CORRECTION

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Survival of 125iodine-labeled Factor VIII in normals and patients with classic hemophilia. Observations on the heterogeneity of human Factor VIII.

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Radiolabeled human Factor VIII was used to study its survival in normals and patients with classic hemophilia, and to study the heterogeneity of Factor VIII; Purified Factor VIII was radiolabeled with 125iodine (125I-VIII) without loss of its structural integrity. The survival of 125I-VIII was studied in six normals and six hemophiliacs of whom four of the hemophiliacs had received transfusions with normal cryoprecipitate before the 125I-VIII infusion. No significant difference was observed between the disappearance of Factor VIII coagulant activity and radioactivity in these hemophiliacs. 125I-VIII in plasma showed a biphasic disappearance with an average t1/2 of 2.9 +/- 0.4 h (SEM) for the first phase and 18.6 +/- 0.7 h (SEM) for the second phase, respectively. The survival of 125I-VIII was similar comparing normals and hemophiliacs. The highest molecular weight forms of Factor VIII disappear more rapidly than the lower molecular weight ones. This was established by analysis of the fractions obtained by gel chromatography of plasma collected at several times after infusion and by analysis of the in vivo disappearance of three subfractions of Factor VIII. The fraction of 125I-VIII binding to platelets in the presence of ristocetin (containing the highest molecular weight forms of Factor VIII including the ristocetin cofactor) represented about 50% of the radioactivity present in plasma after infusion and showed a t 1/2 of 11.7 +/- 0.9 h (SEM) for the second phase. The fraction, which was recovered in cryoprecipitate of the recipient's plasma, represented about 90% of the initial radioactivity and showed a t 1/2 of 16.3 +/- 0.8 h (SEM) for the second phase. The fraction of 125I-VIII remaining in the cryosupernatant plasma (containing low molecular weight forms of Factor (VIII) showed a t 1/2 of 27.2 +/- 1.1 h (SEM). The first phase of the disappearance of 125I-VIII is caused in part by the disappearance of the highest molecular weight forms, which are possibly removed by the reticuloendothelial system.

Authors

J Over, J J Sixma, M H Bruïne, M C Trieschnigg, R A Vlooswijk, N H Beeser-Visser, B N Bouma

A common uptake system for serotonin and dopamine in human platelets.

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Kinetic and pharmacologic properties of uptake of serotonin and dopamine by normal human platelets have been investigated to test whether platelets can be employed as a model system for the reuptake of serotonin and dopamine in brain. Uptake of serotonin into platelets closely resembles reuptake of serotonin into serotonergic neurons. In contrast, uptake of dopamine into platelets appears to be mediated inefficiently via the specific serotonin uptake mechanism, based upon several lines of evidence. Serotonin and dopamine compete with each other, Antidepressant drugs, which are competitive inhibitors of uptake of both of these neurotransmitters, act at the same concentration of drug despite large differences in the Km values. Serotonin antagonists inhibit both serotonin and dopamine uptake. Finally, a serotonin-specific uptake inhibitor (fluoxetine) blocks dopamine, as well as serotonin, uptake.

Authors

G S Omenn, L T Smith

Gentamicin blockade of slow Ca++ channels in atrial myocardium of guinea pigs.

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Cardiac dysfunction is occasionally detected in patients undergoing treatment with amino-glycoside antibiotics, however, the mechanism responsible for the negative inotropic effect of these agents has not been identified. In the present investigation electrically driven left atria of guinea pigs were used to study the effects of gentamicin on calcium ion (Ca++)-dependent contractile events in heart muscle isolated from in vivo influences. When atria were first inactivated by excess potassium ion (K+; 22mM) and contractions were then restored by isoproterenol (an experimental model that accentuates the contractile dependence of myocardial fibers on influx of Ca++ through specific "slow channels" of the sarcolemma), the cardiac depressant activity of gentamicin (0.1 mM) was profoundly augmented. Conversely, the negative inotropic effect of tetrodotoxin (23.5 micron) was abolished by the same experimental conditions. Also, gentamicin (1 mM) and La+++ (0.5 mM) markedly decreased the positive inotropic response to increased frequency of stimulation; whereas, D600 (1.05 micron) converted the positive frequency-force relationship to a negative relationship. Present data indicate a direct cardiac depressant action of gentamicin, and suggest that this antibiotic adversely affects either the transport system responsible for Ca++ movement through slow channels of the sarcolemma, the availability of Ca++ for translocation to these sites, or both.

Authors

H R Adams, L R Durrett

Inheritance of Low Serum Immunoglobulin D

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Previous studies have shown that log IgD levels in normal individuals are distributed in a nonunimodal manner. Therefore, in this study we tested whether inheritance might play a role in determination of IgD levels. IgD levels were measured in serum or plasma from 301 randomly selected children aged 6-18 yr, 245 consecutive adult blood donors, and 134 first-degree relatives of subjects with low IgD levels. Comparison of serum and plasma from five individuals revealed no difference, so the two were used interchangeably. The distributions of log IgD levels in randomly selected populations of both adults and children were nonunimodal with nadirs at 2.15 IU/ml. In both of these randomly selected populations, 13-14% of the subjects had low IgD values (<2.15 IU/ml). In addition, there was a significant sibling-sibling correlation of log IgD values (r = 0.56, n = 72, P <0.01). Because of the nonunimodality of the frequency distribution histogram for IgD values and because of the familial aggregation of these values, the study was extended to include first-degree relatives of subjects with low plasma IgD. Blood samples from 92% of living first-degree relatives, 134 individuals, were analyzed for their level of IgD, and the results of segregation and pedigree analyses of these data were compatible with autosomal recessive inheritance of an allele for low plasma IgD levels. IgD values in plasma from siblings of probands for low IgD were also non-unimodal in distribution with a nadir at ≅2.15 IU/ml. The results suggest that there is autosomal recessive inheritance of an allele for low plasma IgD.

Authors

Sandra L. Dunnette, Gerald J. Gleich, Richard M. Weinshilboum

Selective Uptake of the Synthetic Amino Terminal Fragment of Bovine Parathyroid Hormone by Isolated Perfused Bone

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Studies from our laboratory have shown that the metabolic clearance rate of carboxy terminal immunoreactive parathyroid hormone (i-PTH) can be accounted for by extraction of i-PTH by liver and kidney. In contrast, there was no demonstrable hepatic uptake of the synthetic amino terminal bovine PTH fragment (syn b-PTH 1-34) and the kidney accounted for only 45% of the metabolic clearance rate of amino terminal i-PTH. This suggested that another major site, presumably bone, played a role in the metabolism of syn b-PTH 1-34. Extraction of i-PTH by isolated perfused bone was studied during infusion of purified bovine PTH (b-PTH) 1-84 or syn b-PTH 1-34. In five studies during infusion of syn b-PTH 1-34 the arteriovenous difference for i-PTH across bone was 36%. In contrast, no significant uptake of carboxy terminal i-PTH was observed in nine studies during infusion of b-PTH 1-84. In addition, when H2O2-oxidized (biologically inactive) syn b-PTH 1-34 was used no arteriovenous difference was observed. These findings correlated with the ability of these PTH preparations to stimulate cyclic AMP production by the perfused bone. Syn b-PTH 1-34 increased cyclic AMP production at perfusate PTH concentrations of 1-5 ng/ml, whereas b-PTH 1-84 evoked only a minimal response at concentrations of 10-20 ng/ml. We conclude that bone is a major site of metabolism of the amino terminal PTH fragment, syn b-PTH 1-34. In addition, these data suggest that cleavage of the intact hormone, with the production of amino terminal PTH fragments by peripheral organs (liver and kidney), may play a major role in the regulation of PTH effects on bone.

Authors

Kevin J. Martin, Jeffrey J. Freitag, Mary B. Conrades, Keith A. Hruska, Saulo Klahr, Eduardo Slatopolsky

Effects of Methylprednisolone on Hydrogen Ion Absorption in the Canine Stomach

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The effect of methylprednisolone (2 mg/kg per day given parenterally for 3 doses, 2 wk or 12 wk) on the permeability of mammalian gastric mucosa to hydrogen ion (H+) was examined with denervated fundic pouches in dogs with antrectomies. Transmucosal electric potential difference (PD) and net fluxes of H+ and Na+ were determined for luminal [H+] from 20 to 160 mM and [Na+] from 1 to 140 mM ([H+] and [Na+] were varied reciprocally). The PD was 50-60 mV lumen negative and was constant over the entire range of Na+ and H+ concentration tested. Net H+ flux varied linearly with [H+]. Extrapolation indicated apparent H+ loss at zero luminal concentration, suggesting a basal HCO3− secretion. Addition of acetylsalicylic acid (ASA) or taurocholate decreased the PD to 30-40 mV and increased threefold the slope of the relation between net H+ flux and [H+] (kH). Calculation of PD-independent permeability constants for H+ (PH) with the Goldman constant field equation indicated that this increase in kH could not be attributed solely to the associated decrease in PD. Prednisolone administered for 3 doses had no effect on either the basal mucosal permeability to H+ or the altered permeability induced by ASA or taurocholate. Chronic administration induced a low rate of basal acid secretion (at 12 wk) but had no effect on either PD or kH. However, the increase in kH and PH that developed upon addition of ASA or taurocholate in chronically treated dogs was more than one and a half times that of controls. These data suggest that prolonged treatment with glucocorticoids increases susceptibility of the gastric mucosa to damage by agents that increase permeability to H+.

Authors

Raphael S. K. Chung, Michael Field, William Silen

Maturation of Jejunum and Ileum in Rats: WATER AND ELECTROLYTE TRANSPORT DURING IN VIVO PERFUSION OF HYPERTONIC SOLUTIONS

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During osmotic diarrhea, loss of water and electrolytes appears to be greater in infants than in adults. In 2-, 3-, and 7-wk-old rats, we studied net transport of H2O, Na, and Cl, during in vivo perfusion of segments of the jejunum and ileum, from solutions with osmolalities of 300, 375, 500, or 700 mosmol/kg. In the jejunal segments, from the hypertonic solutions net transport of H2O, Na, and Cl was into the lumen and greater in the 2- than 7-wk-old rats. In the ileal segments, transport of water was into the lumen, transport of Na was minimal and variable, whereas transport of Cl was usually out the lumen. In 3-wk-old rats, transport rates were intermediate between those in 2- and 7-wk-old rats. The calculated filtration coefficient (microliters of H2O transported per hour per unit osmolality gradient—lumen-serum—per gram dry weight) of water suggested that the resistance to water flow did not increase with rise in luminal hypertonicity in the jejunum of the 2- and 3-wk-old rats, whereas in jejunum of the 7-wk-old rats and in ileum of rats in all three ages, the resistance to water flow increased with the rise in luminal osmolality. The differences in the transport rates and the resistance to water flow, between segments of the 2-, 3-, and 7-wk-old rats, suggested a maturational phenomenon that appears to continue beyond the 3rd wk of life and could have been due to differences in some physical property of the mucosal membrane.

Authors

M. K. Younoszai, R. S. Sapario, M. Laughlin

Activation of Adenylate Cyclase by Heat-Labile Escherichia Coli Enterotoxin: EVIDENCE FOR ADP-RIBOSYLTRANSFERASE ACTIVITY SIMILAR TO THAT OF CHOLERAGEN

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Highly purified, polymyxin-released, low molecular weight Escherichia coli heat-labile enterotoxin (LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. This NAD glycohydrolase activity was stimulated by dithiothreitol and was independent of cellular components. Nicotinamide formation was enhanced by arginine methyl ester > d-arginine ≅ l-arginine ≅ guanidine. A 20-fold increase in activity was noted with arginine methyl ester, and maximal activity again required dithiothreitol. When the reaction was initiated with toxin, a delay was observed before a constant rate was established. The reaction products found after incubation of [adenine-U-14C]NAD and l-[3H]arginine or unlabeled arginine methyl ester with the enterotoxin had mobilities on thin-layer chromatograms similar to the reaction products obtained after incubation of choleragen with these substrates and are consistent with the formation of ADP-ribose-l-arginine and ADP-ribose-l-arginine methyl ester, respectively. Both toxins, which catalyze the NAD-dependent activation of adenylate cyclase, thus appear to possess NAD glycohydrolase and ADP-ribosyltransferase activities. Although the activities of both toxins are dependent on dithiothreitol, Escherichia coli enterotoxin exhibited optimal activity in Tris (Cl−) (pH 7.5) and was inhibited by high concentrations of potassium phosphate (pH 7.0) or low pH (sodium acetate, pH 6.2). It appears that the optimal assay conditions as well as the kinetic constants for the reactants differ from those previously noted with choleragen. It is probable therefore that although the two toxins catalyze similar reactions, they differ in primary structure. The presence of transferase and glycohydrolase activities in structurally distinct toxins that activate adenylate cyclase strengthens our hypothesis that the ADP-ribosylation of arginine is a model for the NAD-dependent activation of adenylate cyclase; activation may result from ADP-ribosylation of the cyclase itself or of a protein that regulates its activity.

Authors

Joel Moss

The Role of Acetaldehyde in Mediating the Deleterious Effect of Ethanol on Pyridoxal 5′-Phosphate Metabolism

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Previous studies in vivo and with isolated perfused rat livers have suggested that the deleterious effect of ethanol on hepatic pyridoxal 5′-phosphate metabolism is mediated by acetaldehyde. Inasmuch as acetaldehyde has no effect on the synthesis of pyridoxal phosphate, it has also been postulated that acetaldehyde accelerates pyridoxal phosphate degradation by displacing this coenzyme from binding proteins, which protect it against hydrolysis. To test these hypotheses, studies have been performed with isolated rat hepatocytes, subcellular fractions of rat liver, and human erythrocytes. Ethanol oxidation lowered the pyridoxal phosphate content of isolated liver cells when acetaldehyde oxidation was inhibited by either disulfiram or prior treatment of rats with cyanamide. Additions of 7.5 mM acetaldehyde alone at 40-min intervals to cell suspensions decreased hepatic pyridoxal phosphate content only slightly because acetaldehyde was rapidly metabolized. However, when acetaldehyde oxidation and reduction were inhibited by cyanamide treatment and by 4-methyl-pyrazole and isobutyramide, respectively, a 40% decrease in hepatic pyridoxal phosphate content was observed in 80 min of incubation.

Authors

Lawrence Lumeng

Human erythrocyte hexokinase deficiency. Characterization of a mutant enzyme with abnormal regulatory properties.

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In the erythrocytes of a patient with hereditary nonspherocytic hemolytic anemia, a homozygous expression of hexokinase deficiency was detected. The mutant enzyme was characterized by normal kinetic parameters with respect to its substrates, glucose and MgATP2-, normal pH optimum, normal heat stability at 40 degrees C, but abnormal behavior with respect to its regulation by glucose-1,6-diphosphate and inorganic phosphate, and an altered electrophoretic pattern. Interpretation of the results revealed the presence of two different hexokinases type I in normal human erythrocytes: one enzyme with a high affinity for glucose-1,6-diphosphate, the inhibition of which is regulated by inorganic phosphate; and another enzyme with a lower affinity for the inhibitor, not regulated by inorganic phosphate. The former enzyme was not detectable in the erythrocytes of the patient, whereas the presence of the latter enzyme could be demonstrated.

Authors

G Rijksen, G E Staal

Bone marrow-derived lymphoid cell lines from patients with agammaglobulinemia.

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In vitro infection with Epstein-Barr virus of bone marrow-derived (B) lymphocytes from blood of patients with X-linked or common varied agammaglobulinemia resulted in the establishment of long-term B-lymphoid cell lines (LCL). LCL were established only with B lymphocytes from patients whose B cells failed to respond to in vitro mitogenic stimulation. In contrast, lymphocytes from patients without small B lymphocytes or with B cells that synthesized but failed to secrete Ig failed uniformly to establish LCL. Analysis of the "nonsecretory" B lymphocytes demonstrated the presence of receptors for C3b and C3d as well as surface Ig, but absence of detectable receptors for Epstein-Barr virus. All of the six cell lines that were established formed rosettes with EAC3 but not with sheep erythrocytes. Four of the six LCL were principally surface IgD bearing and did not synthesize detectable Ig for secretion. Two of the cell lines were indistinguishable from cell lines from normal individuals: they had surface IgG, A, M, and D and synthesized and secreted Ig.

Authors

J Schwaber, H Lazarus, F S Rosen

Further Studies on Segmental Sodium Transport in the Rat Kidney during Expansion of the Extracellular Fluid Volume

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The present studies were designed to further investigate the possibility of heterogeneity of nephron function during Ringer loading in the rat, and to determine the specific nephron segment responsible for this finding. As in previous studies from this laboratory with smaller rats (50-125 g), net addition of sodium between late distal tubule and papillary base (6.9 vs. 10.4% of the filtered load, respectively, P <0.005) was found in more mature rats (170-230 g). In contrast, there was net reabsorption of sodium between these two segments in nonvolume-expanded animals, 1.70 vs. 0.45% of the filtered sodium load, P <0.005. Because nephron heterogeneity of sodium transport during extracellular volume expansion is the most likely explanation for these findings, further studies were performed to determine the specific juxtamedullary nephron segment responsible for the net addition pattern between late distal tubule and papillary base in Ringer-loaded animals. First, a comparison was made of sodium delivery to the late proximal tubule of superficial nephrons vs. the delivery rate to the bend of Henle's loop of juxtamedullary nephrons in both hydropenia and Ringer loading. Fractional sodium delivery was quite comparable between the superficial and juxtamedullary nephrons in both hydropenia and Ringer loading although the absolute level was much greater in both groups of nephrons in the Ringer studies. Chlorothiazide (15 mg/kg loading and 15 mg/kg per h) given during Ringer loading markedly increased late distal sodium delivery, 19% of the filtered load, but did not prevent net addition of sodium at the papillary base. In contrast, furosemide (5 mg/kg loading and 5/mg/kg per h) given during Ringer loading completely reversed the segmental pattern, 35.5 and 28.8% at late distal tubule and papillary base, respectively, P <0.005. These studies demonstrate that the net addition of sodium between late distal tubule and papillary base during Ringer loading is not limited to immature rats and that the segmental pattern does not occur in non-volume-expanded animals. Further, the reversal of the net addition pattern with furosemide, but not chlorothiazide, and the comparable proximal nephron delivery rates in Ringer loading suggest that the loop of Henle of juxtamedullary nephrons reabsorbs less sodium than the same portion of superficial nephrons in this setting. A model is proposed to explain this finding.

Authors

Richard W. Osgood, H. John Reineck, Jay H. Stein

ATP Depletion, a Possible Role in the Pathogenesis of Hyperuricemia in Glycogen Storage Disease Type I

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Other investigators have shown that fructose infusion in normal man and rats acutely depletes hepatic ATP and Pi and increases the rate of uric acid formation by the degradation of preformed nucleotides. We postulated that a similar mechanism of ATP depletion might be present in patients with glucose-6-phosphatase deficiency (GSD-I) as a result of ATP consumption during glycogenolysis and resulting excess glycolysis. The postulate was tested by measurement of: (a) hepatic content of ATP, glycogen, phosphorylated sugars, and phosphorylase activities before and after increasing glycolysis by glucagon infusion and (b) plasma urate levels and urate excretion before and after therapy designed to maintain blood glucose levels above 70 mg/dl and thus prevent excess glycogenolysis and glycolysis.

Authors

Harry L. Greene, Frederick A. Wilson, Patrick Hefferan, Annie B. Terry, Jose Roberto Moran, Alfred E. Slonim, Thomas H. Claus, Ian M. Burr

Monovalent cation transport in irreversibly sickled cells.

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Using discontinuous density gradients of Stractan II, we have separated sickle cell blood into discrete subpopulations of reticulocytes, mature discoid cells, and irreversibly sickled cells (ISCs). We have measured active and passive fluxes of monovalent cations in mature discoid cells, ISCs, and normal control cells, also separated upon density gradients. These measurements revealed a decreased active cation transport in ISC-rich populations. However, parallel measurements of Na, K-ATPase activity showed normal ouabain-sensitive ATPase activity in ISCs. Passive permeability to external Rb was also normal in ISCs. The observation of depressed pump activity in intact ISCs, contrasted with normal ATPase activity in ISC membranes, suggests the presence of factors in the intact cell which inhibit the active transport of Na and K in ISCs.

Authors

M R Clark, C E Morrison, S B Shohet

Direct determination of PCO2 in the rat renal cortex.

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The mechanism by which the kidney reabsorbs sodium bicarbonate could be a result of (a) H+ secretion, (b) direct HCO3- reabsorption, or (c) a combination of both processes. Most of the studies which have supported the H+ secretory theory have involved the assumption that tubular fluid and arterial PCO2 were equal. We have utilized a new PCO2 microelectrode to directly determine in situ PCO2 of tubular fluid and stellate vessel blood in the cortex of the rat kidney during control conditions and after alterations in acid-base status. In 21 control rats, proximal tubular fluid PCO2 exceeded systemic arterial PCO2 (deltaCO2) by 25.9 +/- 0.92 mm Hg (P less than 0.001). The values obtained for both distal tubular fluid and stellate vessel blood were not significantly different from proximal tubular PCO2. Evaluation of PCO2 in the proximal tubules of Munich-Wistar rats did not reveal evidence for a declining profile for PCO2 along the length of the nephron. When proximal bicarbonate reabsorption was increased or decreased acutely by alterations in acid-base status, deltaPCO2 changed in paralle. Furthermore, benzolamide administration significantly reduced deltaPCO2. We conclude: (a) that the PCO2 in tubular fluid is significantly greater than systemic arterial PCO2, (b) that there is no tendency for the observed PCO2 to fall along the proximal tubule, (c) the mean PCO2 in the proximal and distal tubules as well as the stellate vessle is not significantly different, thereby rendering the concept of a "diffusion barrier" for CO2 in the proximal tubule unlikely, and (d) the level of renal cortical PCO2 appears to vary directly with the magnitude of bicarbonate reabsorption.

Authors

T D DuBose Jr, L R Pucacco, D W Seldin, N W Carter

Requirements for the solubilization of immune aggregates by complement. The role of the classical pathway.

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In this paper we examine the role of the classical pathway in the complement-mediated solubilization of immune precipitates (CRA). Serum reagents were depleted of the alternative pathway components properdin and factor D. Both depleted reagents lack CRA although they have almost intact hemolytic activity. Also, immune complexes were not solubilized when incubated with high concentrations of the classical pathway components (C1, C4, C2, and C3. We conclude that CRA is not mediated by the classical pathway alone. Activation of the classical pathway by the immune aggregates greatly enhances CRA. The effect of the classical pathway is to deposit C3b on the antigen-antibody lattice and promote the assembly of a lattice-associated, properdin-dependent C3-convertase. Although C3, C4, and properdin were detected on complexes solubilized by serum in the presence of Ca++ and Mg++, only C3 and properdin were found on the complexes when Ca++ had been chelated by ethylene glycol-bis-(beta-aminoethyl ether), N,N'-tetraacetic acid. In both situations the aggregates were capable of converting C5 in the fluid phase. However, no C5 was found on the solubilized complexes. These findings suggest that in contrast to nascent C3b and C4b, nascent C5-9 lacks binding affinity for immune aggregates.

Authors

M Takahashi, S Takahashi, V Brade, V Nussenzweig

Passive Transfer by Cells of Type II Collagen-Induced Arthritis in Rats

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To investigate the role of immunologic hypersensitivity to collagen in the causation of type II collagen-induced arthritis in rats, passive transfer experiments were performed. Wistar/Lewis rats used in these experiments were demonstrated to be histocompatible by prolonged skin graft survival and mixed lymphocyte cultures. Popliteal lymph node weight assays excluded a potential for graft-vs.-host reactivity in this strain. 9 of 32 naive rats developed arthritis after intravenous receipt of pooled spleen and lymph node cells from donors that had been injected intradermally with type II collagen emulsified in incomplete Freund's adjuvant. This passively transferred synovitis was evident clinically as well as histologically. In control cell transfer experiments involving a total of 97 recipients, transfer of arthritis was shown to require viable cells sensitized to type II collagen. These controls included 17 rats receiving cells from unimmunized donors, 20 recipients of cells from donors injected with incomplete Freund's adjuvant alone, and 24 recipients of cells from rats injected with type I collagen in adjuvant. Deliberate addition of solubilized type II collagen to unsensitized cells at the time of transfer or injection of heat-killed sensitized cells also did not cause arthritis in a total of 36 recipients. These latter two control groups indicate that disease transfer was not the result of antigen carry-over. Intravenous injection of sera from arthritic donors was incapable of passively transferring clinical or histologic synovitis in 30 recipients. Thus, these studies directly implicate immunologic sensitivity to the cartilage type of collagen in the etiology of this autoimmune disease.

Authors

David E. Trentham, Roselynn A. Dynesius, John R. David

Multiple forms of human plasma renin substrate.

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The objective of this investigation was to determine whether heterogeneity of plasma renin substrate could be observed in states of steroid excess and various forms of hypertensive disease. In states of stimulated renin substrate production by estrogens or glucocorticoids, multiple forms of renin substrate were apparent when stimulation was excessive. Stimulation of substrate production caused by uremia associated with hypertension showed similar results. None, or only trace quantities of the additional forms of renin substrate were evident in subjects with normal or suppressed levels of plasma renin substrate. The additional forms of renin substrate could be distinguished from the normal form on the basis of cross-reactivity with a specific antiserum to the normal form, electrophoretic mobility, and kinetic rate constants. Differences in rate constants of the various forms of plasma renin substrate may account for the altered rate of the renin reaction associated with several states of hypertension. In plasma of patients with renovascular hypertension, significant quantities of a protein which cross-reacted with the antiserum but could not generate angiotensin I were observed.

Authors

P Eggena, H Hidaka, J D Barrett, M P Sambhi

The effect of complement depletion on lung clearance of bacteria.

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We have investigated the effect of hypocomplementemia on early pulmonary clearance of four species of bacteria. The experiments were performed in an inbred animal model to minimize immunologic variability. Complement was depleted by cobra venom factor, and activity in serum was monitored with a phagocytic assay. Bacterial specific antibodies were examined by an indirect radioimmunoassay, and animals with high levels of activity were excluded from anaysis. 4 h after aerosolization with Streptococcus pneumoniae, complement-depleted animals had cleared only 75% of the initial number of organisms, whereas saline-treated controls cleared 91% (P less than 0.01). Aerosolization with Pseudomonas aeruginosa was followed at 4 h by a twofold greater growth of organisms in the complement-depleted animals (446% of original deposition) as compared to the saline-treated controls (211% of original deposition) (P less than 0.02). Clearance of Klebsiella pneumoniae and Staphylococcus aureus were similar in complement-depleted animals and saline-treated controls. These experiments suggest that hypocomplementemia predisposes to bacterial pneumonia and may explain the high incidence of pulmonary infections in patients having impaired complement activity. Our results further indicate that varying defense mechanisms may be involved with clearing the lung of differing bacterial species.

Authors

G N Gross, S R Rehm, A K Pierce

Myocardial Blood Flow Distribution in Concentric Left Ventricular Hypertrophy

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Regional myocardial blood flow during both control conditions and ischemia-induced vasodilatation was studied in eight chronically instrumented awake dogs. Seven of these animals had coarctation-banding of the ascending aorta performed at 6 wk of age, and the other dog had congenital subvalvular aortic stenosis. The mean left ventricular weight for the group was 157±7.6 g, and the left ventricular body weight ratio was 8.76±0.47 g/kg. None of the animals exhibited signs of congestive heart failure.

Authors

Judith C. Rembert, Leonard H. Kleinman, John M. Fedor, Andrew S. Wechsler, Joseph C. Greenfield Jr.

Sodium, phosphate, glucose, bicarbonate, and alanine interactions in the isolated proximal convoluted tubule of the rabbit kidney.

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Interactions among the transport systems involved with sodium, bicarbonate, glucose, phosphate, and alanine absorption in isolated segments of the rabbit proximal convoluted tubule were examined with radioisotopic techniques to measure glucose, phosphate, and fluid absorption rates. The composition of the perfusate and bath varied from normal, physiological fluids to fluids deficient in a single solute. The deletion of glucose from the perfusate increased the lumen-to-bath flux of phosphate from 5.51 +/- 1.15 to 8.32 +/- 1.34 pmol/mm-min (P less than 0.01). Similar changes occurred when glucose transport was inhibited by phlorizin 10 micron in the perfusate, The deletion of alanine from the perfusate increased the lumen-to-bath flux of phosphate from 6.55 +/- 1.08 to 9.00 +/- 1.30 pmol/mm-min (P less than 0.01) but did not affect glucose transport significantly, 80.1 +/- 10.1 vs. 72.5 +/- 5.4 pmol/mm-min. Replacement of intraluminal sodium with choline, elimination of potassium from the bath, and removal of bicarbonate from the lumen and bath each reduced glucose, phosphate, and fluid absorption. These data indicate that the proximal absorptive processes for glucose and for phosphate include elements that are dependent upon some function of sodium transport. Additionally, the effects on phosphate transport of deleting glucose or alanine occur independent of any changes in net sodium transport and are opposite the effects of deleting bicarbonate. These differences may relate to the observations that the transport of glucose and alanine is electrogenic while that of bicarbonate is not. Regardless of possible mechanisms, the data demonstrate that important changes in the absorption rates of different solutes handled significantly by the proximal convoluted tubule may occur in response to changes in specific components of proximal sodium transport.

Authors

V W Dennis, P C Brazy

Glucose and Alanine Metabolism in Children with Maple Syrup Urine Disease

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In vitro studies have suggested that catabolism of branched chain amino acids is linked with alanine and glutamine formed in, and released from, muscle. To explore this possibility in vivo, static and kinetic studies were performed in three patients with classical, and one patient with partial, branched chain α-ketoacid decarboxylase deficiency (maple syrup urine disease, MSUD) and compared to similar studies in eight age-matched controls. The subjects underwent a 24-30-h fast, and a glucose-alanine flux study using stable isotopes. Basal plasma leucine concentrations were elevated (P <0.001) in patients with MSUD (1,140±125 μM vs. 155±18 μM in controls); and in contrast to the controls, branched chain amino acid concentrations in plasma increased during the fast in the MSUD patients. Basal plasma alanine concentrations were lower (P <0.01) in patients with classical MSUD (153±8 μM vs. 495±27 μM in controls). This discrepancy was maintained throughout the fast despite a decrease in alanine concentrations in both groups. Plasma alanine and leucine concentrations in the patient with partial MSUD were intermediate between those of the controls and the subjects with the classical form of the disease. Circulating ketone bodies and glucoregulatory hormones concentrations were similar in the MSUD and normal subjects during the fast.

Authors

Morey W. Haymond, Ehud Ben-Galim, Karen E. Strobel

Interaction of Diphenylhydantoin (Phenytoin) and Phenobarbital with Hormonal Mediation of Fetal Rat Bone Resorption In Vitro

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Abstract

Chronic administration of high doses of anticonvulsant drugs frequently produces classic osteomalacia with bone histologic changes characteristic of increased parathyroid hormone (PTH) effect in man. However, several reports have documented defects in calcified tissue metabolism suggestive of an end-organ resistance to PTH after chronic anticonvulsant drug therapy. To examine the direct action of anticonvulsant drugs on bone resorption, we investigated the effects of diphenylhydantoin (phenytoin) (DPH) (100-200 μg/ml) and phenobarbital (10-400 μg/ml) on basal and hormonally mediated resorption 5-day cultures of fetal rat forelimb rudiments. In this system both drugs significantly inhibited basal and PTH-stimulated 45Ca and [3H]hydroxyproline release, as well as 1,25-dihydroxyvitamin D3-stimulated 45Ca release. The effects of DPH and phenobarbital were additive, with DPH exhibiting a several-fold more potent inhibitory effect than phenobarbital. Whereas DPH exhibited a striking synergism with the inhibitory effects of human calcitonin (HCT) on PTH-induced resorption, the effect of phenobarbital was merely additive to that of HCT. PTH and PTH plus HCT-induced increases in bone cyclic AMP (cAMP) content were significantly inhibited by DPH but not by phenobarbital. However, in contrast to effects on 45Ca release, DPH inhibition of cAMP generation was not accentuated in the presence of HCT. It is concluded that: (a) both DPH and phenobarbital can directly inhibit basal and hormonally stimulated bone resorption, with DPH being much more potent in this regard; (b) DPH appears to inhibit bone resorption via a cAMP-independent mechanism and has an additional suppressive effect on PTH-induced cAMP generation; and (c) the synergistic interaction of DPH and HCT in inhibiting 45Ca release occurs at a site independent of cAMP generation.

Authors

Theodore J. Hahn, Cheryl R. Scharp, Catherine A. Richardson, Linda R. Halstead, Arnold J. Kahn, Steven L. Teitelbaum

The Influence of Fasting, Diabetes, and Several Pharmacological Agents on the Pathways of Thyroxine Metabolism in Rat Liver

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Abstract

As judged from both paper and column chromatography, slices or homogenates of liver from rats fasted for 48 h displayed a lesser rate of generation of 125I-labeled 3,5,3′-triiodothyronine (T3) from 125I-labeled thyroxine (T4) added to incubation media than did preparations from normal chow-fed animals. A similar defect in the conversion of T4 to T3 in the livers of fasted animals was observed when preparations were incubated with substrate concentrations of T4 so that T3 generation could be assessed by radioimmunoassay. The effect of fasting could be prevented, wholly or in part, by administration of glucose in the drinking water to otherwise fasted animals, and the degree of prevention appeared to be proportional to the concentration of glucose employed. Diminished generation of T3 from T4 was similarly evident in the livers of animals with streptozotocin-induced diabetes mellitus, and this defect was overcome by the provision of insulin in vivo, but not in vitro. Decreased formation of T3 from T4 was also observed in preparations of liver from animals given dexamethasone, amiodarone, and propylthiouracil. In no case could these effects on the net formation of T3 from T4 be explained by effects of the experimental conditions on the degradation of the T3 generated, as judged from the rate of degradation of exogenous 125I-T3 measured in parallel incubates.

Authors

Alan Balsam, Sidney H. Ingbar, Franklin Sexton

Glucose Intolerance in Uremia: QUANTIFICATION OF PANCREATIC BETA CELL SENSITIVITY TO GLUCOSE AND TISSUE SENSITIVITY TO INSULIN

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Abstract

The relative contributions of impaired insulin secretion and of tissue insensitivity to insulin to the carbohydrate intolerance of uremia were investigated in 10 chronically uremic subjects. Two types of glucose-clamp experiments were performed in each patient before and after 10 wk of thrice weekly hemodialysis. In both types the blood glucose concentration was maintained at a constant level by the periodic adjustment of a variable glucose infusion with a negative feedback formula.

Authors

Ralph A. Defronzo, Jordan D. Tobin, John W. Rowe, Reubin Andres

Responsiveness of neoplastic and hyperplastic parathyroid tissues to calcium in vitro.

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Abstract

Secretory and biosynthetic responses of adenomatous, carcinomatous, and hyperplastic parathyroid tissues to variable concentrations of extracellular calcium were assessed in vitro. Tissues, obtained at the time of parathyroidectomy, were incubated for 4 h in media containing radioactive amino acids and varying (0.5-5.0 mM) concentrations of calcium. Amounts of newly synthesized and total parathyroid hormone and proparathyroid hormone in extracts of tissues and media were measured by polyacrylamide gel electrophoresis and by radioimmunoassay, respectively. All tissues studied (six adenomas, two specimens of chief-cell hyperplasia, one carcinoma, and normal bovine and human glands) responded to changes in calcium concentrations; decreasing concentrations of calcium stimulated release and decreased tissue storage of hormone. Six of the abnormal tissues required greater than normal concentrations of calcium (1.8-2.4 mM for 50% of effect) to elicit secretory responses comparable with those of normal glands (1.4 mM). Maximum effects of calcium on release of hormone varied from 2- to 10-fold among different tissues. Release of some hormone persisted even in concentrations of calcium as high as 5.0 mM. Relative amounts of hormone released from and retained in the tissues varied greatly among the tissues, as did the absolute amounts of hormone produced; newly synthesized, labeled hormone ranged between 0.6 and 12% of total labeled protein, and immunoreactive hormone ranged between 0.015 and 0.9% of total tissue protein. Effects of calcium on hormone biosynthesis, as determined by analyses of amounts of proparathyroid hormone in the tissues, were variable among tissues and in many cases were negligible. These results indicate that neoplastic and hyperplastic parathyroid tissues retain secretory responsiveness to changes in extracellular concentrations of calcium. Responses, however, are highly variable among different tissues, and in many instances are abnormal, inasmuch as greater than normal concentrations of calcium are required to alter release and synthesis of hormone. A combination of both increased mass of glandular tissue and abnormal regulations of hormone secretion appear to contribute to the hypersecretion of hyperparathyroidism.

Authors

J F Habener

The Roles of Intracellular and Extracellular Ca⁺⁺ in Glucose-Stimulated Biphasic Insulin Release by Rat Islets

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Abstract

Verapamil, an agent known rapidly to block calcium uptake into islets of Langerhans, has been used to study the roles of intra- and extracellular calcium in the two phases of glucose-induced insulin release. Rates of calcium uptake and insulin release during the first phase were measured simultaneously over 5 min in rat islets after maintenance in tissue culture for 2 days. Rates of 45Ca++ efflux and insulin release during the first and second phases were also measured simultaneously under perifusion conditions. For this, islets were loaded with 45Ca++ during the entire maintenance period to complete isotopic equilibrium. Under static incubation conditions 5 μM Verapamil had no effect upon Ca++ uptake or insulin release in the presence of 2.8 mM glucose. By contrast, glucose-stimulated calcium influx was totally abolished without there being any significant effect upon first phase insulin release. Thus first phase insulin release is independent of increased uptake of extracellular calcium. The lack of effect of 5 μM Verapamil blockade on first phase insulin release was confirmed, under perifusion conditions, and was in marked contrast to the observed 55% inhibition of second phase release. 45Ca++ efflux was inhibited during both phases of the insulin release response.

Authors

Claes B. Wollheim, Masatoshi Kikuchi, Albert E. Renold, Geoffrey W. G. Sharp

Effects of Alpha Adrenergic Blockade upon Coronary Hemodynamics

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Abstract

The effect of alpha adrenergic block-ade on coronary blood flow regulation at rest was studied in 11 normally innervated patients and 8 cardiac allograft recipients by measuring arterial pressure and coronary sinus blood flow by thermodilution before and after alpha adrenergic blockade with phentolamine. Coronary vascular resistance was calculated by using coronary sinus blood flow and mean arterial pressure, and metabolic demand was estimated by the product of systolic arterial pressure and heart rate. In addition, the coronary sinus blood flow response to tachycardia was examined in 9 innervated patients and 12 denervated patients, with measurements repeated after phentolamine in 8 of the 9 innvervated patients and 6 of the 12 denervated patients. There was a 7.3±4.4% increase in coronary sinus blood flow in the innervated patients in response to alpha blockade, whereas the transplanted patients had an 8.2±1.8% fall in coronary sinus blood flow, despite equivalent changes in rate pressure product. The innervated patients also demonstrated a significantly greater increase in coronary sinus blood flow than did the transplanted patients during the first 5 s of an abrupt increase in heart rate (26±4 vs. 8±2.5 ml/min, P <0.001). This early response was blunted after alpha adrenergic blockade. We conclude that there is basal alpha adrenergic tone present on the coronary vasculature in man that is withdrawn by a sudden increase in heart rate.

Authors

Arthur E. Orlick, Donald R. Ricci, Edwin L. Alderman, Edward B. Stinson, Donald C. Harrison

The Metabolic Response to Hypocaloric Protein Diets in Obese Man

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Abstract

Exogenous protein in the absence of other calories can cause protein-sparing, but the mechanisms involved are controversial. It has been postulated that low insulin and high fat-derived substrate levels are necessary and sufficient conditions for such protein-sparing. We therefore established such conditions with differing protocols of protein input to define the role of protein input in mediating the response. Three groups of obese, nondiabetic subjects received the following diets: (1) 82.5±1.0 g protein/day (400 cal/day) for 21 days, n = 7; (2) the same, but as a refeeding diet for 7 days after 21-28 days of total fasts, n = 7; and (3) commencing with the same input, but with daily stepwise decrements over 14 days to 19.4±2.2 g/day, then maintained an additional 7 days, n = 4. Diet 3 gave approximately the amount and pattern of protein lost during total fasting.

Authors

Errol B. Marliss, Frederick T. Murray, Azima F. Nakhooda

Aminoglycoside-Inactivating Enzymes in Clinical Isolates of Streptococcus Faecalis: AN EXPLANATION FOR RESISTANCE TO ANTIBIOTIC SYNERGISM

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Abstract

Clinical isolates of enterococci (Streptococcus faecalis) with high-level resistance to both streptomycin and kanamycin (minimal inhibitory concentration >2,000 μg/ml), and resistant to synergism with penicillin and streptomycin or kanamycin were examined for aminoglycoside-inactivating enzymes. All of the 10 strains studied had streptomycin adenylyltransferase and neomycin phosphotransferase activities; the latter enzyme phosphorylated amikacin as well as its normal substrates, such as kanamycin. Substrate profiles of the neomycin phosphotransferase activity suggested that phosphorylation occurred at the 3′-hydroxyl position, i.e., aminoglycoside 3′-phosphotransferase. A transconjugant strain, which acquired high-level aminoglycoside resistance and resistance to antibiotic synergism after mating with a resistant clinical isolate, also acquired both enzyme activities. Quantitative phosphorylation of amikacin in vitro by a sonicate of the transconjugant strain inactivated the antibiotic, as measured by bioassay, and the phosphorylated drug failed to produce synergism when combined with penicillin against a strain sensitive to penicillin-amikacin synergism.

Authors

Donald J. Krogstad, Thomas R. Korfhagen, Robert C. Moellering Jr., Christine Wennersten, Morton N. Swartz

Glucose Disposal during Insulinopenia in Somatostatin-Treated Dogs: THE ROLES OF GLUCOSE AND GLUCAGON

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Abstract

The first aim of this study was to determine whether the plasma glucose level can regulate hepatic glucose balance in vivo independent of its effects on insulin and glucagon secretion. To accomplish this, glucose was infused into conscious dogs whose basal insulin and glucagon secretion had been replaced by exogenous intraportal insulin and glucagon infusion after somatostatin inhibition of endogenous pancreatic hormone release. The acute induction of hyperglycemia (mean increment of 121 mg/dl) in the presence of basal levels of insulin (7±1 μU/ml) and glucagon (76±3 pg/ml) resulted in a 56% decrease in net hepatic glucose production but did not cause net hepatic glucose uptake.

Authors

Gerald I. Shulman, John E. Liljenquist, Phillip E. Williams, William W. Lacy, Alan D. Cherrington

Analysis of linkage between the major histocompatibility system and juvenile, insulin-dependent diabetes in multiplex families. Reanalysis of data.

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Abstract

Linkage analysis between the major histocompatibility system (HLA) and juvenile, insulin-dependent diabetes, assuming an autosomal recessive mode and 50% penetrance was performed on 21 juvenile, insulin-dependent diabetic multiplex families (two or more diabetics per sibship) with phenotypically normal parents. The total lod score was the highest (3.98) at a recombination fraction of 13%. For a penetrance of 100%, the highest total lod score was 2.92 at a recombination fraction of 18%. These results are compatible with the existence of linkage between an autosomal recessive diabetic gene with 50% penetrance and the HLA in some of the families studied. Our ascertainment strategy would be expected to increase the likelihood of selecting for genetically homogenous diabetes and against sporadic forms of the disease. Thus, our findings may apply only to a small proportion of all cases of juvenile, insulin-dependent diabetes.

Authors

J Barbosa, M M Chern, H Noreen, V E Anderson

Factor VIII/von Willebrand factor protein. Galactose a cryptic determinant of von Willebrand factor activity.

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Abstract

The normal Factor VIII/von Willebrand factor protein has the ability to agglutinate or aggregate normal platelets in the presence of ristocetin (von Willebrand factor activity). Removal of greater than 95% of the sialic acid from this protein by neuraminidase did not affect the von Willebrand factor or procoagulant activity. However, oxidation of the penultimate galactose of the asialo Factor VIII/von Willebrand factor protein with galactose oxidase resulted in a progressive loss of von Willebrand factor activity with no effect on procoagulant activity. Reduction of the 6-aldehydo intermediate by potassium borohydride caused full regeneration of von Willebrand factor activity. These studies confirm the identification of the intact penultimate galactose moiety as a critical determinant of von Willebrand factor activity.

Authors

H R Gralnick