Plasma high density lipoproteins. Metabolism and relationship to atherogenesis.

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Authors

A R Tall

Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors.

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Propagation of venous thrombi or rethrombosis after coronary thrombolytic therapy can occur despite heparin administration. To explore potential mechanisms, we set out to determine whether clot-bound thrombin is relatively protected from inhibition by heparin-antithrombin III but susceptible to inactivation by antithrombin III-independent inhibitors. Using plasma fibrinopeptide A (FPA) levels as an index of thrombin activity, we compared the ability of thrombin inhibitors to block FPA release mediated by fluid-phase thrombin with their activity against the clot-bound enzyme. Incubation of thrombin with citrated plasma results in concentration-dependent FPA generation, which reaches a plateau within minutes. In contrast, there is progressive FPA generation when fibrin clots are incubated with citrated plasma. Heparin, hirudin, hirudin dodecapeptide (hirugen), and D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK) produce concentration-dependent inhibition of FPA release mediated by fluid-phase thrombin. However, heparin is much less effective at inhibiting thrombin bound to fibrin because a 20-fold higher concentration is necessary to block 70% of the activity of the clot-bound enzyme than is required for equivalent inhibition of fluid-phase thrombin (2.0 and 0.1 U/ml, respectively). In contrast, hirugen and PPACK are equally effective inhibitors of fluid- and solid-phase thrombin, while hirudin is only 50% as effective against the clot-bound enzyme. None of the inhibitors displace bound 125I-labeled thrombin from the clot. These studies indicate that (a) clot-bound thrombin is relatively protected from inhibition by heparin, possibly because the heparin binding site on thrombin is inaccessible when the enzyme is bound to fibrin, and (b) clot-bound thrombin is susceptible to inactivation by antithrombin III-independent inhibitors because the sites of their interaction are not masked by thrombin binding to fibrin. For these reasons, antithrombin III-independent inhibitors may be more effective than heparin in certain clinical settings.

Authors

J I Weitz, M Hudoba, D Massel, J Maraganore, J Hirsh

HLA antigens in Japanese patients with myasthenia gravis.

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HLA antigens in 104 Japanese patients and 41 families with myasthenia gravis (MG) were investigated. The frequencies of DR9 and DRw13 were significantly increased in the patients who developed MG before 3 yr of age. The DQw3 antigen was positive for all the patients that developed MG before 15 yr with only one exception. All the examined cases that developed MG before 3 yr (including this DQw3 negative patient) had the same DQA and DQB DNA restriction fragments. These HLA frequencies decreased as the age of onset increased, and no significant association was observed in adult-onset MG. No patients had B8, DR3, and DQw2. The relative risk was higher for the DR9/DRw13 heterozygotes (37.4) than for DR9 (16.4) or DRw13 (7.1) in the childhood-onset MG. Statistical analysis suggested that DR9 and DRw13 (or DQw1 and DQw3) act synergistically in the disease development. Family study revealed diverse DR9 haplotypes. The most frequent DRw13 haplotype was Bw44-BFF-C4A3B1-DRw13-DQw1, which may be evolutionarily related to the caucasian B8-DR3-DQw2 haplotype. These results showed that MG in early childhood in Japanese individuals is genetically different from that in adulthood and that in caucasians.

Authors

K Matsuki, T Juji, K Tokunaga, M Takamizawa, H Maeda, M Soda, Y Nomura, M Segawa

Aspirin potentiates prestimulated acid secretion and mobilizes intracellular calcium in rabbit parietal cells.

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The effects of aspirin on gastric acid secretion were studied in isolated rabbit parietal cells (PC). Aspirin (10(-5) M) potentiated histamine-, dibutyryl cyclic AMP (dbcAMP)-, forskolin- and 3-isobutyl-1-methylxanthine-stimulated acid secretion without affecting basal acid secretion. Augmentation of secretagogue-stimulated acid secretion by aspirin was dependent on calcium (Ca2+) since potentiation was blocked by removal of extracellular Ca2+ ([Ca2+]o) or addition of the calcium antagonist lanthanum chloride. Using the Ca2+ probe fura-2, aspirin (10(-6) - 2 X 10(-5) M) rapidly increased intracellular free Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. The source of released Ca2+ was intracellular as demonstrated by depletion of intracellular Ca2+ and [Ca2+]o with EGTA washing. Aspirin did not affect several other signal transduction sites involved in stimulus-secretion coupling, including the H2 receptor, intracellular cyclic AMP (cAMP), inositol 1,4,5, triphosphate (IP3) and H+,K(+)-ATPase. Aspirin decreased PC prostaglandin E2 (PGE2) content by 98%. Exogenous dimethyl PGE2 (dmPGE2) inhibited both histamine-stimulated acid secretion and its enhancement by aspirin. In contrast, dmPGE2 abolished aspirin-induced potentiation of dbcAMP-stimulated acid secretion by augmenting the dbcAMP-stimulated response. These results indicate that aspirin acts at a site beyond the adenylate cyclase/cAMP system and before the proton pump, presumably by releasing Ca2+ from an IP3-independent intracellular storage pool and by inhibiting PGE2 generation.

Authors

R A Levine, J Nandi, R L King

Identification of activated T cell receptor gamma delta lymphocytes in the liver of tumor-bearing hosts.

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T cell receptor (TcR)gamma delta cells are known to be a minor population of T lymphocytes in the blood (less than 10%) and other peripheral lymphoid organs in healthy donors. We demonstrated here that a large proportion of TcR gamma delta cells, i.e., up to 30% of mononuclear cells (MNC) were detectable in the liver, but not other lymphoid organs of cancer patients. More importantly, the majority of such TcR gamma delta cells (greater than 70%) were shown to be lymphoblastic by electron microscopy. An activation marker of T lymphocytes, Leu-19 (CD56) was also highly expressed on the hepatic TcR gamma delta cells. The possibility of hepatic TcR gamma delta cells being activated was further examined in mice. C3H/He mice injected with syngeneic tumor cells were demonstrated to have an increased number of liver MNC; such MNC showed an ability to proliferate in vitro. These mice eventually had a considerable proportion of TcR gamma delta cells in the liver, showing activation markers, the Ia and LFA-1 antigens. These results suggest that the liver may be an important organ for activation and probably expansion of TcR gamma delta cells especially in tumor bearing hosts.

Authors

S Seki, T Abo, T Masuda, T Ohteki, A Kanno, K Takeda, H Rikiishi, H Nagura, K Kumagai

Soluble Fc gamma receptor III in human plasma originates from release by neutrophils.

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FcRIII (the CD16-antigen), a low affinity receptor for IgG, is expressed by neutrophils, natural killer lymphocytes, and macrophages. We have developed a sensitive radioimmunoassay to quantify FcRIII. A soluble form of FcRIII was identified in human plasma. Immunoprecipitation of FcRIII from plasma showed that the plasma form of FcRIII has an identical electrophoretic mobility as the FcRIII expressed by neutrophils. Moreover, the plasma form of FcRIII exhibited the same polymorphism as does the neutrophil FcRIII. The neutrophil expresses the phosphatidylinositol-linked form of FcRIII, encoded by the gene FcRIII-1. Because it is not known whether this gene is also active in nonhematopoietic cells, we analyzed patients with an acquired clonal disorder of their hematopoietic cells, paroxysmal nocturnal hemoglobinuria (PNH). PNH patients appeared to have a strongly reduced expression of FcRIII on their neutrophils. The concentration of FcRIII in the plasma of these patients was also reduced, indicating that plasma FcRIII originates from neutrophils. A patient deficient in FcRIII-1 but with a normal expression of FcRIII-2 had no soluble FcRIII in her plasma, also indicating that plasma FcRIII originates from neutrophils. The electrophoretic mobility of the protein backbone of plasma FcRIII and FcRIII released by activated neutrophils was identical, whereas deglycosylated FcRIII obtained from a lysate of neutrophils migrated slower. This indicates that plasma FcRIII originates from activation-induced release by neutrophils. Stimulation of neutrophils or neutrophil cytoplasts (closed membrane vesicles filled with cytoplasm) with low concentrations of FMLP (10(-9)-10(-8) M) or phorbol myristate acetate (1-10 ng/ml) induced a dose-dependent release of FcRIII. The plasma concentration of FcRIII was relatively constant (range 40-280% of the mean). Soluble FcRIII was also detected in inflamed joint fluids of arthritis patients, suggesting that FcRIII is also released by activated neutrophils in vivo.

Authors

T W Huizinga, M de Haas, M Kleijer, J H Nuijens, D Roos, A E von dem Borne

Role of alpha adrenoceptors in opossum internal anal sphincter.

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The purpose of the present investigation was to examine the role of alpha adrenoceptors in the internal anal sphincter (IAS). Studies wer performed on alpha-chloralose anesthetized opossums. Resting pressure in the IAS (IASP) was recorded using low compliant continuously perfused catheters. The effects of the alpha-1 adrenoceptor agonist phenylephrine and alpha-2 adrenoceptor agonist clonidine and their corresponding selective antagonists, prazosin and yohimbine, respectively, were examined on the resting IASP, and on rectal balloon distension (RBD)-mediated IAS relaxation. Phenylephrine caused a rise in the IASP that was blocked by prazosin and not by yohimbine. Phenylephrine had no effect on IAS relaxation caused by RBD. Clonidine on the other hand caused significant suppression of IAS relaxation in response to RBD, but caused minimal changes in the resting IASP. The suppression of IAS relaxation by clonidine was selectively antagonized by yohimbine but not by prazosin. From these studies we conclude that alpha-2 adrenoceptors exert important neuromodulatory influences on rectoanal inhibitory reflex, while alpha-1 adrenoceptors may exert modulatory effects on the resting IAS tone.

Authors

S Yamato, S Rattan

Acute metabolic acidosis enhances circulating parathyroid hormone, which contributes to the renal response against acidosis in the rat.

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Abstract

Acute PTH administration enhances final urine acidification in the rat. HCl was infused during 3 h in rats to determine the parathyroid and renal responses to acute metabolic acidosis. Serum immunoreactive PTH (iPTH) concentration significantly increased and nephrogenous adenosine 3H,5H-cyclic monophosphate tended to increase during HCl loading in intact and adrenalectomized (ADX) rats despite significant increments in plasma ionized calcium. Strong linear relationships existed between serum iPTH concentration and arterial bicarbonate or proton concentration (P less than 0.0001). Serum iPth concentration and NcAMP remained stable in intact time-control rats and decreased in CaCl2-infused, nonacidotic animals. Urinary acidification was markedly reduced in parathyroidectomized (PTX) as compared with intact rats during both basal and acidosis states; human PTH-(1-34) infusion in PTX rats restored in a dose-dependent manner the ability of the kidney to acidify the urine and excrete net acid. Acidosis-induced increase in urinary net acid excretion was observed in intact, PTX, and ADX, but not in ADX-thyroparathyroidectomized rats. We conclude that (a) acute metabolic acidosis enhances circulating PTH activity, and (b) PTH markedly contributes to the renal response against acute metabolic acidosis by enhancing urinary acidification.

Authors

M Bichara, O Mercier, P Borensztein, M Paillard

Paradoxical expression of adenosine deaminase in T cells cultured from a patient with adenosine deaminase deficiency and combine immunodeficiency.

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T lymphocytes cultured from a patient (T.D.) with adenosine deaminase (ADA) deficiency expressed ADA activity in the normal range, inconsistent with her severe immunodeficiency, metabolic abnormalities, and with the absence of ADA activity in her B lymphocytes and other nucleated hematopoietic cells. ADA from T.D. T cells had normal Km, heat stability, and sensitivity to ADA inhibitors. Examination of HLA phenotype and polymorphic DNA loci indicated that T.D. was neither chimeric nor a genetic mosaic. Amplified and subcloned ADA cDNA from ADA+ T.D. T cells was shown by allele-specific oligonucleotide hybridization to possess the same mutations (Arg101----Trp, Arg211----His) previously found in the ADA-T.D. B cell line GM 2606 (Akeson, A. L., D. A. Wiginton, M. R. Dusing, J. C. States, and J. J. Hutton. 1988. J. Biol. Chem. 263:16291-16296). Our findings suggest that one of these mutant alleles can be expressed selectively in IL-2-dependent T cells as stable, active enzyme. Cultured T cells from other patients with the Arg211----His mutation did not express significant ADA activity, while some B cell lines from a patient with an Arg101----Gln mutation have been found to express normal ADA activity. We speculate that Arg101 may be at a site that determines degradation of ADA by a protease that is under negative control by IL-2 in T cells, and is variably expressed in B cells. Il-2 might increase ADA expression in T cells of patients who possess mutations of Arg101.

Authors

F X Arredondo-Vega, J Kurtzberg, S Chaffee, I Santisteban, E Reisner, M S Povey, M S Hershfield

Elevated expression of transforming growth factor-beta and proteoglycan production in experimental glomerulonephritis. Possible role in expansion of the mesangial extracellular matrix.

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Glomerular accumulation of extracellular matrix is a prominent feature of progressive glomerulonephritis. Previously, we have shown that transforming growth factor-beta (TGF-beta) is unique among growth factors in regulating the production of the proteoglycans biglycan and decorin by glomerular mesangial cells in vitro. We now provide evidence of an elevated expression of TGF-beta, proteoglycans, and fibronectin in glomerulonephritis induced in rats by injection of anti-thymocyte serum (ATS). Glomeruli were cultured from rat kidneys at 1, 4, 7, 14, and 28 d after ATS administration. Increased proteoglycan synthesis was detected beginning on day 4, which peaked at a 4,900% increase compared with control on day 7, and returned toward control levels by day 28. The increased proteoglycan synthesis by cultured nephritic glomeruli, as well as that of fibronectin, were greatly reduced by addition of antiserum raised against a synthetic peptide from TGF-beta. Conditioned media from ATS glomerular cultures, when added to normal cultured mesangial cells, induced elevated proteoglycan synthesis that also peaked on day 7 and that mimicked the response to added exogenous TGF-beta. The stimulatory activity of the conditioned media was blocked by addition of TGF-beta antiserum. Prior addition of the immunizing peptide to the antiserum abolished the blocking effect. The main induced proteoglycans were identified as biglycan and decorin by immunoprecipitation with antiserum made against synthetic peptides from the proteoglycan core proteins. Glomerular histology showed mesangial matrix expansion in a time course that roughly paralleled both the elevated proteoglycan synthesis by the ATS glomeruli and the ability of the conditioned media from these glomeruli to induce proteoglycan synthesis. At the same time there was an increased expression of TGF-beta mRNA and TGF-beta protein in the glomeruli. These results suggest a central role for TGF-beta in the accumulation of pathological extracellular matrix in glomerulonephritis.

Authors

S Okuda, L R Languino, E Ruoslahti, W A Border

Role of lipoprotein lipase in the regulation of high density lipoprotein apolipoprotein metabolism. Studies in normal and lipoprotein lipase-inhibited monkeys.

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Mechanisms that might be responsible for the low levels of high density lipoprotein (HDL) associated with hypertriglyceridemia were studied in an animal model. Specific monoclonal antibodies were infused into female cynomolgus monkeys to inhibit lipoprotein lipase (LPL), the rate-limiting enzyme for triglyceride catabolism. LPL inhibition produced marked and sustained hypertriglyceridemia, with plasma triglyceride levels of 633-1240 mg/dl. HDL protein and cholesterol and plasma apolipoprotein (apo) AI levels decreased; HDL triglyceride (TG) levels increased. The fractional catabolic rate of homologous monkey HDL apolipoproteins injected into LPL-inhibited animals (n = 7) was more than double that of normal animals (0.094 +/- 0.010 vs. 0.037 +/- 0.001 pools of HDL protein removed per hour, average +/- SEM). The fractional catabolic rate of low density lipoprotein apolipoprotein did not differ between the two groups of animals. Using HDL apolipoproteins labeled with tyramine-cellobiose, the tissues responsible for this increased HDL apolipoprotein catabolism were explored. A greater proportion of HDL apolipoprotein degradation occurred in the kidneys of hypertriglyceridemic than normal animals; the proportions in liver were the same in normal and LPL-inhibited monkeys. Hypertriglyceridemia due to LPL deficiency is associated with low levels of circulating HDL cholesterol and apo AI. This is due, in part, to increased fractional catabolism of apo AI. Our studies suggest that variations in the rate of LPL-mediated lipolysis of TG-rich lipoproteins may lead to differences in HDL apolipoprotein fractional catabolic rate.

Authors

I J Goldberg, W S Blaner, T M Vanni, M Moukides, R Ramakrishnan

Elevated von Willebrand factor antigen is an early plasma predictor of acute lung injury in nonpulmonary sepsis syndrome.

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Abstract

In this prospective study of 45 patients, we tested the hypothesis that markedly elevated levels of plasma von Willebrand antigen (vWf-Ag) a marker of endothelial cell injury, might predict the development of acute lung injury in patients with nonpulmonary sepsis syndrome. Acute lung injury was quantified on a four-point scoring system. At the time of entry into the study, none of the 45 patients had evidence of lung injury. Subsequently, 15 patients developed lung injury and 30 patients did not develop lung injury. The mean plasma vWf-Ag level was markedly elevated in the 15 patients who developed lung injury compared with the 30 patients who did not develop lung injury (588 +/- 204 vs. 338 +/- 196, percentage of control, P less than 0.01). Furthermore, a plasma vWf-Ag level greater than or equal to 450 was 87% sensitive and 77% specific for predicting the development of acute lung injury in the setting of nonpulmonary sepsis. In addition, the combination of a plasma vWf-Ag greater than 450 and nonpulmonary organ failure at the time of entry into the study had a positive predictive value of 80% for acute lung injury. Also, a plasma vWf-Ag level greater than 450 had a positive predictive value of 80% for identifying nonsurvivors. Thus, in patients with nonpulmonary sepsis, an elevated level of plasma vWf-Ag is a useful, early biochemical marker of endothelial injury and it has both predictive and prognostic value.

Authors

D B Rubin, J P Wiener-Kronish, J F Murray, D R Green, J Turner, J M Luce, A B Montgomery, J D Marks, M A Matthay

Abnormal cardiac function in the streptozotocin-diabetic rat. Changes in active and passive properties of the left ventricle.

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To provide an integrated assessment of changes in systolic and diastolic function in diabetic rats, we measured conscious hemodynamics and performed ex vivo analysis of left ventricular passive-elastic properties. Rats given streptozotocin (STZ) 65 mg/kg i.v. (n = 14) were compared with untreated age-matched controls (n = 15) and rats treated with insulin after administration of STZ (n = 11). After 7 d, diabetic rats exhibited decreases in heart rate and peak developed left ventricular (LV) pressure during aortic occlusion. After 26 d of diabetes there were significant decreases in resting LV systolic pressure, developed pressure, and maximal +dP/dt, whereas LV end-diastolic pressure increased and the time constant of LV relaxation was prolonged. The passive LV pressure-volume relationship was progressively shifted away from the pressure axis, and the overall chamber stiffness constant was decreased. However, "operating chamber stiffness" calculated at end-diastolic pressure was increased at 7 d, and unchanged at 26 d. LV cavity/wall volume and end-diastolic volume were increased after 26 d of diabetes. Myocardial stiffness was unchanged at both time intervals. All of the above abnormalities were reversed by the administration of insulin. We conclude that the hemodynamic and passive-elastic changes that occur in diabetic rats represent an early dilated cardiomyopathy which is reversible with insulin.

Authors

S E Litwin, T E Raya, P G Anderson, S Daugherty, S Goldman

Failure of substrate-induced gluconeogenesis to increase overall glucose appearance in normal humans. Demonstration of hepatic autoregulation without a change in plasma glucose concentration.

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It has been proposed that increased supply of gluconeogenic precursors may be largely responsible for the increased gluconeogenesis which contributes to fasting hyperglycemia in non-insulin-dependent diabetes mellitus (NIDDM). Therefore, to test the hypothesis that an increase in gluconeogenic substrate supply per se could increase hepatic glucose output sufficiently to cause fasting hyperglycemia, we infused normal volunteers with sodium lactate at a rate approximately double the rate of appearance observed in NIDDM while clamping plasma insulin, glucagon, and growth hormone at basal levels. In control experiments, sodium bicarbonate was infused instead of sodium lactate at equimolar rates. In both experiments, [6-3H]-glucose was infused to measure glucose appearance and either [U-14C]lactate or [U-14C]alanine was infused to measure the rates of appearance and conversion of these substrates into plasma glucose. Plasma insulin, glucagon, growth hormone, C-peptide, and glycerol concentrations, and blood bicarbonate and pH in control and lactate infusion experiments were not significantly different. Infusion of lactate increased plasma lactate and alanine to 4.48 +/- 3 mM and 610 +/- 33 microM, respectively, from baseline values of 1.6 +/- 0.2 mM and 431 +/- 28 microM, both P less than 0.01; lactate and alanine rates of appearance increased to 38 +/- 1.0 and 8.0 +/- 0.3 mumol/kg per min (P less than 0.01 versus basal rates of 14.4 +/- 0.4 and 5.0 +/- 0.5 mumol/kg per min, respectively). With correction for Krebs cycle carbon exchange, lactate incorporation into plasma glucose increased nearly threefold to 10.4 mumol/kg per min and accounted for about 50% of overall glucose appearance. Alanine incorporation into plasma glucose increased more than twofold. Despite this marked increase in gluconeogenesis, neither overall hepatic glucose output nor plasma glucose increased and each was not significantly different from values observed in control experiments (10.8 +/- 0.5 vs. 10.8 +/- 0.5 mumol/kg per min and 5.4 +/- 0.4 vs. 5.3 +/- 0.3 mM, respectively). We, therefore, conclude that in normal humans there is an autoregulatory process independent of changes in plasma glucose and glucoregulatory hormone concentrations which prevents a substrate-induced increase in gluconeogenesis from increasing overall hepatic glucose output; since this process cannot be explained on the basis of inhibition of gluconeogenesis from other substrates, it probably involves diminution of glycogenolysis. A defect in this process could explain at least in part the increased hepatic glucose output found in NIDDM.

Authors

T Jenssen, N Nurjhan, A Consoli, J E Gerich

Enhancement of electrogenic Na+ transport across rat inner medullary collecting duct by glucocorticoid and by mineralocorticoid hormones.

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We have investigated the effect of steroid hormones on Na+ transport by rat renal inner medullary collecting duct (IMCD) cells. These cells, grown on permeable supports in primary culture, grow to confluence and develop a transmonolayer voltage oriented such that the apical surface is negative with respect to the basal surface. The results of these experiments demonstrate that this voltage is predominantly (or exclusively) the result of electrogenic Na+ absorption. Na+ transport can be stimulated two- to fourfold by exposure to either dexamethasone or aldosterone (100 nM). Experiments using specific antagonists of the glucocorticoid and mineralocorticoid receptors indicate that activation of either receptor stimulates electrogenic Na+ transport; electroneutral Na+ transport is undetectable. Two other features of the IMCD emerge from these studies. (a) These cells appear to have the capacity to metabolize the naturally occurring glucocorticoid hormone corticosterone. (b) The capacity for K+ secretion is minimal and steroid hormones do not induce or stimulate conductive K+ secretion as they do in the cortical collecting duct.

Authors

R F Husted, J R Laplace, J B Stokes

Effects of chronic growth hormone hypersecretion on intrinsic contractility, energetics, isomyosin pattern, and myosin adenosine triphosphatase activity of rat left ventricle.

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We studied papillary muscle mechanics and energetics, myosin phenotype, and ATPase activities in left ventricles from rats bearing a growth hormone (GH)--secreting tumor. 18 wk after tumor induction, animals exhibited a dramatic increase in body weight (+101% vs. controls) but no change in the ventricular weight/body weight ratio. The maximum isometric force of papillary muscles normalized per cross-sectional area rose markedly (+42%, P less than 0.05 vs. controls), whereas the maximum unloaded shortening velocity did not change. This was observed despite a marked isomyosin shift towards V3 (32 +/- 5% vs. 8 +/- 2% in controls, P less than 0.001). Increased curvature of the force-velocity relationship (+64%, P less than 0.05 vs. controls) indicated that the muscles contracted more economically, suggesting the involvement of V3 myosin. Total calcium- and actin-activated myosin ATPase activities assayed on quickly frozen left ventricular sections were similar in tumor-bearing rats and in controls. After alkaline preincubation, these activities only decreased in tumor-bearing rats, demonstrating that V3 enzymatic sites were involved in total ATPase activity. These data demonstrate that chronic GH hypersecretion in the rat leads to a unique pattern of myocardial adaptation which allows the muscle to improve its contractile performance and economy simultaneously, thanks to myosin phenoconversion and an increase in the number of active enzymatic sites.

Authors

J Timsit, B Riou, J Bertherat, C Wisnewsky, N S Kato, A S Weisberg, J Lubetzki, Y Lecarpentier, S Winegrad, J J Mercadier

Molecular analysis of insertion/deletion mutations in protein 4.1 in elliptocytosis. I. Biochemical identification of rearrangements in the spectrin/actin binding domain and functional characterizations.

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Protein 4.1 (80 kD) interacts with spectrin and short actin filaments to form the erythrocyte membrane skeleton. Mutations of spectrin and protein 4.1 are associated with elliptocytosis or spherocytosis and anemia of varying severity. We analyzed two mutant protein 4.1 molecules associated with elliptocytosis: a high molecular weight 4.1 (95 kD) associated with mild elliptocytosis without anemia, and a low molecular weight 4.1 (two species at 68 and 65 kD) associated with moderate elliptocytosis and anemia. 4.1(95) was found to contain a approximately 15-kD insertion adjacent to the spectrin/actin binding domain comprised, at least in part, of repeated sequence. 4.1(68/65) was found to lack the entire spectrin-actin binding domain. The mechanical stability of erythrocyte membranes containing 4.1(95) was identical to that of normal membranes, consistent with the presence of an intact spectrin-actin binding domain in protein 4.1. In contrast, membranes containing 4.1(68/65) have markedly reduced mechanical stability as a result of deleting the spectrin-actin binding domain. The mechanical stability of these membranes was improved following reconstitution with normal 4.1. These studies have thus enabled us to establish the importance of the spectrin-actin binding domain in regulating the mechanical stability of the erythrocyte membrane.

Authors

S L Marchesi, J Conboy, P Agre, J T Letsinger, V T Marchesi, D W Speicher, N Mohandas

Molecular analysis of insertion/deletion mutations in protein 4.1 in elliptocytosis. II. Determination of molecular genetic origins of rearrangements.

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Protein 4.1 is an approximately 80-kD structural protein in the membrane skeleton which underlies and supports the erythrocyte plasma membrane. The preceding companion paper presents a biochemical study of two abnormal protein 4.1 species from individuals with the red blood cell disorder, hereditary elliptocytosis. These variants, "protein 4.1(68/65)" and "protein 4.1(95)," have altered molecular weights due to internal deletions and duplications apparently localized around the spectrin-actin binding domain. Here we use polymerase chain reaction (PCR) techniques to clone and sequence the corresponding mutant reticulocyte mRNAs, and correlate the deletion/duplication end points with exon boundaries of the gene. Protein 4.1(68/65) mRNA lacks sequences encoding the functionally important spectrin-actin binding domain due to a 240 nucleotide (nt) deletion spanning the codons for Lys407-Gly486. Protein 4.1(95) mRNA encodes a protein with two spectrin-actin binding domains by virtue of a 369 nt duplication of codons for Lys407-Gln529. These deletions and duplications correspond to gene rearrangements involving three exons encoding 21, 59, and 43 amino acids, respectively. The duplicated 21 amino acid exon in the 4.1(95) gene retains its proper tissue-specific expression pattern, being spliced into reticulocyte 4.1 mRNA and out of lymphocyte 4.1 mRNA.

Authors

J Conboy, S Marchesi, R Kim, P Agre, Y W Kan, N Mohandas

Proenkephalin system in human polymorphonuclear cells. Production and release of a novel 1.0-kD peptide derived from synenkephalin.

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In the hematopoietic system a pluripotent stem cell generates precursors for lymphoid and myeloid lineages. Proenkephalin-derived peptides were previously detected in differentiated lymphoid cells. We have studied whether the proenkephalin system is expressed in a typical differentiated cell of the myeloid lineage, the neutrophil. Human peripheral polymorphonuclear cells contain and release proenkephalin-derived peptides. The opioid portion of proenkephalin (met-enkephalin-containing peptides) was incompletely processed, resulting in the absence of low molecular weight products. The nonopioid synenkephalin (proenkephalin 1-70) molecule was completely processed to a 1.0-kD peptide derived from the COOH-terminal. This molecule was characterized in neutrophils by biochemical and immunocytochemical methods. The chemotactic peptide FMLP and the calcium ionophore A23187 induced the release of the proenkephalin-derived peptides, and this effect was potentiated by cytochalasin B. The materials secreted were similar to those present in the cell, although in the supernatant a higher proportion corresponded to more processed products. The 1.0-kD peptide was detected in human, bovine, and rat neutrophils, but the chromatographic pattern of synenkephalin-derived peptides suggests a differential posttranslational processing among species. These findings demonstrate the existence of the proenkephalin system in human neutrophils and the production and release of a novel 1.0-kD peptide derived from the synenkephalin molecule. The presence of opioid peptides in neutrophils suggests their participation in the inflammatory process, including a local analgesic effect.

Authors

O Vindrola, M R Padrós, A Sterin-Prync, A Ase, S Finkielman, V Nahmod

Inhibition of human colony-forming-unit erythroid by tumor necrosis factor requires accessory cells.

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Recombinant tumor necrosis factor (rTNF) inhibits erythropoiesis in vivo and in vitro. To study the mechanism of this inhibition, the effect of rTNF on highly purified human CFU-erythroid (E) (mean purity 63.5%), which were generated from peripheral blood burst-forming units-erythroid (BFU-E), was compared to its effect on unpurified human marrow CFU-E (mean purity 0.21%). Although growth of colonies from marrow CFU-E was inhibited by rTNF, no significant effect on purified BFU-E-derived CFU-E colony growth was found. Removal of accessory marrow cells by soy bean agglutinin (SBA) ablated the inhibition of marrow CFU-E colonies by rTNF. Inhibition of colony growth was then restored by adding back SBA+ cells, but not by adding T lymphocytes or adherent cells. Conditioned medium prepared from bone marrow mononuclear cells stimulated by rTNF inhibited the growth of colonies from highly purified BFU-E derived CFU-E resistant to direct inhibition by rTNF. These findings indicate that rTNF does not directly inhibit CFU-E, but requires accessory cells to decrease erythropoiesis. These accessory cells reside in the SBA+ cell fraction, but are neither T cells nor adherent cells. Therefore, in order to produce anemia, TNF must induce release or production of a factor that directly inhibits erythroid colony growth.

Authors

R T Means Jr, E N Dessypris, S B Krantz

Effects of altered glucose homeostasis on glucose transporter expression in skeletal muscle of the rat.

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Previous studies have suggested that alteration in the expression of the insulin-regulatable glucose transporter of muscle (GLUT-4 protein) may be an important determinant of insulin action. In the present studies, we have examined GLUT-4 mRNA and protein concentrations in muscle after variations in the metabolic status of the intact animal (i.e., 7 d streptozotocin-induced diabetes, 7 d insulin-induced hypoglycemia, and 3 d fasting). These changes in glucose homeostasis were associated with the following changes in GLUT-4 gene products: a decrease of approximately 30% in both mRNA and protein with diabetes; a 50% increase in mRNA and a 2.4-fold increase in protein with insulin injection; and normal mRNA in spite of a 2.7-fold increase in protein with fasting. Fasted diabetics exhibited an increase of 50% in GLUT-4 mRNA and a 2.4-fold increase in protein relative to fed diabetics. In diabetic and insulin-injected groups, the changes in GLUT-4 protein were similar to changes in mRNA, but in fasting, GLUT-4 protein increased without a concomitant change in mRNA. Overall there was no correlation between muscle concentrations of GLUT-4 protein and mRNA. Muscle GLUT-4 protein concentration tended to correlate with plasma glucose (r = -0.57, P less than 0.001), but not with plasma insulin. These results indicate that (a) chronic changes in glucose homeostasis are associated with changes in expression of GLUT-4 protein in muscle; (b) GLUT-4 protein increased in fasted soleus muscle without change in mRNA, thereby differing from fasted adipocytes in which both GLUT-4 products diminish; and (c) no simple relationship exists between total muscle GLUT-4 protein content and whole-body insulin sensitivity.

Authors

R E Bourey, L Koranyi, D E James, M Mueckler, M A Permutt

Two elliptocytogenic alpha I/74 variants of the spectrin alpha I domain. Spectrin Culoz (GGT----GTT; alpha I 40 Gly----Val) and spectrin Lyon (CTT----TTT; alpha I 43 Leu---Phe).

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Abstract

Spectrin alpha I/74 elliptocytosis results from abnormalities involving the "head" region of spectrin dimer. Increased susceptibility to trypsin enhances cleavage of the alpha spectrin chain, yielding an increased amount of the alpha I 74-kD fragment at the expense of the alpha I 80-kD parent fragment. Recently we showed that the mutations causing the Sp alpha I/74 abnormality may lie in the alpha- or the beta-chain, and that spectrin Culoz and spectrin Lyon were two (alpha I/74) alpha-variants, respectively. We now show that the spectrin Culoz alpha I domain undergoes prominent tryptic cleavage after Lys 42, whereas cleavage prevails after Arg 39 in spectrin Lyon. Applying the polymerase chain reaction (PCR) technique to exon 2 of the spectrin alpha I domain, we have established that the mutation responsible for spectrin Culoz is alpha I 40 Gly----Val; GGT----GTT. Applying the PCR technique to the cDNA derived from reticulocyte mRNA, we have shown that the mutation responsible for spectrin Lyon is alpha I 43 Leu----Phe; CTT----TTT. Studies of normal controls and of family members using dot blot hybridization with allele-specific oligonucleotide probes confirmed these results. Variants such as spectrin Culoz and spectrin Lyon should provide insight into a region that participates in spectrin dimer self-association and whose susceptibility to proteolysis must reflect subtle conformational changes.

Authors

L Morlé, A F Roux, N Alloisio, B Pothier, J Starck, L Denoroy, F Morlé, R C Rudigoz, B G Forget, J Delaunay

Mast cell chymase potentiates histamine-induced wheal formation in the skin of ragweed-allergic dogs.

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Abstract

Skin mast cells release the neutral protease chymase along with histamine during degranulation. To test the hypothesis that chymase modulates histamine-induced plasma extravasation, we measured wheal formation following intradermal injection of purified mast cell chymase and histamine into the skin of ragweed-allergic dogs. We found that chymase greatly augments histamine-induced wheal formation. The magnitude of the potentiating effect increases with increasing doses of chymase and becomes maximal approximately 30 min after administration. Injection of chymase without histamine does not evoke wheal formation. The chymase potentiation of histamine-induced skin responses is prevented completely by pretreatment with the H1-receptor antagonist pyrilamine, and is prevented by inactivation of chymase with soybean trypsin inhibitor, suggesting that both histamine and preserved catalytic activity are required for the effects of chymase. To examine the effects of histamine and chymase released in situ in further experiments, we measured wheal size following local degranulation of mast cells by intradermal injection of ragweed antigen or compound 48/80. We found that pretreatment with either soybean trypsin inhibitor or pyrilamine markedly reduces ragweed antigen- or 48/80-induced wheal formation, supporting the results obtained by injection of exogenous chymase and histamine. These findings suggest a novel and important proinflammatory role for chymase in modulating the effects of histamine on vascular permeability during mast cell activation.

Authors

I Rubinstein, J A Nadel, P D Graf, G H Caughey

Insulin-like growth factor-I enhances luteinizing hormone binding to rat ovarian theca-interstitial cells.

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Abstract

We tested the hypothesis that insulin-like growth factor-I (IGF-I) stimulates ovarian androgen production by increasing theca-interstitial cell luteinizing hormone (LH) binding affinity and/or binding capacity. We then investigated the role of transcriptional and translational events in mediating these actions of IGF-I. LH bound to saturable, high affinity binding sites on rat ovarian theca-interstitial cells. Preincubation with LH produced a decrease in LH binding capacity with no effect on LH binding affinity. Treatment with IGF-I, both in the absence and presence of LH, increased LH binding capacity 1.5- to 2-fold with no change in LH binding affinity. Androgen production was increased progressively by LH, suggesting that LH-stimulated steroidogenesis is not tightly coupled to LH receptor downregulation. IGF-I increased androgen synthesis in proportion to its upregulation of LH binding capacity. Transcriptional inhibition with dichlorobenzimidazole riboside inhibited the IGF-I-mediated increase in LH binding capacity but had no effect on androgen production. Translational inhibition with cycloheximide inhibited both the IGF-I-mediated increase in LH binding and stimulation of androgen synthesis. We conclude that IGF-I increases theca-interstitial cell LH binding capacity and reverses the LH-induced downregulation of LH binding sites. The enhancement of LH binding by IGF-I is compatible with transcriptional mediation whereas the effect of IGF-I on androgen synthesis appears to be mediated by a direct effect of the peptide on the translational process(es) involved in steroidogenesis.

Authors

J F Cara, J Fan, J Azzarello, R L Rosenfield

Enhanced prostaglandin synthesis after ultraviolet injury is mediated by endogenous histamine stimulation. A mechanism for irradiation erythema.

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Abstract

Acute ultraviolet light B (UVB) injury is associated with dermal mast cell histamine release. The possibility that histamine-stimulated prostaglandin (PG) synthesis could be a mechanism for irradiation erythema was therefore examined using human skin explants. Explants responded to UV irradiation (120 mJ/cm2) with a fivefold increase in synthesis of prostaglandins E2, F2 alpha and 6-keto PGF1 alpha. Incubating explants with the H1 antihistamines brompheniramine (50 microM) or pyrilamine (30 microM) inhibited PG release from irradiated explants 63 +/- 4.9% (mean +/- SEM) 6 h after UV exposure. Antihistamines did not affect PG synthesis in control explants. Irradiation increased the histamine concentration in explant conditioned medium only 50% over basal values, suggesting that irradiation enhanced histamine responsiveness. Explants were therefore incubated with exogenous histamine. In irradiated explants, PG synthesis was stimulated threefold by 3 microM histamine. Unirradiated explants' PG synthesis was unaffected by histamine. Enhanced histamine sensitivity was also examined in epidermal cell cultures. In irradiated cultures, histamine sensitivity was again markedly potentiated: as little as 1 microM histamine stimulated significant PGE2 release and the response to 10-30 microM histamine was increased six to eight times compared with that of unirradiated cultures. These studies demonstrate that endogenous histamine stimulates PG synthesis in human skin after UV injury by potentiation of histamine-induced prostaglandin release. Potentiated agonist responses induced by UV exposure may contribute to the effects of UVB irradiation injury and in particular to irradiation erythema.

Authors

A P Pentland, M Mahoney, S C Jacobs, M J Holtzman

Evidence for altered epicardial coronary artery autoregulation as a cause of distal coronary vasoconstriction after successful percutaneous transluminal coronary angioplasty.

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Abstract

To determine whether vasoconstriction distal to the site of successful percutaneous transluminal coronary angioplasty (PTCA) is a result of altered autoregulation in a hypoperfused coronary artery, we examined the association of this distal vasoconstriction with lesion severity in 20 patients. Lesion severity was classified as moderate, severe or critical (greater than 1.0, 0.5-1.0, and less than 0.5 mm, respectively). Quantitative coronary measurements were made at 3, 15, and 30 min after PTCA, and then after intracoronary (IC) nitroglycerin, in coronary segments distal to the dilated lesion (distal) and in a nonmanipulated vessel (control). Coronary vasoconstriction in the Distal segment after PTCA correlated with lesion severity, with 14 +/- 4%, 28 +/- 2%, and 41 +/- 5% vasoconstriction (vs. IC nitroglycerin, 30 min after PTCA) in the moderate, severe and critical lesion severity subgroups, respectively (P less than 0.01 for critical or severe vs. moderate). This vasoconstriction was significantly greater than that observed in the corresponding control segment for patients with severe (P less than 0.01), and critical (P less than 0.001) lesions. These findings suggest that hypoperfused human epicardial coronary arteries "reset" their autoregulatory responsiveness and that distal vasoconstriction after PTCA is the result of this altered autoregulation. These findings have clinical implications concerning the etiology, prophylaxis and treatment of coronary spams after PTCA and coronary bypass surgery.

Authors

T A Fischell, K N Bausback, T V McDonald

Inhibition of Na/H exchange in avian intestine by atrial natriuretic factor.

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Abstract

Effects of 8-bromo-cGMP (8-Br-cGMP) and synthetic rat atriopeptin III (APIII) on sodium absorption by isolated chicken villus enterocytes and intact chicken ileal mucosa were determined. In isolated cells, both agents significantly decreased initial rates of influx of 22Na and caused a persistent decrease in intracellular pH (pHi); effects that are not additive to those caused by amiloride (10(-3) M). The ED50 for APIII was 0.3 nM. In intact mucosa, both 8-Br-cGMP (10(-4) M) and 5-(N-methyl-N-isobutyl)amiloride (MIBA) (10(-5) M) reduced JNams and JNa.net, their effects were not additive. APIII (10(-7) M) significantly increased cellular cGMP but not cAMP. Both 8-Br-cGMP (10(-4) M) and APIII (10(-7) M) stimulated a persistent increase in cytosolic calcium (Cai), which could be prevented by pretreating the cells with the cytosolic calcium buffering agent MAPTAM or with H-8, an inhibitor of cyclic nucleotide-dependent protein kinases. Furthermore, pretreatment of cells with H-8 or the calmodulin inhibitor, calmidazolium (CM), prevented the effects of 8-Br-cGMP and APIII on pHi. However, the pHi response to subsequent addition of the calcium-ionophore ionomycin was blocked only by CM and not by H-8. These data suggest that APIII and 8-Br-cGMP inhibit amiloride-sensitive Na/H exchange by increasing Cai, an event requiring activation of cGMP-dependent protein kinase.

Authors

C E Semrad, E J Cragoe Jr, E B Chang

Dysregulated interleukin 6 expression produces a syndrome resembling Castleman's disease in mice.

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Abstract

Interleukin 6 (IL-6) is an important regulator of the acute phase response, T cell function, and terminal B cell differentiation. Excessive or inappropriate production of this cytokine may be involved in a variety of autoimmune and neoplastic disorders. To investigate the consequences of dysregulated synthesis of IL-6 in vivo, a high-titer recombinant retroviral vector produced in psi-2 packaging cells was used to introduce the coding sequences of murine IL-6 into mouse hematopoietic cells. Congenitally anemic W/Wv mice reconstituted with bone marrow cells transduced with the retroviral vector developed a syndrome characterized by anemia, transient granulocytosis, hypoalbuminemia, and polyclonal hypergammaglobulinemia, with marked splenomegaly and peripheral lymphadenopathy. Extensive plasma cell infiltration of lymph nodes, spleen, liver, and lung was noted. The similarity of these findings to those of multicentric Castleman's disease, taken together with the observation that lymph nodes from these patients elaborate large amounts of this cytokine, suggest that the inappropriate synthesis of IL-6 has a primary role in the pathogenesis of this systemic lymphoproliferative disorder.

Authors

S J Brandt, D M Bodine, C E Dunbar, A W Nienhuis

Proteinuria, not altered albumin metabolism, affects hyperlipidemia in the nephrotic rat.

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Abstract

It has been established previously that nephrotic hyperlipidemia is characterized by both an increase in lipid synthesis and a defect in removal of lipoproteins. The relationship between these defects and altered albumin metabolism is uncertain. One hypothesis is that hepatic lipogenesis increases in parallel with albumin synthesis. To test this hypothesis, albumin synthesis was increased in nephrotic rats fed an 8.5% protein diet (LPN) by increasing dietary protein to 40% (HPN). Proteinuria was modulated in half of the rats fed 40% protein by enalapril (HPE). Albumin synthesis was the same in both HPN and HPE, but proteinuria was reduced in HPE compared to HPN, and so were serum cholesterol and triglycerides (TG). To examine the effect of serum albumin on lipid clearance in the absence of proteinuria, plasma clearance of chylomicrons (CM) and VLDL was measured in Nagase analbuminemic rats (NAR) and found to be no different than in normal SD rats. When proteinuria was induced in NAR and in SD rats, a severe and identical defect in both CM and VLDL clearance was acquired in both groups and blood lipid levels were increased to a similar degree in both groups. Neither hyperlipidemia nor defective removal of lipoproteins from the circulation are linked to albumin synthesis or serum albumin concentration but result, at least in part, from proteinuria. Postheparin lipoprotein lipase (LPL) activity was reduced slightly in nephrotic animals compared to nonnephrotic controls, but the most striking finding was a highly significant decrease in postheraprin LPL activity in normal NAR compared to SD rats (P less than 0.001), suggesting that reduced LPL activity is not responsible for reduced clearance of CM and VLDL in nephrotic rats.

Authors

R W Davies, I Staprans, F N Hutchison, G A Kaysen

HLA-DQ gene complementation and other histocompatibility relationships in man with the anti-Ro/SSA autoantibody response of systemic lupus erythematosus.

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Abstract

A strong gene interaction between HLA-DQ1 and DQ2 alleles has been associated with anti-Ro/SSA autoantibodies (Harley, J.B., M. Reichlin, F. C. Arnett, E. L. Alexander, W. B. Bias, and T. T. Provost. 1986. Science [Wash. DC]. 232:1145-1147; Harley, J. B., A. S. Sestak, L. G. Willis, S. M. Fu, J. A. Hansen, and M. Reichlin. 1989. Arthritis Rheum. 32:826-836; Hamilton, R. G., J. B. Harley, W. B. Bias, M. Roebber, M. Reichlin, M. C. Hochberg, and F. C. Arnett. 1988. Arthritis Rheum. 31:496-505). To test a gene complementation mechanism for these results, restriction fragment length polymorphisms (RFLP) of the DQ alpha and DQ beta genes have been related to Ro/SSA precipitins in patients with systemic lupus erythematosus. In this study Ro/SSA precipitins are related to the simultaneous presence of a particular pair of RFLPs. A DQ alpha RFLP associated with HLA-DQ1 and a DQ beta RFLP associated with HLA-DQ2 predict that the alpha beta heterodimer in HLA-DQ1/DQ2 heteroxygotes is most closely related to anti-Ro/SSA autoantibodies, thereby supporting a gene complementation mechanism. Beyond this effect, an RFLP associated with HLA-DQ2 and/or DR7 is also related to Ro/SSA precipitins. Multiple molecular histocompatibility mechanisms are implicated, therefore, in the production of anti-Ro/SSA autoantibodies in autoimmune disease. For anti-Ro/SSA autoantibodies in SLE, and perhaps more generally, these data show that the histocompatibility antigens are among the elements that confer autoimmune response specificity and restrict the production of particular autoantibodies among patients with systemic lupus erythematosus.

Authors

A Fujisaku, M B Frank, B Neas, M Reichlin, J B Harley

Simultaneous synthesis and degradation of rat liver glycogen. An in vivo nuclear magnetic resonance spectroscopic study.

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Abstract

Using 13C nuclear magnetic resonance spectroscopic methods we examined in vivo the synthesis of liver glycogen during the infusion of D-[1-13C]glucose and the turnover of labeled glycogen during subsequent infusion of D-[1-13C]glucose. In fasted rats the processes of glycogen synthesis and degradation were observed to occur simultaneously with the rate of synthesis much greater than degradation leading to net glycogen synthesis. In fed rats, incorporation of infused D-[1-13C]glucose occurred briskly; however, over 2 h there was no net glycogen accumulated. Degradation of labeled glycogen was greater in the fed versus the fasted rats (P less than 0.001), and the lack of net glycogen synthesis in fed rats was due to degradation and synthesis occurring at similar rates throughout the infusion period. There was no indication that suppression of phosphorylase a or subsequent activation of glycogen synthase was involved in modulation of the flux of tracer into liver glycogen. We conclude that in both fed and fasted rats, glycogen synthase and phosphorylase are active simultaneously and the levels of liver glycogen reached during refeeding are determined by the balance between ongoing synthetic and degradative processes.

Authors

M David, W A Petit, M R Laughlin, R G Shulman, J E King, E J Barrett

Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum.

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Abstract

The human malaria parasite Plasmodium falciparum demonstrates variability in its dependence upon erythrocyte sialic acid residues for invasion. Some lines of P. falciparum invade neuraminidase-treated or glycophorin-deficient red blood cells poorly, or not at all, while other lines invade such cells at substantial rates. To explore the molecular basis of non-sialic acid dependent invasion, we selected parasite lines from a clone (Dd2) that initially exhibited low invasion of neuraminidase-treated erythrocytes. After maintaining Dd2 for several cycles in neuraminidase-treated erythrocytes, parasite lines were recovered that invaded both untreated and neuraminidase-treated erythrocytes at equivalently high rates (Dd2/NM). The change in phenotype was maintained after removal of selection pressure. Four subclones of Dd2 were isolated and each readily converted from sialic acid dependence to non-sialic acid dependence during continuous propagation in neuraminidase-treated erythrocytes. The neuraminidase-selected lines and the Dd2 clone demonstrated identical restriction fragment length polymorphism markers indicating that the Dd2 clone was not contaminated during the selection process. Parasite proteins that bound to neuraminidase-treated and untreated erythrocytes were indistinguishable among the parent Dd2 clone and the neuraminidase-selected lines. The ability of the Dd2 parasite to change its invasion requirements for erythrocyte sialic acid suggests a switch mechanism permitting invasion by alternative pathways.

Authors

S A Dolan, L H Miller, T E Wellems

Mechanism of decreased baroreceptor activity in chronic hypertensive rabbits. Role of endogenous prostanoids.

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Abstract

We examined the contribution of endogenous prostanoids to baroreceptor activation in chronic renal hypertension. Baroreceptor activity was recorded from the vascularly isolated carotid sinus during slow ramp increases in pressure in rabbits anesthetized with pentothal and chloralose. Mean arterial pressure averaged 133 +/- 4 mmHg in hypertensive rabbits (one kidney, one wrap, n = 12) and 85 +/- 3 mmHg in normotensive rabbits (one kidney, no wrap, n = 13). Baroreceptor activity was decreased significantly (P less than 0.05) in the hypertensive compared with the normotensive rabbits. The decreased baroreceptor activity could not be explained by decreased distensibility of the carotid sinus (sonomicrometers). Inhibition of the endogenous formation of prostanoids with intrasinus administration of indomethacin (50 microM) decreased baroreceptor activity in normotensive (P less than 0.05) but not in hypertensive rabbits over a wide range of pressures. At a pressure of 120 mmHg, activity declined from 61 +/- 14 spikes/s before indomethacin to 47 +/- 12 spikes/s with indomethacin, i.e., a drop of 24 +/- 4%. In contrast, corresponding values in hypertensive rabbits averaged 41 +/- 13 and 40 +/- 12 spikes/s (-1 +/- 2%). Intrasinus prostacyclin, on the other hand, increased activity in both groups: at 120 mmHg activity increased from 62 +/- 9 to 92 +/- 15 spikes/s (51 +/- 17%) in normotensive rabbits and from 29+/- 7 to 47 +/- 14 spikes/s (68 +/- 23%) in hypertensive rabbits. Neither indomethacin nor prostacyclin (n = 5) influenced the pressure-diameter relation of the carotid sinus. The increase in prostacyclin (6-keto-PGF 1 alpha) formation by the sinus in response to its exposure to arachidonic acid (10 microM) was significant (P less than 0.05) in the normotensives (1,627 +/- 344%; n = 5) but not in the hypertensives (583 +/- 353%; n = 5). We conclude that the decreased baroreceptor activity in chronic hypertension may not be caused by decreased distensibility of the vascular wall of the sinus and that endogenous prostanoids that contribute to baroreceptor activation in normotensive rabbits fail to do so in hypertensive rabbits. This appears to be due to decreased formation of prostacyclin rather than decreased sensitivity of the baroreceptors to prostacyclin. The results suggest a new mechanism that contributes to chronic baroreceptor resetting in hypertension.

Authors

P L Xie, M W Chapleau, T S McDowell, G Hajduczok, F M Abboud

Separation of sublethal and lethal effects of polymorphonuclear leukocytes on Escherichia coli.

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Abstract

Escherichia coli ingested by PMN promptly stop growing and form no colonies in nutrient agar, but metabolize near normally for up to several hours. The bactericidal/permeability increasing protein (BPI) of PMN also inhibits E. coli growth without initial metabolic impairment. We recently showed that BPI-treated E. coli, although unable to grow in normal nutrient agar, can form colonies in this medium plus 0.1% BSA, as long as their metabolism is maintained, indicating that biochemical impairment is a better indicator of death than growth arrest (1990. J. Clin. Invest. 85:853-860). We have now reexamined the fate of ingested E. coli. Rabbit PMN ingest greater than 85% of several rough E. coli strains in 15 min, but greater than 80% of these bacteria, while unable to form colonies in conventional agar, grow normally on agar plus 0.1% BSA. Thus, the PMN under these conditions promptly stop growth of ingested E. coli without killing. Adding nonlethal concentrations of normal human serum (NHS) before, but not after ingestion, accelerates killing and, in parallel, loss of bacterial metabolism (t1/2 less than 0.5 h vs. greater than 3 h, respectively, with and without NHS). The rapid killing of both rough and smooth E. coli pretreated with NHS is lost after C7 depletion (C7-D) and restored when C7 is replenished. Similar results are obtained with human PMN. In contrast, ingested Staphylococcus epidermidis, opsonized with either NHS or C7-D serum rapidly stop metabolizing and do not form colonies in nutrient agar with or without BSA. Respiratory burst activity is the same during ingestion of E. coli (with or without NHS) and S. epidermidis. Killing of E. coli J5 (however, not of O111-B4) by BPI is also accelerated by pretreatment with NHS but not C7-D human serum. These findings indicate that late complement components are needed for efficient killing of both rough and smooth E. coli by PMN, and that BPI is the principal intracellular agent acting on ingested rough E. coli.

Authors

B A Mannion, J Weiss, P Elsbach

Effect of a high-carbohydrate, low-saturated-fat diet on apolipoprotein B and triglyceride metabolism in Pima Indians.

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Abstract

The mechanisms by which high-carbohydrate, low-saturated-fat diets lower LDL cholesterol (LDLC) concentrations are unknown. In this study, kinetics of VLDL, intermediate density lipoprotein (IDL), and LDL apoprotein B and VLDL triglyceride were determined in seven nondiabetic (ND) and seven non-insulin-dependent diabetic (NIDDM) Pima Indian subjects on high-fat and high-carbohydrate (HICHO) diets. Metabolic changes were similar in ND and NIDDM. On the HICHO diet, LDLC decreased (131 +/- 8 vs. 110 +/- 7 mg/dl, P less than 0.0001) in all subjects. Mean fasting and 24-h triglyceride (TG) concentrations were unchanged, as were mean production rates and fractional clearance rates (FCR) of VLDL apoB and VLDL TG. The mean VLDL apoB pool size (303 +/- 20 vs. 371 +/- 38 mg, P = 0.01) increased owing to a decrease in the mean transport rate (10.7 +/- 1.1 vs. 8.4 +/- 0.9 mg/kg fat-free mass (ffm) per day, P less than 0.0001) and the mean rate constant (2.3 +/- 0.2 vs. 1.5 +/- 0.2, P less than 0.001) for the VLDL apoB to IDL apoB conversion pathway. The mean transport rate of VLDL apoB to LDL apoB via IDL (10.2 +/- 0.9 vs. 8.0 +/- 0.8 mg/kg ffm per day, P less than 0.001) decreased. Mean LDL apoB concentrations decreased (70 +/- 5 vs. 61 +/- 5 mg/dl, P less than 0.001) on the HICHO diet. Means for total LDL apoB transport rate, LDL apoB FCR, and LDLC/apoB ratios were unchanged. In summary, the HICHO diet decreased the activity of mechanisms that convert VLDL to LDL, which contributed to the decrease in LDLC in all subjects. There was also evidence in some subjects for increased activity of LDL apoB clearance mechanisms, and a decrease in the LDLC to apoB ratio.

Authors

W G Abbott, B Swinburn, G Ruotolo, H Hara, L Patti, I Harper, S M Grundy, B V Howard

Pharmacodynamic study of F(ab')2 fragments of murine monoclonal antibody 7E3 directed against human platelet glycoprotein IIb/IIIa in patients with unstable angina pectoris.

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Abstract

The pharmacodynamics of intravenous bolus injections of 0.05, 0.10, 0.15, and 0.20 mg/kg of F(ab')2 fragments of the murine monoclonal antibody 7E3, 7E3-F(ab')2, directed against the glycoprotein IIb/IIIa (GPIIb/IIIa) receptor of human platelets, were studied in groups of four patients with unstable angina pectoris. With 0.20 mg/kg, the template bleeding time prolonged from 6.3 +/- 1.9 (mean +/- SD) to greater than 30 min; it subsequently decreased to 13 +/- 7.8 min after 12 h and to 8.3 +/- 1.5 min after 24 h. The number of unblocked GPIIb/IIIa receptors (preinfusion value, 32,000 +/- 3,000 per platelet) decreased to 13 +/- 7% of the preinfusion value 1 h after infusion, and then increased to 33 +/- 10% at 12 h, 44 +/- 8% at 24 h and 67 +/- 7% at 72 h. The logarithm of the bleeding time was inversely proportional with the residual GPIIb/IIIa receptors (r = 0.73, P less than 0.0001). ADP-induced platelet aggregation (measured by changes in light transmittance in percent) decreased from 60 +/- 5% before infusion to 1.5 +/- 3% 1 h after infusion; it then increased to 29 +/- 3% after 24 h and 39 +/- 6% after 72 h. Platelet counts decreased by 16% at 1 h and returned to control values within 24 h. Proportionally smaller effects were seen at lower doses of 7E3-F(ab')2. Antibody injection did not induce spontaneous bleeding. Angina was not observed during the first 12 h when the bleeding time was significantly prolonged, but occurred in 6 of the 16 patients within the next 3 d. 2 of the 16 patients developed low titers of IgG antibodies specific for 7E3-F(ab')2. Thus 7E3-F(ab')2 induces dose-related inhibition of platelet function; at a dose of 0.20 mg/kg, it causes profound inhibition of platelet aggregation and prolongation of the bleeding time, but no spontaneous bleeding.

Authors

H K Gold, L W Gimple, T Yasuda, R C Leinbach, W Werner, R Holt, R Jordan, H Berger, D Collen, B S Coller

Expression and localization of gastrin messenger RNA and peptide in spermatogenic cells.

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Abstract

In previous studies we have shown that the gene encoding cholecystokinin (CCK) is expressed in spermatogenic cells of several mammalian species. In the present study we show that a gene homologous to the CCK-related hormone, gastrin, is expressed in the human testis. The mRNA hybridizing to a human gastrin cDNA probe in the human testis was of the same size (0.7 kb) as gastrin mRNA in the human antrum. By in situ hybridization the gastrinlike mRNA was localized to seminiferous tubules. Immunocytochemical staining of human testis revealed gastrinlike peptides in the seminiferous tubules primarily at a position corresponding to spermatids and spermatozoa. In ejaculated spermatozoa gastrinlike immunoreactivity was localized to the acrosome. Acrosomal localization could also be shown in spermatids with electron microscopy. Extracts of the human testis contained significant amounts of progastrin, but no bioactive amidated gastrins. In contrast, ejaculated sperm contained mature carboxyamidated gastrin 34 and gastrin 17. The concentration of gastrin in ejaculated human spermatozoa varied considerably between individuals. We suggest that amidated gastrin (in humans) and CCK (in other mammals) are released during the acrosome reaction and that they may be important for fertilization.

Authors

M Schalling, H Persson, M Pelto-Huikko, L Odum, P Ekman, C Gottlieb, T Hökfelt, J F Rehfeld

Oxygen tension regulates the expression of the platelet-derived growth factor-B chain gene in human endothelial cells.

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Abstract

Hypoxic states are associated with abnormal proliferation and constriction of the smooth muscle cells surrounding the distal vessels of the lung. In hypoxic as well as in normal states, the endothelial cell layer may play a key role in controlling smooth muscle tone by secreting a number of vasoactive agents. Platelet-derived growth factor (PDGF), produced by endothelial cells, is a major growth factor for vascular smooth muscle cells and a powerful vasoconstrictor. It consists of a disulfide-linked dimer of two related peptides, A and B, that are products of two different genes. We found that hypoxic conditions (0-3% oxygen environments) significantly increased PDGF-B mRNA in cultured human umbilical vein endothelial cells by enhancing the transcriptional rate of this gene. This increase was inversely proportional to oxygen tension and was reversible upon reexposure of cells to a 21% oxygen atmosphere. mRNA levels of PDGF-A were not affected nor was the overall rate of cellular gene transcription increased in response to hypoxia. These studies indicate that endothelial cells are not only capable of sensing oxygen tension, but are also able to discriminate and respond to even small differences in oxygen tension resulting in dramatic upregulation of the PDGF-B chain gene.

Authors

S Kourembanas, R L Hannan, D V Faller

Uniparental isodisomy 6 associated with deficiency of the fourth component of complement.

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Abstract

We identified an extremely rare condition, isolated complete deficiency of the fourth component of complement, in a child with systemic lupus erythematosus. The genes for C4 are located within the major histocompatibility complex (MHC) on the short arm of chromosome 6. The patient expressed only paternal phenotypes for proteins encoded by the MHC (HLA and GLO), yet was 46XX with no detectable 6p deletion. Genomic DNA from patient, parents, and sibling was digested with restriction enzymes, and blots were probed for five chromosome 6 markers. At all loci, maternal and paternal RFLPs could be distinguished, and the patient showed only paternal bands. RFLP analysis of markers from four other chromosomes showed maternal and paternal contribution. The data are consistent with uniparental isodisomy 6 (inheritance of two identical chromosome 6 haplotypes from the father and none from the mother). Direct analysis of genetic material from both parents, as well as detection of multiple protein polymorphisms encoded on chromosome 6, clearly demonstrates this novel mechanism for the expression of a recessive genetic condition.

Authors

T R Welch, L S Beischel, E Choi, K Balakrishnan, N A Bishof