Demonstration of free radical generation in „stunned” myocardium of intact dogs with the use of the spin trap α-phenyl N-tert-butyl nitrone

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Authors

Acylation of monocyte and glomerular mesangial cell proteins. Myristyl acylation of the interleukin 1 precursors.

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Acylation of cellular proteins with the fatty acids myristate or palmitate represents an important mechanism for the co- or posttranslational modification of proteins. Lipid A, the biologically active component of bacterial endotoxin, exerts a number of biochemical effects on responsive cell types. Evidence is presented that lipid A stimulates the synthesis and subsequent myristyl acylation of intracellular monocyte and glomerular mesangial cell proteins. Two of the myristylated monocyte proteins were identified by specific immunoprecipitation as the 33-kD IL 1 alpha and beta precursors; a similar myristylated protein was found in mesangial cells. The 17-kD secretory form of monocyte IL 1 beta did not contain covalently linked myristate. Myristyl acylation of the IL 1 precursor proteins may facilitate the processing or membrane localization of these proteins, which lack characteristic hydrophobic signal sequences. The acylated 33-kD IL 1 alpha may remain preferentially associated with the membrane in an active form, whereas limited proteolysis may convert the biologically inactive IL 1 beta precursor into the extracellular, nonacylated, active 17-kD protein.

Authors

S L Bursten, R M Locksley, J L Ryan, D H Lovett

Donor splice site mutation in the apolipoprotein (Apo) C-II gene (Apo C-IIHamburg) of a patient with Apo C-II deficiency.

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The DNA, RNA, and protein of apo C-II have been analyzed in a patient with apo C-II deficiency (apo C-IIHamburg). Markedly reduced levels of plasma and intrahepatic C-II apolipoprotein were demonstrated by immunoblotting and immunohistochemical analysis. Northern, slot blot, and in situ hybridization studies revealed low levels of a normal-sized apo C-II mRNA. No major rearrangement of the apo C-II gene was detected by Southern blotting. Sequence analysis of apo C-II genomic clones revealed a G-to-C substitution within the donor splice site of intron II. This base substitution resulted in the formation of a new Dde I and loss of a Hph I restriction enzyme cleavage site. Amplification of the mutant sequence by the polymerase chain reaction and digestion with Dde I and Hph I restriction enzymes established that the patient was homozygous for the G-to-C mutation. This is the initial report of the DNA sequence of an abnormal apo C-II gene from a patient with deficiency of apo C-II. We propose that this donor splice site mutation is the primary genetic defect that leads to defective splicing and ultimately to an apo C-II deficiency in this kindred.

Authors

S S Fojo, U Beisiegel, U Beil, K Higuchi, M Bojanovski, R E Gregg, H Greten, H B Brewer Jr

Augmentation of hypoxic pulmonary vasoconstriction in the isolated perfused rat lung by in vitro antagonists of endothelium-dependent relaxation.

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The role of the endothelium in hypoxic constriction of the intact pulmonary vascular bed has not been clearly elucidated. To test for a possible role for endothelium-derived relaxing factor(s) (EDRF) in the hypoxic pressor response, isolated, whole blood-perfused rat lungs from male Sprague-Dawley rats treated with meclofenamate were prepared. Three protocols were performed, including: (a) normal saline (control); (b) the putative EDRF inhibitors, eicosatetraynoic acid (ETYA, 1 X 10(-4) M) or nordihydroguaiaretic acid (NDGA, 1 X 10(-4) M) versus vehicle DMSO; and (c) the putative EDRF inhibitor hydroquinone (HQ, 1 X 10(-4) M) versus vehicle ethyl alcohol (ETOH). The pulmonary pressor response to angiotensin II (Ang II, 0.25 micrograms) injections alternated with 6-min periods of hypoxic ventilation (3% O2, 5% CO2) was measured before and after the administration of saline, inhibitors, or vehicles. The administration of the EDRF inhibitors ETYA, NDGA, and HQ resulted in a marked accentuation of the hypoxic pressor response that was not seen in the controls (P less than 0.05). In separate experiments, lungs precontracted with norepinephrine (1 X 10(-6) M) were pretreated with edrophonium (1 X 10(-4) M) and then observed for endothelium-dependent vasodilator responses to acetylcholine at increasing doses (1 X 10(-7)-1 X 10(-4) M). Administration of ETYA, NDGA, or HQ abrogated the observed vasodilatation to acetylcholine, which was not seen with vehicles alone (P less than 0.01). These studies suggest an important role for the endothelium in pulmonary vascular responsiveness to alveolar hypoxia through possible release of a relaxing factor(s) that attenuates the degree of pulmonary arterial constriction.

Authors

V L Brashers, M J Peach, C E Rose Jr

Impaired insulin-stimulated muscle glycogen synthase activation in vivo in man is related to low fasting glycogen synthase phosphatase activity.

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Insulin-mediated glycogen synthase activity in skeletal muscle correlates with the rate of insulin-mediated glycogen deposition and is reduced in human subjects with insulin resistance. To assess the role of glycogen synthase phosphatase as a possible mediator of reduced glycogen synthase activity, we studied 30 Southwestern American Indians with a broad range of insulin action in vivo. Percutaneous biopsies of the vastus lateralis muscle were performed before and during a 440-min euglycemic clamp at plasma insulin concentrations of 89 +/- 5 and 1,470 +/- 49 microU/ml (mean +/- SEM); simultaneous glucose oxidation was determined by indirect calorimetry. After insulin stimulation, glycogen synthase activity was correlated with the total and nonoxidative glucose disposal at both low (r = 0.73, P less than 0.0001; r = 0.68, P less than 0.0001) and high (r = 0.75, P less than 0.0001; r = 0.74, P less than 0.0001) plasma insulin concentrations. Fasting muscle glycogen synthase phosphatase activity was correlated with both total and nonoxidative glucose disposal rates at the low (r = 0.48, P less than 0.005; r = 0.41, P less than 0.05) and high (r = 0.47, P less than 0.05; r = 0.43, P less than 0.05) plasma insulin concentrations. In addition, fasting glycogen synthase phosphatase activity was correlated with glycogen synthase activity after low- (r = 0.47, P less than 0.05) and high- (r = 0.50, P less than 0.01) dose insulin stimulations. These data suggest that the decreased insulin-stimulated glucose disposal and reduced glycogen synthase activation observed in insulin resistance could be secondary to a low fasting glycogen synthase phosphatase activity.

Authors

D Freymond, C Bogardus, M Okubo, K Stone, D Mott

Hemoglobin-mediated oxidant damage to the central nervous system requires endogenous ascorbate.

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Hemorrhage within the central nervous system (CNS) may be associated with subsequent development of seizure states or paralysis. Prior investigations indicate that hemoglobin, released from extravasated erythrocytes, may be toxic to the CNS by promoting peroxidation of lipids and inhibition of Na,K-ATPase. These deleterious effects are blocked both in vitro and in vivo by the Fe3+ chelator, desferrioxamine, indicating the involvement of free iron derived from hemoglobin. We now report that the Fe2+ chelator, ferene, also inhibits methemoglobin- and ferric iron-mediated CNS lipid oxidation, reflecting the reduction of Fe3+ by some component of the CNS. This reduction is apparent in the accumulation of the highly chromophoric ferene: Fe2+ chelate after the addition of Fe3+ salts to supernatants of murine brain homogenates. Because large amounts of ascorbic acid occur in mammalian CNS, we suspected that this reducing substance might be responsible. Indeed, the peroxidative effects of hemoglobin and iron on murine brain are blocked by washing of CNS membranes or by preincubation of crude homogenates with ascorbate oxidase. Furthermore, the addition of ascorbate to washed CNS membranes fully restores hemoglobin/iron-driven peroxidation. We conclude that posthemorrhagic CNS dysfunction may stem from damaging redox reactions between hemoglobin iron, ascorbic acid, and oxidizable components of the nervous system.

Authors

S M Sadrzadeh, J W Eaton

Clostridium difficile toxin A perturbs cytoskeletal structure and tight junction permeability of cultured human intestinal epithelial monolayers.

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Toxin A of Clostridium difficile causes severe inflammatory enterocolitis in man and animals that appears to be mediated in part by acute inflammatory cells that migrate into the toxin A-exposed mucosa. To determine the direct effects of toxin A on intestinal epithelial permeability and structure in the absence of other modulating factors, we used cultured monolayers of a human intestinal epithelial cell line (T84). A toxin A concentration of 7 x 10(-1) micrograms/ml (3 x 10(-9) M) nearly abolished monolayer transepithelial resistance within 6-8 h. This marked permeability defect occurred while the monolayers were still confluent. Dual sodium-mannitol flux studies localized the permeability defect to the intercellular tight junction. Cytotoxicity assays and morphological evaluation using Nomarski optics and electron microscopy failed to demonstrate any evidence of cell damage at the time the maximum resistance response was observed. Fluorescent staining for F actin, however, revealed a marked decrease in fluorescent intensity in toxin-treated monolayers versus controls. These data show that toxin A can directly affect the barrier function of this model intestinal epithelium and initially does so by selectively enhancing tight junction permeability. Furthermore, cytoskeletal structure is markedly altered over the same time course, although the integrity of individual cells is maintained. Because the cytoskeleton of intestinal epithelial cells is known to be capable of regulating tight junction permeability, we speculate that the above effects of toxin A on epithelial barrier function result from alterations of the cytoskeleton.

Authors

G Hecht, C Pothoulakis, J T LaMont, J L Madara

Neutrophil actin dysfunction is a genetic disorder associated with partial impairment of neutrophil actin assembly in three family members.

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A male infant with a severe neutrophil motility disorder and poorly polymerizable actin in PMN extracts was reported over a decade ago to have neutrophil actin dysfunction (NAD) (1974. N. Engl. J. Med. 291:1093-1099). Polymerized actin (F-actin) content of fixed and permeabilized intact neutrophils from the father, mother, and sister of the NAD index case have been measured using nitrobenzoxadiazole-phallacidin, a fluorescent compound which binds specifically to actin filaments. F-actin content of unstimulated PMN from all three family members was significantly lower than unstimulated control PMN (mean 23.6 +/- 0.4 SEM fluorescent units vs. 32.6 +/- 0.6 for controls). After stimulation with the chemotactic peptide FMLP, maximal F-actin content of NAD family member PMN was below that of controls (52.7 +/- 1.3 vs. 72.6 +/- 1.8). F-actin content of detergent insoluble cytoskeletons after stimulation with FMLP was also significantly lower in PMN from NAD family members as compared with controls (21 +/- 6% vs. 73 +/- 8%). PMN extracts from the father and mother, when treated with 0.6 M KCl, polymerized half as much actin as controls. Whereas diisopropylfluorophosphate treatment of normal PMN decreased actin polymerizability in cell extracts, this treatment increased the assembly of actin in parental PMN extract. Addition of purified actin to NAD extracts failed to reveal an abnormal actin polymerization inhibitory activity, and no obvious structural defect in actin purified from the father's PMNs was noted by HPLC and two dimensional thin layer chromatography of tryptic digests. The present studies of actin assembly in intact PMNs confirm that NAD is associated with a true defect in PMN actin assembly and is a genetic disorder that is recessively inherited.

Authors

F S Southwick, G A Dabiri, T P Stossel

Assessment of clonality in gastrointestinal cancer by DNA fingerprinting.

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DNA fingerprinting with three different probes (33.15, 33.6, and alpha-globin 3'HVR) was investigated as a method for the determination of clonality in gastrointestinal tumors. In 29/44 carcinomas the tumor DNA showed clonal somatic mutations that were not seen in the corresponding peripheral blood and normal mucosa samples. The changes consisted of either novel fingerprint bands, losses of bands, or both. The probe 33.15 yielded the highest rate of abnormal DNA fingerprints (21/44 carcinomas). Sequential use of the probes increased the number of cases where clonal fingerprint markers could be detected. One out of five colorectal adenomas also showed a clonal loss of a fingerprint band. In two cases of gastric cancer, DNA from the metastatic tumor had a different DNA fingerprint from that found in the primary carcinoma. DNA fingerprinting offers a novel approach to determining clonality in tumors and may prove useful for the study of tumor progression.

Authors

M F Fey, R A Wells, J S Wainscoat, S L Thein

Pancreatic noradrenergic nerves are activated by neuroglucopenia but not by hypotension or hypoxia in the dog. Evidence for stress-specific and regionally selective activation of the sympathetic nervous system.

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To determine if acute stress activates pancreatic noradrenergic nerves, pancreatic norepinephrine (NE) output (spillover) was measured in halothane-anesthetized dogs. Central neuroglucopenia, induced by intravenous 2-deoxy-D-glucose [( 2-DG] 600 mg/kg + 13.5 mg/kg-1 per min-1) increased pancreatic NE output from a baseline of 380 +/- 100 to 1,490 +/- 340 pg/min (delta = +1,110 +/- 290 pg/min, P less than 0.01). Surgical denervation of the pancreas reduced this response by 90% (delta = +120 +/- 50 pg/min, P less than 0.01 vs. intact innervation), suggesting that 2-DG activated pancreatic nerves by increasing the central sympathetic outflow to the pancreas rather than by acting directly on nerves within the pancreas itself. These experiments provide the first direct evidence of stress-induced activation of pancreatic noradrenergic nerves in vivo. In contrast, neither hemorrhagic hypotension (50 mmHg) nor hypoxia (6-8% O2) increased pancreatic NE output (delta = +80 +/- 110 and -20 +/- 60 pg/min, respectively, P less than 0.01 vs. neuroglucopenia) despite both producing increases of arterial plasma NE and epinephrine similar to glucopenia. The activation of pancreatic noradrenergic nerves is thus stress specific. Furthermore, because both glucopenia and hypotension increased arterial NE, yet only glucopenia activated pancreatic nerves, it is suggested that a regionally selective pattern of sympathetic activation can be elicited by acute stress, a condition in which sympathetic activation has traditionally been thought to be generalized and nondiscrete.

Authors

P J Havel, R C Veith, B E Dunning, G J Taborsky Jr

Demonstration of the production and physiological role of insulin-like growth factor II in rat thyroid follicular cells in culture.

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Insulin-like growth factors (IGFs) are potent mitogens for FRTL5 rat thyroid follicular cells. IGFs also synergize the independent mitogenic effects of thyrotropin-stimulating hormone (TSH) and other agents that increase intracellular AMP concentration. We examined whether FRTL5 cells and M12 cells, a TSH-independent mutant cell line derived therefrom, secrete IGF that regulates the growth of rat thyroid follicular cells. Immunoreactive IGF-II, but not IGF-I, was found in media conditioned by FRTL5 cells; media from M12 cells contained four- to fivefold higher concentrations. Medium conditioned by FRTL5 and M12 both stimulated [3H]thymidine incorporation in FRTL5 and amplified the mitogenic effects of TSH. M12-conditioned medium was more potent than FRTL5-conditioned medium. Sm-1.2, a monoclonal antibody that recognizes IGF-I and IGF-II but not insulin, inhibited basal DNA synthesis in FRTL5 and M12 cells and the mitogenic effects in FRTL5 of agents that are synergized by IGF, such as TSH, forskolin, Bt2cAMP, and Graves'-IgG. Sm-1.2 did not inhibit the mitogenic response to insulin. Thus, rat insulin-like growth factor II (rIGF-II) is an autocrine growth factor that regulates FRTL5 growth, in part by amplifying the mitogenic response to TSH. Results with M12 cells raise the possibility that endogenous rIGF-II may partially mediate the TSH-independent growth of these cells.

Authors

R M Maciel, A C Moses, G Villone, D Tramontano, S H Ingbar

Modulation of the human monocyte binding site for monomeric immunoglobulin G by activated Hageman factor.

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Macrophage Fc gamma receptors play a significant role in inflammation and host defense. One monocyte/macrophage Fc gamma receptor, Fc gamma RI, the binding site for monomeric IgG, appears to be especially responsive to modulatory signals by hormones and mediators. Since Factor XIIa is generated during inflammation, we studied the effect of XIIa on Fc gamma RI. Factor XIIa, in a concentration-dependent manner (0.01-0.19 microM), reduced the number of monocyte binding sites for monomeric IgG up to 80% without altering the affinity of binding. Its precursor, Factor XII, and the low molecular weight fragment of XIIa, lacking most of the heavy chain region, did not reduce the expression of Fc gamma RI. Neither corn trypsin inhibitor (36 microM) nor diisopropylfluorophosphate (3.6 mM) diminished the effect of Factor XIIa on Fc gamma RI, although each completely inhibited the coagulant and amidolytic activity contained on the light chain of Factor XIIa. Protein synthesis was not a requirement for this effect of Factor XIIa, nor was internalization of Fc gamma RI necessary. In contrast to similar concentrations of IgG, Factor XIIa failed to displace significantly monomeric IgG from the monocyte surface, suggesting that Factor XIIa does not directly compete for Fc gamma RI. The data suggest that the heavy chain of XIIa, which contains domains that may have cell hormone activity, also contains a domain that regulates Fc gamma RI on monocytes. In addition to other hormones and mediators, Factor XIIa may serve a regulatory function in modulating Fc gamma receptor expression during inflammation.

Authors

P Chien, R A Pixley, L G Stumpo, R W Colman, A D Schreiber

A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum.

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To obtain free amino acids for protein synthesis, trophozoite stage malaria parasites feed on the cytoplasm of host erythrocytes and degrade hemoglobin within an acid food vacuole. The food vacuole appears to be analogous to the secondary lysosomes of mammalian cells. To determine the enzymatic mechanism of hemoglobin degradation, we incubated trophozoite-infected erythrocytes with peptide inhibitors of different classes of proteinases. Leupeptin and L-transepoxy-succinyl-leucyl-amido-(4-guanidino)-butane (E-64), two peptide inhibitors of cysteine proteinases, inhibited the proteolysis of globin and caused the accumulation of undegraded erythrocyte cytoplasm in parasite food vacuoles, suggesting that a food vacuole cysteine proteinase is necessary for hemoglobin degradation. Proteinase assays of trophozoites demonstrated cysteine proteinase activity with a pH optimum similar to that of the food vacuole and the substrate specificity of lysosomal cathepsin L. We also identified an Mr 28,000 proteinase that was trophozoite stage-specific and was inhibited by leupeptin and E-64. We conclude that the Mr 28,000 cysteine proteinase has a critical, perhaps rate-limiting, role in hemoglobin degradation within the food vacuole of Plasmodium falciparum. Specific inhibitors of this enzyme might provide new means of antimalarial chemotherapy.

Authors

P J Rosenthal, J H McKerrow, M Aikawa, H Nagasawa, J H Leech

Binding of straight-chain saturated dicarboxylic acids to albumin.

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Dicarboxylic acids are prominent features of several diseases, including Reye's syndrome. Long-chain dicarboxylic acids have profound effects on the function and structure of isolated mitochondria, suggesting that they could contribute to the mitochondrial dysfunction in Reye's syndrome. Binding of fatty acids to albumin and the intracellular fatty acid-binding proteins is important in regulating the transport and metabolism of fatty acids and protects against the toxic effects of unbound fatty acids. We studied the binding of dicarboxylic acids to defatted albumin using equilibrium dialysis to assess to what extent dicarboxylic acids are likely to be bound in the plasma of patients. Dicarboxylic acids bind weakly to albumin in a molar ratio of 3.8, 4.2, 1.6, 0.8, and 0.7 to 1 for octadecanedioic, hexadecanedioic, tetradecanedioic, dodecanedioic, and decanedioic acid, respectively. The dissociation constants for long-chain dicarboxylic acids are 100-1,000-fold larger than those of comparable monocarboxylic acids. Oleate competes with dicarboxylic acid and reduces the moles of dicarboxylic acid bound per mol of albumin to less than 1. Octanoate inhibits dicarboxylic acid binding. Our observations indicate that in Reye's syndrome, substantial concentrations of dicarboxylic acids of patients may be free and potentially toxic to mitochondria and other cellular processes.

Authors

J H Tonsgard, S A Mendelson, S C Meredith

Potentiation of pentazocine analgesia by low-dose naloxone.

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The analgesia produced by combinations of low-dose naloxone with pentazocine or morphine was studied in 105 patients with moderately severe postoperative pain after standardized surgery for removal of impacted third molars. Pain intensity was quantified using a visual-analogue scale. To eliminate the release of endogenous opioids produced by the placebo component of open drug administration, all injections were made by a preprogrammed infusion pump. The analgesia produced by pentazocine, an agonist-antagonist opiate-analgesic acting predominantly at the kappa opiate receptor, was potentiated by low-dose naloxone, whereas the analgesia produced by morphine, a mu-agonist, was attenuated by low-dose naloxone. To evaluate whether similar potentiation would be present in an animal model, and specifically, in the absence of diazepam, which patients receive, we performed an analogous experiment in rats in which nociceptive threshold was determined using the Randall-Selitto paw-withdrawal test. The results were completely analogous to the clinical results: pentazocine analgesia was potentiated by low-dose naloxone, whereas morphine analgesia was attenuated by low-dose naloxone. These data demonstrate a novel interaction between opiates, and suggest a rationale for opiate combinations to produce potent analgesia with fewer autonomic side effects and less abuse potential than presently available analgesics.

Authors

J D Levine, N C Gordon, Y O Taiwo, T J Coderre

Differential effect of DPI 201-106 on the sensitivity of the myofilaments to Ca2+ in intact and skinned trabeculae from control and myopathic human hearts.

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The effects of DPI, a new inotropic agent, were compared in trabeculae carneae from control and myopathic human hearts loaded with aequorin, a bioluminescent calcium indicator that emits light when it combines with calcium, and in saponin-skinned trabeculae carneae from the same hearts. The force-pCa curves in saponin-skinned fibers and the peak force-peak Ca2+ curves in aequorin-loaded preparations were not significantly different between the control and myopathic tissues. The force-pCa curve in the skinned and aequorin-loaded preparations from the same control hearts displayed no significant shifts with the addition of DPI. In contrast, a leftward shift was present in the force-calcium relationship in the presence of DPI in aequorin-loaded and skinned muscles from myopathic hearts, indicating an increase in the sensitivity of the myofilaments to calcium. These differences in the modulation of calcium activation between myopathic and control tissues indicate that pharmacological agents may produce differential effects in normal and diseased hearts.

Authors

R J Hajjar, J K Gwathmey, G M Briggs, J P Morgan

Mechanism of hypercalciuria in genetic hypercalciuric rats. Inherited defect in intestinal calcium transport.

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Excessive urine calcium excretion in patients with idiopathic hypercalciuria may involve a primary increase in intestinal calcium absorption, overproduction of 1,25-dihydroxyvitamin D3 or a defect in renal tubular calcium reabsorption. To determine the mechanism of hypercalciuria in an animal model, hypercalciuria was selected for in rats and the most hypercalciuric animals inbred. Animals from the fourth generation were utilized to study mineral balance and intestinal transport in relation to levels of serum 1,25(OH)2D3. Both urine calcium excretion and net intestinal calcium absorption were greater in hypercalciuric males (HM) than in normocalciuric males (NM) and in hypercalciuric females (HF) than in normocalciuric females (NF). However, serum 1,25(OH)2D3 was lower in HM than in NM and not different in HF than in NF. Net calcium balance was more positive in HM than in NM and in HF than in NF. In vitro duodenal calcium net flux was correlated with serum 1,25(OH)2D3 in HM and HF and in NM and NF. However, with increasing serum 1,25(OH)2D3 there was greater calcium net flux in hypercalciuric rats than in normocalciuric controls. Hypercalciuria in this colony of hypercalciuric rats is due to a primary intestinal overabsorption of dietary calcium and not an overproduction of 1,25(OH)2D3 or a defect in the renal tubular reabsorption of calcium.

Authors

D A Bushinsky, M J Favus

Role of intracellular sodium in the regulation of intracellular calcium and contractility. Effects of DPI 201-106 on excitation-contraction coupling in human ventricular myocardium.

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Experiments were performed to investigate the mechanism of action of DPI 201-106 on human heart muscle. In both control and myopathic muscles, DPI produced concentration-dependent increases in action potential duration, resting muscle tension, peak isometric tension, and duration of isometric tension. These changes were associated with increases in resting intracellular calcium and peak calcium transients as measured by aequorin. At higher concentrations of DPI, a second delayed Ca2+ transient (L') appeared. L' was inhibited by tetrodotoxin and ryanodine, suggesting that DPI acts at both the sarcolemma and the sarcoplasmic reticulum. DPI toxicity was manifested by after-glimmers and after-contractions reflecting a Ca2+-overload state: DPI effects were mimicked by veratridine, a Na+ channel agonist, and reversed by tetrodotoxin, yohimbine, and cadmium, Na+ channel antagonists. These results suggest that DPI acts primarily as a Na+ channel agonist. DPI may produce an increase in intracellular Ca2+ by increasing intracellular Na+ and altering Na+-Ca2+ exchange across the sarcolemma. DPI may also increase intracellular Ca2+ by directly altering sarcoplasmic reticulum Ca2+ handling.

Authors

J K Gwathmey, M T Slawsky, G M Briggs, J P Morgan

Regulation of leucine catabolism by caloric sources. Role of glucose and lipid in nitrogen sparing during nitrogen deprivation.

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Previously we showed that hypocaloric amounts of glucose reduce leucine catabolism while an isocaloric amount of fat does not (1985. J. Clin. Invest. 76:737.). This study was designed to investigate whether the same difference exists when the entire caloric need is provided either as glucose or lipid. Rats were maintained for 3 d on total parenteral nutrition (350 cal/kg per d), after which the infusion of amino acids was discontinued and rats received the same amount of calories entirely as glucose or lipid for three more days. A third group of rats was infused with saline for 3 d. In comparison to glucose, lipid infusion resulted in higher urinary nitrogen excretion (55 +/- 3 vs. 37 +/- 2 mg N/24 h, P less than 0.05), muscle concentrations of tyrosine (95 +/- 8 vs. 42 +/- 8 microM, P less than 0.01), and leucine (168 +/- 19 vs. 84 +/- 16 microM, P less than 0.01), activity of BCKA dehydrogenase in muscle (2.2 +/- 0.2 vs. 1.4 +/- 0.04 nmol/mg protein per 30 min, P less than 0.05), and whole body rate of leucine oxidation (3.3 +/- 0.5 vs. 1.4 +/- 0.2 mumol/100 g per h, P less than 0.05). However, all these parameters were significantly lower in lipid-infused than starved rats. There was no significant difference between leucine incorporation into liver and muscle proteins of lipid and glucose-infused rats. On the other hand, starved rats showed a lower leucine incorporation into liver proteins. The data show that under conditions of adequate caloric intake lipid has an inhibitory effect on leucine catabolism but not as great as that of glucose. The mechanism of this difference may be related to a lesser inhibition of muscle protein degradation by lipid than glucose, thereby increasing the leucine pool, which in turn stimulates leucine oxidation.

Authors

J A Vazquez, H S Paul, S A Adibi

"Intact nephrons" as the primary origin of proteinuria in chronic renal disease. Study in the rat model of subtotal nephrectomy.

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Single nephron filtration rate of albumin (SNGFRAlb) was measured in remnant nephrons of Munich-Wistar rats 4-6 wk after subtotal nephrectomy (NPX). Serial thin-section histological analysis was then conducted on the same glomeruli by light microscopy. SNGFRAlb ranged from 1 to 15 times normal. However, a direct relationship between abnormalities of structure and function was not seen, e.g. the glomeruli with the fewest structural abnormalities and marked hyperfiltration often had the highest SNGFRAlb. Moreover, the majority of glomeruli had minimal structural abnormalities. Normalization of the markedly elevated glomerular capillary pressure (PGC) in these glomeruli was accomplished by acute intravenous infusion of verapamil, which decreased SNGFRAlb by 9-83% without affecting the single nephron filtration rate of water (SNGFRH2O). 1-2 wk after subtotal NPX, all glomeruli were hyperfiltering and had elevated PGC. The fractional clearance of larger (greater than 36 A) dextrans was selectively increased in these glomeruli that lacked discernible damage by light microscopy. Verapamil normalized PGC, reduced proteinuria to 48 +/- 4% of baseline, and improved glomerular size selectivity without altering SNGFRH2O. Proteinuria after subtotal NPX thus originates largely from glomeruli with minimal structural abnormalities. The defect in size selectivity is largely attributed to the prevailing high PGC, producing large, nonselective channels on the glomerular capillary wall. The observations raise the possibility that in chronic renal diseases, the reduction in proteinuria often seen after therapeutic measures, including antihypertensive medication, may reflect their functional effect on the relatively intact glomeruli rather than their structure-sparing effect on severely damaged glomeruli, which contribute little to the proteinuria.

Authors

T Yoshioka, H Shiraga, Y Yoshida, A Fogo, A D Glick, W M Deen, J R Hoyer, I Ichikawa

Effect of selective aldosterone deficiency on acidification in nephron segments of the rat inner medulla.

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Mineralocorticoid plays a role in urinary acidification and acid-base balance, but the response of the inner medulla to aldosterone has not been elucidated. A model of selective aldosterone deficiency (SAD) with hyperkalemia and hyperchloremic metabolic acidosis was employed to assess segmental acidification by measuring in situ pH, titratable acidity (TA) and total ammonia (Am). Hydrogen ion secretion was also examined as a function of the increment in in situ PCO2 in the collecting duct during bicarbonate loading. SAD rats were compared to ADX controls that received adrenalectomy and chronic replacement of gluco- and mineralocorticoid and to rats with chronic metabolic acidosis induced by oral NH4Cl (CMA). Both fractional and absolute delivery of Am to the loop of Henle was lower in SAD vs. CMA rats (1.34 to 3.63 mM, P less than 0.01). Delivery of Am to the base and tip collecting duct (BCD and TCD) was also markedly lower in SAD (1.50 vs. 0.52 and 1.77 vs. 0.47 mM, respectively, P less than 0.01). Net addition of Am and net acid between BCD and TCD, observed in CMA rats, was not observed in SAD despite equivalent degrees of systemic metabolic acidosis. Similarly, the concentration gradient favoring transfer of NH3 between loop of Henle and CD was reduced in SAD. During bicarbonate loading the increment in PCO2 at BCD, TCD and in final urine was significantly lower in SAD rats than in adrenal intact bicarbonate-loaded rats. Therefore, the acidification defect in this model of SAD appears to be a result of a decrease in ammonia production and delivery to the loop of Henle, impaired transfer from loop to collecting duct and reduction in the rate of H+ secretion by the collecting duct.

Authors

T D DuBose Jr, C R Caflisch

Distinct murine macrophage receptor pathway for human triglyceride-rich lipoproteins.

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Murine P388D1 macrophages have a receptor pathway that binds human hypertriglyceridemic very low density lipoproteins (HTG-VLDL) that is fundamentally distinct from the LDL receptor pathway. Trypsin-treated HTG-VLDL (tryp-VLDL), devoid of apolipoprotein (apo)-E, fail to bind to the LDL receptor, yet tryp-VLDL and HTG-VLDL cross-compete for binding to P388D1 macrophage receptors, indicating that these lipoproteins bind to the same sites. The specific, high affinity binding of tryp-VLDL and HTG-VLDL to macrophages at 4 degrees C is equivalent and at 37 degrees C both produce rapid, massive, curvilinear (receptor-mediated) triglyceride accumulation in macrophages. Ligand blots show that P388D1 macrophages express a membrane protein of approximately 190 kD (MBP190) that binds both tryp-VLDL and HTG-VLDL; this binding is competed by HTG-VLDL, trypsinized HTG-VLDL, and trypsinized normal VLDL but not by normal VLDL or LDL. The macrophage LDL receptor (approximately 130 kD) and cellular uptake of beta-VLDL, but not MBP 190 nor uptake of tryp-VLDL, are induced when cells are exposed to lipoprotein-deficient medium and decreased when cells are cholesterol loaded. Unlike the macrophage LDL receptor, MBP 190 partitions into the aqueous phase after phase separation of Triton X-114 extracts. An anti-LDL receptor polyclonal antibody blocks binding of HTG-VLDL to the LDL receptor and blocks receptor-mediated uptake of beta-VLDL by P388D1 cells but fails to inhibit specific cellular uptake of tryp-VLDL or to block binding of tryp-VLDL to MBP 190. Human monocytes, but not human fibroblasts, also express a binding protein for HTG-VLDL and tryp-VLDL similar to MBP 190. We conclude that macrophages possess receptors for abnormal human triglyceride-rich lipoproteins that are distinct from LDL receptors in ligand specificity, regulation, immunological characteristics, and cellular distribution. MBP 190 shares these properties and is a likely receptor candidate for the high affinity uptake of TG-rich lipoproteins by macrophages.

Authors

S H Gianturco, A H Lin, S L Hwang, J Young, S A Brown, D P Via, W A Bradley

Repair of protease-damaged elastin in neonatal rat aortic smooth muscle cell cultures.

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Abstract

The objective of this study was to investigate the elastin repair process in the rat aortic smooth muscle cell culture after proteolytic injury. Although little studied in vivo, elastin repair is thought to occur through a sequential process involving enzymatic removal (debridement) of damaged fibers followed by synthesis of tropoelastin, its subsequent processing, and eventual incorporation into new insoluble elastin. A second repair mechanism of proteolytically damaged elastin in a culture system is reported here. Repair in this system relates directly to restoration of resistance to elastin solubilization by hot alkali. As expected, severe injuries were observed with porcine pancreatic elastase (PPE). Using PPE, only 6% of the elastin, relative to control, was resistant to hot alkali immediately after elastase treatment. 4 wk later, resistance to hot alkali had increased dramatically to a mean of 90%. Repair took longer after injury with 75 micrograms of PPE as compared with 50 micrograms of PPE. The limited elastic fiber proteolysis induced by either human neutrophil elastase or porcine trypsin was repaired in culture within 2 wk. Elastin that had been radiolabeled with [3H]lysine 4-5 wk before injury was converted from a hot NaOH-susceptible to a NaOH-resistant elastin fraction during recovery from PPE injury. At the same time, the frayed elastic fibers that were seen with the electron microscope immediately after PPE treatment were replaced by continuous bands of elastin that resembled those in control cultures. Restoration of NaOH resistance did not require a net increase in total cell layer elastin, suggesting that relatively little new tropoelastin incorporation into the cell layer was required for this type of repair. These results suggested a salvage repair mechanism for proteolytically damaged elastin.

Authors

P J Stone, S M Morris, B M Martin, M P McMahon, B Faris, C Franzblau

Murine glomerular leukotriene B4 synthesis. Manipulation by (n-6) fatty acid deprivation and cellular origin.

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Abstract

Leukotriene (LT) B4 is an important pro-inflammatory autocoid. In order to investigate the potential role of this eicosanoid in renal inflammation, in this study we determined the capability of glomeruli to synthesize this mediator. Glomeruli were able to synthesize LTB4 when provided with exogenous substrate in a dose-dependent fashion in the presence of ionophore A23187. Ionophore, although by itself a weak agonist for LTB4 formation, was required for LTB4 production from exogenous arachidonate. The identity of LTB4 was confirmed by specific radioimmunoassay, high pressure liquid chromatography, and gas chromatography/mass spectrometry. The synthesis of LTB4 was inhibited by BW755C (a lipoxygenase/cyclooxygenase inhibitor) but not indomethacin. Essential fatty acid (EFA) deficiency, obtained by the deprivation of (n-6) fatty acids, is known to exert a protective effect in renal inflammatory states. This dietary manipulation markedly attenuated the ability of glomeruli to synthesize LTB4. In contrast, the synthesis of cyclooxygenase products from exogenous arachidonate was increased by EFA deficiency. Because EFA deficiency has been shown to deplete glomeruli of resident mesangial macrophages, it was hypothesized that this effect accounted for the diminished LTB4 synthesis. To test this hypothesis, glomeruli were depleted of macrophages using x-irradiation. Glomeruli from these animals exhibited a marked decrease in LTB4 synthesis. Glomerular synthesis of cyclooxygenase products was unaffected by irradiation. In sum, glomeruli have the capability to synthesize LTB4, and this capacity is correlated with the presence of glomerular macrophages. EFA deficiency attenuates the ability of glomeruli to synthesize LTB4 by depleting them of macrophages.

Authors

J B Lefkowith, A R Morrison, G F Schreiner

Depression of systolic and diastolic myocardial reserve during atrial pacing tachycardia in patients with dilated cardiomyopathy.

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Abstract

Previous reports have shown that increases in heart rate may result in enhanced left ventricular (LV) systolic and diastolic performance. To assess whether this phenomenon occurs in the presence of depressed LV function, the effects of pacing on LV pressure and volume were compared in seven patients with dilated cardiomyopathy (LV ejection fraction 0.19 +/- 0.11) and six patients with no or minimal coronary artery disease (LV ejection fraction 0.69 +/- 0.11). Patients with normal LV function demonstrated significant increases in LV peak-positive dP/dt, LV end-systolic pressure-volume ratio, LV peak filling rate, and a progressive leftward and downward shift of their pressure-volume diagrams, compatible with increased contractility and distensibility in response to pacing tachycardia. There was no change in LV peak-negative dP/dt or tau. Patients with dilated cardiomyopathy, in contrast, demonstrated no increase in either LV peak-positive dP/dt or the end-systolic pressure-volume ratio, and absence of a progressive leftward shift of their pressure-volume diagrams. Moreover, cardiomyopathy patients demonstrated no increase in LV peak-negative dP/dt or LV peak filling rate and a blunted downward shift of the diastolic limb of their pressure-volume diagrams. Tau, as determined from a derivative method, became abbreviated although never reaching control values. We conclude that patients with dilated cardiomyopathy may demonstrate little or no significant enhancement in systolic and diastolic function during atrial pacing tachycardia, suggesting a depression of both inotropic and lusitropic reserve.

Authors

M D Feldman, J D Alderman, J M Aroesty, H D Royal, J J Ferguson, R M Owen, W Grossman, R G McKay

Human serum amyloid A. Three hepatic mRNAs and the corresponding proteins in one person.

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Abstract

Serum amyloid A protein (SAA) is a major acute-phase protein in humans and most other mammals. In addition, it is the serum precursor of the major protein constituent of reactive amyloid fibrils. Sequence analyses have identified a number of polymorphic forms of human SAA and amyloid A protein (AA), but the question of the number of genes encoding SAA in the human has not been addressed. In addition, there are insufficient data to predict whether one form of SAA predisposes to amyloid fibril formation. In the present study three separate SAA proteins have been isolated from the plasma of one individual and completely sequenced. While two of the SAA forms (SAA2 alpha and SAA2 beta) differ from each other only at position 71, they differ from the most abundant form (SAA1) at seven and eight other positions, respectively. Nucleotide sequencing of cDNAs from a liver library of this individual identified all three mRNs coding for these proteins and proved that: (a) the often-reported absence of arginine at the amino terminus of SAA proteins must result from proteolytic processing of the protein; (b) the polymorphism involving histidine and arginine at position 71 is present at the DNA level and therefore is not due to an event at the translational level; (c) there are at least two genes coding for human SAA. Comparison of these data to published sequences of SAA and AA proteins may help in identifying genetically determined forms of SAA which predispose to reactive amyloid fibril formation.

Authors

B Kluve-Beckerman, F E Dwulet, M D Benson

Local extrahepatic expression of complement genes C3, factor B, C2, and C4 is increased in murine lupus nephritis.

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Abstract

Systemic lupus erythematosus (SLE) is associated with the presence of complement proteins and immune complexes in affected organs. Since complement proteins are synthesized in hepatic and extrahepatic sites, we studied a murine model of SLE to ascertain the relative importance of local and humoral (liver) synthesis of complement. C3, C4, and C2 mRNA increase in kidney coincident with the development of nephritis in the MRL lpr/lpr mouse, a strain that spontaneously develops SLE. Two factor B messenger RNA transcripts are expressed in kidney and intestine; SLE nephritis is associated with decrease in the long factor B mRNA and increase in the short form. Increased local synthesis of C3 and B protein and a concomitant glomerular and renal interstitial macrophage infiltrate paralleled the increase in mRNA content in the (lpr/lpr) mice. In addition to kidney, an increase in C3, C4, C2 and factor B mRNA was noted in the lung, heart and intestine and to a lesser extent in liver of (lpr/lpr) in comparison to the MRL (+/+) animals. These results suggest that in SLE local expression of complement genes plays a role in the pathogenesis of chronic glomerulonephritis and in the autoimmune arteritis of other organs.

Authors

J Passwell, G F Schreiner, M Nonaka, H U Beuscher, H R Colten

Alveolar macrophages release an insulin-like growth factor I-type molecule.

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Abstract

Human alveolar macrophages, when activated, release a progression-type growth factor for fibroblasts that signals "competent" fibroblasts to replicate. The present study demonstrates that this growth activity is an insulin-like growth factor I (IGF-I)-type molecule. Partial purification of medium conditioned by activated alveolar macrophages using ion exchange and gel filtration chromatography revealed an IGF-I molecule as detected by an anti-IGF-I polyclonal antibody and that the specific activity of the progression-type growth activity tracked with the amount of IGF-I present. In a serum-free complementation test, the increase in fibroblast proliferation by alveolar macrophage IGF-I was reduced in a dose-response manner with an anti-IGF-I monoclonal antibody. The alveolar macrophage IGF-I displaced 125I-IGF-I from its receptor in a binding assay utilizing human lung fibroblasts and it stimulated type I IGF receptors purified from human lung fibroblasts to phosphorylate a tyrosine-containing artificial substrate. In contrast to the 7.6-kD serum IGF-I, gel chromatography revealed that the alveolar macrophage IGF-I had an apparent molecular mass of 26 kD, similar to other tissue IGF-Is. Alveolar macrophages expressed IGF-I mRNA transcripts as detected by solution hybridization using a 32P-labeled riboprobe complementary to exons I-II-III of the IGF-I gene. In the context of the known functions of the family of IGF-I molecules in cell growth, IGF-I released by activated alveolar macrophages may play a role in acute and chronic inflammatory disorders.

Authors

W N Rom, P Basset, G A Fells, T Nukiwa, B C Trapnell, R G Crysal

Metabolic and functional consequences of inhibiting adenosine deaminase during renal ischemia in rats.

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Abstract

The concentrations of renal ATP have been measured by 31P-nuclear magnetic resonance (NMR) before, during, and after bilateral renal artery occlusion. Using in vivo NMR, the initial postischemic recovery of ATP increased with the magnitude of the residual nucleotide pool at the end of ischemia. ATP levels after 120 min of reflow correlated with functional recovery at 24 h. In the present study the effect of blocking the degradation of ATP during ischemia upon the postischemic restoration of ATP was investigated. Inhibition of adenosine deaminase by 80% with the tight-binding inhibitor 2'-deoxycoformycin led to a 20% increase in the residual adenine nucleotide pool. This increased the ATP initial recovery after 45 min of ischemia from 52% (in controls) to 62% (in the treated animals), as compared to the basal levels. The inhibition also caused an accelerated postischemic restoration of cellular ATP so that at 120 min it was 83% in treated rats vs. 63% in untreated animals. There was a corresponding improvement in the functional recovery from the insult (increase of 33% in inulin clearance 24 h after the injury). Inhibition of adenosine deaminase during ischemia results in a injury similar to that seen after a shorter period of insult.

Authors

M E Stromski, A van Waarde, M J Avison, G Thulin, K M Gaudio, M Kashgarian, R G Shulman, N J Siegel

Human tissue-type plasminogen activator releases fibrinopeptides A and B from fibrinogen.

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Abstract

In five patients with venous thromboembolic disease treated with recombinant tissue-type plasminogen activator (rt-PA), there was a marked increase in the mean concentrations of fibrinopeptide A (from 0.6 to 5.9 nM; P less than 0.0001) and desarginine fibrinopeptide B (from 5.6 nM to 24.1 nM; P less than 0.01) 30 min after a bolus of rt-PA (0.6 mg/kg). Thrombin was unlikely to be responsible because the levels of desarginine fibrinopeptide B exceeded those of fibrinopeptide A and the changes occurred despite concomitant heparin therapy. The purpose of this study therefore, was to determine whether rt-PA directly releases the fibrinopeptides from fibrinogen. Incubation of rt-PA with heparinized plasma or purified fibrinogen resulted in time and dose-dependent release of both fibrinopeptide A and B. Contaminating thrombin was not responsible for this activity by the following criteria: the rate of rt-PA mediated fibrinopeptide B release was considerably faster than that of fibrinopeptide A, and fibrinopeptide release was unaffected by heparin, hirudin, or a monospecific antithrombin IgG. Aprotinin also had no effect on fibrinopeptide release, indicating that this activity was not plasmin mediated. Fibrinopeptide release was shown to be due to rt-PA because this activity was completely blocked by a monoclonal antibody against the enzyme. Further, the specificity of rt-PA for the thrombin cleavage sites on fibrinogen was confirmed by the demonstration that rt-PA released fibrinopeptide A or fibrinopeptide B from fibrinopeptide A or B-containing substrates, respectively. These studies thus demonstrate that (a) rt-PA releases fibrinopeptides A and B from fibrinogen thereby indicating that this enzyme is not specific for plasminogen, and (b) plasma fibrinopeptide A and desarginine fibrinopeptide B levels are not specific markers of thrombin action on fibrinogen in patients receiving rt-PA.

Authors

J I Weitz, M K Cruickshank, B Thong, B Leslie, M N Levine, J Ginsberg, T Eckhardt

Prostaglandin endoperoxides modulate the response to thromboxane synthase inhibition during coronary thrombosis.

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Abstract

Prostaglandin endoperoxides (PGG2/PGH2), precursors of thromboxane (TX) A2 and prostaglandins, may accumulate sufficiently in the presence of a TXA2 synthase inhibitor to exert biological activity. To address whether this modulates the response to TXA2 synthase inhibition in the setting of thrombosis in vivo, we examined the interaction of a TXA2 synthase inhibitor (U63,557a) and a TXA2/prostaglandin endoperoxide receptor antagonist (L636,499) in a canine model of coronary thrombosis after electrically induced endothelial injury. U63,557a exerted little inhibitory effect in this model despite a marked reduction in serum TXB2 and urinary 2,3-dinor-TXB2, an index of TXA2 biosynthesis. Combination of the two drugs was more effective than either drug alone. The enhanced effect achieved upon addition of the TXA2/prostaglandin endoperoxide receptor antagonist to the TXA2 synthase inhibitor suggests that the response to the latter compound was limited by the proaggregatory effects of prostaglandin endoperoxides. The increased effect of the combination over the receptor antagonist alone may reflect metabolism of PGG2/PGH2 to platelet inhibitory prostaglandins. This is supported by the following findings: (a) urinary 2,3-dinor-6-keto-PGF1 alpha, an index of prostacyclin biosynthesis, increased after administration of the synthase inhibitor, an effect that was exaggerated in the presence of thrombosis; (b) inhibition of arachidonate-induced platelet aggregation by U63,557a was dependent on the formation of a platelet-inhibitory prostaglandin; and (c) pretreatment with aspirin abolished the synergism between these compounds. These studies demonstrate that prostaglandin endoperoxides modulate the response to TXA2 synthase inhibition in vivo and identify a drug combination of potential therapeutic efficacy in the prevention of thrombosis.

Authors

D J Fitzgerald, J Fragetta, G A FitzGerald

Bacterial lipopolysaccharide-induced intestinal microvascular lesions leading to acute diarrhea.

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Abstract

Subcutaneous challenge of mice with lipopolysaccharide (LPS) from gram negative bacteria, produced an intestinal microvascular lesion causing fluid exudation into the lumen of the intestine and diarrhea. The microvascular lesion was characterized by endothelial cell damage and microthrombi in the venules and capillaries of the intestinal lamina propria. Marker organisms, given orally to challenged mice, grew in the exuded fluid and could invade the mucosa. Intravenous transfer of postchallenge plasma produced the lesion in normal mice and absorption of such plasma by Sepharose coupled to LPS-antibody abolished this effect. Instillation of large quantities of LPS into the lumen of the intestine produced scattered microvascular lesions, although none of these animals developed diarrhea. Since a similar microvascular lesion has been described in the rectal mucosal lamina propria of adults with acute diarrhea, it is suggested that LPS-induced vascular damage may be a novel mechanism in the pathogenesis of acute diarrhea.

Authors

V I Mathan, G R Penny, M M Mathan, D Rowley

Amplification of altered self-reactive cytolytic T lymphocyte responses by cloned, allospecific human Th cells.

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Abstract

The effect of a cloned allospecific human Th cell, termed 86, on the in vitro generation of altered self-reactive cytolytic T lymphocytes (CTL) was investigated. Utilizing the induction of hapten altered self-reactive CTL as a model for virus or tumor-specific cell-mediated immunity, we determined that the presence of small numbers of clone 86 cells markedly amplified the generation of hapten altered self-reactive CTL. The killer cells induced belong to the CD4-, CD8+ subset, are specific for the hapten-modified autologous stimulator cells present in culture, and are MHC class I restricted. The CTL induced under these culture conditions are readily expanded in the presence of IL-2 with maintenance of efficient and specific altered self-killing. Of interest, clone 86 cells preferentially enhance the growth of CD8+ T cells and selectively amplify altered self-cytolysis but not NK cell activity. Although in vitro clone 86 cells mediate help for CTL generation via the production of lymphokines (IL-4 but little IL-2), one can envision immunotherapeutic strategies for human disease that involve the adoptive transfer of Th cells functionally analogous to clone 86.

Authors

S M Friedman, A Green, C Russo, D N Posnett, D Diffley, M K Crow

Dendritic cell-lymphocyte clusters that form spontaneously in rheumatoid arthritis synovial effusions differ from clusters formed in human mixed leukocyte reactions.

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Abstract

Lymphocytes cluster about dendritic cells (DC) spontaneously in 48 h cultures of rheumatoid arthritis synovial fluid (RA SF) mononuclear cells and in peripheral blood autologous or allogeneic mixed leukocyte reactions. In the latter case, the clusters are predominantly CD4+ T cells (T4/T8 greater than 5) and with time progress in blastic cells that express IL-2 (Tac) and/or transferrin (T9) receptors. In contrast, the clusters in RA SF cultures have a T4/T8 ratio of less than 1 and a majority of the T8 cells coexpress the Leu 7 marker. T cells in these clusters remain inert and with time the clusters disintegrate. Addition of IL-1, IL-2, or IFN-gamma alone or in combination had no effect on RA SF clusters but T cells became blastic when exposed to 10% RA SF. Mixing experiments using RA SF DC with normal T cells and RA T cells with normal DC show that both RA SF DC and T cells are immunofunctional. In addition, clusters of RA SF from a patient with active tuberculosis proliferated vigorously to PPD. Therefore, the unique RA SF cluster profile may reflect the memory nature of the RA SF T cells resulting in a paucity of T cells that are responsive to autologous stimulation. However, an immunosuppressive role for the double-labeled (CD8 and Leu 7) cells has not been excluded.

Authors

V Tsai, N J Zvaifler

Recognition of an endothelial determinant for CD 18-dependent human neutrophil adherence and transendothelial migration.

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Abstract

Human neutrophil (PMN) attachment to human umbilical vein endothelial cells (HUVEC) was evaluated in vitro using two MAbs, R6-5-D6 and RR1/1, that recognize intercellular adhesion molecule-1 (ICAM-1), and one MAb, TS1/18, that recognizes CD18. Pretreatment of the HUVEC with anti-ICAM-1 MAbs produced greater than 50% inhibition of attachment to HUVEC, and IL-1 (0.5 U/ml)- or lipopolysaccharide (LPS) (10 ng/ml)-stimulated HUVEC, and greater than 99% inhibition of f-Met-Leu-Phe (0.5 nM) enhanced adherence. Anti-ICAM-1 MAbs also inhibited by greater than 85% the transendothelial migration induced by 4-h IL-1 (0.5 U/ml) and LPS (10 ng/ml) activation of the HUVEC. That these effects involved a CD18-dependent mechanism is supported by the following results: pretreatment of PMN with TS1/18 produced the same degree of inhibition of attachment and migration as seen with R6-5-D6. In addition, the use of both MAbs together did not further increase the inhibition of cell attachment to stimulated HUVEC. The attachment of PMN from patients with CD18 deficiency to stimulated HUVEC was not reduced by R6-5-D6, and both R6-5-D6 and TS1/18 revealed the same time course for appearance and disappearance of an adherence component on stimulated HUVEC not blocked by either MAb. These results demonstrate that attachment and transendothelial migration of PMN in vitro depend substantially on both CD18 on the PMN and ICAM-1 on the endothelial cell.

Authors

C W Smith, R Rothlein, B J Hughes, M M Mariscalco, H E Rudloff, F C Schmalstieg, D C Anderson

Mechanisms underlying transition from acute glomerular injury to late glomerular sclerosis in a rat model of nephrotic syndrome.

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Abstract

Functional and morphologic measurements were performed in Munich-Wistar rats after a single central venous injection of puromycin aminonucleoside (PA) or saline vehicle (sham). During phase I, PA rats exhibited overt nephrotic syndrome and impaired glomerular filtration, primarily due to a reduction in the glomerular capillary ultrafiltration coefficient. The morphologic counterpart of the latter consisted of effacement of glomerular epithelial cell foot processes and decrease in the number of filtration slit diaphragms. Administration of the angiotensin I converting enzyme inhibitor (CEI) enalapril to PA rats did not ameliorate glomerular dysfunction. During phase II, PA rats exhibited spontaneous resolution of proteinuria, impaired function, and morphologic abnormalities. However, PA rats now demonstrated marked glomerular capillary hypertension and continued, albeit lesser, reductions in the ultrafiltration coefficient. Concurrent CEI administration modestly lowered systemic arterial pressure, and normalized the glomerular capillary hydraulic pressure and ultrafiltration coefficient. Additional rats were studied during phase III, 70 wk after injection. In PA rats, prior glomerular hypertension was associated with development of recurrent proteinuria and extensive glomerular sclerosis, whereas concurrent CEI administration limited these parameters to values comparable to those in sham rats. Glomerular hypertension thus may explain the development of glomerular sclerosis and renal failure long after an episode of acute glomerular injury.

Authors

S Anderson, J R Diamond, M J Karnovsky, B M Brenner

Endothelial cell-mediated conversion of Glu-plasminogen to Lys-plasminogen. Further evidence for assembly of the fibrinolytic system on the endothelial cell surface.

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Abstract

Lysine-plasminogen (Lys-PLG), the plasmin-modified form of native glutamic acid-plasminogen (Glu-PLG), displays enhanced binding affinity for fibrin and also enhanced activation by urokinase and tissue plasminogen activator. We previously demonstrated high-affinity, specific, and functional binding of Glu-PLG as well as tissue plasminogen activator to cultured human umbilical vein endothelial cells (HUVEC). In the present study, we demonstrate binding of Lys-PLG to HUVEC, as well as conversion of Glu-PLG to Lys-PLG at the cell surface. Binding of Lys-PLG to HUVEC was saturable, reversible, epsilon-aminocaproic acid-sensitive, and involved two saturable sites with Kd's of 142 pM and 120 nM, respectively. Upon incubation with Glu-PLG, HUVEC, as well as endothelium in situ, partially converted the ligand to a Lys-PLG-like species. Conversion by HUVEC was blocked by diisopropyl-fluorophosphate, but not by other serine protease inhibitors, including alpha 2-plasmin inhibitor. Eluates of intact umbilical cord vessels contained Lys-PLG by immunoblot analysis. Lys-PLG was also identified immunohistochemically on the endothelial surface of vessels from a variety of normal and inflamed tissues. Thus, endothelial cells appear to actively modify circulating Glu-PLG, bind Lys-PLG to their surface, and thus enhance the fibrinolytic potential of the blood vessel wall.

Authors

K A Hajjar, R L Nachman

Synergistic regulation of human megakaryocyte development.

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Abstract

Little information exists concerning differing levels of regulation occurring during human megakaryocyte development. We hypothesize that megakaryocytic proliferation and maturation is controlled by two, synergistic regulatory factors. One, megakaryocyte colony-stimulating activity, is an obligate requirement for colony formation and drives the development of relatively immature cells. Megakaryocyte colony-stimulating activity is a functional component of the human recombinant proteins, interleukin 3 or GM-CSF. Human recombinant growth factors, interleukin 1, interleukin 6, or crythropoietin, do not effect megakaryocyte development either alone or in combination with interleukin 3. Full maturation requires a second synergistic activity which increases megakaryocyte number, size, and cytoplasmic and antigenic content. In culture, this synergistic regulator augments maturation by increasing the number of colonies, colony cellularity, and size. In suspension cultures, this cofactor increases megakaryocyte cytoplasmic and antigenic content, and shifts the morphological distribution from immature to mature megakaryocytes. Finally, this activity also increases the number of antigen positive megakaryocytes, either by stimulating proliferation or conversion of antigen-negative to antigen-positive cells. Comparative studies of megakaryocytic regulation suggests that this in vitro regulator mimicks some of the known effects of thrombopoietin in vivo.

Authors

M W Long, R J Hutchinson, L L Gragowski, C H Heffner, S G Emerson

Interleukin 1 binds to specific receptors on human keratinocytes and induces granulocyte macrophage colony-stimulating factor mRNA and protein. A potential autocrine role for interleukin 1 in epidermis.

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Abstract

Cultured human keratinocytes have been shown to produce IL-1 alpha and beta mRNA and protein. IL-1 biological activity has been identified in normal human epidermis; in vitro, most biologically active IL-1 resides in a cell-associated compartment. The potential for autocrine effects of IL-1 on human keratinocytes was assessed by measurement of keratinocyte IL-1 receptors. Both high- and low-affinity cell surface receptors that bound recombinant (r) IL-1 alpha and beta with comparable affinities could be identified on cultured human keratinocytes, using 125I-labeled rIL-1. Chemical crosslinking experiments identified a cell surface molecule of roughly 72,500 Mr that bound 125I-labeled IL-1, similar to the molecular weight of previously described IL-1 receptors on fibroblasts, B cells, and T cells. To assess the biological consequences of keratinocyte IL-1 binding, granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression was measured. The addition of exogenous rIL-1 alpha led to a dose-dependent increase in the accumulation of GM-CSF mRNA, as measured by a sensitive and specific S1 nuclease assay. This increase in mRNA was reflected in a marked increase in GM-CSF biological activity as measured by proliferation of blast cells from chronic myelogenous leukemia patients. The biological activity was completely inhibitable by an antibody to human rGM-CSF. GM-CSF activates mature neutrophils and macrophages and appears to enhance the efficiency of Langerhans cell antigen presentation to T cells. Release of IL-1 from injured or activated keratinocytes may lead to enhanced epidermal GM-CSF gene expression via an autocrine mechanism, thus enhancing local host defense.

Authors

T S Kupper, F Lee, N Birchall, S Clark, S Dower

Regulation of lipolysis during the neonatal period. Importance of thyrotropin.

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Abstract

We investigated the lipolytic effect of several hormones on isolated human adipocytes obtained at different donor ages. In neonates, noradrenaline and adrenaline had an insignificant lipolytic effect (70% over basal). In this age group only thyrotropin (TSH) had a significant effect in physiological concentrations, and the maximal lipolytic effect (700% over basal) was the same as that of isoprenaline. The lipolytic effect of TSH was the same in premature 4-10-wk-old infants with a gestational age of 27-33 wk as in neonates, but fat cells from infants 4-10 wk old, born at term, showed a significantly lower effect. In children and adults, the lipolytic effect of TSH gradually decreased further and was present only in unphysiological concentrations. The catecholamine-induced lipolysis was pronounced and was similar in children and adults (350% over basal). TSH is the dominating lipolytic hormone in vitro during the neonatal period. Thus, the peak elevation of circulating TSH, which is seen immediately after birth, may be essential to lipolysis during this part of life.

Authors

C Marcus, H Ehrén, P Bolme, P Arner

Antibodies to parathyroid hormone-related protein lower serum calcium in athymic mouse models of malignancy-associated hypercalcemia due to human tumors.

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Abstract

A parathyroid hormone-related protein (PTHrP) has recently been isolated from tumors associated with hypercalcemia. In the present study, we tested the effects of neutralizing antisera to the PTHrP on serum calcium and urine cAMP in two animal models of malignancy-associated hypercalcemia. The animal models consisted of (a) a human squamous cell lung cancer and (b) a human laryngeal cancer, both serially carried in athymic mice. The antisera specifically reduced the elevated serum calcium and urinary cAMP levels in the tumor-bearing animals. We conclude that PTHrP plays a major role in the pathogenesis of malignancy-associated hypercalcemia.

Authors

S C Kukreja, D H Shevrin, S A Wimbiscus, P R Ebeling, J A Danks, C P Rodda, W I Wood, T J Martin

Genetic evidence from two families that the apolipoprotein B gene is not involved in abetalipoproteinemia.

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Abstract

Abetalipoproteinemia (ABL) is a recessive disorder in which affected individuals have extremely low or undetectable levels of serum apo B-containing lipoproteins. Using restriction fragment length polymorphisms, we have studied two families, each with two children with classical ABL born of normal parents. In each of these families, the two affected children have inherited different apo B alleles from at least one parent, whereas the siblings would be anticipated to share common alleles if this disorder were due to an apo B gene mutation. This linkage study shows that in these families, the apo B gene is discordant with ABL and therefore the disorder is caused by a defect in another gene, which is important for the normal synthesis or secretion of apo B-containing lipoproteins from both the liver and intestine.

Authors

P J Talmud, J K Lloyd, D P Muller, D R Collins, J Scott, S Humphries