PURPOSES IN MEDICAL RESEARCH AN INTRODUCTION TO THE JOURNAL OF CLINICAL INVESTIGATION

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Alfred E. Cohn

THE JOURNAL OF CLINICAL INVESTIGATION 1974

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Jean D. Wilson

Canalicular Bile Secretion in Man STUDIES UTILIZING THE BILIARY CLEARANCE OF [¹⁴C]MANNITOL

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[14C]Mannitol was administered i.v. as a bolus injection to five postcholecystectomy patients with indwelling T-tubes and re-established enterohepatic circulations to evaluate the biliary clearance of [14C]mannitol as a means of estimating canalicular bile flow in man. [14C]Mannitol appeared in collections of bile 9-22.5 min after intravenous injection, rose to a peak, and thereafter paralleled the plasma [14C]mannitol disappearance curve. Bile-plasma [14C]mannitol ratios and [14C]mannitol clearances were determined during control and choleretic periods after correction of the bile [14C]mannitol points for the transit time of a given sample. After i.v. injection of sodium dehydrocholate in five studies, bile flow and mannitol clearance increased proportionately. However, when ductular secretion was stimulated with an i.v. bolus of secretin in three other studies, [14C]mannitol clearance remained essentially unchanged, indicating that [14C]mannitol entered bile at the level of the hepatocyte and could be utilized as a marker of canalicular flow in man.

Authors

James L. Boyer, Joseph R. Bloomer

Resolution of Experimental Pulmonary Emboli with Heparin and Streptokinase in Different Dosage Regimens

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Thrombolytic agents may be useful in acute pulmonary embolism, but their optimal dosage remains uncertain. We have examined the relative efficacy of heparin and different doses of streptokinase, either alone or in combination, in acute experimental pulmonary embolism. A standardized massive embolus of autologous blood clot incorporating canine [125I]-fibrinogen was given to 40 dogs; the degree of resolution after 24 h was quantitated by measuring the radioactivity in the lungs and was compared with detailed postmortem observations.

Authors

J. F. Cade, J. Hirsh, E. Regoeczi, M. Gent, M. R. Buchanan, D. M. Hynes

Increased Sheep Lung Vascular Permeability Caused by Pseudomonas Bacteremia

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In awake sheep, we compared the responses of lung lymph flow and lymph and plasma protein concentrations to steady state elevations of pulmonary vascular pressures made by inflating a left atrial balloon with those after an intravenous infusion of 105-1010Pseudomonas aeruginosa. Lymph flow increased when pressure was increased, but lymph-plasma protein concentration ratios always fell and lymph protein flow (lymph flow × lymph protein concentration) increased only slightly. After Pseudomonas, sheep had transient chills, fever, leukopenia, hypoxemia, increased pulmonary artery pressure and lymph flow and decreased left atrial pressure and lymph protein concentration, 3-5 h after Pseudomonas, when vascular pressures and lymph protein concentrations had returned to near base line, lymph flow increased further to 3-10 times base line and remained at a steady level for many hours. During this steady state period, lymph-plasma protein concentration ratios were similar to base line and lymph protein flow was higher than in the increased pressure studies. Two sheep died of pulmonary edema 7 and 9 h after Pseudomonas, but in 16 studies, five other sheep appeared well during the period of highest lymph flow and all variables returned to base line in 24-72 h. Six serial indicator dilution lung water studies in five sheep changed insignificantly from base line after Pseudomonas. Postmortem lung water was high in the two sheep dead of pulmonary edema and one other, but six sheep killed 1-6 h after Pseudomonas had normal lung water. Because of the clear difference between the effects of increased pressure and Pseudomonas on lymphplasma protein concentration ratios and lymph protein flow, we conclude that Pseudomonas causes a prolonged increase in lung vessel permeability to protein. Because we saw lung lymph flow as high as 10 times base line without pulmonary edema, we conclude that lung lymphatics are a sensitive high-capacity mechanism for removing excess filtered fluid. An equivalent pore model of sheep lung vessels suggests that the changes we saw after Pseudomonas could result from small changes in the structure of exchanging vessel walls.

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Kenneth L. Brigham, William C. Woolverton, Lynn H. Blake, Norman C. Staub

Preferential Binding of β^S Globin Chains Associated with Stroma in Sickle Cell Disorders

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Sickle cell anemia (SS) is associated with abnormalities of the red cell membrane and decreased red cell deformability. The present study assesses globin chain binding to stroma in SS, sickle cell trait (AS), and nonsickling (AA) cells. The results indicate that there is preferential binding of newly synthesized βS globin to red cell stroma in SS cells and preferential binding of βS to stroma compared to βA in AS cells. These studies show that βS globin binding to stroma accompanies the membrane abnormalities in SS and AS patients.

Authors

Arthur Bank, Gregory Mears, Robert Weiss, Joyce V. O'Donnell, Clayton Natta

Formation and Excretion of Pyrrole-2-Carboxylate in Man

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A detailed investigation of the purification of pyrrole-2-carboxylate (PCA) from human and rat urine indicates that previously reported mean values overestimate the correct quantity of free PCA by a factor of approximately three for rat urine and approximately five for human urine. Although several criteria of purity were satisfied by a previous method, pyrrole-reactive impurities were still present in the final fractions. These impurities are separated from PCA by chromatography through an amino acid analyzer ion-exchange resin. With the corrected method, normal human values for endogenous urinary PCA in 16 individuals averaged 0.51 μmol/day, with a range of 0.20-1.3 μmol and a SD of 0.31 μmol. The probable source of human PCA is free hydroxy-L-proline, as inferred from the high value for PCA in the urine of a subject with hereditary hydroxyprolinemia, and from the threeto eightfold elevation in PCA excretion by two normal subjects after a large oral load of hydroxyl-L-proline. Subcutaneous administration of [2-14C]PCA to a single human subject indicated almost complete conversion of the exogenous compound to derivatives, which are largely excreted in the urine. Data are discussed suggesting that much or all of the PCA in human urine may be formed in urine from a labile precursor, presumably Δ1-pyrroline-4-hydroxy-2-carboxylate.

Authors

Anne M. Heacock, Elijah Adams

Glucose Modulation of Amino Acid-Induced Glucagon and Insulin Release in the Isolated Perfused Rat Pancreas

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Interactions between glucose and arginine and a mixture of 20 amino acids found in normal rat serum were studied in the isolated perfused rat pancreas of normal rats, with release of immunoreactive glucagon and insulin as parameters. Secretion of both pancreatic hormones was low during the steady state, whether glucose (5 mM) was included in the perfusion medium or not. This glucose concentration significantly stimulated insulin release twofold and resulted in an 80% inhibition of basal glucagon release. Arginine and the amino acid mixture were potent stimulants of both hormones. Secretion of both hormones followed identical biphasic response patterns after addition of arginine or the amino acid mixture. However, stimulation of insulin release occurred only when glucose was included, whereas both phases of glucagon release were elicited in the absence of glucose and markedly reduced in its presence. The dose-dependency curves of hormone release due to arginine on one hand and the amino acid mixture on the other differed substantially: with arginine, release of insulin and glucagon was linear between a concentration of 0.3 and 20 mM. In contrast, the amino acid mixture resulted in half-maximal release for both hormones between a concentration of 3 and 4.5 mM, and maximal release between 6 and 8 mM. The dose-dependencies of glucose modulation of α- and β-cell activity were also different: when the amino acid mixture was maintained at 15 mM and glucose varied (0-6.25 nM), no insulin release occurred until glucose was above 2.5 mM, whereas incremental inhibition of glucagon occurred through the complete dose range. It was also observed that glucose inhibition of amino acid-stimulated glucagon release was dissociated from glucose-dependent increase of insulin release.

Authors

Anthony S. Pagliara, Susan N. Stillings, Barbara Hover, Duane M. Martin, Franz M. Matschinsky

Characterization of the Effects of Arginine and Glucose on Glucagon and Insulin Release from the Perfused Rat Pancreas

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To characterize the mechanisms by which arginine and glucose affect pancreatic alpha and beta cell function, the effects of these agents over their full dose response, both alone and in various combinations, were studied using the perfused rat pancreas. Arginine (0-38 mM), in the absence of glucose, stimulated biphasic glucagon (IRG) secretion (Km≃3-4 mM) at concentrations less than 1 mM and caused nonphasic insulin (IRI) release (Km≃12-13 mM) but only at concentrations greater than 6 mM. Glucose (0-27.5 mM) alone stimulated biphasic IRI release (Km≃9-10 mM) at concentrations in excess of 5.5 mM and caused nonphasic inhibition of IRG secretion (Kt≃5-6 mM) at concentrations as low as 4.1 mM. These results demonstrate fundamental differences in pancreatic alpha and beta cell secretory patterns in response to glucose and arginine and suggest that glucagon secretion is more sensitive to the effect of both glucose and arginine. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated biphasic IRG and IRI release: IRG responses were diminished and IRI responses were enhanced compared with those seen with arginine in the absence of glucose. Glucose (0-27.5 mM) in the presence of 3.2 or 19.2 mM arginine caused similar inhibition of IRG secretion (Km≃5-6 mM) and stimulation of IRI release (Km≃9-10 mM) as that seen with glucose alone, although greater IRG and IRI release occurred. This augmentation of IRI secretion was greater than that expected from mere additive effects of glucose and arginine. Classical Lineweaver-Burk analysis of these results indicates that glucose is a non-competitive inhibitor arginine-stimulated glucagon secretion and suggests that glucose and arginine affect pancreatic alpha and beta cell function via different mechanisms. In addition, comparison of simultaneous insulin and glucagon secretion patterns under various conditions suggests that endogenous insulin per se has little or no direct effect on IRG secretion and that endogenous glucagon does not appreciably affect pancreatic beta cell function.

Authors

John E. Gerich, M. Arthur Charles, Gerold M. Grodsky

Profile of Circulating Vasoactive Substances in Hemorrhagic Shock and Their Pharmacologic Manipulation

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(a) Hemorrhage in dogs (to 45-50 mm Hg) was associated with a 10-fold increase in plasma renin activity (PRA) which remained elevated throughout the time-course of shock including the irreversible (decompensation) stage. The presence of angiotensin II (AII) in arterial blood was demonstrated by the bloodbathed organ technique and confirmed by blockade with specific AII antagonists (cysteine8-AII or isoleucine8-AII). The contribution of AII to systemic peripheral resistance during hemorrhage shock in dogs was established by administering AII antagonists which immediately cause a further fall in blood pressure.

Authors

Barbara A. Jakschik, Garland R. Marshall, Janet L. Kourik, Philip Needleman

Thyroid Hormone Action IN VITRO CHARACTERIZATION OF SOLUBILIZED NUCLEAR RECEPTORS FROM RAT LIVER AND CULTURED GH₁ CELLS

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We previously reported that putative nuclear receptors for thyroid hormone can be demonstrated by incubation of hormone either with intact GH1 cells, a rat pituitary tumor cell line, or with isolated GH1 cell nuclei and rat liver nuclei in vitro.

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Herbert H. Samuels, Jir S. Tsai, Juan Casanova, Frederick Stanley

Hereditary Hemolytic Anemia with Human Erythrocyte Pyrimidine 5′-Nucleotidase Deficiency

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A severe deficiency of a red cell pyrimidine 5′-nucleotidase was found to be associated with hereditary hemolytic anemia in four members of three kindreds. The syndrome was characterized by marked increases above normal in red cell basophilic stippling, total nucleotides, and GSH and by a fairly severe deficiency of ribosephosphate pyrophosphokinase (EC 2.7.6.1.). Patient erythrocytes uniquely contained large amounts of pyrimidine 5′-ribonucleotides. In earlier studies, these were erroneously considered to be adenosine phosphates, since all previous investigations of the nucleotides of human red cells and reticulocytes have shown 97% or more to contain adenine. Total nucleotides in patient cells were present in amounts 3-6 times greater than normal, and approximately 80% contained pyrimidine. The ultraviolet spectral curves of deproteinized red cell extracts exhibited a shift in maximum absorbance from the usual 256-257 nm to approximately 266-270 nm, and absorbance at 250, 270, 280, and 290 nm, expressed as a ratio of that at 260 nm, differed greatly from normal. The spectral characteristics of extracts provide the basis of a readily performed screening procedure, which does not require enzyme assay. The nucleotidase activity in deficient red cells assayed less than 14%, and usually less than 10%, of normal and much less in terms of reticulocyte-rich blood, where it was consistently found to be increased. The enzyme has a pH optimum of 7.5-8.0, is inhibited by EDTA, and does not utilize purine 5′-ribonucleotides or β-glycerophosphate as substrates. While comparatively few family members have been available thus far for study, initial data are compatible with an autosomal, recessive mode of transmission of the deficiency. The pyrimidine 5′-ribonucleotides are presumably derived from RNA degradation and, not being diffusible, accumulate when the enzyme catalyzing their dephosphorylation is deficient. It is postulated that the prominent basophilic stippling results from retarded ribosomal RNA degradation secondary to accumulation of degradation products, namely pyrimidine 5′-ribonucleotides. Ribosephosphate pyrophosphokinase deficiency is considered to be an epiphenomenon. The mechanism responsible for increased red cell GSH is unknown.

Authors

William N. Valentine, Kay Fink, Donald E. Paglia, Susan R. Harris, William S. Adams

Increased Collagen Synthesis by Scleroderma Skin Fibroblasts In Vitro A POSSIBLE DEFECT IN THE REGULATION OR ACTIVATION OF THE SCLERODERMA FIBROBLAST

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Cultures of dividing skin fibroblasts from normal and sclerodermatous human skin have permitted estimations of soluble collagen concentration, net collagen accumulation, cell-doubling times, and the comparison of morphologic and ultrastructural characteristics. In vitro, the scleroderma fibroblast produces more soluble collagen, synthesizes collagen more rapidly, and fourfold more of its protein synthetic activity is directed to collagen production than in the normal skin fibroblast. Cell-doubling times and morphologic and ultrastructural observations of cells in culture have not provided clues to the nature of the biologic defect in the regulation or activation of collagen synthesis by the scleroderma fibroblast.

Authors

E. Carwile LeRoy

The Responses of Rat Intestinal Brush Border and Cytosol Peptide Hydrolase Activities to Variation in Dietary Protein Content DIETARY REGULATION OF INTESTINAL PEPTIDE HYDROLASES

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The effects of variation in dietary protein content on small intestinal brush border and cytosol peptide hydrolase activities have been investigated. One group of rats was fed a high protein diet (55% casein) and another group was fed a low protein diet (10% casein). After 1 wk, brush border peptide hydrolase activity (L-leucyl-β-naphthylamide as substrate) and cytosol peptide hydrolase activity (L-prolyl-L-leucine as substrate) were determined in mucosae taken from the proximal, middle, and distal small intestine. As judged by several parameters, brush border peptide hydrolase activity was significantly greater in rats fed the high protein diet when data for corresponding segments were compared. In contrast, no significant difference was seen in cytosol peptide hydrolase activity.

Authors

J. Alex Nicholson, Denis M. McCarthy, Young S. Kim

Neural Control of the Lower Esophageal Sphincter INFLUENCE OF THE VAGUS NERVES

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We performed studies in the opossum to define the influence of the vagi in the control of lower esophageal sphincter (LES) function. Bilateral vagotomy caused transient sphincter hypertension which was prevented by phentolamine and by atropine. Stimulation of the peripheral end of vagus, after bilateral cervical vagotomy, caused relaxation of the LES over a wide range of frequency and intensity of electrical stimulation. The relaxation was less marked at the lower frequencies of stimulation, and atropine treatment did not enhance this relaxation. In other experiments, atropine treatment reversed the rise in gastric (fundic) pressure with the vagal stimulation, but atropine did not enhance the degree of LES relaxation. Stimulation of the central end of the vagus caused an increase in LES pressure due to a centrally mediated reflex; the efferents for this motor response were not present in the vagi, as the reflex contraction persisted after bilateral vagotomy. The LES contraction with the stimulation of the vagal afferents was antagonized by phentolamine as well as by atropine. These studies suggest that: (a) the vagi do not mediate any cholinergic excitatory influences to the LES and the vagal influence of the sphincter is entirely inhibitory; (b) the vagi carry afferent fibres for a centrally mediated neural reflex which contracts the LES, but the efferent path of this reflex arc does not lie in the vagi.

Authors

Satish Rattan, Raj K. Goyal

Sanfilippo A Syndrome SULFAMIDASE DEFICIENCY IN CULTURED SKIN FIBROBLASTS AND LIVER

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The Sanfilippo A syndrome is an autosomal recessive mucopolysaccharidosis characterized clinically by severe mental retardation and biochemically by storage in tissue and excretion in urine of excessive amounts of heparan sulfate. Since sulfamide groups are present in heparan sulfate, a sulfamidase deficiency could explain the impaired degradation of this polysaccharide. To investigate the enzymic basis of this disease, assays for sulfamidase were performed. Extracts of cultured fibroblasts and post-mortem liver were prepared by suspension of tissues in acetate: NaCl buffer, pH 4.5, containing Triton X-100 (Rohm and Haas Co., Philadelphia, Pa.), sonication, and centrifugation at 10,000 g. The supernatant fluid was incubated with [35S]-N-sulfated heparin. The release of inorganic sulfate after 18 h of incubation was determined by chromatography on Sephadex G-25. The liver and fibroblast extracts of patients with the Sanfilippo A syndrome showed a deficiency of sulfamidase.

Authors

Reuben Matalon, Albert Dorfman

Hyperresponse to Thyrotropin-Releasing Hormone Accompanying Small Decreases in Serum Thyroid Hormone Concentrations

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To determine whether pituitary thyrotropin (TSH) responsiveness to thyrotropin-releasing hormone (TRH) is enhanced by small decreases in serum thyroxine (T4) and triiodothyronine (T3), 12 euthyroid volunteers were given 190 mg iodide po daily for 10 days to inhibit T4 and T3 release from the thyroid. Basal serum T4, T3, and TSH concentrations and the serum T4 and TSH responses to 400 μg TRH i.v. were assessed before and at the end of iodide administration. Iodide induced small but highly significant decreases in basal serum T4 (8.0±1.6 vs. 6.6±1.7 μg/100 ml; mean ± SD) and T3 (128±15 vs. 110±22 ng/100 ml) and increases in basal serum TSH (1.3±0.9 vs. 2.1±1.0 μU/ml). During iodide administration, the TSH response to TRH was significantly increased at each of seven time points up to 120 min. The maximum increment in serum TSH after TRH increased from a control mean of 8.8±4.1 to a mean of 13.0±2.8 μU/ml during iodide administration. As evidence of the inhibitory effect of iodide on hormonal release, the increment in serum T3 at 120 min after TRH was significantly lessened during iodide administration (61±42 vs. 33±24 ng/100 ml). These findings demonstrate that small acute decreases in serum T4 and T3 concentrations, resulting in values well within the normal range, are associated both with slight increases in basal TSH concentrations and pronounced increases in the TSH response to TRH. These results demonstrate that a marked sensitivity of TSH secretion and responsiveness to TRH is applicable to decreasing, as well as increasing, concentrations of thyroid hormones.

Authors

Apostolos G. Vagenakis, Basil Rapoport, Fereidoun Azizi, Gary I. Portnay, Lewis E. Braverman, Sidney H. Ingbar

Effects of Steroid Hormones on Human Polymorphonuclear Leukocyte Lysosomes

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Lysosomal membrane stabilization has been proposed as a mechanism for the anti-inflammatory action of corticosteroid hormones. This hypothesis was based on studies with liver organelles. We studied the action of steroids on intact lysosomes isolated from human peripheral blood polymorphonuclear (PMN) leukocytes. Both androstenedione and progesterone, 10-3-10-5 M, caused leakage of acid hydrolase markers from these organelles, thus resembling their effects on liver lysosomes. But none of the anti-inflammatory steroids tested protected organelle membranes from either detergent lysis (Triton X-100) or heat incubation (37°C, 90 min). Hydrocortisone (HC), HC sodium succinate, HC acetate, HC hemisuccinate, prednisone, and dexamethasone were without detectable stabilizing activity at concentrations of 10-3-5 × 10-8 M. Release of the lysosomal marker, β-glucuronidase, was not retarded by any of the compounds studied. In addition, PMN leukocyte lysosomes isolated from human volunteers receiving prednisolone were not more stable than control organelles, nor did serum from steroid-treated humans protect intact lysosomes from detergent lysis.

Authors

Robert H. Persellin, Leighton C. Ku

Mechanism of Impaired Water Excretion in the Hypothyroid Rat

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The ability to excrete an oral water load and the renal diluting mechanism were studied in hypothyroid rats and in age-matched euthyroid controls. Hypothyroid animals excreted a significantly smaller fraction of a 50-ml/kg oral water load than controls, demonstrating the same limited ability to excrete free water as thyroid-deficient man. During hypotonic (0.45%) saline infusion, absolute sodium delivery to the diluting segment and free water clearance were markedly lower in hypothyroid rats. However, both fractional distal sodium delivery and fractional free water clearance were similar in hypothyroid and control animals, suggesting that the reduced absolute free water formation in hypothyroid rats was due to decreased net distal delivery. In support of this hypothesis was the observation that fractional distal sodium reabsorption was equal or higher in thyroid-deficient rats, which indicates that the sodium reabsorptive capacity of the diluting segment was preserved in these animals. The results cannot be attributed to incomplete suppression of antidiuretic hormone (ADH) since they were identical in diabetes insipidus rats, nor to different rates of non-ADH-dependent backflux of filtrate since tissue osmolality and solute concentrations in the cortex, medulla, and papilla were similar in hypothyroid and control rats of both Sprague-Dawley and Brattleboro strains.

Authors

Dimitrios S. Emmanouel, Marshall D. Lindheimer, Adrian I. Katz

Osmotic Extraction of Hypotonic Fluid from the Lungs

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After injections of sucrose, NaCl, and urea solutions, the flow of tissue fluid from the lungs amounted to 0.182, 0.216, and 0.152 × 10-3 ml/s per mosmol/kg of concentration difference between plasma and tissues in each gram of wet tissue weight. The extracted fluid contained less than 20% of the Na+, K+ and urea concentrations of the plasma. It was concluded that this fluid was distinctly hypotonic in comparision with the fluids of the plasma and tissue compartments both before and after the injection of hypertonic solutions.

Authors

Richard M. Effros

Imbalance in α and β Globin Synthesis Associated with a Hemoglobinopathy

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In contrast to findings in the thalasemia syndromes, studies of globin synthesis in subjects with structurally abnormal hemoglobins have generally revealed equal production of α and β polypeptide chains. However, in the present investigation of globin biosynthesis in vitro in blood and marrow from two subjects heterozygous for unstable hemoglobin Leiden, β6 or 7 Glu → O, a significant excess of α-chain production was revealed. A mother and daughter of northern European ancestry with mild compensated hemolytic anemia were found to have 25% hemoglobin Leiden. Increased hemolysis occurred after the ingestion of a sulfonamide and during infections. Normal levels of hemoglobin A2, 3.0 and 2.7%, and hemoglobin F, 0.8 and 0.6%, were found in the two subjects. Similar percentages of the minor hemoglobins were demonstrated in other family members without hemoglobin Leiden. After incubation of peripheral blood with [3H]-leucine, the βA/βLeiden synthesis ratio was 1.3, and the specific activity of βLeiden was 1.3-2 times βA. These results indicate preferential destruction of the unstable hemoglobin Leiden. However, in contrast to previous studies of other unstable hemoglobins, there was excess synthesis of α-chains. The total β/α synthesis ratio was 0.47-0.63 in peripheral blood and 0.82 in marrow. A pool of free α-chains was demonstrated by starch gel electrophoresis and DEAE column chromatography. The synthesis of globin chains was balanced in family members without hemoglobin Leiden. This degree of predominance of α-chain synthesis in subjects with hemoglobin Leiden resembles the findings in heterozygous β-thalassemia. However, the relatively normal hemoglobin content of the cells with this abnormal hemoglobin suggests the possibility of an absolute excess α-chain production in the hemoglobin Leiden syndrome.

Authors

R. F. Rieder, G. W. James III

Influence of Calcium on the Inotropic Actions of Hyperosmotic Agents, Norepinephrine, Paired Electrical Stimulation, and Treppe

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To analyze the interaction of calcium ion concentration with hypertonic agents and with other inotropic interventions, isolated right ventricular cat papillary muscles were studied under isometric conditions in Krebs-Ringer bicarbonate solution. Extracellular calcium concentrations were varied between 2.5 and 11.0 mM. Maximal inotropic effects occurred between 5 and 8.0 mM calcium and further elevation to 11.0 mM was without additional influence. The effect of hyperosmotic sucrose and mannitol on papillary muscle performance was compared with that of 10-6 M norepinephrine at calcium concentrations of 2.5 and 10.0 mM and with paired electrical stimulation in 10.0 mM calcium. Both norepinephrine and the hyperosmotic agents produced significant increases in developed tension and in the maximal rate of tension rise (dT/dt) in Krebs-Ringer in 2.5 and 4.0 mM calcium. In 10 mM calcium norepinephrine increased developed tension and dT/dt, but sucrose and mannitol caused no change or small reductions in both. Paired electrical stimulation, like hyperosmolality, caused no increase in dT/dt in 10 mM calcium.

Authors

James T. Willerson, J. Stanley Crie, Robert C. Adcock, Gordon H. Templeton, Kern Wildenthal

Hematopoiesis in the Grey Collic Dog STUDIES OF THE REGULATION OF ERYTHROPOIESIS

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Hematopoiesis in the grey collie dog undergoes periodic fluctuations which involve reticulocytes, granulocytes, platelets, lymphocytes, and monocytes. This syndrome is inherited in an autosomal recessive manner and can be transmitted or abolished by appropriate bone marrow transplantation experiments, thus demonstrating this to be a primary marrow defect. Investigation of humoral regulation in this setting indicates that serum erythropoietin (ESF) also undergoes cyclic fluctuation and that shortly after the increase and peak in serum ESF levels recognizable red cell precursors appear in the marrow. Erythropoiesis in the grey collie is reciprocally related to the blood O2 carrying capacity. With phlebotomy, ESF activity and reticulocytes increase but continue to cycle, while hypertransfusion eliminates reticulocyte production completely. Neither phlebotomy nor hypertransfusion alter the underlying cycle time (11-12 days) nor influence the peaks of peripheral blood granulocytes. Thus, in these experiments, no direct evidence of competition between reticulocyte and granulocyte production is observed. In vitro studies of canine hemoglobin synthesis fail to demonstrate evidence of an inhibitor to ESF. These results indicate that periodic fluctuation of serum ESF is an integral part of the grey collie syndrome and are most consistent with some form of feedback regulation of ESF production.

Authors

John W. Adamson, David C. Dale, Ronald J. Elin

Nitrogen Sparing Induced by a Mixture of Essential Amino Acids Given Chiefly as Their Keto-Analogues during Prolonged Starvation in Obese Subjects

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11 normal obese subjects were fasted for 33 days. In five, who served as controls, urine urea nitrogen excretion remained constant for 2 wk thereafter. The other six were given seven daily infusions containing 6-8 mmol each of the α-keto-analogues of valine, leucine, isoleucine, phenylalanine, and methionine (as sodium salts) plus 3-4 mmol each of the remaining essential amino acids (lysine, threonine, tryptophan, and histidine). Rapid amination of the infused ketoacids occurred, as indicated by significant increases in plasma concentrations of valine, leucine, isoleucine, alloisoleucine, phenylalanine, and methionine. Glutamine, glycine, serine, glutamate, and taurine fell significantly. Blood glucose, ketone bodies, plasma free fatty acids, and serum immunoreactive insulin concentrations were unaltered. Urine urea nitrogen fell from 1.46 to 0.89 g/day on the last day of infusions; 5 days later it was still lower (0.63 g/day) and in two subjects studied for 9 and 17 days postinfusion it remained below preinfusion control values. Urine ammonia, creatinine, and uric acid were unaltered. Nitrogen balance became less negative during and after infusions. The results indicate that this mixture of essential amino acids and their keto-analogues facilitates nitrogen sparing during prolonged starvation, in part by conversion of the ketoacids to amino acids and in part by altering mechanisms of nitrogen conservation. The latter effect persists after the ketoacids are metabolized.

Authors

Daniel G. Sapir, Oliver E. Owen, Thomas Pozefsky, Mackenzie Walser

Hepatic Ketogenesis and Gluconeogenesis in Humans

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Splanchnic arterio-hepatic venous differences for a variety of substrates associated with carbohydrate and lipid metabolism were determined simultaneously with hepatic blood flow in five patients after 3 days of starvation.

Authors

A. J. Garber, P. H. Menzel, G. Boden, O. E. Owen

Qualitative and Quantitative Aspects of the Human Antibody Response to Streptococcal Group A Carbohydrate

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The lack of impressive quantitative differences in antibody to various streptococcal extracellular and cellular antigens among patients with acute rheumatic fever, acute glomerulonephritis, and following uncomplicated streptococcal infection has prompted investigation of qualitative aspects of the antibody response among these patients. By using a radiolabeled antigenically univalent hapten derived from streptococcal A carbohydrate, affinity of serum antibody to A-carbohydrate (A-antibody) was studied by an ammonium sulfate precipitation technique. The data obtained demonstrate average association constants (K0S) of acute rheumatic fever patient sera to be significantly lower than those of acute glomerulonephritis or streptococcal infection patients (P<0.02 and P<0.001, respectively). Further analysis of the data from hapten binding studies documents the fact that the radioimmune precipitin assay for the determination of A-antibody level is little influenced by K0 but directly correlates with the concentration of antibody binding sites.

Authors

Stanford T. Shulman, Elia M. Ayoub

Immunologic Specificity of Transfer Factor

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This study examined the immunologic specificity of transfer factor using a chromatographically purified transfer factor preparation. The specificity of transfer was examined utilizing immunity to keyhole limpet hemocyanin (KLH) and tuberculin. Transfer factor prepared from a donor immune to KLH successfully transferred KLH skin test reactivity to 10 out of 10 recipients. In contrast, comparable amounts of transfer factor from two donors not immune to KLH failed to transfer immunity to KLH in 11 recipients despite evidence for successful transfer of tuberculin reactivity. Unlike prior studies with a variety of antigens, the immunity to KLH in recipients of KLH immune transfer factor appeared comparable to that of the donor since both could be elicited with the same skin test antigen dose. These observations indicate that transfer factor can initiate a specific immune response to an antigen not previously encountered by the recipient and that in certain circumstances this immune response can be comparable to that of the donor. These observations on specificity and potency of transfer factor have important implications for the clinical use of this material.

Authors

Kenneth S. Zuckerman, James A. Neidhart, Stanley P. Balcerzak, Albert F. LoBuglio

Evaluation of Leukocyte Chemotaxis In Vitro in Thermally Injured Patients

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Leukocyte chemotaxis in vitro was studied by a modification of the Boyden technic in 46 thermally injured patients. All patients demonstrated a decrease in leukocyte migration through a Nuclepore filter toward a standard casein-serum chemotactic agent. Leukocyte chemotaxis was inversely correlated with burn size during the first 72 h after injury. After 72 h, leukocyte chemotaxis directly correlated with clinical status and was highly predictive for ultimate mortality. Since mortality was largely due to infection, these findings suggest that suppression of leukocyte chemotaxis may explain the susceptibility to opportunistic infection in thermally injured patients.

Authors

Glenn D. Warden, Arthur D. Mason Jr., Basil A. Pruitt Jr.

Superoxide Dismutases in Polymorphonuclear Leukocytes

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Isolated human polymorphonuclear leukocytes were found to contain two distinct superoxide dismutases, electrophoretically identical to the superoxide dismutases found in other human tissues. One is inhibited by cyanide and is located in the cytosol. The other is not inhibited by cyanide and is mitochondrial. These findings disagree with reports by other laboratories, which did not find the cyanide-sensitive cytoplasmic enzyme. The superoxide dismutase-containing preparation of leukocytes released large amounts of superoxide into the medium when the cells were phagocytizing, suggesting that, if the cell population is homogeneous, superoxide production probably takes place on the outer surface of the cell membrane, in addition to inside the phagocytic vacuole.

Authors

Marvin L. Salin, Joe M. McCord