The insulin receptor and the molecular mechanism of insulin action.

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Abstract

Authors

C R Kahn, M F White

Expression of platelet-derived growth factor (PDGF)-related transcripts and synthesis of biologically active PDGF-like proteins by human malignant epithelial cell lines.

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Human malignant epithelial cell lines were analyzed for expression of platelet-derived growth factor (PDGF) genes. Of the 12 cell lines tested, 9, derived from breast, lung, gastric, and ovarian carcinomas, were found to express both PDGF-1 and PDGF-2 genes. The levels of both PDGF-1 and PDGF-2 transcripts were superinduced when these cells were treated with cycloheximide, an inhibitor of protein synthesis. These cells also released an activity that in studies with BALB-c/3T3 cells, inhibited binding of 125I-labeled PDGF and stimulated incorporation of [3H]thymidine. This stimulating activity was inhibited after reduction of the conditioned media by mercaptoethanol or after preincubation with antibodies to PDGF. Moreover, this activity was not affected by heat treatment. Immunoprecipitation studies revealed that breast, lung, and gastric carcinoma cells produced PDGF-like proteins that migrated as 30- and 32-kD species under nonreducing conditions and as 15- and 16-kD species under reducing conditions. In contrast, malignant cells of ovarian origin produced 14-16-kD PDGF-like proteins that were unchanged in mobility after reduction. As PDGF receptors were not detected on these malignant epithelial cells, the production of PDGF-like proteins may affect other cells in the microenvironment by paracrine mechanisms and may contribute to excessive cell proliferation, inflammatory reactions, and connective tissue remodeling seen in certain carcinomas.

Authors

E Sariban, N M Sitaras, H N Antoniades, D W Kufe, P Pantazis

Calcium/calmodulin-mediated action of calcitonin on lipid metabolism in rats.

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The effects of calcitonin on lipid metabolism were investigated in three kinds of rats, one strain of rabbits, and a primary culture of rat hepatocytes. In a short-term experiment, calcitonin decreased serum cholesterol and triglycerides after injection in rats on either an ordinary or high-fat diet. In a long-term experiment, calcitonin decreased the serum cholesterol and triglycerides in uremic rats, hypothalamic obese rats, and Watanabe-heritable hyperlipidemic rabbits. In cultured hepatocytes, calcitonin reduced the incorporation of [14C]acetate into cholesterol and triglycerides in a dose-dependent way. Treatment with W7, a calmodulin inhibitor, overcame the decrease caused by calcitonin in serum lipids in rats and in the synthesis of triglycerides from acetate or palmitate in the hepatocytes, but did not alter the intracellular cAMP level or incorporation of [32P]Pi into PI in the cells. The results suggest that calcitonin lowers serum lipid levels and lipogenesis in hepatocytes in a calcium/calmodulin-dependent way.

Authors

Y Nishizawa, Y Okui, M Inaba, S Okuno, K Yukioka, T Miki, Y Watanabe, H Morii

Immunoperoxidase localization of bile salts in rat liver cells. Evidence for a role of the Golgi apparatus in bile salt transport.

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The mechanisms of intracellular transport of bile acids from the sinusoidal pole to the canalicular pole of the hepatocyte are poorly understood. There is physiological and autoradiographic evidence for a vesicular pathway. The purpose of this study was to determine the localization of natural bile acids in the liver using antibodies against cholic acid conjugates and ursodeoxycholic acid. An indirect immunoperoxidase technique was used on rat liver sections fixed either with paraformaldehyde (PF) and saponin, a membrane-permeabilizing agent that allows penetration of antibodies into the cell, or with PF alone. Retention of taurocholate in the liver after tissue processing was 26 +/- SD 15% of the bile acid initially present. When sections fixed with PF and saponin were incubated with the antibody against cholic acid conjugates, a granular cytoplasmic staining was observed by light microscopy in all hepatocytes. By electron microscopy, strong electron-dense deposits were observed mostly on vesicles of the Golgi apparatus (GA) and, sometimes, in the smooth endoplasmic reticulum (SER). After taurocholate infusion, the intensity of the reaction increased. When the liver was fixed with PF alone, almost no reaction was visible on light microscopy, but on electron microscopy the label was localized on the hepatocyte plasma membrane, mainly on the bile canalicular domain and to a lesser extent on the sinusoidal domain. With the antibody against ursodeoxycholic acid, no staining was observed in three of four livers, and a slight staining was observed in one. However, after infusion of ursodeoxycholic acid, staining of GA and SER vesicles was observed when the liver was fixed with PF and saponin. With PF alone, the reaction was intense on the canalicular membrane. These results support the view that, within the limits of the method, vesicles from the GA and possibly vesicles of the SER are involved in the intracellular transport of bile acids before canalicular secretion.

Authors

Y Lamri, A Roda, M Dumont, G Feldmann, S Erlinger

Bias toward use of a specific T cell receptor beta-chain variable region in a subgroup of individuals with sarcoidosis.

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To evaluate the concept that biases in the usage of T cell antigen receptor beta variable (V) regions may be manifested in T lymphocytes that accumulate in nonmalignant, T cell-mediated human disorders, a V beta 8-specific antibody (anti-Ti3A, 5REX9H5) was used to evaluate lung and blood T cells in pulmonary sarcoidosis, a chronic granulomatous disorder of unknown etiology. Whereas normal patients had less than 5% Ti3A+ lung (n = 7) and/or blood (n = 9) lymphocytes, strikingly, a subgroup (8 of 21) with active pulmonary sarcoidosis had greater than 7% Ti3A+ lung and/or blood T cells and a higher proportion of Ti3A+ lymphocytes in the lung compared with blood. Dual-color flow cytometry demonstrated compartmentalization of Ti3A+ CD4+ lymphocytes to lung and Ti3A+ CD8+ lymphocytes to blood. Analysis with a 32P-labeled V beta 8 probe revealed that sarcoid lung T lymphocytes contained higher amounts of V beta 8+ mRNA than autologous blood T cells. However, Southern analysis of sarcoid lung and blood T cell DNA demonstrated no evidence of clonal rearrangements of V beta 8 genes. These observations demonstrate a clear bias toward the use of at least one V beta region in sarcoidosis, and suggests T cells accumulate secondary to external selective pressure, rather than in a random polyclonal fashion or by clonal expansion of one or few T cell clones.

Authors

D R Moller, K Konishi, M Kirby, B Balbi, R G Crystal

Studies on the molecular mechanisms of human Fc receptor-mediated phagocytosis. Amplification of ingestion is dependent on the generation of reactive oxygen metabolites and is deficient in polymorphonuclear leukocytes from patients with chronic granulomatous disease.

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Human PMN and monocytes both possess a mechanism for amplifying Fc receptor-mediated phagocytic function, which is dependent on activation of the respiratory burst. The pathway for augmentation of phagocytosis requires superoxide anion, hydrogen peroxide, and lactoferrin and is independent of the hydrogen peroxide-MPO-halide system. In neither cell type is this mechanism induced upon exposure to the opsonized target. PMN require an additional signal for stimulation of the respiratory burst; this is not true of monocytes. On the other hand, monocytes require an exogenous source of lactoferrin in order to activate this pathway for enhanced ingestion. The dependence of this pathway for both PMN and monocytes on superoxide anion, hydrogen peroxide, and cell-bound lactoferrin is consistent with a role for locally generated reactive oxygen metabolites, possibly hydroxyl radicals, in phagocytosis amplification. Patients with chronic granulomatous disease, who are genetically deficient in the ability to activate the respiratory burst, are unable to amplify Fc receptor-mediated phagocytosis. Thus, these patients may have a previously unrecognized defect in the recruitment of phagocytic function at inflammatory sites.

Authors

H D Gresham, J A McGarr, P G Shackelford, E J Brown

Serum thymulin in human zinc deficiency.

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The activity of thymulin (a thymic hormone) is dependent on the presence of zinc in the molecule. We assayed serum thymulin activity in three models of mildly zinc-deficient (ZD) human subjects before and after zinc supplementation: (a) two human volunteers in whom a specific and mild zinc deficiency was induced by dietary means; (b) six mildly ZD adult sickle cell anemia (SCA) subjects; and (c) six mildly ZD adult non-SCA subjects. Their plasma zinc levels were normal and they showed no overt clinical manifestations of zinc deficiency. The diagnosis of mild zinc deficiency was based on the assay of zinc in lymphocytes, granulocytes, and platelets. Serum thymulin activity was decreased as a result of mild zinc deficiency and was corrected by in vivo and in vitro zinc supplementation, suggesting that this parameter was a sensitive indicator of zinc deficiency in humans. An increase in T101-, sIg-cells, decrease in T4+/T8+ ratio, and decreased IL 2 activity were observed in the experimental human model during the zinc depletion phase, all of which were corrected after repletion with zinc. Similar changes in lymphocyte subpopulation, correctable with zinc supplementation, were also observed in mildly ZD SCA subjects. Inasmuch as thymulin is known to induce intra- and extrathymic T cell differentiation, our studies provide a possible mechanism for the role of zinc on T cell functions.

Authors

A S Prasad, S Meftah, J Abdallah, J Kaplan, G J Brewer, J F Bach, M Dardenne

Cultured human endothelial cells stimulated with cytokines or endotoxin produce an inhibitor of leukocyte adhesion.

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Activation of cultured human endothelial cells (HEC) by inflammatory stimuli, such as interleukin 1 (IL-1), tumor necrosis factor (TNF), and bacterial endotoxin (lipopolysaccharide, LPS), increases their surface adhesiveness for blood leukocytes and related cell lines. We now report that activated HEC also generate a soluble leukocyte adhesion inhibitor (LAI), which accumulates in conditioned media from IL-1-, TNF-, or LPS-treated, but not sham-treated, HEC cultures. LAI significantly inhibits the adhesion of PMN and monocytes to activated, but not unactivated, HEC. In contrast, LAI has no effect on the adhesion of lymphocytes, the promyelocytic cell line HL-60 or the monocyte-like cell line U937 to HEC monolayers. LAI appears to act directly on the leukocyte, but does not inhibit either agonist-induced responses in PMN (membrane depolarization, changes in cytosolic calcium concentration, superoxide production) or PMN attachment to serum-coated plastic surfaces. Endothelial generation of LAI is blocked by actinomycin D but not by aspirin or indomethacin. Preliminary biochemical characterization indicates that LAI is a soluble, protein-containing molecule that is heat- and acid-stable. Fractionation by HPLC gel filtration yields a single peak of LAI activity (14,000 less than Mr greater than 24,000). Thus, in addition to proadhesive cell surface changes, the endothelium may also actively contribute to the regulation of endothelial-leukocyte interactions at sites of inflammation in vivo through the production of soluble adhesion inhibitors such as LAI.

Authors

M E Wheeler, F W Luscinskas, M P Bevilacqua, M A Gimbrone Jr

Interleukin 1 stimulates its own receptor expression on human fibroblasts through the endogenous production of prostaglandin(s).

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The regulation of interleukin 1 receptor (IL 1R) expression on human dermal fibroblasts was investigated. On exposure to IL 1 for 3 h at 37 degrees C, the capacity of fibroblasts to bind 125I-labeled human recombinant IL 1 alpha (125I-IL 1 alpha) was reduced by 75%. The IL 1 binding capability of the fibroblasts was restored to control levels by 16 h after removal of unbound IL 1, and then increased to about twofold over that of control cells by 48 h. This later enhancement of IL 1 receptor expression after IL 1 treatment was abolished by indomethacin. Addition of exogenous (PGE1 and PGE2, also analogues of AMP, or forskolin increased the specific binding of 125I-IL 1 alpha to fibroblasts. Scatchard analysis indicated that PGE2 increased the number of IL 1R from approximately 1.6 X 10(3) to 5.4 X 10(3) per cell without change in the binding affinity. These data suggest that the later IL 1-induced up-regulation of IL 1R is mediated by IL 1 stimulation of endogenous prostaglandin production. The combination of PGE2 and prednisolone increased the number of IL 1R on fibroblasts in an additive manner.

Authors

T Akahoshi, J J Oppenheim, K Matsushima

Platelets mediate neutrophil-dependent immune complex nephritis in the rat.

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Neutrophils and platelets are frequently present in glomeruli in immune glomerulonephritis (GN). No role for the platelet in acute neutrophil-mediated renal injury has been defined. We investigated a neutrophil-mediated model of subendothelial immune complex GN in the rat. Rats were platelet-depleted (mean platelet less than 10,000/microliter) with goat anti-platelet IgG before induction of GN by the renal artery perfusion of concanavalin A followed by anti-concanavalin A IgG. Platelet-depletion resulted in a significant reduction in albuminuria (7 +/- 2 vs. 55 +/- 10 mg/24 h) and fractional albumin excretion (0.045 +/- 0.01 vs. 0.410 +/- 0.09) compared with controls. The decrease in albuminuria was not due to differences in blood or glomerular neutrophil counts, complement, renal function, or glomerular antibody binding. Platelet-depleted rats had equivalent subendothelial deposits and glomerular endothelial cell injury but had minimal platelet infiltrates and fibrin deposition compared with controls. These studies demonstrate a role for platelets in mediating acute neutrophil-induced glomerular injury and proteinuria in this model of GN.

Authors

R J Johnson, C E Alpers, P Pritzl, M Schulze, P Baker, C Pruchno, W G Couser

Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor.

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Activated protein C (APC) acts as a potent anticoagulant enzyme by inactivating Factor V and Factor VIII. In this study, protein S was shown to increase the inactivation of purified Factor VIII by APC ninefold. The reaction rate was saturated with respect to the concentration of protein S when protein S was present in a 10-fold molar excess over APC. The heavy chain of Factor VIII was cleaved by APC and protein S did not alter the degradation pattern. Factor VIII circulates in a complex with the adhesive protein von Willebrand factor. When purified Factor VIII was recombined with von Willebrand factor, the inactivation of Factor VIII by APC proceeded at a 10-20-fold slower rate as compared with Factor VIII in the absence of von Willebrand factor. Protein S had no effect on the inactivation of the Factor VIII-von Willebrand factor complex by APC. After treatment of this complex with thrombin, however, the actions of APC and protein S towards Factor VIII were completely restored. In hemophilia A plasma, purified Factor VIII associated with endogenous von Willebrand factor, resulting in a complete protection against APC (4 nM). By mixing hemophilic plasma with plasma from a patient with severe von Willebrand's disease, we could vary the amount of von Willebrand factor. 1 U of von Willebrand factor was needed to provide protection of 1 U Factor VIII. Also in plasma from patients with the IIA-type variant of von Willebrand's disease, Factor VIII was protected. In von Willebrand's disease plasma, which was depleted of protein S, APC did not inactivate Factor VIII. These results indicate that protein S serves as a cofactor in the inactivation of Factor VIII and Factor VIIIa by APC and that von Willebrand factor can regulate the action of these two anticoagulant proteins.

Authors

J A Koedam, J C Meijers, J J Sixma, B N Bouma

Gamma heavy chain disease in man. Genomic sequence reveals two noncontiguous deletions in a single gene.

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A genomic clone was isolated from a human lymphoid cell line which synthesized an NH2-terminally deleted gamma 3 heavy chain disease protein. Nucleotide sequence analysis revealed a normal sequence from 310 bp 5' to the initiator ATG through the codon for VH amino acid 14. Amino acid 15 was derived from the codon for the last J4 amino acid. Thus, the clone contained a deletion of the codons for the VH region beyond amino acid 14, as well as those for the entire D region and most of the J coding region. Some sequence abnormalities were observed in the 400 bp after the deletion. Beyond this, there was excellent homology to published J and intervening sequences, including those containing the enhancer elements. The 1,200-bp switch region was abruptly interrupted by a sequence corresponding to the 3' one-third of CH1. Thus, a second deletion eliminated the acceptor splice site at the 5' end of CH1. When splicing of the primary RNA transcript occurred, the truncated VH region was joined via the J4 donor splice site to the next available acceptor site 5' to the first hinge exon. Hence, the aberrant serum protein was the product of two deletions and a splice correction as well as postsynthetic NH2-terminal proteolysis.

Authors

A Alexander, I Anicito, J Buxbaum

Glomerular macrophages express augmented procoagulant activity in experimental fibrin-related glomerulonephritis in rabbits.

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Glomerular fibrin deposition and augmentation of procoagulant activity (PCA) are dependent on glomerular macrophage infiltration in anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM GN) in rabbits. Expression of PCA on the surface of glomerular macrophages and/or augmentation of intrinsic glomerular cell PCA by macrophage cytokines (such as IL 1) are potential mechanisms by which macrophages may augment glomerular PCA. Macrophages were isolated from glomeruli of rabbits developing anti-GBM GN to measure their PCA expression. These macrophages were characterized by morphological and functional criteria. Glomerular macrophages expressed markedly augmented PCA (2.8 +/- 0.7 mU/10(3) cells) compared with blood monocytes (0.05 +/- 0.02 mU/10(3) cells) and alveolar macrophages (0.09 +/- 0.02 mU/10(3) cells) from the same rabbits. Glomerular macrophage PCA was functionally identical to the PCA of whole glomeruli, and was consistent with that of tissue factor. Supernatants from nephritic glomeruli contained IL 1 bioactivity and augmented endothelial cell PCA in vitro. However, these supernatants and purified IL 1 failed to augment the PCA of normal and macrophage-depleted nephritic glomeruli. These studies demonstrate that, in this model of anti-GBM GN, glomerular macrophages contribute directly to the augmented glomerular PCA by their expression of surface membrane PCA, and have the potential to indirectly augment glomerular PCA by their production of cytokines capable of enhancing endothelial cell PCA.

Authors

P G Tipping, M G Lowe, S R Holdsworth

Alveolar fluid neutrophil elastase activity in the adult respiratory distress syndrome is complexed to alpha-2-macroglobulin.

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We characterized the elastase and antielastase activity of the alveolar fluid of seven patients with the adult respiratory distress syndrome (ARDS) and thirteen normal volunteers. Alpha-1-antitrypsin (A1AT) concentrations were 60-fold higher in ARDS as compared to normal lavage fluid (2,140 +/- 498 nM; 36.1 +/- 4.2 nM, respectively). ARDS fluid antineutrophil elastase activity was also considerably higher than that of normals (979 +/- 204 nM; 31.3 +/- 2.9 nM, respectively). Despite the antineutrophil elastase excess, 5 of 7 ARDS lavage samples contained elastase activity (mean, 6.1 +/- 2.4 pM) as assayed using low-molecular-mass substrate, while only 1 of 13 normal subjects had detectable elastase activity (0.2 pM) (P less than 0.01, compared with ARDS). That this activity was due to alpha-2-macroglobulin (A2MG)-complexed neutrophil elastase was evidenced by (a) the Sephadex G-75 elution profile; (b) the inactivity against insoluble [3H]elastin; (c) the inhibitory profile with phenylmethylsulfonyl fluoride, methoxy-succinyl-alanyl-alanyl-prolyl-valyl-chloromethylketone, ethylene diamine tetraacetic acid, and A1AT; and (d) the immobilization by A2MG antibody bound to polystyrene plates. Furthermore, in agreement with the predicted affinity of A1AT and A2MG for neutrophil elastase, the ratio of A2MG to A1AT in the fluid (0.57%) coincided with the ratio of the A2MG- to A1AT-complexed elastase (0.36%). These findings suggest that the net lung protease-antiprotease balance in ARDS is shifted largely in favor of the antiproteases (chiefly A1AT), and that the antiproteases, A1AT and A2MG, have similar affinities for neutrophil elastase in vivo.

Authors

M D Wewers, D J Herzyk, J E Gadek

Bone turnover in postmenopausal osteoporosis. Effect of calcitonin treatment.

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To investigate the effectiveness of calcitonin treatment of postmenopausal osteoporosis in relation to bone turnover, we examined 53 postmenopausal osteoporotic women before and after one year of therapy with salmon calcitonin (sCT), at the dose of 50 IU every other day. Baseline evaluation revealed that 17 (32%) patients had high turnover (HTOP), and 36 (68%) normal turnover osteoporosis (NTOP) as assessed by measurement of whole body retention (WBR) of 99mTc-methylene diphosphonate. The two groups did not differ in terms of bone mineral content (BMC) measured by dual photon absorptiometry at both lumbar spine and femoral diaphysis. However, HTOP patients had higher levels of serum osteocalcin (OC) and urinary hydroxyproline excretion (HOP/Cr). Multivariate regression analysis showed no correlation between parameters of bone turnover (WBR, OC, HOP/Cr) and both femoral and vertebral bone density; the latter being negatively correlated only with the years elapsed since menopause (R2 = 0.406). Treatment with sCT resulted in a significant increase of vertebral BMC in the 53 patients taken as a whole group (+/- 7%, P less than 0.001). When the results obtained in HTOP and NTOP were analyzed separately, only those with HTOP showed a marked increment of spinal BMC (+22%, P less than 0.001), NTOP subjects neither gained nor lost bone mineral during the study. Femoral BMC decreased in the whole group after sCT therapy (-3%, P less than 0.003). However, HTOP patients maintained initial BMC values, whereas those with NTOP lost a significant amount of bone during the study period (-5%, P less than 0.001). The increase of vertebral bone mass was associated with a marked depression of bone turnover detectable in both subsets of patients and in the whole group. In conclusion: (a) assessment of bone turnover cannot help predict the severity of bone loss in postmenopausal osteoporosis; (b) calcitonin therapy appears to be particularly indicated for patients with high-turnover osteoporosis, resulting in a net gain of bone mineral in the axial skeleton and a slowing of bone loss in the appendicular bones.

Authors

R Civitelli, S Gonnelli, F Zacchei, S Bigazzi, A Vattimo, L V Avioli, C Gennari

Gene for the rat atrial natriuretic peptide is regulated by glucocorticoids in vitro.

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Glucocorticoids regulate the expression of the gene for atrial natriuretic peptide (ANP) in neonatal cardiocytes. Dexamethasone (Dex) increased cytoplasmic ANP mRNA levels and media ANP immunoreactivity in a dose-dependent fashion. These effects were not shared by the other classes of steroid hormones and were reversed by the glucocorticoid antagonist RU 38486. The effect on ANP mRNA levels resulted, at least in part, from enhanced transcription of the gene. Dex effected a two-fold increase in ANP gene activity assessed using a run-on transcription assay. The turnover of the ANP transcript was approximated using a standard pulse-chase technique. The half-life of the ANP mRNA was 18 h in hormone-free media. In the presence of Dex this half-life increased modestly to 30 h, although the increase relative to the control did not reach statistical significance. The effect of Dex at the level of the individual myocardial cell was assessed by in situ hybridization analysis using a specific [3H]cRNA probe. These studies demonstrated a significant level of ANP expression within a subpopulation of cells in the cultures. Exposure of the cells to Dex for 24 h did not recruit additional cells into the expressing pool (27.3% cells/high power field vs. 31.3% for the control) but did increase the level of expression (i.e., grain density) within individual cells. These findings indicate that glucocorticoids stimulate expression of the ANP gene directly at the level of the myocardial cell. This results predominantly from transcriptional activation in cells already expressing the gene rather than through recruitment of previously quiescent cells.

Authors

D G Gardner, B J Gertz, C F Deschepper, D Y Kim

Human recombinant granulocyte-macrophage colony stimulating factor and interleukin 3 have overlapping but distinct hematopoietic activities.

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The hematopoietic stimulatory activities of human recombinant IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF) were directly compared using highly enriched human bone marrow progenitor target cells. IL-3 supported a larger number of erythroid and megakaryocytic progenitor cells than did GM-CSF, while GM-CSF supported more myeloid progenitors. IL-3 directly stimulated the division and migration of primitive erythroid burst forming units, while GM-CSF merely sustained their net survival in culture without promoting division and expansion. IL-3 promoted the formation of larger numbers of multipotential granulocyte-erythroid-macrophage-megakaryocyte colony forming unit--derived colonies than did GM-CSF. These data indicate that human IL-3 and GM-CSF have overlapping but distinct hematopoietic activities, and suggest a potential role for the clinical application of combined IL-3/GM-CSF therapy.

Authors

S G Emerson, Y C Yang, S C Clark, M W Long

Defective expression of the 2H4 molecule after autologous mixed lymphocyte reaction activation in systemic lupus erythematosus patients.

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Previous studies demonstrated that patients with active systemic lupus erythematosus (SLE), especially those with active renal disease, had a marked reduction in T4+2H4+ suppressor inducer cells in their peripheral blood. However, it was puzzling to find that active SLE patients without renal diseases often had normal percentages of T4+2H4+ cells. In the present study, we attempted to determine whether active SLE patients bearing normal percentages of T4+2H4+ cells had a defect in their expression of the 2H4 molecule on T4+ cells after autologous mixed lymphocyte reaction (AMLR) activation. The peripheral blood lymphocytes (PBL) from 50 SLE patients with normal percentages of T4+2H4+ cells (greater than or equal to 7% in PBL) were studied and the results were compared with those of 40 normal individuals. The density of the 2H4 molecule on T4 cells from normal controls increased during the 7-d AMLR; in contrast T4 cells from patients with SLE, especially those with active SLE, had defective expression of the 2H4 antigen after AMLR activation. Patients with inactive SLE, like normals, showed an increase in the 2H4 molecule after AMLR activation. Moreover, a strong correlation was observed between percent suppression of pokeweed mitogen (PWM)-driven IgG synthesis and the density of the 2H4 antigen on AMLR-activated T4 cells. Serial analysis of patients with SLE showed that the density of the 2H4 antigen expression and the suppressor inducer activity of AMLR-activated T4 cells were inversely correlated with disease activity. Thus, defective expression of the 2H4 antigen may be an important mechanism for the failure of active SLE patients with normal percentages of T4+2H4+ cells to generate suppression.

Authors

T Takeuchi, S Tanaka, A D Steinberg, T Matsuyama, J Daley, S F Schlossman, C Morimoto

A juxta-membrane epitope on the human acetylcholine receptor recognized by T cells in myasthenia gravis.

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T cell proliferative responses to synthetic peptides taken from the human nicotinic acetylcholine receptor (AChR) alpha-chain sequence, or to whole AChR purified from electric fish (Torpedo marmorata), have been studied, using blood, thymus, and lymph node cells, from 34 patients with myasthenia gravis (MG) and 17 controls mostly with other neurological diseases. Peptides were selected because they contained amino acid motifs that recur in most defined T cell epitopes. Peptide 257-269 (from the extracellular loop of the AChR alpha-chain between the second and third trans-membrane domains) stimulated cells from six patients and no controls. Peptides from region 125-143 (from the main extracellular 1-210 stretch), which is thought to be an important T cell epitope in rats, provoked responses in 26% of patients and 41% of controls. Two patients responded both to these peptides and to peptide 257-269, thereby implying some heterogeneity of their reacting T cells. Whereas the initial blood T cell samples sometimes responded both to Torpedo AChR and to the 125-143 peptides, T cell lines selected with either antigen subsequently showed no response to the other. This observation suggests that it may be essential to use human AChR sequences for studying truly autoreactive T cells in MG. Finally, no strong association was found between any of the responses to peptides and the HLA types of the responding individuals.

Authors

G C Harcourt, N Sommer, J Rothbard, H N Willcox, J Newsom-Davis

Sympathetic nerve discharge is coupled to muscle cell pH during exercise in humans.

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We used phosphorus nuclear magnetic resonance spectroscopy (31P-NMR) to probe the cellular events in contracting muscle that initiate the reflex stimulation of sympathetic outflow during exercise. In conscious humans, we performed 31P-NMR on exercising forearm muscle and simultaneously recorded muscle sympathetic nerve activity (MSNA) with microelectrodes in the peroneal nerve to determine if the activation of MSNA is coupled to muscle pH, an index of glycolysis, or to the concentrations (II) of inorganic phosphate (Pi) and adenosine diphosphate (ADP) which are modulators of mitochondrial respiration. During both static and rhythmic handgrip, the onset of sympathetic activation in resting muscle coincided with the development of cellular acidification in active muscle. Furthermore, increases in MSNA were correlated closely with decreases in intracellular pH but dissociated from changes in phosphocreatine [( PCr]), [Pi], and [ADP]. The principal new conclusion is that activation of muscle sympathetic outflow during exercise in humans is coupled to the cellular accumulation of protons in contracting muscle.

Authors

R G Victor, L A Bertocci, S L Pryor, R L Nunnally

Kappa/lambda immunoglobulin distribution in Graves' thyroid-stimulating antibodies. Simultaneous analysis of C lambda gene polymorphisms.

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From patients with untreated Graves' disease 11 sera showing high cAMP release in the FRTL-5 cell assay were studied for relative proportions of kappa or lambda Ig molecules showing cAMP releasing activity. Immunoabsorption of gamma-globulins was performed using monoclonal murine anti-kappa or anti-lambda antibodies linked to cyanogen bromide-activated sepharose. Specific kappa- or lambda-adsorbed fractions were also eluted from immunoabsorbents using chaotrophic thiocyanate buffers and equilibrated with pH 7.4 low salt buffer by dialysis. Immunoabsorption and elution experiments showed that five Graves' sera contained predominant cAMP-releasing activity within lambda Ig fractions, whereas two Graves' sera showed predominant cAMP-releasing activity in kappa Ig fractions. Four sera showed cAMP release approximately equally divided between kappa and lambda Ig both after immunoabsorption and specific anti-kappa or anti-lambda eluates were studied. C lambda genotypes were examined by Southern blotting and restriction fragment length polymorphism analysis of Eco RI-digested genomic DNA from 158 patients with Graves' disease in parallel with 112 normal controls and 29 patients with autoimmune hypothyroidism. Notable shifts in proportions of 8/8 and 18/18 genotypes were present when Graves' patients were compared with normal controls. Allelic frequencies and ratios of genotype 8 to 18 were significantly different (P less than 0.05) when Graves' patients were compared either to normal controls or to patients with autoimmune hypothyroidism.

Authors

R C Williams Jr, N J Marshall, K Kilpatrick, J Montano, P M Brickell, M Goodall, P A Ealey, B Shine, A P Weetman, R K Craig

Epidermal growth factor inhibits the hydroosmotic effect of vasopressin in the isolated perfused rabbit cortical collecting tubule.

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Epidermal growth factor (EGF) is a 53-amino acid polypeptide which is a potent mitogen for cultured cells. The kidney has recently been shown to be a major site of synthesis for the EGF precursor. EGF infusions in sheep result in a diuresis and natriuresis despite a fall in GFR, suggesting a direct tubular effect. Using in vitro microperfusion of rabbit cortical collecting tubules (CCTs) at 37 degrees C, we examined the effect of EGF on the transepithelial voltage (Vt) and arginine vasopressin (AVP)-stimulated hydraulic conductivity (Lp). Pretreatment with peritubular EGF at concentrations from 10(-8) to 10(-12) M resulted in a 50% inhibition of both AVP- and 8-chlorophenythio-cyclic AMP-stimulated peak Lp. This effect was reversed by the protein kinase C inhibitor, staurosporine, but unaffected by indomethacin. CCTs with an initially negative Vt, depolarized after exposure to bath EGF. 10(-8) M EGF applied from the lumen had no effect on either Lp or Vt. Specific binding of 20 nM 125I-EGF to microdissected CCTs was also demonstrated. These results suggest that EGF can modulate both salt and water transport in the CCT via a receptor linked to protein kinase C activation.

Authors

M D Breyer, H R Jacobson, J A Breyer

Tumor necrosis factor and the acute metabolic response to tissue injury in man.

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Tumor necrosis factor (cachectin), a protein produced by monocytes and macrophages, has been implicated as an important mediator of the lethal effects of endotoxic shock and the cachexia of chronic infection. Recombinant human tumor necrosis factor alpha (rTNF) was given intravenously to patients as part of an antineoplastic trial. Fever, tachycardia, and at higher doses, hypotension occurred after a single injection of rTNF. Metabolic effects after rTNF administration were dose related and included enhanced energy expenditure with elevated CO2 production, increased whole body protein metabolism and peripheral amino acid efflux from the forearm, and decreased total arterial amino acid levels associated with a significant increase in plasma cortisol. Elevated serum triglycerides, as well as increased glycerol and free fatty acid turnover were seen, suggesting increased whole body lipolysis and fat utilization after rTNF. These findings indicate that administration of TNF in man reproduces many of the acute physiologic and metabolic responses to tissue injury, including energy substrate mobilization.

Authors

H F Starnes Jr, R S Warren, M Jeevanandam, J L Gabrilove, W Larchian, H F Oettgen, M F Brennan

Adenosine triphosphate depletion induces a rise in cytosolic free calcium in canine renal epithelial cells.

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Abstract

An elevation in cytosolic free calcium (Cai) produced by cellular ATP depletion may contribute to the initiation of cytotoxic events in renal ischemia. To evaluate whether ATP depletion results in a rise in Cai we examined the effect of cyanide and 2-deoxy-D-glucose on the Cai of Madin-Darby canine kidney cells. Exposure to the metabolic inhibitors resulted in a rise in Cai from 112 +/- 11 to 649 +/- 99 nM in 15 min. This combination of metabolic inhibitors also resulted in a decrement of cell ATP to 11 +/- 2% of control by 15 min. Experiments that were performed with other metabolic inhibitors confirm that the increment in Cai is due to inhibition of ATP synthesis. With the removal of cyanide and 2-deoxy-D-glucose, Cai recovered to 101 +/- 16 nM. In the absence of extracellular calcium activity (Ca0), Cai declined from 127 +/- 7 to 38 +/- 6 nM, whereas with cyanide plus 2-deoxy-D-glucose in the absence of Ca0 the Cai rose from 108 +/- 21 to 151 +/- 28 nM. Because the rise in Cai produced by ATP depletion in the absence of Ca0 is significantly less than that which occurs in the presence of Ca0, influx of Ca0 is necessary for the maximal rise of Cai. The rise in Cai that occurred in the absence of Ca0 suggests that the release of calcium from intracellular stores contributes to the increment in Cai seen with ATP depletion. TMB-8, an inhibitor of calcium release from intracellular stores, blunted the rise in Cai by nearly 50%. Neither verapamil nor nifedipine inhibited the rise in Cai. This study demonstrates that ATP depletion induced by the metabolic inhibitors cyanide and 2-deoxy-D-glucose is associated with a rapid and reversible increase in Cai. Both Ca0 influx and Cai redistribution contribute to this rise.

Authors

C E McCoy, A M Selvaggio, E A Alexander, J H Schwartz

Inhibition of the release of arachidonic acid prevents the development of sarcolemmal membrane defects in cultured rat myocardial cells during adenosine triphosphate depletion.

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Abstract

Previous studies have suggested that phospholipid degradation is closely associated with the development of sarcolemmal membrane injury. This study was initiated to characterize the effects of synthetic inhibitors of phospholipase activities using a cultured myocardial cell model in which arachidonic acid is liberated after treatment with the metabolic inhibitor, iodoacetate. Pretreatment with a steroidal diamine (U26,384) blocked the degradation of labeled phosphatidylcholine and the release of arachidonic acid in cultured myocardial cells during ATP depletion. Inhibition of phospholipid degradation by U26,384 prevented the development of sarcolemmal membrane defects and the release of creatine kinase from the cultured myocardial cells during ATP depletion. Pretreatment with U26,384 had no significant effect on the extent of ATP depletion after iodoacetate treatment, which indicates that the activity of this compound could not be simply ascribed to a sparing effect on ATP concentration. These results support the hypothesis that the development of sarcolemmal membrane injury and the associated loss of cell viability are causally related to progressive phospholipid degradation. In addition, these studies indicate that the release of arachidonic acid during ATP depletion is associated with the net loss of the phosphatidylcholine molecule.

Authors

A Sen, J C Miller, R Reynolds, J T Willerson, L M Buja, K R Chien

Haemophilus influenzae lipopolysaccharide-induced blood brain barrier permeability during experimental meningitis in the rat.

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Abstract

The factors responsible for blood-brain barrier (BBB) injury during bacterial meningitis are incompletely defined. We evaluated the role of Haemophilus influenzae type b (Hib) lipopolysaccharide (LPS) in the alteration of blood-brain barrier permeability (BBBP) in an adult, normal and leukopenic, rat model of meningitis. Intracisternal inoculation of Hib LPS resulted in (a) dose-dependent increases in BBBP from 2 pg to 20 ng, with significant attenuation in the peak response after challenge with 500 ng and 1 microgram; (b) time-dependent increases in BBBP, with a delayed onset of at least 2 h, maximum alteration at 4 h, and complete reversal at 18 h; (c) greater BBBP than after challenge with the live parent strain; (d) and a close correlation (r = 0.86) between CSF pleocytosis and BBBP at 4 h. The LPS effect was significantly inhibited by preincubation with Polymyxin B and neutrophil acyloxyacyl hydrolase, however two different oligosaccharide-specific monoclonal antibodies did not inhibit activity. No change in BBBP after inoculation with Hib LPS occurred in leukopenic rats. Hib LPS, in the setting of an intact leukocyte response, exerts profound effects on BBBP.

Authors

B Wispelwey, A J Lesse, E J Hansen, W M Scheld

Evaluation of the antiinflammatory and phospholipase-inhibitory activity of calpactin II/lipocortin I.

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Abstract

We have examined the ability of a highly purified 38-kD phospholipase-inhibitory protein (p38) isolated from human placental membranes that is also a preferred substrate for the epidermal growth factor-urogastrone (EGF-URO) receptor/kinase, to block the release of arachidonate from zymosan-stimulated murine peritoneal macrophages in vitro and to exhibit antiinflammatory activity in a carrageenin rat paw edema test in vivo. The ability of glucocorticoids to increase the amounts of this protein in macrophage cultures was also examined. p38 represents the naturally occurring, intact, NH2-terminally blocked human placental form of the protein termed calpactin II (or lipocortin I), for which partial amino acid sequence data and a complete amino acid sequence deduced from cDNA analysis have been reported. Our data demonstrated that, whereas p38 was an effective inhibitor of pancreatic phospholipase A2 in vitro, it was unable to inhibit either the release of arachidonate from cultured zymosan-stimulated mouse peritoneal macrophages or inflammation in a rat paw edema test. At comparatively high protein concentrations, p38 enhanced either arachidonate release from intact macrophages in vitro (0.5-10 micrograms/ml) or carrageenin-induced paw swelling in vivo (2.5 or 25 micrograms per injection). Furthermore, we were unable to detect induced amounts of p38 in cultures of glucocorticoid-treated peritoneal macrophages obtained from either mice or rats. Our data indicate that the antiphospholipase activity of p38 in vitro and the ability of p38 to serve as a receptor/kinase substrate may in no way relate to the putative ability of the protein to modify eicosanoid release from macrophages in vivo, so as to modulate the inflammatory process. Our data also raise the possibility that p38 (calpactin II) may not be a true representative of the lipocortin family of glucocorticoid-inducible antiinflammatory proteins, despite its ability to inhibit phospholipase A2 in vitro.

Authors

J K Northup, K A Valentine-Braun, L K Johnson, D L Severson, M D Hollenberg

Characterization of point mutations in the same arginine codon in three unrelated patients with ornithine transcarbamylase deficiency.

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Abstract

Point mutations in the X-linked ornithine transcarbamylase (OTC) gene have been detected at the same Taq I restriction site in 3 of 24 unrelated probands with OTC deficiency. A de novo mutation could be traced in all three families to an individual in a prior generation, confirming independent recurrence. The DNA sequence in the region of the altered Taq I site was determined in the three probands. In two unrelated male probands with neonatal onset of severe OTC deficiency, a guanine (G) to adenine (A) mutation on the sense strand (antisense cytosine [C] to thymine [T]) was found, resulting in glutamine for arginine at amino acid 109 of the mature polypeptide. In the third case, where the proband was a symptomatic female, C to T (sense strand) transition converted residue 109 to a premature stop. These results support the observation that Taq I restriction sites, which contain an internal CG, are particularly susceptible to C to T transition mutation due to deamination of a methylated C in either the sense or antisense strand. The OTC gene seems especially sensitive to C to T transition mutation at arginine codon 109 because either a nonsense mutation or an extremely deleterious missense mutation will result.

Authors

A Maddalena, J E Spence, W E O'Brien, R L Nussbaum

Insulin receptor function in fibroblasts from patients with leprechaunism. Differential alterations in binding, autophosphorylation, kinase activity, and receptor-mediated internalization.

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Abstract

Insulin receptor function was examined in cultured skin fibroblasts from three patients with leprechaunism (Ark-1, Minn-1, and Can-1), a rare syndrome of severe insulin resistance and neonatal growth retardation. All three patients cell lines demonstrated insulin binding less than 15% of control. This was primarily due to reduced affinity of the receptor in Can-1 and due to reduced number of receptors in the other two cell lines (Ark-1 and Minn-1). When expressed as a fraction of total insulin bound, the percentage of cell-associated insulin internalized and degraded did not differ between the patient cell lines and the controls. However, chloroquine, which inhibited degradation by 50% in the control cells, had no effect in the cells from the patients. When normalized to insulin binding, insulin receptor autophosphorylation was normal in cells from Can-1, but reduced in those of Ark-1 and Minn-1. In contrast, the receptor-associated tyrosine kinase activity toward exogenous substrates was decreased in all three patient cell lines. These results suggest that leprechaunism is a biochemically heterogenous disease associated with a variety of alterations in receptor function. Cells from Ark-1 and Minn-1 exhibit parallel alterations in receptor autophosphorylation and kinase activity. Cells from Can-1 demonstrate normal receptor autophosphorylation but reduced kinase activity, thus displaying a unique form of a mutant insulin receptor. Despite reduced kinase activity, all three cell lines exhibit normal rates of insulin internalization, but decreased lysosomal-mediated degradation. Our data imply that receptor autophosphorylation and kinase activity may be regulated separately and that kinase activity may be linked to insulin degradation, but not necessarily internalization.

Authors

S S Reddy, V Lauris, C R Kahn

5-(N,N-dimethyl)amiloride-sensitive Na-Li exchange in isolated specimens of human atrium.

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Abstract

To examine if a transmembrane Na-Li exchange similar to that reported to occur in human blood cells can be demonstrated in the heart, we incubated specimens of human atrium in cold (2-3 degrees C) Li-Tyrode's solution. The Li-loaded, Na-depleted specimens were then transferred to warm (30 degrees C) Na-Tyrode's solution. After transfer the membrane potential hyperpolarized to a level more negative than the equilibrium potential for K+. The hyperpolarization was inhibited by acetylstrophanthidin or K+-free solution indicating that it was due to current produced by the Na, K-pump responding to a Na load. This suggested that intracellular Li+ had been exchanged for Na+. The hyperpolarization was abolished by 10 microM 5-(N,N-dimethyl)amiloride while 10 microM bumetanide had no effect, findings that are consistent with the notion that the exchange of intracellular Li+ for extracellular Na+ occurs via an operational mode of the Na-H exchanger rather than being mediated through a mechanism involving the Na/K/2Cl cotransporter.

Authors

H H Rasmussen, R D Harvey, E J Cragoe Jr, R E ten Eick

Botulinum C2 toxin ADP-ribosylates actin and enhances O2- production and secretion but inhibits migration of activated human neutrophils.

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Abstract

The binary botulinum C2 toxin ADP-ribosylated the actin of human neutrophils. Treatment of human neutrophils with botulinum C2 toxin for 45 min increased FMLP-stimulated superoxide anion (O2-) production 1.5-5-fold, whereas only a minor fraction of the cellular actin pool (approximately 20%) was ADP-ribosylated. Effects of botulinum C2 toxin depended on toxin concentrations, presence of both components of the toxin, and incubation time. Cytochalasin B similarly enhanced O2- production. The effects of botulinum C2 toxin and cytochalasin B were additive at submaximally, but not maximally effective concentrations and incubation time of either toxin. Botulinum C2 toxin also enhanced stimulation of O2- production by Con A and platelet-activating factor, but not by phorbol 12-myristate 13-acetate (PMA). Botulinum C2 toxin increased FMLP-induced release of N-acetyl-glucosaminidase by 100-250%; release of vitamin B12-binding protein induced by FMLP and PMA was enhanced by approximately 150 and 50%, respectively. Botulinum C2 toxin blocked both random migration of neutrophils and migration induced by FMLP, complement C5a, leukotriene B4, and a novel monocyte-derived chemotactic agent. The data suggest that botulinum C2 toxin-catalyzed ADP-ribosylation of a minor actin pool has a pronounced effect on the activation of human neutrophils by various stimulants.

Authors

J Norgauer, E Kownatzki, R Seifert, K Aktories

Atrial natriuretic factor inhibits vasopressin-stimulated osmotic water permeability in rat inner medullary collecting duct.

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Abstract

The inner medullary collecting duct (IMCD) has been proposed to be a site of atrial natriuretic factor (ANF) action. We carried out experiments in isolated perfused terminal IMCDs to determine whether ANF (rat ANF 1-28) affects either osmotic water permeability (Pf) or urea permeability. In the presence of a submaximally stimulating concentration of vasopressin (10(-11) M), ANF (100 nM) significantly reduced Pf by an average of 46%. Lower concentrations of ANF also significantly inhibited vasopressin-stimulated Pf by the following percentages: 0.01 nM ANF, 18%; 0.1 nM, 46%; 1 nM, 48%. Addition of exogenous cyclic GMP (0.1 mM) mimicked the effect of ANF, decreasing Pf by an average of 48%. ANF also inhibited cyclic AMP-stimulated Pf by an average of 31%. ANF did not affect urea permeability, nor did it alter vasopressin-stimulated cyclic AMP accumulation. We conclude that ANF at physiological concentrations causes a large inhibition of vasopressin-stimulated Pf in the rat terminal IMCD, and that cyclic GMP is the second messenger mediating the effect. ANF appears to act at a site distal to cyclic AMP generation in the chain of events linking vasopressin receptor binding to an increase in osmotic water permeability.

Authors

H Nonoguchi, J M Sands, M A Knepper

Structural and kinetic comparison of recombinant human single- and two-chain tissue plasminogen activator.

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Abstract

We examined the similarities and differences in conformation between recombinant human single-chain tissue plasminogen activator (sct-PA) and two-chain tissue plasminogen activator (tct-PA), and compared these structural data with measurement of enzymatic activity. The intrinsic protein fluorescence of native tct-PA was 54% that of sct-PA. Differences in steady state protein fluorescence were also noted with denaturation of these plasminogen activators, as well as in the quenching of intrinsic fluorescence of the reduced, alkylated species by iodide. Using the chromogenic substrate H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288), the catalytic efficiency of sct-PA was found to be 26% that of tct-PA, and this was primarily a reflection of the difference in Km. On addition of soluble fibrin monomer prepared with the tetrapeptide glycyl-L-prolyl-L-arginyl-L-proline (GPRP), the catalytic efficiency of both species increased by 13-fold for sct-PA and by 3.5-fold for tct-PA to approximately the same value. Using the fluorophore eosin iodoacetamide covalently coupled to the single free cysteine in the molecule, Cys 83, the microenvironment of the fibrin-binding site located near this residue was studied. On addition of soluble fibrin monomer to eosin-labeled tct-PA, no effect on eosin fluorescence was noted. Eosin-labeled tct-PA had 16% less eosin fluorescence than did sct-PA and on addition of soluble fibrin monomer to eosin-labeled sct-PA, a decrease in eosin fluorescence, approaching that of eosin coupled to tct-PA, was observed. Together, these structural and kinetic data suggest that sct-PA undergoes a conformational change on binding to fibrin monomer that leads to dramatic differences in catalytic efficiency of the single-chain species. In so doing, sct-PA bound to fibrin assumes the kinetic profile of tct-PA bound to fibrin.

Authors

J Loscalzo

Reversibility of defective adipocyte insulin receptor kinase activity in non-insulin-dependent diabetes mellitus. Effect of weight loss.

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Abstract

Insulin-stimulated kinase activity of adipocyte-derived insulin receptors is reduced in subjects with non-insulin-dependent diabetes mellitus (NIDDM) but normal in obese nondiabetics. To assess the reversibility of the kinase defect in NIDDM, insulin receptor kinase activity was measured before and after weight loss in 10 NIDDM and 5 obese nondiabetic subjects. Peripheral insulin action was also assessed in vivo by glucose disposal rates (GDR) measured during a hyperinsulinemic (300 mU/M2 per min) euglycemic clamp. In the NIDDMs, insulin receptor kinase activity was reduced by 50-80% and rose to approximately 65-90% (P less than 0.01) of normal after 13.2 +/- 2.0 kg (P less than 0.01) weight loss; comparable weight loss (18.2 +/- 1.5 kg, P less than 0.01) in the nondiabetics resulted in no significant change in insulin receptor kinase activity. Relative to GDR measured in lean nondiabetics, GDR in the NIDDMs was 35% of normal initially and 67% (P less than 0.01) of normal after diet therapy; weight loss in the nondiabetics resulted in an increase in GDR from 53 to 76% of normal (P less than 0.05). These results indicate that the insulin receptor kinase defect that is present in NIDDM is largely reversible after weight reduction. In contrast, the improvement in GDR, in the absence of any change in insulin receptor kinase activity in the nondiabetics, suggests that the main cause of insulin resistance in obesity lies distal to the kinase.

Authors

G R Freidenberg, D Reichart, J M Olefsky, R R Henry

Interaction of atriopeptin III and vasopressin on calcium kinetics and contraction of aortic smooth muscle cells.

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Abstract

The cellular mechanism of the vasodilatory action of atriopeptin III (APIII) on vasopressin (AVP)-induced Ca2+ mobilization and cell shape change in cultured vascular smooth muscle cells (VSMC) was studied. APIII (10(-8) M) attenuated the increase of intracellular free Ca2+, [Ca2+]i, induced by 10(-8) M AVP (234.0 +/- 14.8 vs. 310.0 +/- 28.4 nM, P less than 0.01). Similar results were obtained in 45Ca2+ efflux experiments. APIII (10(-7) M), however, did not alter AVP-induced inositol trisphosphate (IP3) production, although the levels of inositol-1-phosphate were significantly reduced. The effect of APIII to block or attenuate AVP-induced Ca2+ mobilization was associated with an inhibition of AVP-stimulated cell shape change. The effect of atrial natriuretic factor (ANF) on cell shape, however, occurred at lower ANF concentrations than the effect on the Ca2+ mobilization. APIII stimulated production of cyclic guanosine monophosphate (cGMP) in VSMC. The effect of APIII on AVP-stimulated Ca2+ mobilization was partially mimicked by the stable nucleotide 8-bromo cGMP and was not affected by the soluble guanylate cyclase inhibitor, methylene blue (10(-4) M). These results suggest that APIII exerts its vasodilatory effect, in part, by interference with vasopressor-stimulated Ca2+ mobilization in vascular smooth muscle cells, perhaps by stimulating particulate guanylate cyclase and cGMP. However, an effect of ANF on the contractile mechanism at a site independent of Ca2+ release is also suggested by the present results.

Authors

H Meyer-Lehnert, C Caramelo, P Tsai, R W Schrier

Downregulation of interferon alpha but not gamma receptor expression in vivo in the acquired immunodeficiency syndrome.

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Abstract

Interferons (IFN) elicit antiviral and antineoplastic activities by binding to specific receptors on the cell surface. In evaluating the role of IFN as therapeutic agents in AIDS, we investigated the expression of IFN alpha and gamma receptors on peripheral blood mononuclear cells (PBM) from patients with AIDS, ARC, and heterosexual control subjects using radioiodinated IFN alpha 2 and IFN gamma. The binding characteristics of the 125I-IFN alpha and gamma to PBM were analyzed to determine receptor numbers and dissociation constants. PBM from controls expressed 498 +/- 247 IFN alpha receptor sites/cell (n = 17). However, eight patients with ARC and seven patients with AIDS had a mean number of IFN alpha receptor/cell of 286 +/- 235 (P less than 0.05) and 92 +/- 88 (P less than 0.001), respectively. This was consistent with elevated levels of serum acid-labile IFN alpha and cellular 2-5A synthetase activity in patients. Treatment of PBM from the AIDS patients with exogenous IFN alpha in vitro resulted in minimal 2-5A synthetase induction in comparison to controls. In contrast, the expression of IFN gamma receptors in ARC (n = 5) and AIDS (n = 4) patients remained normal. Thus the decrease in IFN alpha receptor expression and consequent hyporesponsiveness to IFN alpha raises the question of the usefulness of IFN alpha therapy in end-stage AIDS. The normal expression of IFN gamma receptors in AIDS patients suggests that IFN gamma may prove useful in attempts to provide immune reconstitution.

Authors

A S Lau, S E Read, B R Williams

Relationship of pleural effusions to increased permeability pulmonary edema in anesthetized sheep.

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Abstract

We studied anesthetized sheep to determine the relationship between increased permeability pulmonary edema and the development and mechanism of pleural effusion formation. In 12 sheep with intact, closed thoraces, we studied the time course of pleural liquid formation after 0.12 ml/kg i.v. oleic acid. After 1 h, there were no pleural effusions, even though extravascular lung water increased 50% to 6.0 +/- 0.7 g/g dry lung. By 3 h pleural effusions had formed, they reached a maximum at 5 h (48.5 +/- 16.9 ml/thorax), and at 8 h there was no additional accumulation of pleural liquid (45.5 +/- 16.9 ml). Morphologic studies by light and electron microscopy demonstrated subpleural edema but no detectable injury to the visceral pleura, suggesting that the pleural liquid originated from the lung and not the pleura. In nine sheep, we quantified the rate of formation of pleural liquid by enclosing one lung in a plastic bag. By comparing in the same sheep the volume of pleural liquid collected from the enclosed lung to the volume found in the opposite intact chest, we estimated the rate of liquid absorption from the intact chest to be 0.32 ml/(kg.h); we had previously reported a liquid absorption rate of 0.28 ml/(kg.h) in normal sheep. These studies also supported the conclusion that the majority of the pleural liquid originated from the lung because we could account for all of the pleural liquid that was formed and cleared. The volume of pleural liquid collected from the enclosed lungs was equal to 21% of the excess lung liquid that formed after oleic acid-induced lung injury. Thus, the pleural space and parietal pleural lymphatic pathways are important pathways for the clearance of pulmonary edema liquid after experimentally induced increased permeability pulmonary edema.

Authors

J P Wiener-Kronish, V C Broaddus, K H Albertine, M A Gropper, M A Matthay, N C Staub

Interleukin 1-dependent paracrine granulopoiesis in chronic granulocytic leukemia of the juvenile type.

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Abstract

Marrow and peripheral blood cells from nine children with juvenile chronic granulocytic leukemia (JCGL) demonstrated intense (94 +/- 16% maximum) spontaneous granulocyte/macrophage colony growth but cells from five children with the adult variety of CGL did not. This unusual pattern of colony growth depended upon a stimulatory protein(s) produced by mononuclear phagocytes. No GM-CSA activity was found in any chromatofocused fraction of JCGL monocyte-conditioned media but an activity that induced GM-CSA in umbilical vein endothelial cells was detected at pI 6.9-7.2. Moreover, the CSA-inducing monokine was neutralized by an anti-IL-1 antibody in vitro and, in the one case so tested, the same antibody also inhibited "spontaneous" colony growth. Therefore granulocyte/macrophage colony growth in JCGL is characteristically abnormal and distinguishes JCGL from the adult form of the disease. This abnormality depends upon the production, by mononuclear phagocytes, of IL-1 which, in turn, stimulates the release of high levels of colony stimulating activity by other cells. The high proliferative activity of CFU-GM we found in JCGL patients, and the high levels of GM-CSA found in their serum are compatible with the view that the in vitro abnormality reflects a similar abnormality in vivo.

Authors

G C Bagby Jr, C A Dinarello, R C Neerhout, D Ridgway, E McCall

Inhibitory guanosine triphosphate-binding protein-mediated regulation of vasopressin action in isolated single medullary tubules of mouse kidney.

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Abstract

Vasopressin (AVP) plays a key role in maximal urine concentration by stimulating NaCl reabsorption in the medullary thick ascending limbs of Henle (MAL) and by increasing water permeability in the medullary collecting tubules (MCT). These effects of AVP in MAL and MCT are mediated by cAMP. Alpha 2-adrenergic stimulation in MCT, and high ambient Ca2+ and PGE2 in MAL inhibit AVP-dependent cAMP production and thereby modulate urine concentration. The present study was undertaken to clarify the mechanisms underlying the inhibition of AVP-dependent cAMP production by these agents using microdissected mouse MAL and MCT. Preincubation of MCT and MAL with 1 microgram/ml pertussis toxin for 3 and 6 h, respectively, resulted in ADP-ribosylation of an approximately 41-kD protein, which was presumably an alpha subunit of the inhibitory GTP-binding protein Gi. Epinephrine, 10(-6) M, via alpha 2-adrenergic stimulation, inhibited AVP-dependent cAMP production in MCT. Preincubation of MCT for 3 h with pertussis toxin abolished the inhibition of AVP-dependent cAMP production by epinephrine. High ambient Ca2+ and PGE2 both inhibited AVP-dependent cAMP production in MAL. Preincubation of MAL for 6 h with pertussis toxin abolished the inhibition by high ambient Ca2+ and attenuated the inhibition by PGE2. Preincubation of MCT or MAL with pertussis toxin for 1 h was ineffective in ADP-ribosylation and did not modify the inhibition of AVP-dependent cAMP production by these agents in both nephron segments. Our data suggest that the inhibition of AVP-dependent cAMP production by alpha 2-adrenergic stimulation in MCT, and by high ambient Ca2+ and adrenergic stimulation in MCT, and by high ambient Ca2+ and PGE2 in MAL, is mediated, at least in part, through activation of Gi.

Authors

K Takaichi, K Kurokawa

Chronic metabolic acidosis causes an adaptation in the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter in the rat proximal convoluted tubule.

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Abstract

The effect of chronic dietary acid on the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter was examined in the in vivo microperfused rat proximal tubule. Transporter activity was assayed with the epifluorescent measurement of cell pH using the intracellular, pH-sensitive fluorescent dye, (2'7')-bis(carboxyethyl)-(5,6)-carboxy-fluorescein (BCECF). BCECF was calibrated intracellularly, demonstrating similar pH-sensitivity of the dye in control and acidotic animals. In subsequent studies, lumen and peritubular capillaries were perfused to examine Na/H and Na(HCO3)3 transporter activity in the absence of contact with native fluid. The initial rate of change in cell pH (dpHi/dt) was 97, 50, and 44% faster in tubules from acidotic animals when peritubular [HCO3] was changed from 25 to 10 mM in the presence or absence of chloride, or peritubular [Na] was changed from 147 to 50 mM, respectively. dpHi/dt was 57% faster in tubules from acidotic animals when luminal [Na] was changed from 152 to 0 mM. Buffer capacities, measured using NH3/NH+4 addition, were similar in the two groups. The results demonstrate that chronic metabolic acidosis causes an adaptation in the intrinsic properties of both the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter.

Authors

P A Preisig, R J Alpern

Phase I study of granulocyte colony-stimulating factor in patients with transitional cell carcinoma of the urothelium.

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Abstract

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) was administered at a dose of 1-60 micrograms/kg of body weight to 22 patients with transitional cell carcinoma before chemotherapy as part of a Phase I/II study. In all patients, a specific dose-dependent increase in the absolute neutrophil count (ANC) of 1.8-12 fold was seen. In addition, this augmentation in the ANC was accompanied by an increase in leukocyte alkaline phosphatase, a marker of secondary granule formation. In six of eight patients analyzed, an increase in bone marrow myeloid to erythroid cell ratio was seen. Day 14 peripheral blood cell derived colony forming unit granulocyte macrophage were also increased by day 6 of rhG-CSF treatment. Circulating levels of eosinophils and basophils were unchanged; however, a 10-fold increase in monocytes was observed in patients treated at the highest doses. There was also a small increase in CD3+ lymphocytes that was not dose dependent. Hemoglobin, hematocrit, and platelet count remained near baseline throughout the period of rhG-CSF administration. These findings demonstrate that rhG-CSF is a potent stimulus for normal neutrophil proliferation and maturation.

Authors

J L Gabrilove, A Jakubowski, K Fain, J Grous, H Scher, C Sternberg, A Yagoda, B Clarkson, M A Bonilla, H F Oettgen

Differential inhibitory effects of forskolin, isoproterenol, and dibutyryl cyclic adenosine monophosphate on phosphoinositide hydrolysis in canine tracheal smooth muscle.

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Abstract

A characteristic feature of airway smooth muscle is its relative sensitivity to relaxant effects of beta adrenergic agonists when contracted by inflammatory mediators, such as histamine, vs. resistance to these relaxant effects when contracted by muscarinic agonists. Because contractions presumably depend upon the hydrolysis of membrane phosphoinositides (PI) and the generation of inositol phosphates (IP), our goal was to test for the effects of forskolin, isoproterenol, and dibutyryl cAMP on histamine- vs. methacholine-induced IP accumulation in canine tracheal smooth muscle. Methacholine (10(-3) M) was a more effective stimulant of IP accumulation (9.6 +/- 2.1-fold increase) than equimolar histamine (3.6 +/- 0.5-fold increase) in this tissue. When responses to equieffective methacholine (4 x 10(-6) M) and histamine (10(-3) M) were compared, neither forskolin, isoproterenol, nor dibutyryl cAMP significantly decreased IP accumulation in response to methacholine. In contrast, each of these three agents significantly decreased responses to histamine (by 56 +/- 9, 52 +/- 2, and 61 +/- 2%, respectively). We concluded that, in canine tracheal smooth muscle, increased cAMP is associated with inhibition of PI hydrolysis in response to histamine but not methacholine. The findings suggest a novel mechanism for selective modulation by cAMP of receptor-mediated cellular activation.

Authors

J M Madison, J K Brown

Interleukin 1 promotes tumor cell adhesion to cultured human endothelial cells.

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Abstract

We report that IL 1 acts on the endothelium, inducing a long-lasting increase in its adhesivity to tumor cells. Selective pretreatment of cultured human umbilical vein endothelial cells (EC) with IL 1 caused a significant increase in adhesion of three human colorectal carcinoma (HT-29, HCC-P2988, and HCC-M1410) cell lines and one human melanoma (A-375) cell line. Tumor necrosis factor (TNF) was as effective as IL 1 in promoting tumor cell adhesion to EC, whereas IFN gamma and IL 2 were inactive. The IL 1 and TNF induction of EC adhesivity was both concentration (threshold concentration 1 U/ml) and time dependent (peak 4-6 h), reversible within 24 h, and blocked by a protein synthesis inhibitor. The IL 1 and TNF action on EC may play a role in tumor cell lodgement.

Authors

E Dejana, F Bertocchi, M C Bortolami, A Regonesi, A Tonta, F Breviario, R Giavazzi

Correction of the molecular defect in B lymphocytes from X-linked agammaglobulinemia by cell fusion.

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Abstract

The X chromosome-linked antibody deficiency disease, X-linked agammaglobulinemia (XLA), results from failure of B lymphoid development. In the minor form of XLA, B lymphoid development terminates at the stage of immature B lymphocytes that produce truncated Ig heavy (H) chains composed of D-J-C(mu/delta), resulting from failure of VH gene rearrangement. Fusion of B cells from a patient with the minor form of XLA with mouse myeloma results in complementation of this defect; hybrid cells produce full-length H chains composed of VH-D-JH-C. The VH gene is of human origin. Complementation occurs independent of retention or loss of the human X (XLA) chromosome in the hybrid cells. These results indicate that the D-JH-C structure of the XLA B cells is fully functional for the subsequent rearrangement of a VH gene element, and that failure of immunoglobulin expression is susceptible to correction.

Authors

J Schwaber, N Koenig, J Girard