/liter) was less than in control cells (9.0 mmol/liter). Pyruvate kinase activity of carbamylated cells was decreased (∼25%) but the activities of other glycolytic enzymes were similar to those of control cells. Oxygen affinity of carbamylated sickle, normal, and DPG-depleted normal cells increased, and was a sensitive index of the degree and duration of reaction with cyanate. The reactivity of carbamylated cells to DPG was similar to control cells. DPG-depleted carbamylated cells regenerated DPG and increased the P50 when incubated with pyruvate, inosine, and phosphate. The Bohr effect of normal and of sickle cells was not affected (Δlog P50/Δ pH=-0.48 and -0.53, respectively) after carbamylation. The reserve buffering capacity of plasma offset the slightly diminished (∼15%) CO2 capacity of carbamylated cells so that whole blood CO2 capacity, pH, and PCO2 were normal. These studies provide further support for the potential clinical use of cyanate in treating and preventing the anemia and painful crises of sickle cell disease.

Authors

Frank G. De Furia, Denis R. Miller, Anthony Cerami, James M. Manning

Role of Antibody and Complement in the Immune Clearance and Destruction of Erythrocytes I. IN VIVO EFFECTS OF IgG AND IgM COMPLEMENT-FIXING SITES

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Abstract

A model which permits evaluation in molecular terms of the role of antibody and of complement in the immune destruction of erythrocytes was established in the guinea pig. IgM and IgG immunoglobulins were isolated from rabbit anti-guinea pig erythrocyte antisera and were used to sensitize 51Cr-labeled guinea pig erythrocytes. The average number of complement-fixing sites per erythrocyte formed by antibody was determined for each of the various preparations by the Cla fixation and transfer test. The rate of clearance and of organ localization was determined for cells sensitized with either IgM or IgG antibodies, and dose-response curves were established in normal guinea pigs and guinea pigs with a genetically controlled, complete absence of the fourth component of complement (C4).

Authors

Aland D. Schreiber, Michael M. Frank

Role of Antibody and Complement in the Immune Clearance and Destruction of Erythrocytes II. MOLECULAR NATURE OF IgG AND IgM COMPLEMENT-FIXING SITES AND EFFECTS OF THEIR INTERACTION WITH SERUM

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Abstract

A model for the immune clearance and destruction of homologous erythrocytes has been further explored. In this model, every IgM anti-erythrocyte antibody molecule in an antibody preparation was shown to fix Cl. About 2000 IgG antibody molecules were required to form a Cl-fixing site on the guinea pig erythrocyte surface. 60 IgM complement-fixing sites per erythrocyte were required for the immune clearance of IgM-sensitized erythrocytes. This number of sites could be detected by a direct agglutination test. 1.4 complement-fixing sites were required for immune clearance of IgG-sensitized cells, a number of molecules which could not be detected by direct agglutination. This number could, however, be detected with the use of a Coombs antiglobulin reagent.

Authors

Alan D. Schreiber, Michael M. Frank

Fibrinogen St. Louis: A New Inherited Fibrinogen Variant, Coincidentally Associated with Hemophilia A

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Abstract

A patient with classical hemophilia (factor VIII deficiency) was found to have a new abnormal fibrinogen (fibrinogen St. Louis). Other family members exhibited either defect alone. Fibrinogen St. Louis was inherited as an autosomal dominant and was not associated with clinical bleeding. When compared with normal fibrinogen, fibrinogen St. Louis was found to have defective fibrin polymerization and possibly a slower release of fibrinopeptides. The prolonged thrombin times were partially corrected by calcium chloride and protamine sulfate. Ultracentrifugal sedimentation, electrophoretic mobility, DEAE chromatographic pattern, carbohydrate content, N-terminal amino acids, immunodiffusion, and immunoelectrophoretic patterns and electrophoresis of reduced and alkylated fragments were all normal. In contrast to fibrinogen St. Louis, the most similar other fibrinogen variant (fibrinogen Zurich) was found to be heterogeneous by several criteria and to have reduced hexose content.

Authors

Laurence A. Sherman, Lamont W. Gaston, Manuel E. Kaplan, Alan R. Spivack

Early Increase in Left Ventricular Compliance after Myocardial Infarction

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Abstract

The left ventricular (LV) pressure-volume (P-V) relationship is a resultant of several determinants, including initial ventricular volume, geometry, and wall stiffness. A quantitative index of one of these determinants, LV wall stiffness, was developed from a mathematical analysis of the isolated P-V relationship. Since this relationship was exponential, stiffness (dP/dV) could be expressed by the equation dP/dV = aP + b, where a and b are constants. The a constant, termed the passive elastic modulus, was independent of both pressure and volume, was modified only slightly by changes in geometry, and thus was primarily affected by changes in wall stiffness. LV wall stiffness was assessed by determination of the passive elastic modulus in eight normal canine hearts and in five hearts 1 hr after acute myocardial infarction. The value of the passive elastic modulus for the normal canine LV was found to be 0.099±0.006 cc-1. In the five infarcted hearts there was a modest, but statistically insignificant, shift of the P-V curves from control, such that for the same pressure the infarcted hearts contained greater volume. However, the passive elastic modulus decreased 41% to 0.057±0.006 cc-1 (P < 0.001). Thus, although LV wall stiffness may increase later in the course of myocardial infarction, it is concluded that it was significantly decreased 1 hr after infarction. Calculation of the passive elastic modulus provided a sensitive means of detecting such changes, whereas P-V curves alone were generally insensitive.

Authors

James S. Forrester, George Diamond, William W. Parmley, H. J. C. Swan

Isolation and Properties of Phagocytic Vesicles II. ALVEOLAR MACROPHAGES

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Phagocytic vesicles were obtained by density gradient centrifugation of homogenized rabbit alveolar macrophages that had ingested emulsified paraffin oil contained Oil Red O. The phagocyte vesicles floated and thereby were separated from the soluble fraction and from other cell components which sedimented. The purity of the isolated vesicles was documented by electron microscopy, chemical and enzyme analysis. The vesicles contained 87% of the cell-associated Oil Red O, and were essentially free of DNA, RNA, succinic dehydrogenase, and glucose-6-phosphatase. Acid phosphatase, β-glucuronidase, and catalase were transferred from the sedimenting fraction to the phagocytic vesicle fraction during phagocytosis, whereas enzyme activities of the soluble fraction remained unchanged. Half of the catalase of resting macrophages was in the pellet fraction and, compared with acid phosphatase, greater amounts of digitonin were required to release full activity. Such differential latency has been described for enzymes of peroxisomes vs. those of lysosomes. Compared with polymorphonuclear leukocyte vesicles studied previously, phagocytic vesicles of macrophages had more electron-dense material and lower Oil Red O:protein, phospholipid:protein, and enzyme:protein ratios. It is thus probable that secondary lysosomes become part of the macrophage vesicle. When paraffin oil particles, the stimulus for phagocytic vesicle formation, were washed away from the macrophages, acquisition of hydrolases by preformed vesicles ceased, i.e. transfer of these enzymes into phagocytic vesicles occurred only during or shortly after the formation of new vesicles. As noted previously by others, the content of acid hydrolases of stimulated alveolar macrophages was doubled in comparison to normal cells. The difference between stimulated and normal macrophages was even more marked when isolated phagocytic vesicles were analyzed. Vesicles from stimulated macrophages had 3-5 times more enzyme activity (per milligram of vesicle protein or per amount of paraffin oil ingested) than did vesicles from normal cells.

Authors

Thomas P. Stossel, Robert J. Mason, Thomas D. Pollard, Martha Vaughan

Quantitative Studies of Phagocytosis by Polymorphonuclear Leukocytes: Use of Emulsions to Measure the Initial Rate of Phagocytosis

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Abstract

Polymorphonuclear leukocytes suspended in Krebs-Ringer phosphate medium ingest paraffin oil containing Oil Red O emulsified with a variety of substances. Spectrophotometric determination of Oil Red O in the cells after uningested particles have been removed by differential centrifugation provides a quantitative measure of phagocytosis. This system has been used to investigate the effects of several drugs and hormones on the initial rate of phagocytosis and to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. The rate of uptake of paraffin oil emulsified with bovine albumin was constant for 6 min and was proportional to cell concentration when saturating concentrations of paraffin oil emulsion were used. At lower concentrations of substrate, the initial rate of phagocytosis was directly proportional to paraffin oil concentration. The increment in glucose oxidation associated with phagocytosis varied directly with the initial rate of particle uptake. The rate of ingestion of the albumin emulsion was not altered by serum (2-20%, v/v), glucose (5-20 mM), or omission of potassium from the medium. The rate of phagocytosis was decreased 65% if magnesium was omitted, and was essentially zero in the absence of divalent cations. The initial rate of uptake was inhibited by inhibitors of glycolysis, by N-ethylmaleimide (0.05-1 mM), colchicine (0.001-0.1 mM), theophylline (1 and 2 mM), dibutyryl cyclic AMP (1 mM), hydrocortisone (2.1 mM), and ethanol (85 mM). Inhibitors of oxidative phosphorylation and dexamethasone (0.01 mM) were without effect, while insulin (2 mU/ml) slightly stimulated the phagocytic rate. Paraffin oil emulsified with different agents was used to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. Emulsions prepared with nonionic detergents, methylated proteins, and proteins with a weak net charge at pH 7.4 were poorly ingested. On the other hand emulsions prepared with agents of strong net positive or negative charge were rapidly taken up. The effect of divalent cations on the rate of phagocytosis varied with the nature of the emulsifier, but was not related in any simple, direct fashion to the net surface charge of the particles. However, it has not been conclusively established that charge was the only variable of the emulsion particles employed.

Authors

Thomas P. Stossel, Robert J. Mason, John Hartwig, Martha Vaughan

A Micropuncture Study of Postobstructive Diuresis in the Rat

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Abstract

In order to investigate the syndrome of postobstructive diuresis, clearance and micropuncture studies were carried out in rats after relief of 24 hr of bilateral (BUL) or unilateral (UUL) ureteral ligation. In rats with BUL, a striking diuresis and natriuresis occurred when the obstruction to one kidney (the experimental kidney) was relieved. The results were not influenced by administration of vasopressin or d-aldosterone. Whole kidney clearances of inulin and p-aminohippuric acid (PAH) in the experimental kidney were reduced to 10% and 20% of normal, respectively. Superficial nephron inulin and PAH clearances were also reduced, but only to 40% and 45%, respectively. These findings suggest a heterogeneity of nephron function in which deep nephrons were functioning poorly or not at all. To investigate the site of impaired tubular reabsorption in the surface nephrons, absolute and fractional water reabsorption was measured. Absolute reabsorption was found to be decreased all along the nephron. Fractional reabsorption in proximal tubules was normal, as indicated by an average endproximal tubular fluid per plasma inulin (TF/PIn) of 2.16 vs. 2.30 in controls. TF/PIn was markedly decreased in distal tubules (2.91 vs. 8.02) and final urine (5.56 vs. 263). These observations indicate that the major sites of impaired sodium reabsorption leading to the diuresis were beyond the proximal tubule.

Authors

William E. Yarger, Hagop S. Aynedjian, Norman Bank

Biologic Activity of Digoxin-Specific Antisera

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Digoxin-specific antibodies are capable of removing essentially all intracellular digoxin from rat renal cortical slices or from human erythrocytes. In removing digoxin from erythrocytes, these antibodies are capable of reversing an effect of the drug on cellular potassium transport. This study provides direct evidence that antibodies are capable of removing, and thereby reversing the biological effect of, physiologically active low molecular weight substances after they have been taken up by mammalian cells. This biologic property of digoxin-specific antibodies suggests that autidigoxin sera may prove useful in the reversal of digoxin toxicity.

Authors

John F. Watson, Vincent P. Butler Jr.

Abnormal Bactericidal, Metabolic, and Lysosomal Functions of Chediak-Higashi Syndrome Leukocytes

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Abstract

Phagocytic, antimicrobial, and metabolic functions were studied in leukocytes obtained from three patients with the Chediak-Higashi syndrome (CHS) and compared to normals, individuals, heterozygous for Chediak-Higashi syndrome, and two subjects with chronic granulomatous disease of childhood (CGD). Chediak-Higashi syndrome leukocytes showed normal ingestion of a variety of bacteria, Candida albicans, and polystyrene latex particles. Intracellular destruction was significantly impaired for Staphylococcus aureus, Group D streptococci, and a rough strain of Type II pneumococci over a 2 hr incubation. Killing of Serattia marcescens was consistently delayed at 1 hr whereas that of Escherichia coli and C. albicans appeared normal, unless the incubations were shortened to 20 min. Examination of the rates of killing indicated that the greatest defect occurred in the first 20 min of contact between Chediak-Higashi syndrome cells and bacteria. Separation of Chediak-Higashi syndrome granulocytes from monocytes revealed that the former were most defective in bactericidal activity. After phagocytosis, Chediak-Higashi syndrome granulocytes displayed a normal burst in oxygen consumption and oxidation of glucose-1-14C and glucose-6-14C and formate-14C. Oxidation of glucose-1-14C by non-phagocytizing Chediak-Higashi syndrome granulocytes and monocytes averaged 2-3 times normal, whereas glucose-6-14C and formate-14C oxidation were not significantly increased by resting cells. Iodination of intracellular protein by Chediak-Higashi syndrome leukocytes was significantly increased above normal in both the resting and phagocytizing state. Electron microscopic histochemistry revealed that almost all peroxidase activity was localized to the giant granules in Chediak-Higashi granulocytes, and after bacterial ingestion there was a failure of delivery of peroxidase to many phagosomes. Upon longer incubation more phagosomes acquired peroxidase activity, presumably through a fusion process, although many giant granules remained intact. The contrasting patterns and kinetics of the killing defects and the differing metabolic properties of Chediak-Higashi syndrome and chronic granulomatous disease leukocytes emphasize the pleiomorphic nature of inherited disorders of leukocyte function.

Authors

Richard K. Root, Alan S. Rosenthal, Dominic J. Balestra

Hemoglobin Malmö β-97 (FG-4) Histidine→Glutamine: A Cause of Polycythemia

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Abstract

A striking history of familial polycythemia led to a search for an abnormal hemoglobin. None could be demonstrated by routine electrophoretic methods, but the propositus' hemolysate had increased oxygen affinity. Manipulation of the conditions of electrophoresis, and chromatographic methods, permitted identification of hemoglobin Malmö. Studies of hemolysates demonstrated a normal Bohr effect, decreased heme-heme interaction (n=1.58), and a p50 of 1.3 mm Hg at 10°C and pH 7.2. The amino acid substitution occurs in the same position (FG-4) as that of hemoglobin Chesapeake, but in the β-chain rather than the α-chain. The two types of hemolysate have different pathophysiologic properties, and carriers of hemoglobin Malmö exhibit more striking hematologic abnormalities.

Authors

Samuel H. Boyer, Samuel Charache, Virgil F. Fairbanks, Jorge E. Maldonado, Andrea Noyes, Esther E. Gayle

Immunological Studies of Y Protein. A MAJOR CYTOPLASMIC ORGANIC ANION-BINDING PROTEIN IN RAT LIVER

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An antibody produced against rat Y protein, the major cytoplasmic organic anion-binding protein in liver, was characterized. The antibody precipitated Y protein from liver supernatant fractions and specifically removed the organic anion-binding capacity from this fraction.

Authors

Gerald Fleischner, John Robbins, Irwin M. Arias

Platelet Interaction with Polymerizing Fibrin

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Abstract

Interaction of washed pig, rabbit, or human platelets with fibrinogen was studied during its transition to fibrin using photometric, isotopic, and electron microscopic techniques. Untreated fibrinogen and fully polymerized fibrin had no detectable effect on platelets. Fibrinogen, incubated with low concentrations of reptilase or thrombin, formed intermediate products which readily became associated with platelets and caused their aggregation. Neutralization of the thrombin did not prevent this interaction. In the absence of fibrinogen, reptilase did not affect platelets.

Authors

Stefan Niewiarowski, Erwin Rogoeczi, Gwendolyn J. Steward, Andrew F. Senyi, J. Fraser Mustard

Immunoglobulins on the Surface of Lymphoid Cells in Waldenström's Macroglobulinemia

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Abstract

Immunofluorescence study of viable and fixed cells was performed on marrow and blood samples from 25 patients with Waldenström's macroglobulinemia. Whereas intracytoplasmic staining for IgM was restricted to the plasma cells and to a limited number of lymphocytic cells, the vast majority of the pleomorphic “lymphoid” proliferating cells in the marrow were shown to bear the monoclonal IgM on their surface. All IgM-secreting plasma cells displayed membrane positivity. Most lymphocytic proliferating cells carried the monoclonal IgM on their surface in the absence of detectable amounts of intracytoplasmic IgM. Despite the usual absence of increase in blood lymphocyte counts, a large number of circulating lymphocytes were shown to bear membrane-bound monoclonal IgM in patients with active disease. The number of fluorescent spots, their size and brightness varied greatly from cell to cell in marrow and blood samples of a given patient, and this finding is in contrast to the homogeneous fluorescent pattern observed in chronic lymphocytic leukemia. The data suggest that macroglobulinemia represents the proliferation of a clone of B cells which continues to mature and differentiate.

Authors

Jean-Louis Preud'homme, Maxime Seligmann

Functional Evaluation of Prolactin Secretion: a Guide to Therapy

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Abstract

Stimulation and inhibition tests are proposed for evaluating prolactin secretion. Thyrotropin-releasing hormone (TRH) stimulates the release of prolactin from the pituitary. Chlorpromazine acts presumably at the hypothalamic level to increase prolactin secretion. L-Dopa (D,L-α-hydrazino-α-methyl-β-[3,4-di-hydroxyphenyl]) has the opposite effect; it inhibits prolactin secretion and may be effective in suppressing galactorrhea.

Authors

H. Friesen, H. Guyda, P. Hwang, J. E. Tyson, A. Barbeau

Erythrocytosis in Spontaneously Hypertensive Rats

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Abstract

During the study of an inbred strain of Wistar rats which spontaneously develop hypertension when they reach a weight of approximately 150 g, it was found that these animals also develop an erythrocytosis. A significant increase in red cell count was observed in spontaneously hypertensive (SH) rats (8-11 × 106 RBC/mm3) when compared with normotensive rats (6-7 × 106 RBC/mm3) of the same strain. This increase in red cell count paralleled the increase in body weight and the rise in blood pressure.

Authors

Subha Sen, George C. Hoffman, Nicholas T. Stowe, Robert R. Smeby, F. Merlin Bumpus