Cytomegalovirus (CMV) is a major cause of inflammatory organ disease in immunosuppressed persons. To elucidate the mechanisms of CMV-induced inflammation, we investigated whether tumor necrosis factor-alpha (TNF-alpha) was involved in the pathogenesis of CMV colitis in patients with AIDS. In in situ hybridization experiments, TNF-alpha mRNA was shown to be abundantly present in colonic mucosa from AIDS patients with CMV colitis but not in colonic mucosa from control (AIDS and normal) subjects. The TNF-alpha transcripts, identified in macrophage-like cells containing cytomegalic inclusions, were positively associated with CMV, but not HIV-1, within the mucosa. In in vitro experiments, a patient-derived isolate of CMV, but not HIV-1Ba-L, induced human monocytes to express TNF-alpha mRNA and to release increased levels of TNF-alpha peptide following stimulation. CMV induction of TNF-alpha may play a critical role in CMV-induced inflammation and, since TNF-alpha upregulates expression of HIV-1, offers a mechanism by which CMV could serve as a co-factor for HIV-1 expression without both viruses infecting the same cell.
P D Smith, S S Saini, M Raffeld, J F Manischewitz, S M Wahl
Early passaged bovine pulmonary artery endothelial cells exposed to 0.1-2.0 ng/ml transforming growth factor-beta 1 (TGF-beta 1) showed concentration-dependent growth inhibition, as assessed by [3H]thymidine labeling and cell counts, over a 96-h interval. Most of the inhibition of [3H]thymidine labeling measured at 96 h persisted when the medium was replaced with TGF-beta 1-free medium after 24 h, but the inhibition of labeling was prevented by the presence of anti-TGF-beta 1 antibody in the replacement medium. Additions of 2 mM cysteine, 1 mM cystine, or 2 mM N-acetylcysteine at the time of the initial addition of TGF-beta 1 blocked the inhibitory effect of TGF-beta 1 on [3H]-thymidine labeling when this was assessed after 72-96 h, but not at earlier times. Prevention of the inhibitory effect on cellular proliferation produced by cysteine, cystine and N-acetylcysteine was associated with elevation of cellular glutathione that was present at 48-96 h. There was no evidence for direct inactivation of TGF-beta 1 by the thiol-amino acids. Conditioned medium from TGF-beta 1-treated endothelial cells inhibited proliferation of mink lung carcinoma (CCL64) cells, supporting a previously reported concept of autocrine production of TGF-beta 1 by the endothelial cells. The inhibitory action of the conditioned medium was partially prevented when 1 mM cysteine was added during conditioning. Thus, TGF-beta 1 treatment of endothelial cells appears to set off autocrine production by these cells of TGF-beta 1 that perpetuates the inhibition of cellular proliferation. Replenishment of cellular glutathione with thiol-amino acids counteracts the growth-inhibitory effect of TGF-beta 1 through a currently undefined mechanism.
S K Das, A C White, B L Fanburg
Acute hypotension is an important complication of hemodialysis, but the underlying mechanisms remain poorly understood. Because hemorrhage-induced hypovolemia can trigger a sudden decrease in sympathetic activity resulting in bradycardia and vasodilation, we hypothesized that hemodialysis-induced hypovolemia also can trigger the same type of vasodepressor reaction, which would exacerbate the volume-dependent fall in blood pressure. We therefore measured blood pressure, vascular resistance, and sympathetic nerve activity (intraneural microelectrodes) during sessions of maintenance hemodialysis in 7 patients with and 16 patients without a history of hemodialysis-induced hypotension. During hemodialysis, blood pressure at first remained unchanged as calf resistance increased in both hypotension-resistant (from 37 +/- 4 to 49 +/- 5 U, P < 0.05) and hypotension-prone (from 42 +/- 6 to 66 +/- 12 U, P < 0.05) patients; sympathetic activity increased comparably in the subset of patients in whom it could be measured. With continued hemodialysis, calf resistance and sympathetic activity increased further in the hypotension-resistant patients, but in the hypotension-prone patients the precipitous decrease in blood pressure was accompanied by decreases in sympathetic activity, vascular resistance, and heart rate as well as symptoms of vasodepressor syncope. On an interdialysis day, both groups of patients increased vascular resistance normally during unloading of cardiopulmonary baroreceptors with lower body negative pressure and increased heart rate normally during unloading of arterial baroreceptors with infusion of nitroprusside. These findings indicate that in a group of hemodialysis patients without diabetes or other conditions known to impair autonomic reflexes, hemodialysis-induced hypotension is not caused by chronic uremic impairment in arterial or cardiopulmonary baroreflexes but rather by acute, paradoxical withdrawal of sympathetic vasoconstrictor drive producing vasodepressor syncope.
R L Converse Jr, T N Jacobsen, C M Jost, R D Toto, P A Grayburn, T M Obregon, F Fouad-Tarazi, R G Victor
Hypertrophic cardiomyopathy occurs as an autosomal dominant familial disorder or as a sporadic disease without familial involvement. While missense mutations in the beta cardiac myosin heavy chain (MHC) gene account for approximately half of all cases of familial hypertrophic cardiomyopathy, the molecular causes of sporadic hypertrophic cardiomyopathy are unknown. To determine whether beta cardiac MHC mutations are also associated with sporadic disease, we screened this gene in seven individuals with sporadic hypertrophic cardiomyopathy. Mutations in the beta cardiac MHC genes were identified in two probands with sporadic disease. In that their parents were neither clinically nor genetically affected, we conclude that mutations in each proband arose de novo. Transmission of the mutation and disease to an offspring occurred in one pedigree, predicting that these are germline mutations. The demonstration of hypertrophic cardiomyopathy arising within a pedigree coincident with the appearance of a de novo mutation provides compelling genetic evidence that beta cardiac MHC mutations cause this disease. We suggest that de novo mutations account for some instances of sporadic hypertrophic cardiomyopathy and that these mutations can be transmitted to children. The clinical benefits of defining mutations responsible for familial hypertrophic cardiomyopathy should also be available to some patients with sporadic disease.
H Watkins, L Thierfelder, D S Hwang, W McKenna, J G Seidman, C E Seidman
Diabetes mellitus is associated with a reduction of lipoprotein lipase (LPL) activity and development of hypertriglyceridemia. In the current experiments the mechanisms involved in the regulation of LPL have been examined in control rats, streptozocin-induced diabetic rats, and diabetic rats treated chronically or with a single injection of insulin. Diabetes decreased adipose tissue LPL activity partially by decreasing immunoreactive LPL protein and the steady-state levels of LPL mRNA, but primarily by reducing the catalytic activity of LPL. Both chronic and acute insulin increased adipose tissue LPL activity by correcting the defect in the catalytic activity of LPL and increasing immunoreactive LPL protein; however, only chronic insulin restored LPL mRNA levels to normal. In the heart, LPL activity tended to be elevated with diabetes in parallel to an increase in immunoreactive LPL protein even though levels of LPL mRNA declined. Both chronic and acute insulin normalized LPL activity and immunoreactive LPL protein, while only chronic insulin corrected the levels of LPL mRNA. No changes in the catalytic activity of LPL in heart were detected among the groups. Thus, diabetes and insulin treatment regulate LPL expression pretranslationally, translationally, and post-translationally, with tissue-specific differences apparent in the mechanisms involved.
K Tavangar, Y Murata, M E Pedersen, J F Goers, A R Hoffman, F B Kraemer
We studied metabolic, polypeptide and genetic variation in eight glutaric acidemia type II (GA II) patients with electron transfer flavoprotein (ETF) deficiency. As measured by 3H-fatty acid oxidations in fibroblasts, beta-oxidation pathway flux correlated well with clinical phenotypes. In six patients with severe neonatal onset GA II, oxidation of [9,10(n)-3H]-palmitate ranged from 2% to 22% of control and of [9,10(n)-3H]myristate, from 2% to 26% of control. Of two patients with late onset GA II, one had intermediate residual activities with these substrates and the other normal activities. Radiolabeling and immunoprecipitation studies revealed that three of the six neonatal onset GA II patients had greatly diminished or absent alpha- and beta-ETF subunits, consistent with a failure to assemble a stable heterodimer. Another neonatal onset patient showed normal synthesis of beta-ETF but decreased synthesis of alpha-ETF. Two neonatal onset and two late onset GA II patients showed normal synthesis of both subunits. Analysis of the pre-alpha-ETF coding sequence revealed seven different mutations in the six patients with neonatal onset GA II. The most common mutation was a methionine for threonine substitution at codon 266 found in four unrelated patients, while all the other mutations were seen in single patients. No mutations were detected in the two patients with late onset GA II.
E Freneaux, V C Sheffield, L Molin, A Shires, W J Rhead
Chemotactic stimulation of neutrophils results in translocation of CD11b/CD18 (Mac-1) from intracellular storage pools to the cell surface. Though results from several laboratories indicate that the newly arrived surface Mac-1 is not involved in the adherence induced by the initial stimulus, the present study addresses the hypothesis that this Mac-1 plays a role in subsequent adherence-dependent functions. The response of human neutrophils to changing concentrations of a chemotactic stimulus was evaluated by determining the amount of newly arrived surface Mac-1, and Mac-1-dependent adhesion and locomotion. Small step-wise increases in the concentration of f-Met-Leu-Phe (FMLP) resulted in proportional stepwise increases in surface Mac-1 that plateaued within 2-4 min. This newly arrived Mac-1 supported adhesion to protein-coated surfaces only when the cells were exposed to an additional increase in the FMLP stimulus level. Adherence-dependent cellular locomotion was evaluated in chambers that allowed rapid changes in the stimulus concentration. Repeated small increments in the stimulus level at 200-s intervals resulted in significantly longer migration paths than a single-step increase in the stimulus. The results support the hypothesis that small increments in the chemotactic stimulus bring Mac-1 to the cell surface, and this newly mobilized Mac-1 is available for adherence-dependent locomotion with subsequent increases in the concentration of the stimulus.
B J Hughes, J C Hollers, E Crockett-Torabi, C W Smith
In the present study, we have investigated the effects of IL-4 on the proliferation and differentiation of leukemic and normal human B cell precursors (BCP). We have demonstrated that IL-4 significantly inhibited spontaneous [3H]thymidine ([3H]-TdR) incorporation by leukemic blasts from some B lineage acute lymphoblastic leukemia (BCP-ALL) patients (8 of 14). Furthermore, IL-4 was found to suppress the spontaneous and factor-dependent (IL-7 and IL-3) proliferation of normal BCP (CD10+ surface [s] IgM- cells) isolated from fetal bone marrow. Maximum growth inhibition of either leukemic or normal BCP was reached at low IL-4 concentrations (10 U/ml), and the effect was specifically neutralized by anti-IL-4 antibody. IL-4 was further found to induce the expression of CD20 antigen on BCP-ALL cells from a number of the cases examined (5 of 8), but in contrast to leukemic cells, IL-4 failed to induce CD20 antigen on normal BCP. Finally, IL-4 was found to induce neither the expression of cytoplasmic mu chain, nor the appearance of sIgM+ cells in cultures of normal or leukemic BCP. Our data indicate that IL-4 has the potential to inhibit cell proliferation in leukemic and normal human B lymphopoiesis but is unable to drive the transition from BCP to mature B cells.
D Pandrau, S Saeland, V Duvert, I Durand, A M Manel, M T Zabot, N Philippe, J Banchereau
Angiotensin II is a vasoactive peptide and may act as a growth factor in vascular smooth muscle cells. Experimental injury of the rat aorta causes rapid migration of medial smooth muscle cells and their proliferation resulting in the formation of neointima. We have examined, using quantitative autoradiography, the expression of angiotensin II receptor subtypes AT1 and AT2, and angiotensin-converting enzyme, in the neointima formed in the rat thoracic aorta 15 d after balloon-catheter injury. In contrast to the normal aortic wall, which contained both AT1 and AT2 receptors (80% and 20%, respectively), neointimal cells expressed almost exclusively angiotensin II AT1 receptors. The apparent number of these receptors was fourfold higher in the neointima compared to that in the normal aortic wall. The affinities of the neointimal receptors to angiotensin II or to the AT1 receptor antagonist, losartan, were not different from those in the normal aortic wall. Angiotensin-converting enzyme binding in the neointima was not different from that in the media of the uninjured aorta. Our data suggest that angiotensin II AT1 receptors may have a significant role in injury-induced vascular smooth muscle proliferation and migration.
M Viswanathan, C Strömberg, A Seltzer, J M Saavedra
We studied a 43 yr-old Spanish patient with homozygous 4.1(-) hereditary elliptocytosis. Any form of protein 4.1 was missing in the red cells. Spectrin and actin were slightly, yet significantly, diminished. Alterations appeared at the level of proteins 4.5 and 4.9. Glycophorin C was sharply reduced. The abnormal allele was associated with the -++-- haplotype (Pvu II, Bgl II, Bgl II, Pvu II, Pvu II). mRNA 4.1(-) had an apparently normal size but was diminished by about two-thirds. Because the abnormal phenotype pertained to the red cell, we sequenced the 4.1 cDNA regions that appear critical to this cell type. The ultimate change turned out to be a point mutation of the downstream translation initiation codon (AUG-->AGG). No disorders in other cell types could be related with certainty to the present 4.1(-) HE allele.
N Dalla Venezia, F Gilsanz, N Alloisio, M T Ducluzeau, E J Benz Jr, J Delaunay
Escherichia coli endotoxin (LPS) can induce the clinical syndrome of septic shock and renal cortical necrosis and can stimulate nitric oxide (NO) production from macrophages, vascular smooth muscle, and glomerular mesangial cells in vitro. NO is an endogenous vasodilator, which also inhibits platelet aggregation and adhesion. We therefore sought to determine whether LPS would stimulate NO production in vivo and, if so, whether this NO would modulate endotoxin-induced glomerular thrombosis. The stable NO end-products, NO2 and NO3, were measured in serum and urine collections from rats during baseline and after injection of LPS, with or without substances that modulate NO synthesis. The urinary excretion of NO2/NO3 was 1,964 +/- 311 nm/8 h during the baseline and increased to 6,833 +/- 776 nm/8 h after a single intraperitoneal injection of 0.1 mg/kg LPS (P < 0.05). The serum concentration of NO2/NO3 also significantly increased after LPS injection. Both the urine and serum stimulation was significantly prevented by the NO synthesis inhibitor, Nw-nitro-L-arginine methyl ester (L-NAME). L-Arginine, given with LPS+L-NAME significantly restored the NO2/NO3 levels in the urine. Ex vivo incubation of tissues from rats treated with LPS demonstrated NO production by the aorta, whole kidney, and glomeruli, but not cortical tubules. Histological examination of kidneys from rats given either LPS or L-NAME alone revealed that 2 and 4.5% of the glomeruli contained capillary thrombosis, respectively. In contrast, rats given LPS+L-NAME developed thrombosis in 55% of glomeruli (P < 0.001), which was significantly prevented when L-arginine was given concomitantly. We conclude that LPS stimulates endogenous production of NO in vivo and that this NO is critical in preventing LPS-induced renal thrombosis.
P J Shultz, L Raij
A human ovarian small cell carcinoma line (BIN-67) expresses abundant calcitonin (CT) receptors (CTR) (143,000 per cell) that are coupled, to adenylate cyclase. The dissociation constants (Kd) for the CTRs on these BIN-67 cells is approximately 0.42 nM for salmon CT and approximately 4.6 nM for human CT. To clone a human CTR (hCTR), a BIN-67 cDNA library was screened using a cDNA probe from a porcine renal CTR (pCTR) that we recently cloned. One positive clone of 3,588 bp was identified. Transfection of this cDNA into COS cells resulted in expression of receptors with high affinity for salmon CT (Kd = approximately 0.44 nM) and for human CT (Kd = approximately 5.4 nM). The expressed hCTR was coupled to adenylate cyclase. Northern analysis with the hCTR cDNA probe indicated a single transcript of approximately 4.2 kb. The cloned cDNA encodes a putative peptide of 490 amino acids with seven potential transmembrane domains. The amino acid sequence of the hCTR is 73% identical to the pCTR, although the hCTR contains an insert of 16 amino acids between transmembrane domain I and II. The structural differences may account for observed differences in binding affinity between the porcine renal and human ovarian CTRs. The CTRs are closely related to the receptors for parathyroid hormone-parathyroid hormone-related peptide and secretin; these receptors comprise a distinct family of G protein-coupled seven transmembrane domain receptors. Interestingly, the hCTR sequence is remotely related to the cAMP receptor of Dictyostelium discoideum (21% identical), but is not significantly related to other G protein-coupled receptor sequences now in the data bases.
A H Gorn, H Y Lin, M Yamin, P E Auron, M R Flannery, D R Tapp, C A Manning, H F Lodish, S M Krane, S R Goldring
In humans and in several animal species, puberty results from changes in pulsatile gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus. In particular, the frequency of pulsatile GnRH secretion increases at the onset of puberty, as can be shown by using hypothalamic explants of male rats of 15 and 25 d. Previous observations from us and others suggested that the initiation of puberty could involve a facilitatory effect of excitatory amino acids mediated through N-methyl-D-aspartate (NMDA) receptors. We found that GnRH secretion could be activated through NMDA receptors only around the time of onset of puberty (25 d). The aim of this study was to clarify why this activation did not occur earlier (at 15 d) and could no longer be observed by the end of puberty (at 50 d). We studied GnRH secretion in the presence of MK-801, a noncompetitive antagonist of NMDA receptors or AP-5, a competitive antagonist. We showed that, in the hypothalamus of immature male rats (15 d), a highly potent inhibitory control of pulsatile GnRH secretion in vitro was mediated through NMDA receptors. These data were confirmed in vivo because administration of the antagonist MK-801 (0.001 mg/kg) to immature male rats resulted in early pubertal development. Onset of puberty (25 d) was characterized by the disappearance of that NMDA receptor-mediated inhibition, thus unmasking a facilitatory effect also mediated through NMDA receptors. During puberty, there was a reduction in activity of this facilitatory control which was no longer opposed by its inhibitory counterpart. We conclude that a sequential reduction in activity of inhibitory and facilitatory NMDA receptors provides a developmental basis for the neuroendocrine mechanism of onset of puberty.
J P Bourguignon, A Gérard, M L Alvarez Gonzalez, P Franchimont
A left-to-right shunt is accompanied by an increased plasma and blood volume. Since this is likely realized through renin/aldosterone-mediated salt and water retention, other body fluid compartments may be changed too. Therefore, we studied blood volume and body fluid compartments by a single-injection, triple-indicator dilution technique in nine 8-wk-old lambs with an aortopulmonary left-to-right shunt (55 +/- 3% of left ventricular output; mean +/- SEM) and in 11 control lambs, 2.5 wk after surgery. Systemic blood flow was maintained at the same level as in control lambs, but the aortic pressure of the shunt lambs was lower. Blood volume in shunt lambs was larger than in control lambs (110 +/- 6 vs. 84 +/- 7 ml/kg, P < 0.001) through an increase in plasma volume, which correlated significantly with the magnitude of the left-to-right shunt (r = 0.81, P < 0.01). Red blood cell volume was equal to that of control lambs. Evidence was obtained that the increase in plasma volume was induced by a transient increase in renin (8.0 +/- 2.2 vs. 1.6 +/- 0.2 nmol.l-1.h-1; P < 0.02) and aldosterone (0.51 +/- 0.14 vs. 0.24 +/- 0.09 nmol/liter) concentrations. Interstitial water volume, however, was not significantly different from that in control lambs. The amount of intravascular protein was significantly higher than in control lambs (5.0 +/- 0.3 vs. 3.5 +/- 0.2 g/kg body mass, P < 0.001). There were no significant differences in intracellular and total body water volumes between the two groups. We conclude that the increased amount of intravascular protein confines the fluid retained by the kidneys to the vascular compartment.
J W Gratama, M Dalinghaus, J J Meuzelaar, A M Gerding, J H Koers, W G Zijlstra, J R Kuipers
Autoantibodies specifically directed to U1RNA were found in patients suffering from systemic lupus erythematosus (SLE) overlap syndromes. To obtain more insight in the mechanism responsible for this U1RNA-specific antibody formation and to use the antibodies eventually as a tool to study U1RNA-protein (U1RNP) interactions, the B cell epitopes on U1RNA were mapped. Using in vitro synthesized domains of U1RNA, the main epitope regions were found in stemloops II and IV. Furthermore, 3'-end or 5'-end truncation of both stemloop II and stemloop IV showed that the conformation of the stemloops is critical for antibody recognition. Mutant studies on both stemloops indicated that in the case of stemloop II the stem is the main antigenic region, whereas in stemloop IV, the loop (E-loop) is a main target. The results of this study support the idea that the anti-U1RNA autoantibody could be the result of a process driven by the human U1RNP complex itself (antigen-driven process).
R M Hoet, P De Weerd, J K Gunnewiek, I Koornneef, W J Van Venrooij
Chronic administration of alpha 1-receptor antagonists is associated with loss of clinical efficacy, especially in congestive heart failure, although the mechanism is uncertain. To evaluate changes in venous alpha 1-adrenoceptor responsiveness during chronic alpha 1-adrenoceptor blockade, dose-response curves to phenylephrine and angiotensin II were constructed in 10 healthy subjects before, during, and after administration of terazosin 1 mg orally for 28 d. Terazosin initially shifted the dose-response curve of phenylephrine to the right, with a significant increase in ED50 for phenylephrine from a control value of 102 to 759 ng/min on day 1 of terazosin (P < 0.001). However, by day 28, the dose-response curve had shifted back towards baseline with an ED50 of 112 ng/min. After discontinuing terazosin, the ED50 for phenylephrine remained near the baseline value, indicating no evidence of supersensitivity to phenylephrine. There was no change in responsiveness to angiotensin II during the course of treatment with terazosin. Plasma terazosin concentrations were stable throughout the period of drug administration. The mean Kd of terazosin was estimated as 11 +/- 15 nM in the first few days of treatment. This study demonstrates that pharmacological tolerance to the alpha 1-adrenoceptor blocking action of terazosin occurs in man and may be responsible for loss in efficacy with chronic therapy.
J Vincent, W Dachman, T F Blaschke, B B Hoffman
It is generally believed that glucose production (GP) cannot be adequately suppressed in insulin-treated diabetes because the portal-peripheral insulin gradient is absent. To determine whether suppression of GP in diabetes depends on portal insulin levels, we performed 3-h glucose and specific activity clamps in moderately hyperglycemic (10 mM) depancreatized dogs, using three protocols: (a) 54 pmol.kg-1 bolus + 5.4 pmol.kg-1.min-1 portal insulin infusion (n = 7; peripheral insulin = 170 +/- 51 pM); (b) an equimolar peripheral infusion (n = 7; peripheral insulin = 294 +/- 28 pM, P < 0.001); and (c) a half-dose peripheral infusion (n = 7), which gave comparable (157 +/- 13 pM) insulinemia to that seen in protocol 1. Glucose production, use (GU) and cycling (GC) were measured using HPLC-purified 6-[3H]- and 2-[3H]glucose. Consistent with the higher peripheral insulinemia, peripheral infusion was more effective than equimolar portal infusion in increasing GU. Unexpectedly, it was also more potent in suppressing GP (73 +/- 7 vs. 55 +/- 7% suppression between 120 and 180 min, P < 0.001). At matched peripheral insulinemia (protocols 2 and 3), not only stimulation of GU, but also suppression of GP was the same (55 +/- 7 vs. 63 +/- 4%). In the diabetic dogs at 10 mM glucose, GC was threefold higher than normal but failed to decrease with insulin infusion by either route. Glycerol, alanine, FFA, and glucagon levels decreased proportionally to peripheral insulinemia. However, the decrease in glucagon was not significantly greater in protocol 2 than in 1 or 3. When we combined all protocols, we found a correlation between the decrements in glycerol and FFAs and the decrease in GP (r = 0.6, P < 0.01). In conclusion, when suprabasal insulin levels in the physiological postprandial range are provided to moderately hyperglycemic depancreatized dogs, suppression of GP appears to be more dependent on peripheral than portal insulin concentrations and may be mainly mediated by limitation of the flow of precursors and energy substrates for gluconeogenesis and by the suppressive effect of insulin on glucagon secretion. These results suggest that a portal-peripheral insulin gradient might not be necessary to effectively suppress postprandial GP in insulin-treated diabetics.
A Giacca, S J Fisher, Z Q Shi, R Gupta, H L Lickley, M Vranic
After acute lung injury, mesenchymal cells migrate into the alveolar airspace where they proliferate and deposit connective tissue macromolecules. Early in the disease process, inflammatory cell-derived trophic factors modulate these mesenchymal cell functions. However, in those patients who die, even as the inflammatory response abates, the fibroproliferative response continues, resulting in extensive intraalveolar fibrosis. We therefore hypothesized that lung mesenchymal cells obtained from individuals dying with acute alveolar fibrosis would manifest an enhanced proliferative capacity that was independent of persistent exogenous signals. To examine this hypothesis, the in vitro growth properties of mesenchymal cells prepared from patients dying with acute lung injury (n = 3) were analyzed in defined medium and compared with those of mesenchymal cells similarly prepared from patients dying with histologically normal lungs (n = 3). Isolates were characterized as mesenchymal cells by using morphological and immunohistochemical criteria. In accord with the hypothesis, mesenchymal cells isolated from lung-injured patients doubled within 3 d in the complete absence of exogenous peptide growth factors, reaching a saturation density of approximately 15 x 10(3) cells/cm2. As expected, lung mesenchymal cells from normal individuals failed to significantly increase in number. Consistent with this proliferative phenotype, the immediate early cell division cycle genes c-fos and c-jun were constitutively expressed in each cell strain prepared from injured lungs, but not in those from control lungs. The observed proliferative phenotype was stable through the fifth subcultivation of the cells. Despite these proliferative properties, three separate criteria indicated the mesenchymal cells from injured lungs were not transformed: normal karyotype; finite lifespan in vitro (9-10 subcultivations); and inability to disseminate in mice with severe combined immunodeficiency. These data support the hypothesis that mesenchymal cells manifest an enhanced proliferative state after acute lung injury.
B Chen, V Polunovsky, J White, B Blazar, R Nakhleh, J Jessurun, M Peterson, P Bitterman
Cytokines have been implicated in the pathogenesis of a number of brain diseases in which neurological dysfunction has been attributed to a change in amino acid neurotransmitter metabolism. In the present in vitro study, we investigated the effects of cytokines on astrocyte glutamine synthetase (GS) activity and subsequently on N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity. Proinflammatory cytokines IL-1 alpha, IL-1 beta, and IL-6 at a concentration of 20 ng/ml did not affect GS activity; however, tumor necrosis factor-alpha inhibited this activity by 20% in mixed neuronal/astrocyte cultures. Treatment for 24 h with transforming growth factor (TGF)-beta 1 or -beta 2 inhibited up to 60% GS activity. TGF-beta 2 also inhibited GS in enriched astrocyte cultures with an ED50 of 10 pg/ml. Antibodies specific to TGF-beta 2 blocked this effect. Treatment of astrocytes with TGF-beta 2 (250 pg/ml) resulted in markedly dilated rough endoplasmic reticulum. Since astrocyte GS may play a protective role in NMDA receptor-mediated neurotoxicity, we treated mixed neuronal/astrocyte cultures with TGF-beta 2 (250 pg/ml) and found a threefold potentiation of NMDA receptor-mediated neurotoxicity. These data suggest that TGF-beta impairs astrocyte GS function and enhances neurotoxicity, thus providing insight into understanding one mechanism of cytokine-mediated central nervous system disease.
C C Chao, S Hu, M Tsang, J Weatherbee, T W Molitor, W R Anderson, P K Peterson
Coronary artery stenosis or occlusion results in reduced coronary flow and myocardial contractile depression. At severe flow reductions, increased inorganic phosphate (Pi) and intracellular acidosis clearly play a role in contractile depression. However, during milder flow reductions the mechanism(s) underlying contractile depression are less clear. Previous perfused heart studies demonstrated no change of Pi or pH during mild flow reductions, suggesting that changes of intravascular pressure (garden hose effect) may be the mediator of this contractile depression. Others have reported conflicting results regarding another possible mediator of contractility, the cytosolic free calcium (Cai). To examine the respective roles of Cai, Pi, pH, and vascular pressure in regulating contractility during mild flow reductions, Indo-1 calcium fluorescence and 31P magnetic resonance spectroscopy measurements were performed on Langendorff-perfused rat hearts. Cai and diastolic calcium levels did not change during flow reductions to 50% of control. Pi demonstrated a close relationship with developed pressure and significantly increased from 2.5 +/- 0.3 to 4.2 +/- 0.4 mumol/g dry weight during a 25% flow reduction. pH was unchanged until a 50% flow reduction. Increasing vascular pressure to superphysiological levels resulted in further increases of developed pressure, with no change in Cai. These findings are consistent with the hypothesis that during mild coronary flow reductions, contractile depression is mediated by an altered relationship between Cai and pressure, rather than by decreased Cai. Furthermore, increased Pi and decreased intravascular pressure may be responsible for this altered calcium-pressure relationship during mild coronary flow reductions.
V M Figueredo, R Brandes, M W Weiner, B M Massie, S A Camacho
We undertook studies focused on folate acquisition by Chlamydia trachomatis L2, Chlamydia psittaci 6BC, and C. psittaci francis. Results from in situ studies, using wild-type host cells, confirmed that C. trachomatis L2 and C. psittaci 6BC are sensitive to sulfonamides whereas C. psittaci francis is resistant. In addition C. trachomatis L2 and C. psittaci francis were inhibited by methotrexate in situ whereas C. psittaci 6BC was not. In contrast to C. trachomatis, neither C. psittaci strain was affected by trimethoprim. Surprisingly our results indicate that all three strains are capable of efficient growth in folate-depleted host cells. When growing in folate-depleted cells C. psittaci francis becomes sensitive to sulfonamide. The ability of all three strains to carry out de novo folate synthesis was demonstrated by following the incorporation of exogenous [3H]pABA into intracellular folates and by detecting dihydropteroate synthase activity in reticulate body crude extract. Dihydrofolate reductase activity was also detected in reticulate body extract. In aggregate the results indicate that C. trachomatis L2, C. psittaci francis, and C. psittaci 6BC can all synthesize folates de novo, however, strains differ in their ability to transport preformed folates directly from the host cell.
H Fan, R C Brunham, G McClarty
The alveolar surface of the lung is a major target for oxidant injury. After injury, repair of the alveolar epithelium is dependent on the ability of epithelial type 2 (T2) cells to proliferate. The regulation of T2 cell proliferation and the effect of reactive oxygen (O2) species on this lung cell proliferation have not been well defined. To investigate this process we focused on the regulation of two late cell cycle genes, histone and thymidine kinase, in T2 cells and fibroblasts exposed in vitro to varying periods of hyperoxia (95% O2). Hyperoxia for 24 to 48 h arrested cell proliferation in a SV40T-immortalized T2 cell line we have developed and in primary and SV40T-immortalized lung fibroblasts. Despite the cessation of proliferation, histone and TK mRNA continued to be expressed at high levels; mRNA half-lives were markedly prolonged but neither protein was translated. Thus proliferation arrest induced by hyperoxia was associated with posttranscriptional control of at least two late cell cycle-related genes. This form of proliferation arrest is also seen when primary and SV40T-T2 cells but not fibroblasts are serum deprived, suggesting that T2 cells in vitro may be uniquely sensitive to alterations in their redox state and that these alterations in turn affect translational control of a subset of proliferation-related genes.
A Clement, M Edeas, K Chadelat, J S Brody
Silica particles (quartz dust) are toxic to macrophages after their uptake into these cells. These experiments describe the opsonization mechanism(s) and macrophage receptor(s) involved in silica uptake. Freshly isolated rat liver macrophages (Kupffer cells) were incubated at 37 degrees C with silica particles in the presence or absence of autologous or heterologous plasma or purified plasma fibronectin and cell viability was assessed at various times. Within 60 min of coincubation, > 80% of macrophages were lysed in the presence of plasma or purified fibronectin but not in their absence (viability > 90%). Lysis was slower with defibronectinized plasma (28% in 60 min). Macrophages could be protected from lysis by addition of the monosaccharide N-acetyl-D-galactosamine but not by N-acetyl-D-glucosamine. Galactosylated serum albumin but not mannosylated albumin or native albumin exerted full protection from lysis. The pentapeptide GRGDS also prevented macrophage lysis in synergy with N-acetyl-galactosamine. Enzymatic deglycosylation of fibronectin reduced lysis significantly. These findings indicate an important opsonizing activity for fibronectin and dual recognition via the lectin-like galactose-specific binding activity of membrane-associated C-reactive protein and by integrin receptor(s). Binding experiments (at 4 degrees C) revealed initial binding as primarily galactose-inhibitable, suggesting integrin-mediated binding as a later event necessary for effective uptake.
Generalized resistance to thyroid hormone (GRTH) is a syndrome of hyposensitivity to triiodothyronine (T3) that displays autosomal dominant inheritance. The genetic defect commonly lies in the ligand-binding domain of one of the TR beta alleles. Since there are two major thyroid hormone receptor (TR) isoforms, TR alpha and TR beta, it is not known how the mutant receptor mediates a dominant negative effect. Previously, we showed that T3 caused dissociation of TR homodimers and TR alpha/TR beta dimers from several thyroid hormone response elements (TREs). Hence, we used the electrophoretic mobility shift assay to compare the effect of T3 on the DNA binding of mutant TR beta-1 (Mf-1) from a kindred with GRTH with normal TR beta. Mf-1 bound better as a homodimer than TR beta, but dissociated from DNA only at high T3 concentrations. Both receptors heterodimerized with nuclear auxiliary proteins. They also dimerized with TR alpha and with each other. Surprisingly, T3 disrupted the DNA binding of the Mf-1/TR isoform dimers. Thus, mechanisms for the dominant negative effect by mutant TRs likely involve either increased binding to TREs by mutant homodimers that cannot bind T3 (hence cannot dissociate from DNA) and/or the formation of inactive mutant TR/nuclear protein heterodimers.
P M Yen, A Sugawara, S Refetoff, W W Chin
Although viremia is a hallmark of disseminated cytomegalovirus (CMV) infection, not all viremic patients have visceral organ CMV disease. We used blot hybridization with a cloned subgenomic probe to quantitate viral DNA in blood leukocytes of 60 viremic patients (25 with solid organ transplants, 20 with AIDS, and 15 marrow recipients) who had different clinical manifestations of CMV infection. The results are expressed as pg of viral DNA/10 micrograms of leukocyte DNA. Patients with AIDS or with solid organ transplants who had CMV visceral organ disease had the largest amounts of viral DNA in their granulocytes (median 632 and 237 pg, respectively). These amounts were significantly greater than those in similar viremic patients without CMV visceral disease (17 and 21 pg; P < 0.005 and 0.002, respectively). All patients in the study with > 150 pg of CMV DNA in their granulocytes had visceral CMV disease. The amounts of viral DNA in granulocytes of AIDS and organ transplant patients with CMV retinitis were low (median 22 pg). Marrow transplant patients were unique in that the amounts of CMV DNA in granulocytes were low whether CMV visceral organ disease was present (17 pg) or absent (14 pg). We conclude that high levels of circulating CMV DNA in viremic AIDS and solid organ transplant patients reflect viral involvement of visceral organs but not the retina. In marrow recipients, the severity of CMV disease, even when fatal, is not reflected quantitatively in peripheral blood leukocytes.
R L Saltzman, M R Quirk, M C Jordan
Insulin rapidly stimulates tyrosine phosphorylation of a protein of approximately 185 kD in most cell types. This protein, termed insulin receptor substrate-1 (IRS-1), has been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the present study we have examined the levels of IRS-1 and the phosphorylation state of insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo in two rat models of insulin resistance, i.e., insulinopenic diabetes and fasting, and a mouse model of non-insulin-dependent diabetes mellitus (ob/ob) by immunoblotting with anti-peptide antibodies to IRS-1 and anti-phosphotyrosine antibodies. As previously described, there was an increase in insulin binding and a parallel increase in insulin-stimulated receptor phosphorylation in muscle of fasting and streptozotocin-induced (STZ) diabetic rats. There was also a modest increase in overall receptor phosphorylation in liver in these two models, but when normalized for the increase in binding, receptor phosphorylation was decreased, in liver and muscle of STZ diabetes and in liver of 72 h fasted rats. In the hyperinsulinemic ob/ob mouse there was a decrease in insulin binding and receptor phosphorylation in both liver and muscle. The tyrosyl phosphorylation of IRS-1 after insulin stimulation reflected an amplification of the receptor phosphorylation in liver and muscle of hypoinsulinemic animals (fasting and STZ diabetes) with a twofold increase, and showed a significant reduction (approximately 50%) in liver and muscle of ob/ob mouse. By contrast, the levels of IRS-1 protein showed a tissue specific regulation with a decreased level in muscle and an increased level in liver in hypoinsulinemic states of insulin resistance, and decreased levels in liver in the hyperinsulinemic ob/ob mouse. These data indicate that: (a) IRS-1 protein levels are differentially regulated in liver and muscle; (b) insulin levels may play a role in this differential regulation of IRS-1; (c) IRS-1 phosphorylation depends more on insulin receptor kinase activity than IRS-1 protein levels; and (d) reduced IRS-1 phosphorylation in liver and muscle may play a role in insulin-resistant states, especially of the ob/ob mice.
M J Saad, E Araki, M Miralpeix, P L Rothenberg, M F White, C R Kahn
To investigate the mechanism of intracellular Ca2+ ([Cai]) increase in human burst-forming unit-erythroid-derived erythroblasts by erythropoietin, we measured [Cai] with digital video imaging, cellular phosphoinositides with high performance liquid chromatography, and plasma membrane potential and currents with whole cell patch clamp. Chelation of extracellular free Ca2+ abolished [Cai] increase induced by erythropoietin. In addition, the levels of inositol-1,4,5-trisphosphate did not increase in erythropoietin-treated erythroblasts. These results indicate that in erythropoietin-stimulated cells, Ca2+ influx rather than intracellular Ca2+ mobilization was responsible for [Cai] rise. Both Ni2+ and moderately high doses of nifedipine blocked [Cai] increase, suggesting involvement of ion channels. Resting membrane potential in human erythroblasts was -10.9 +/- 1.0 mV and was not affected by erythropoietin, suggesting erythropoietin modulated a voltage-independent ion channel permeable to Ca2+. No voltage-dependent ion channel but a Ca(2+)-activated K+ channel was detected in human erythroblasts. The magnitude of erythropoietin-induced [Cai] increase, however, was insufficient to open Ca(2+)-activated K+ channels. Our data suggest erythropoietin modulated a voltage-independent ion channel permeable to Ca2+, resulting in sustained increases in [Cai].
J Y Cheung, M B Elensky, U Brauneis, R C Scaduto Jr, L L Bell, D L Tillotson, B A Miller
Neuroblastoma, a malignant neoplasm that arises in the adrenal medulla or sympathetic ganglion, is one of the most common solid tumors of childhood. Reports that neuroblastomas spontaneously mature to form benign ganglioneuromas have prompted investigations into the efficacy of using agents that induce neuronal differentiation in the treatment of this malignancy. Retinoic acid is one agent in particular that has been shown to induce growth inhibition and terminal differentiation of neuroblastoma cell lines in vitro. Using the human neuroblastoma cell line SMH-KCNR, we have investigated the role of the extracellular matrix protein thrombospondin in retinoic acid induced neuroblastoma differentiation. Treatment with retinoic acid results in a rapid induction (within 4 h) of thrombospondin (TSP) message which is independent of intervening protein synthesis and superinducible in the presence of cycloheximide. This suggests that TSP functions as a retinoic acid inducible immediate early response gene. A concomitant increase in both cell associated and soluble forms of TSP protein can be detected within 24 h of retinoic acid treatment. A functional role for TSP in SMH-KCNR differentiation was established in experiments which showed that exposure to anti-TSP monoclonal antibodies delay retinoic acid differentiation for 48 h. At the time the cells overcome the effects of TSP inhibition, laminin production becomes maximal. Treatment of the cells with a combination of anti-TSP and antilaminin antibodies results in complete inhibition of differentiation.
V P Castle, X Ou, S O'Shea, V M Dixit
Generalized peroxisome-deficient disorders including cerebro-hepato-renal Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease are autosomal recessive diseases, where catalase-containing particles (peroxisomes) are morphologically absent. We previously isolated two Chinese hamster ovary (CHO) cell mutants (Z24 and Z65) that resemble the fibroblasts from patients with such diseases, in their defective peroxisome assembly (Tsukamoto, T., S. Yokota, and Y. Fujiki. 1990. J. Cell Biol. 110:651-660). Here we report isolation by the P9OH/UV method of a peroxisome-deficient CHO mutant, ZP92, of the third complementation group distinct from those of Z24 and Z65. Peroxisomal membrane ghosts were noted by immunochemical staining in all of the CHO mutants. Complementation analysis by cell fusion of the CHO mutants with cultured fibroblasts from patients with generalized peroxisomal disorders revealed that two CHO mutants (Z24 and ZP92) represent the human complementation groups, E (the same as group 1 in the U.S.) and C (the same as group 4), respectively. These CHO cell mutants are an apparently relevant animal cell model for studies on the molecular bases and primary defects of human peroxisome-deficient diseases.
N Shimozawa, T Tsukamoto, Y Suzuki, T Orii, Y Fujiki
Enzymes within the P450IIIA (CYP3A) subfamily appear to account for significant "first pass" metabolism of some drugs in the intestine. To identify which of the known P450IIIA genes are expressed in intestine, enterocyte RNA was hybridized on Northern blots with synthetic oligonucleotides complementary to hypervariable regions of hepatic P450IIIA4, P450IIIA5, and P450IIIA7 cDNAs. Hybridization was detected only with the P450IIIA4-specific oligonucleotide. The identity of the hybridizing mRNA was confirmed to be P450IIIA4 by direct sequencing of a DNA fragment amplified from enterocyte cDNA by the polymerase chain reaction. To determine if enterocyte P450IIIA4 is inducible, biopsies of small bowel mucosa were obtained from five volunteers before and after they received 7d of treatment with rifampin, a known inducer of P450IIIA4 in liver. Rifampin treatment resulted in a five- or eightfold mean increase (P < 0.05) in the biopsy concentration of P450IIIA4 mRNA when normalized for content of sucrase isomaltase or intestinal fatty acid binding protein mRNAs, respectively. Rifampin also induced P450IIIA immunoreactive protein in enterocytes in each of the subjects, as judged by immunohistochemistry, and resulted in a 10-fold increase in P450IIIA4-specific catalytic activity (erythromycin N-demethylation) in the one patient studied. Our identification of inducible P450IIIA4 in enterocytes may in part account for drug interactions characteristic of P450IIIA4 substrates and suggests a strategy for controlling entry into the body of a major class of xenobiotics.
J C Kolars, P Schmiedlin-Ren, J D Schuetz, C Fang, P B Watkins
IL-3 is a glycoprotein cytokine involved in the hematopoietic response to infectious, immunologic, and inflammatory stimuli. In addition, clinical administration of recombinant IL-3 augments recovery in states of natural and treatment-related marrow failure. IL-3 acts by binding to high affinity cell surface receptors present on hematopoietic cells. To determine the site(s) at which IL-3 binds to it receptor, we analyzed a series of interspecies chimera of the growth factor for species-specific receptor binding and biological activity. The results suggest that IL-3 binds to its receptor and triggers a proliferative stimulus through two noncontiguous helical domains located near the amino terminus and the carboxy terminus of the molecule. To corroborate these findings, we have also mapped the binding epitopes of 10 mAb of human or murine IL-3, and have defined four distinct epitopes. Two of these epitopes comprise the amino-terminal receptor binding domain. A third epitope corresponds to the carboxy-terminal receptor interactive domain, and the fourth epitope, apparently not involved in the interaction of IL-3 and its receptor, lies between these sites. And on the basis of sandwich immunoassays using pairs of these mAbs, the two receptor interactive regions appear to reside in close juxtaposition in the tertiary structure of the molecule. These results provide a correlation of the structure-function relationships of IL-3 that should prove useful in evaluating the details of IL-3-IL-3 receptor interaction and in the rational design of clinically useful derivatives of this growth factor.
K Kaushansky, S G Shoemaker, V C Broudy, N L Lin, J V Matous, E M Alderman, J D Aghajanian, P J Szklut, R E VanDyke, M K Pearce
Hypertriglyceridemia is common in the general population, but its mechanism is largely unknown. In previous work human apo CIII transgenic (HuCIIITg) mice were found to have elevated triglyceride levels. In this report, the mechanism for the hypertriglyceridemia was studied. Two different HuCIIITg mouse lines were used: a low expressor line with serum triglycerides of approximately 280 mg/dl, and a high expressor line with serum triglycerides of approximately 1,000 mg/dl. Elevated triglycerides were mainly in VLDL. VLDL particles were 1.5 times more triglyceride-rich in high expressor mice than in controls. The total amount of apo CIII (human and mouse) per VLDL particle was 2 and 2.5 times the normal amount in low and high expressors, respectively. Mouse apo E was decreased by 35 and 77% in low and high expressor mice, respectively. Under electron microscopy, VLDL particles from low and high expressor mice were found to have a larger mean diameter, 55.2 +/- 16.6 and 58.2 +/- 17.8 nm, respectively, compared with 51.0 +/- 13.4 nm from control mice. In in vivo studies, radiolabeled VLDL fractional catabolic rate (FCR) was reduced in low and high expressor mice to 2.58 and 0.77 pools/h, respectively, compared with 7.67 pools/h in controls, with no significant differences in the VLDL production rates. In an attempt to explain the reduced VLDL FCR in transgenic mice, tissue lipoprotein lipase (LPL) activity was determined in control and high expressor mice and no differences were observed. Also, VLDLs obtained from control and high expressor mice were found to be equally good substrates for purified LPL. Thus excess apo CIII in HuCIIITg mice does not cause reduced VLDL FCR by suppressing the amount of extractable LPL in tissues or making HuCIIITg VLDL a bad substrate for LPL. Tissue uptake of VLDL was studied in hepatoma cell cultures, and VLDL from transgenic mice was found to be taken up much more slowly than control VLDL (P < 0.0001), indicating that HuCIIITg VLDL is not well recognized by lipoprotein receptors. Additional in vivo studies with Triton-treated mice showed increased VLDL triglyceride, but not apo B, production in the HuCIIITg mice compared with controls. Tissue culture studies with primary hepatocytes showed a modest increase in triglyceride, but not apo B or total protein, secretion in high expressor mice compared with controls. In summary, hypertriglyceridemia in HuCIIITg mice appears to result primarily from decreased tissue uptake of triglyceride-rich particles from the circulation, which is most likely due to increased apo CIII and decreased apo E on VLDL particles. the HuCIIITg mouse appears to be a suitable animal model of primary familial hypertriglyceridemia, and these studies suggest a possible mechanism for this common lipoprotein disorder.
K Aalto-Setälä, E A Fisher, X Chen, T Chajek-Shaul, T Hayek, R Zechner, A Walsh, R Ramakrishnan, H N Ginsberg, J L Breslow
Current knowledge of the phenotype of mononuclear cells accumulating in pancreatic islets in insulin-dependent diabetes (IDDM) and factors determining their homing into the pancreas is limited. Therefore, a pancreas obtained at the onset of IDDM was studied in detail. Cryostat sections were stained for mononuclear cell types, T cell receptor subtypes, and adhesion molecules of vascular endothelium and studied by immunofluorescence microscopy, and peripheral blood mononuclear cells were phenotyped using flow cytometry. Monocytes/macrophages (lysozyme- or CD 14-reactive cells) were identified among other mononuclear cell types in islet infiltrates. V beta 8-positive T cells were overrepresented, but T cells with other V beta s studied (V beta 5, V beta 5.1, V beta 6, V beta 12) were also found. The vascular endothelium of the islets and many small vessels nearby islets strongly expressed intercellular adhesion molecule-1, whereas vascular cell adhesion molecule-1 and E-selectin were totally absent. We conclude: (a) that increased expression of intercellular adhesion molecule-1 on vascular endothelium may increase endothelial adhesion of mononuclear cells and enhance their accumulation in the pancreas during diabetic insulitis; (b) that T cells with certain T cell receptors can be enriched in infiltrated pancreatic islets; and (c) that macrophages and antigen-specific CD 8-positive T cells are involved in pancreatic beta cell destruction at the onset of IDDM.
A Hänninen, S Jalkanen, M Salmi, S Toikkanen, G Nikolakaros, O Simell
Among the small round cell tumors differential diagnosis is particularly difficult for their undifferentiated or primitive character. In this mixed group of tumors, only the primitive neuroectodermal tumors, which include Ewing's sarcoma (ES), show the unique and consistent feature of the (11;22)(q24;q12) translocation, which can therefore be considered a hallmark of these neoplasias. We analyzed four primitive neuroectodermal tumor cell lines, one osteosarcoma cell line, and 11 patients by fluorescent in situ hybridization with cosmid clones 23.2 and 5.8, bracketing the t(11;22) at 11q24. Metaphase spreads from tumor cell lines, and from biopsy specimens of three patients with ES were analyzed. In the remaining eight patients comprising five ES, two small cell osteosarcomas and one chronic osteomyelitis, only nuclei preparations were available for analysis. We detected the t(11;22) in interphase nuclei of the four primitive neuroectodermal tumor cell lines, of three patients in which the karyotype demonstrated the translocation and in five cases of ES in which cytogenetic analysis had not been possible. Two cases of small cell osteosarcoma and one chronic osteomyelitis were also analyzed and were both normal with respect to the t(11;22). By analyzing cell lines and small round cell tumor samples by fluorescent in situ hybridization, we established that interphase cytogenetics is a rapid alternative to chromosomal analysis for the detection of the t(11;22) and represents an invaluable tool for the differential diagnosis of small round cell tumors.
M Giovannini, L Selleri, J A Biegel, K Scotlandi, B S Emanuel, G A Evans
Clusterin is a multifunctional protein endowed with cell-aggregating, complement-inhibitory, and lipid-binding properties. Since several studies have demonstrated highly increased clusterin gene expression in epithelial and nervous tissues regressing as a consequence of tissue involution and apoptotic cell death, clusterin is also considered as a specific marker of dying cells. To determine whether clusterin expression is also upregulated during thymocyte death occurring during the negative selection process we analyzed the cellular distribution of clusterin mRNA and protein by in situ hybridization and immunocytochemistry in the human thymus. We observed that the expression of clusterin mRNA was confined to cells present in the thymic medulla, concentrated mainly around Hassal's bodies. Immunostaining of adjacent sections with antikeratin Ab revealed that cells containing clusterin mRNA were predominantly epithelial. By contrast no clusterin mRNA was found in thymocytes by in situ hybridization and Northern blot analysis of total RNA from purified thymocyte populations. Clusterin protein colocalized with the membrane attack complex of complement and vitronectin in the center of the largest Hassal's bodies, but was not detectable by immunocytochemistry in or at the surface of epithelial cells. Our results demonstrate that clusterin gene expression does not take place in apoptotic thymocytes, and therefore that clusterin synthesis by the dying cell is probably not a prerequisite to its death. However, synthesis of clusterin by medullary epithelial cells may be related to their terminal differentiation, and, furthermore, its presence in Hassal's bodies raises the possibility that the secreted protein is involved in the disposal of cell debris resulting from thymocyte apoptosis.
L E French, A P Sappino, J Tschopp, J A Schifferli
Local production of growth factors may play a major role in vascular repair after injury. We examined the regulation of insulin-like growth factor-I (IGF-I) and its specific membrane receptor in balloon-denuded rat aorta. Aortic IGF-I mRNA and radioimmunoassayable IGF-I content increased severalfold after balloon denudation with a peak at 7 d after injury. This coincided with a reciprocal 25% decrease in IGF-I receptor mRNA content and a 40% decrease in total 125I-IGF-I binding. Scatchard analysis indicated a single class of binding sites, with a decrease in receptor number at 7 d compared to control and no change in affinity. By in situ hybridization the predominant site of IGF-I expression in the normal and the denuded vessel wall was the medial smooth muscle cell. After denudation there was a relative decrease in IGF-I receptor mRNA in the medial cells as compared to the neointima, suggesting that the site of IGF-I action was predominantly in the medial layer. These data suggest that local expression and action of IGF-I are significant in the promotion of smooth muscle cell proliferation after arterial injury.
M J Khorsandi, J A Fagin, D Giannella-Neto, J S Forrester, B Cercek
To define the interplay of glomerular hypertension and hypertrophy with mesangial extracellular matrix (ECM) deposition, we examined the effects of glomerular capillary distention and mesangial cell stretching on ECM synthesis. The volume of microdissected rat glomeruli (Vg), perfused ex vivo at increasing flows, was quantified and related to the proximal intraglomerular pressure (PIP). Glomerular compliance, expressed as the slope of the positive linear relationship between PIP and Vg was 7.68 x 10(3) microns 3/mmHg. Total Vg increment (PIP 0-150 mmHg) was 1.162 x 10(6) microns 3 or 61% (n = 13). A 16% increase in Vg was obtained over the PIP range equivalent to the pathophysiological limits of mean transcapillary pressure difference. A similar effect of renal perfusion on Vg was also noted histologically in tissue from kidneys perfused/fixed in vivo. Cultured mesangial cells undergoing cyclic stretching increased their synthesis of protein, total collagen, and key components of ECM (collagen IV, collagen I, laminin, fibronectin). Synthetic rates were stimulated by cell growth and the degree of stretching. These results suggest that capillary expansion and stretching of mesangial cells by glomerular hypertension provokes increased ECM production which is accentuated by cell growth and glomerular hypertrophy. Mesangial expansion and glomerulosclerosis might result from this interplay of mechanical and metabolic forces.
B L Riser, P Cortes, X Zhao, J Bernstein, F Dumler, R G Narins
The production of IL-1 and IL-6 by pituitary cells has recently been demonstrated. In this study we investigated the expression of IL-2 and its receptor (IL-2R) by pituitary cells of different species. In Northern blots, a single hybridizing band of 1 kb, identical to that in normal stimulated lymphocytes, was obtained with specific IL-2 probes. In the mouse AT-20 pituitary tumor cell line, IL-2 mRNA expression was detected after stimulation with corticotropin-releasing hormone or phorbol myristate acetate. In human corticotrophic adenoma cells, basal IL-2 mRNA expression as well as IL-2 secretion were further stimulated by phorbol myristate acetate. Both adenoma and AtT-20 cells showed detectable amounts of IL-2R mRNA and by immunofluorescence, IL-2R membrane expression. In addition, dual immunofluorescence studies in rat anterior pituitary cells demonstrated colocalization of IL-2R with ACTH-positive cells and other cell types expressing the receptor. In addition to the action of lymphocyte-produced IL-2, this cytokine may have a paracrine or autocrine regulatory role within the pituitary. It remains to be established whether IL-2 production occurs in the normal pituitary or is intrinsic to the process of tumor development of these cells. IL-2 may be involved in the growth control of pituitary cells.
E Arzt, G Stelzer, U Renner, M Lange, O A Müller, G K Stalla
To examine the role of metalloproteinases in tissue remodeling associated with wound healing, we used in situ hybridization to localize the expression of collagenase and tissue inhibitor of metalloproteinases (TIMP) in samples of pyogenic granuloma. Strong hybridization for collagenase mRNA was detected in basal keratinocytes near the advancing edge of all ulcerative lesions, but no collagenase mRNA was seen in samples without ulceration. Distinct from the sites of collagenase expression, TIMP mRNA was detected in stromal cells and in cells surrounding proliferating vessels. No collagenase mRNA was found in the epidermis of healthy skin, although occasional stromal cells contained collagenase or TIMP mRNAs, and TIMP mRNA was detected in hair follicles and sebaceous glands. Our results suggest that basal keratinocytes adjacent to wounded epidermis are critically involved in matrix remodeling, much more so than adjacent or underlying dermal fibroblasts. Furthermore, as several reports have suggested, TIMP may play a role in angiogenesis. Finally, in contrast to findings from other models which indicate that collagenase and TIMP proteins are secreted by the same cells, our data also demonstrate that these proteins can be produced in vivo independently of each other.
U K Saarialho-Kere, E S Chang, H G Welgus, W C Parks
Lipoprotein(a) consists of a low-density lipoprotein containing apolipoprotein (apo) B-100 and of the genetically polymorphic apo(a). It is not known where and how lipoprotein(a) is assembled and whether there exists a precursor for lipoprotein(a). We have determined the phenotype, concentration, and distribution of apo(a) in plasma from patients with lipoprotein lipase (LPL) deficiency (type I hyperlipoproteinemia, n = 14), in apo E 2/2 homozygotes with type III hyperlipoproteinemia (n = 12) and in controls (n = 16). In the two genetic conditions, there is grossly impaired catabolic conversion of apo B-100-containing precursor lipoproteins to low-density lipoproteins. Considering apo(a) type, the plasma concentration of apo(a) was normal in type III patients but significantly reduced in LPL deficiency. Despite the defects in the catabolism of other apo B-containing lipoproteins, the distribution of apo(a) was only moderately affected in both metabolic disorders, with 66.7% (type I) and 74.7% (type III) being present as the characteristic lipoprotein(a) in the density range of 1.05-1.125 g/ml (controls 81.6%). The remainder was distributed between the triglyceride-rich lipoproteins (type I 12.4%, type III 8.5%, controls 4.7%) and the lipid-poor bottom fraction (type I 19.3%, type III 15.3%, controls 12.6%). In all conditions most apo(a) (57-88%) dissociated from the triglyceride-rich lipoproteins upon recentrifugation and was recovered as lipoprotein(a). These data suggest that lipoprotein(a) is not generated from a triglyceride-rich precursor. Lipoprotein(a) may be secreted directly into plasma or may be formed by preferential binding of secreted apo(a) to existing low-density lipoprotein.
C Sandholzer, G Feussner, J Brunzell, G Utermann
Functional studies indicate that atrial natriuretic peptide (ANP), a member of the natriuretic peptide family, has direct effects on cardiac muscle cells. However, conventional ligand-binding studies designed to establish the presence of natriuretic peptide-binding sites in the heart have yielded conflicting results. There are discrepancies also between the latter and the receptor distribution predicted from the pattern of the mRNA transcripts localized by in situ hybridization. Here we have employed the technique of cDNA amplification with the polymerase chain reaction to confirm the presence of natriuretic peptide A, B, and C receptor mRNAs in rat and human cardiac tissue. In the rat heart, the distribution of the A and B receptor transcripts appears to be relatively homogeneous; in contrast, the C type mRNA is concentrated principally in the atria, with no difference between the left and right sides of the heart. A and B receptor DNA products were obtained after amplification of left, but not right, ventricular cDNA from the heart of a 16-yr-old male with cystic fibrosis; the yield of C receptor DNA was similar for both ventricles. If these mRNA transcripts are translated into functional receptors in the rat and human heart, ANP and the other natriuretic peptides may have direct effects on cardiac function, including regulation of natriuretic peptide release via a short feedback loop, modulation of contractility of the heart, or activation of cardiac reflexes.
D J Nunez, M C Dickson, M J Brown
Radiolabeled analogues of 2-deoxyglucose are widely used to trace glucose metabolism in cell cultures, whole organs, and intact animals, although kinetic differences in transport and phosphorylation between these compounds and glucose exist. The present studies were undertaken to determine the effects of insulin stimulation on the phosphorylation of 2-deoxyglucose compared to glucose in the intact, saline-perfused working rat heart. Rates of glucose utilization determined from tritiated glucose differed from rates estimated from the accumulation of [14C]2-deoxyglucose in a nonconstant manner when comparing rates in the absence or presence of physiologic levels of insulin (13 microU/ml). The fraction of monophosphorylated hexoses that was accounted for by [14C]2-deoxyglucose 6-phosphate was dramatically decreased in hearts perfused in the presence of insulin. Additionally, hexokinase activity associated with the mitochondrial fraction of tissue extracts was increased in hearts stimulated by insulin. While this redistribution of hexokinase to the mitochondria did not affect the apparent affinity constant for glucose, hexokinase bound to mitochondria exhibited an 8.5-fold decrease in the affinity for 2-deoxyglucose when compared with hexokinase present in the cytosolic fraction. The findings are consistent with an insulin-mediated preferential uptake and phosphorylation of glucose compared to deoxyglucose. The results also imply that the redistribution of hexokinase and the differential effect of insulin on its affinity for tracer and tracee are responsible for changes in the "lumped constant" (i.e., the correction factor used to equate 2-deoxyglucose to glucose uptake). These changes must be taken into account when regional myocardial glucose metabolism is assessed by the 2-deoxyglucose method.
R R Russell 3rd, J M Mrus, J I Mommessin, H Taegtmeyer
We investigated the protein composition of J774-E clone macrophage phagosomes isolated at different stages of phagolysosome biogenesis. Phagosomes formed by internalizing antibody-coated Staphylococcus aureus for 3 min followed by chase for 0, 4, 9, or 15 min were isolated by density gradient centrifugation. Enrichment and purity of the phagosome preparations were quantitated by radiolabeled ligand recovery, enzyme markers, and electron microscopy. One-dimensional SDS-PAGE analyses of the isolated phagosomes revealed virtually identical protein compositions. However, Western blot analyses with antibodies directed against selected proteins of known itineraries along the endocytic pathway demonstrated distinct differences in phagosome protein compositions. Accumulating within the maturing phagosome were the 31-kD subunit of the vacuolar proton pump, cathepsin D,beta-glucuronidase, the cation dependent mannose 6-phosphate receptor, and LAMP-1. Decreasing within the maturing phagosome were the FcII receptor, the mannose receptor, and alpha-adaptin. These results indicate that although the macrophage phagosome's total protein composition changes little during phagolysosome formation, the maturing phagosome both receives and eliminates, possibly by protein recycling, specific membrane and sequestered proteins.
A Pitt, L S Mayorga, P D Stahl, A L Schwartz
Acute gastrointestinal infections due to rotaviruses and other enteric pathogens are major causes of morbidity and mortality in infants and young children throughout the world. Breast-feeding can reduce the rate of serious gastroenteritis in infants; however, the degrees of protection offered against rotavirus infection vary in different populations. The mechanisms associated with milk-mediated protection against viral gastroenteritis have not been fully elucidated. We have isolated a macromolecular component of human milk that inhibits the replication of rotaviruses in tissue culture and prevents the development of gastroenteritis in an animal model system. Purification of the component indicates that the antiviral activity is associated with an acidic fraction (pI = 4.0-4.6), which is free of detectable immunoglobulins. Furthermore, high levels of antiviral activity are associated with an affinity-purified complex of human milk mucin. Deglycosylation of the mucin complex results in the loss of antiviral activity. Further purification indicated that rotavirus specifically binds to the milk mucin complex as well as to the 46-kD glycoprotein component of the complex. Binding to the 46-kD component was substantially reduced after chemical hydrolysis of sialic acid. We have documented that human milk mucin can bind to rotavirus and inhibit viral replication in vitro and in vivo. Variations in milk mucin glycoproteins may be associated with different levels of protection against infection with gastrointestinal pathogens.
R H Yolken, J A Peterson, S L Vonderfecht, E T Fouts, K Midthun, D S Newburg
Early diabetes mellitus is characterized by impaired responses to pressor hormones and pressor receptor downregulation. The present study examined the effect of elevated extracellular glucose concentrations on angiotensin II (AII) and arginine vasopressin (AVP) receptor kinetics in cultured rat vascular smooth muscle cells (VSMC). Scatchard analysis of [3H]AVP and 125I-AII binding to confluent VSMC showed that high glucose concentrations (20 mM) similarly depressed AVP and AII surface receptor Bmax but did not influence receptor Kd. This receptor downregulation was not reproduced by osmotic control media containing either L-glucose or mannitol. Receptor downregulation was maximal at a glucose concentration of 15-20 mM and required 24-48 h for a maximum effect. Normalization of the extracellular glucose concentration allowed complete recovery of AVP and AII binding within 48 h. Receptor downregulation was associated with depressed AVP and AII-stimulated intracellular signaling and cell contraction. High glucose concentrations induced a sustained activation of protein kinase C (PKC) in VSMC, which was prevented by coincubation with H-7. H-7 also markedly attenuated glucose-induced downregulation of AVP and AII receptors on VSMC. This study demonstrates a novel cellular mechanism whereby high extracellular glucose concentrations directly and independently downregulate pressor hormone receptors and their function on vascular tissue via glucose-stimulated PKC activation.
B Williams, P Tsai, R W Schrier
The complement system participates in the immune recognition of foreign antigens, many of which may penetrate the skin by physical injury or transcutaneous adsorption. In this study, we examined the presence of complement components and complement regulatory proteins in the human skin and cultured human keratinocytes. Immunofluorescence studies showed C3, Factor B, decay accelerating factor, the C3b receptor (CR1), and C3d receptor (CR2), distributed among cells of the epidermis as well as on cultured keratinocytes. Immunoblot analysis of keratinocytes supernatants showed the presence of C3 with a molecular weight of approximately 180 kD. The decay accelerating factor was localized as previously reported on elastic fibers; additionally it was observed in the basement membrane zone. In situ hybridization studies suggest the expression of CR1 and CR2 mRNA in human epidermis. These results show the presence in the human epidermis of complement components that are capable of generating the initial C3 convertase of the alternative pathway. The presence of complement regulatory proteins could endow keratinocytes with immune functions such as the regulation of complement activation and endocytosis of C3 opsonized particles.
N Dovezenski, R Billetta, I Gigli
Lipoprotein lipase enhances binding at 4 degrees C of human plasma lipoproteins (chylomicrons, VLDL, intermediate density lipoprotein, LDL, and HDL3) to cultured fibroblasts and hepG-2 cells and to extracellular matrix. Heparinase treatment of cells and matrix reduces the lipoprotein lipase enhanced binding by 90-95%. Lipoprotein lipase causes only a minimal effect on the binding of lipoproteins to heparan sulfate deficient mutant Chinese hamster ovary cells while it promotes binding to wild type cells that is abolished after heparinase treatment. With 125I-LDL, lipoprotein lipase also enhances uptake and proteolytic degradation at 37 degrees C by normal human skin fibroblasts but has no effect in heparinase-treated normal cells or in LDL receptor-negative fibroblasts. These observations prove that lipoprotein lipase causes, predominantly, binding of lipoproteins to heparan sulfate at cell surfaces and in extracellular matrix rather than to receptors. This interaction brings the lipoproteins into close proximity with cell surfaces and may promote metabolic events that occur at the cell surface, including facilitated transfer to cellular receptors.
S Eisenberg, E Sehayek, T Olivecrona, I Vlodavsky
The effects of the calcium channel blocking agent, verapamil, were studied in a murine model of viral myocarditis. Three groups of 8-wk-old DBA/2 mice (n = 25 each) were inoculated with 10 plaque-forming units of encephalomyocarditis virus and randomized to three treatment regimens. Group 1 mice received verapamil intraperitoneally (5 mg/kg per d) for 7 d before infection, followed by verapamil orally (mean dose of 3.5 mg/mouse per d) in drinking water during infection. Group 2 mice received only verapamil orally starting on day 4 after infection, coincident with peak viremia. Group 3 (infected control) received no verapamil in regular drinking water after viral inoculation. Additional control animals were studied in group 4 (n = 21), consisting of uninfected control animals receiving intraperitoneal and oral verapamil at doses identical to group 1, and in group 5 (n = 21), consisting of uninfected and untreated controls. Animals were randomly killed from each group (n = 7) at 7, 14, and 28 d after infection. Routine histology was performed blindly on an apical slice of each heart and semi-quantitatively graded for inflammation, necrosis, calcification, and fibrosis on a scale of 0-4. Digital planimetry was performed to measure the absolute and relative areas of inflammation and necrosis. The pretreated animals in group 1 showed marked reduction in inflammation and necrosis (score of 3.7 +/- 1.4 vs. 8.7 +/- 2.0 in group 3 on day 14, P < 0.05) and were indistinguishable from the posttreated group 2 mice (score of 4.0 +/- 1.5 vs. 8.7 +/- 2.0 in group 3 on day 14, P < 0.05). All the uninfected control animals (groups 4 and 5) showed no myocardial lesions whether treated with verapamil or not. Quantitative planimetry confirmed decreased inflammation and necrosis (2.0 +/- 3.3% in group 1 and 3.5 +/- 3.1% in group 2 vs. 21.9 +/- 22.6% in group 3 on day 14). Untreated infected hearts injected with liquid silicone rubber exhibited extensive areas of focal microvascular constriction and microaneurysm formation; verapamil treatment in either group 1 or 2 completely abolished these abnormalities, resembling uninfected controls in groups 4 or 5. We conclude that verapamil, whether given before infection or after peak viremia in an encephalomyocarditis model of murine myocarditis, significantly reduces the microvascular changes and myocardial necrosis, fibrosis, and calcification leading to cardiomyopathy. This suggests the potentially important role of calcium and microvascular spasm in the pathogenesis of viral myocarditis leading to dilated cardiomyopathy, and may have future therapeutic implications.
R Dong, P Liu, L Wee, J Butany, M J Sole
Leukemia inhibitory factor (LIF) has many biological actions which parallel those of IL-1, IL-6 and tumor necrosis factor-alpha, but its role in the pathogenesis of human disease is unknown. A specific radioreceptor competition assay capable of detecting LIF at concentrations above 1 ng/ml (45 pM) was developed. To identify disease states in which LIF might be involved, a cross-sectional survey of serum and body fluids from approximately 1,500 subjects with a variety of diseases was performed using the LIF radioreceptor competition assay. Serum LIF concentrations were transiently elevated (2-200 ng/ml) in six subjects with meningococcal or Gram-negative septic shock, and in a subject with idiopathic fulminant hepatic failure. Moderately elevated LIF concentrations (> 10 ng/ml) were detected in cerebrospinal fluid from subjects with bacterial meningitis, in effusions associated with pneumonia and peritonitis, and in amniotic fluid from a woman with chorioamnionitis. Low LIF concentrations (1-10 ng/ml) were present in synovial fluid from subjects with inflammatory arthritis, amniotic fluid from women in labor, and some reactive, chronic inflammatory and malignant effusions and cyst fluids, but rarely in transudates. These initial findings suggest that LIF might be involved in the pathogenesis of inflammation and septic shock.
P Waring, K Wycherley, D Cary, N Nicola, D Metcalf
The human Pena/Penb alloantigen system represents a naturally occurring polymorphism of human platelet membrane glycoprotein (GP) IIIa, and has previously been implicated in the onset of two important clinical syndromes, neonatal alloimmune thrombocytopenic purpura and posttransfusion purpura. To investigate the molecular basis of the polymorphism underlying the Pen alloantigen system, we used the polymerase chain reaction to amplify platelet-derived GPIIIa mRNA transcripts. DNA sequence analysis of amplified GPIIIa cDNAs from nucleotides 161 to 1341 (encompassing amino acid residues 22-414) revealed a G526<==>A526 polymorphism that segregated precisely with Pen phenotype in twelve other individuals examined. This nucleotide substitution results in an Arg (CGA) to Gln (CAA) polymorphism at amino acid 143 of GPIIIa. Interestingly, this polymorphic residue is located within the putative RGD binding site (residues 109-171) of GPIIIa. Platelet aggregation patterns of a Penb/b individual, however, were nearly normal in response to all physiological agonists tested, indicating that this polymorphism does not grossly affect integrin function. Short synthetic peptides encompassing residue 143 were unable to mimic either the Pena or Penb antigenic determinants, suggesting that the Pen epitopes are dependent upon proper folding of the polypeptide chain. Finally, we constructed allele-specific recombinant forms of GPIIIa that differed only at amino acid residues 143. Whereas anti-Pena alloantibodies were able to recognize the Arg143 recombinant form of GPIIIa, anti-Penb antibodies were not. Conversely, anti-Penb alloantibodies were reactive only with the Gln143 isoform of the GPIIIa molecule. It thus appears that amino acid 143 of GPIIIa is not only associated with Pen phenotype, but specifically controls the formation and expression of the Pen alloantigenic determinants.
R Wang, K Furihata, J G McFarland, K Friedman, R H Aster, P J Newman
Heparin inhibits smooth muscle cell (SMC) proliferation after arterial injury by mechanisms that have yet to be defined. Since the initiation of SMC proliferation is mediated by basic fibroblast growth factor (bFGF), we have investigated the possibility that heparin inhibits SMC proliferation by displacing bFGF from the arterial wall. Using a rat carotid artery model of balloon catheter injury, we demonstrate that a bolus injection of heparin depletes the arterial wall of both systemically administered bFGF and of endogenous bFGF. Heparin, however, does not reduce the bFGF content of unmanipulated arteries. Further, a single injection of heparin given at the time of balloon injury reduces SMC proliferation by 55% but has no effect when given 6 h after injury. SMC proliferation induced in a denuded artery by injection of bFGF is inhibited almost completely by a bolus injection of heparin; however, pretreatment with a bolus of heparin does not prevent SMC from responding to a subsequent bolus of bFGF. These experiments suggest that heparin can inhibit SMC proliferation in part by removal of released bFGF from sites of injury.
V Lindner, N E Olson, A W Clowes, M A Reidy
Little is known about changes in the amount of iron in the intracellular low molecular weight pool, which catalyzes the Fenton reactions during reperfusion after ischemia. In this study a new approach is presented to measure low molecular weight iron and it is applied to normal hearts during ischemia and to iron-loaded hearts during anoxia and reoxygenation. The results of this study show that (a) during ischemia in normal hearts a progressive 30-fold increase occurs in low molecular weight iron after 45 min of ischemia, whereas (b) during 45 min of anoxic perfusion the low molecular weight iron does not increase. This means that the reductive release from the storage protein ferritin is greatly enhanced by the acidification that occurs during ischemia. (c) Anoxic perfusion of iron-loaded hearts does increase low molecular weight iron and there is a further increase upon reoxygenation, which is prevented by (+)-cyanidanol-3. Based on these findings it is concluded that oxygen deprivation enhances the susceptibility of rat hearts to oxygen radicals by increasing the amount of catalytic, ferrous iron in the low molecular weight pool.
A Voogd, W Sluiter, H G van Eijk, J F Koster
The three isoforms of transforming growth factor-beta (TGF-beta) have previously been implicated in embryonic development of the heart as well as in repair of myocardial damage after ischemia/reperfusion injury. TGF-beta 1 has also been localized intracellularly to both mitochondria and contractile filaments of cardiac myocytes, although its role in these structures has not been defined. We now report that exogenous TGF-beta stabilizes the beating rate of neonatal rat cardiac myocytes cultured on fibroblast matrix, and sustains their spontaneous rhythmic beating in serum-free medium. Moreover, using blocking antibodies to TGF-beta, we show that endogenous TGF-beta secreted by these myocytes acts in an autocrine fashion to maintain their beating rate. In contrast, IL-1 beta, an inflammatory mediator secreted by immune cells during myocardial injury, inhibits the beating of cardiac myocytes, and TGF-beta can overcome this inhibition. The antagonistic effects of TGF-beta and IL-1 were not observed when the myocytes were cultured on gelatin, as compared to native fibroblast matrix. The data indicate that TGF-beta is an important regulator of contractile function of the heart and have significant implications for understanding cardiac physiology in health and disease.
A B Roberts, N S Roche, T S Winokur, J K Burmester, M B Sporn
The effect of ultrasound on the rate of fibrinolysis has been investigated using an in vitro system. Plasma or blood clots containing a trace label of 125I fibrin were suspended in plasma containing plasminogen activator and intermittently exposed to continuous wave 1-MHz ultrasound at intensities up to 8 W/cm2. Plasma clot lysis at 1 h with 1 microgram/ml recombinant tissue plasminogen activator (rt-PA) was 12.8 +/- 1.2% without ultrasound and was significantly (P = 0.0001) increased by exposure to ultrasound with greater lysis at 1 W/cm2 (18.0 +/- 1.4%), 2 W/cm2 (19.3 +/- 0.7%), 4 W/cm2 (22.8 +/- 1.8%), and 8 W/cm2 (58.7 +/- 7.1%). Significant increases in lysis were also seen with urokinase at ultrasound intensities of 2 W/cm2 and above. Exposure of clots to ultrasound in the absence of plasminogen activator did not increase lysis. Ultrasound exposure resulted in a marked reduction in the rt-PA concentration required to achieve an equivalent degree of lysis to that seen without ultrasound. For example, 15% lysis occurred in 1 h at 1 microgram/ml rt-PA without ultrasound or with 0.2 microgram/ml with ultrasound, a five-fold reduction in concentration. Ultrasound at 1 W/cm2 and above also potentiated lysis of retracted whole blood clots mediated by rt-PA or urokinase. The maximum temperature increase of plasma clots exposed to 4 W/cm2 ultrasound was only 1.7 degrees C, which could not explain the enhancement of fibrinolysis. Ultrasound exposure did not cause mechanical fragmentation of the clot into sedimentable fragments, nor did it alter the sizes of plasmic derivatives as demonstrated by SDS polyacrylamide gel electrophoresis. We conclude that ultrasound at 1 MHz potentiates enzymatic fibrinolysis by a nonthermal mechanism at energies that can potentially be applied and tolerated in vivo to accelerate therapeutic fibrinolysis.
C W Francis, P T Onundarson, E L Carstensen, A Blinc, R S Meltzer, K Schwarz, V J Marder
Oral administration of isotretinoin (13-cis retinoic acid) was shown previously (Kraemer, K. H., J. J. DiGiovanna, A. N. Moshell, R. E. Tarone, and G. L. Peck. 1988. N. Engl. J. Med. 318:1633-1637) to reduce the frequency of skin cancers in xeroderma pigmentosum (XP) patients. The mechanism of protection was unclear. In the present study, x-ray-induced chromatid damage in PHA-stimulated blood lymphocytes from five XP patients receiving isotretinoin was approximately half that in blood samples from the same patients before or subsequent to treatment. The x-ray-induced chromatid damage in blood lymphocytes from a normal control was reduced significantly by cocultivation with blood or plasma from an XP patient receiving isotretinoin or by addition of 10(-6) M isotretinoin to cultures 1 h before x-irradiation. A similar reduction in x-ray-induced chromatid damage was reported previously by adding to the culture medium, mannitol, a scavenger of the free hydroxyl radical, or catalase, which decomposes hydrogen peroxide; both of these products are generated during ionizing radiation. The present observations suggest that isotretinoin acts as a scavenger of such radiation products, thereby providing protection against x-ray-induced chromatid damage.
K K Sanford, R Parshad, F M Price, R E Tarone, K H Kraemer
Abnormal processing of immune complexes (IC) may be important in the pathogenesis of systemic lupus erythematosus (SLE). The clearance of large soluble IC (comprising hepatitis B surface antigen (HBsAg)/anti-HBsAg) radiolabeled with 123I was examined in 12 normal subjects and 10 patients with SLE. IC localization was analyzed by static and dynamic gamma-scintigraphy. Initial IC clearance from blood was more rapid in patients (median t1/2 = 2.15 min) than normals (median t1/2 = 5.15 min) due to more rapid uptake in the liver. However, in the SLE group, up to 12% of complexes were released from the liver after 30-50 min. Splenic uptake of immune complexes was reduced in the patients and there was reduced ability to retain IC in this organ. Plasma complement levels and erythrocyte complement receptor type 1 numbers were reduced in the patients, resulting in defective opsonization of IC and reduced red cell binding in vivo. These observations support the hypothesis that IC handling is abnormal in SLE.
K A Davies, A M Peters, H L Beynon, M J Walport
We have reported that transgenic mice overexpressing rat apo E shows marked reduction of plasma cholesterol and triglyceride levels due to the disappearance of VLDL and LDL. In this study, we investigated the metabolism of plasma lipoproteins in transgenic mice. After intravenous injection, the rates of clearance of 125I-VLDL and 125I-LDL were 3.0- and 2.4-fold greater in transgenic mice than in controls, respectively. Furthermore, clearance of chylomicron remnants estimated by oral retinyl palmitate-loading test was markedly enhanced in transgenic mice. The hepatic expression of LDL receptors by immunoblot analysis was similar in both groups. These data suggest that elimination of lipoproteins containing apo B was due to enhanced clearance of these lipoproteins enriched with apo E through hepatic LDL receptors. When fed a high cholesterol diet, controls showed twofold elevation of plasma cholesterol levels with marked increases in VLDL and LDL cholesterol on gel filtration chromatography. In contrast, cholesterol-fed transgenic mice showed resistance against these increases. High cholesterol feeding decreased the activity of hepatic LDL receptors and had no effect on enhancement of chylomicron remnant clearance in transgenic mice. Thus, overexpression of apo E facilitates metabolism of lipoproteins containing apo B presumably primarily via the LDL receptor pathway and possibly through an interaction with the chylomicron remnant receptor.
H Shimano, N Yamada, M Katsuki, K Yamamoto, T Gotoda, K Harada, M Shimada, Y Yazaki
The constitutive endothelial cell nitric oxide synthase (NOS) importantly regulates vascular homeostasis. To gain understanding of this enzyme, a pEF BOS cDNA library of 5 x 10(5) clones was prepared from bovine aortic endothelial cells (BAEC) and screened with a 2.8-kb cDNA BamHI fragment of rat brain NOS. Clone pBOS13 was found to express NO synthase activity when transfected into COS-7 cells. Sequence analysis revealed sequences compatible with binding domains for calcium/calmodulin, flavin mononucleotide, flavin adenine nucleotide and NADPH. The deduced amino acid sequence revealed a protein with a relative mol mass of 133,286, which is 58% homologous to the rat cerebellar NOS and 51% homologous to the mouse macrophage NOS. The amino-terminal portion of the protein exhibits several characteristics peculiar to the endothelial cell NOS. These include a proline-rich region and several potential sites for proline-directed phosphorylation as well as a potential substrate site for acyl transferase. Northern hybridization to mRNA from cultured BAEC revealed an abundant 4.8-kb message, which was not increased by coincubation with tumor necrosis factor alpha, but was markedly increased by exposure to shear stress for 24 h. The unique features of the endothelial cell NO synthase, particularly in the amino terminal portion of the molecule, may provide for novel regulatory influences of enzyme activity and localization.
K Nishida, D G Harrison, J P Navas, A A Fisher, S P Dockery, M Uematsu, R M Nerem, R W Alexander, T J Murphy
We have analyzed the nucleotide sequence of the androgen receptor from 22 unrelated subjects with substitution mutations of the hormone-binding domain. Eleven had the phenotype of complete testicular feminization, four had incomplete testicular feminization, and seven had Reifenstein syndrome. The underlying functional defect in cultured skin fibroblasts included individuals with absent, qualitative, or quantitative defects in ligand binding. 19 of the 21 substitution mutations (90%) cluster in two regions that account for approximately 35% of the hormone-binding domain, namely, between amino acids 726 and 772 and between amino acids 826 and 864. The fact that one of these regions is homologous to a region of the human thyroid hormone receptor (hTR-beta) which is a known cluster site for mutations that cause thyroid hormone resistance implies that this localization of mutations is not a coincidence. These regions of the androgen receptor may be of particular importance for the formation and function of the hormone-receptor complex.
M J McPhaul, M Marcelli, S Zoppi, C M Wilson, J E Griffin, J D Wilson
Unlike the human Fc gamma RII and Fc gamma RIII families, which exhibit considerable diversity at both the nucleic acid and protein levels, the human Fc gamma RI family has only a single recognized product expressed as a 70-kD cell surface receptor with high affinity for monomeric IgG (hFc gamma RIa1). Using both polymerase chain reaction-based amplification and Northern hybridization, we document multiple interferon-gamma-inducible hFc gamma RI RNA transcripts in human mononuclear cells and neutrophils. The sequences of two of these Fc gamma RI related transcripts indicate that they are alternatively spliced products of a second Fc gamma RI family gene, termed Fc gamma RIB. The cDNA derived from the larger of these transcripts, termed hFc gamma RIb1, encodes a surface molecule that is not recognized by Fc gamma RI specific monoclonal antibodies when transfected into COS-7 cells. Unlike the interferon-gamma-inducible hFc gamma RIA gene product, hFc gamma RIb1 does not bind monomeric IgG with high affinity. However, both hFc gamma RIa1 and hFc gamma RIb1 do bind aggregated human IgG. Previously unrecognized diversity within the hFc gamma RI family includes an interferon-gamma-inducible, putative low affinity Fc gamma receptor that may play an important role in the mechanism by which Fc gamma receptors participate in the humoral immune response.
A J Porges, P B Redecha, R Doebele, L C Pan, J E Salmon, R P Kimberly
Fractionated and unfractionated heparins are widely used as antithrombotic agents. Because of their heterogeneous composition, it is difficult to study the pharmacokinetics of these drugs. We now report on a new method for labeling low molecular weight heparins with 131I by binding tyramine to the anhydromannose end of the molecules. We examined the pharmacokinetics of the compound by intravenous injection of 131I-tyramine-heparin into Wistar rats. About 18% of the activity was found in the liver, whereas 33% was detected in urine. Biological activity in terms of Factor Xa inhibition was measurable. Since evidence from cell culture experiments implies that reticuloendothelial cell system receptors might be involved in heparin metabolism, maleylated BSA, a substance known to block scavenger receptors, was injected before the radiolabeled heparin compound. The liver uptake was reduced from 17.4 to 4.8%. Injection of unfractionated heparin before tracer application caused a considerable increase in urine excretion of the tracer substance. To our knowledge, this is the first report that liver uptake of heparins is linked to scavenger receptor mediated mechanisms in vivo. This interaction of heparins with scavenger receptors might play an important role in the biology of the vessel wall.
G Stehle, E A Friedrich, H Sinn, A Wunder, J Harenberg, C E Dempfle, W Maier-Borst, D L Heene
Hybrid receptors were studied in GC rat pituitary cells overexpressing either wild-type 950Tyr (WT) human insulin-like growth factor I (IGF-I) receptors or mutant human IGF-I receptors truncated at position 952 in the beta subunit transmembrane region (952STOP). 125I-IGF-I binding was increased in both 950Tyr (WT) (14-fold) and truncated human IGF-I receptor (952STOP) stable transfectants (50-fold), when compared to untransfected cells that contained endogenous rat IGF-I receptors. Metabolic cell labeling followed by immunoprecipitation with monoclonal alpha and beta subunit-specific antibodies revealed the presence of hybrid rat/truncated human receptors, truncated transfected human receptors, and WT human IGF-I holotetramers. Both mutant and hybrid receptors were degraded slower than 950Tyr (WT) receptors (> 16 h). Despite their markedly increased ligand binding and prolonged receptor half-life, 952STOP transfectants failed to transduce the IGF-I signal to suppress growth hormone (GH). Also, they neither underwent autophosphorylation nor phosphorylated endogenous proteins. The expected suppression of GH by endogenous rat IGF-I receptors was completely abrogated in 952STOP transfectants (P < 0.001 compared to untransfected cells). Mutant 952STOP cells were therefore completely devoid of biological signaling to GH despite the presence of endogenous rat IGF-I receptors. Thus mutant IGF-I receptors block ligand-mediated endogenous rat IGF-I signaling by functioning as a dominant negative forming nonfunctional human/rat hybrid receptors. Defective IGF-I receptors may function therefore as dominant negative phenotypes which suppress normal receptor responses in pituitary cells.
D Prager, H Yamasaki, M M Weber, S Gebremedhin, S Melmed
The hydroxyl radical (OH.) scavenger dimethyl sulfoxide (DMSO) was found to dose-dependently inhibit interleukin 8 (IL-8) production in LPS-stimulated human whole blood. At a concentration of 1% (vol/vol), DMSO blocked IL-8 release by approximately 90% in the presence of 1 microgram/ml LPS at a 24-h time point, but did not affect cell viability or reduce the production of tumor necrosis factor (TNF), interleukin 6, or interleukin-1 beta (IL-1 beta). DMSO was found to directly inhibit IL-8 expression at the level of transcription. Furthermore, this effect was not LPS-specific, in that IL-8 production was reduced by DMSO to a similar extent upon stimulation of blood with phytohemagglutinin, aggregated immune complexes, TNF, or IL-1 beta. Other oxygen radical scavengers that have been shown to inhibit OH.-dependent reactions (dimethyl thiourea, thiourea, mannitol, and ethanol) also inhibited IL-8 production. Conversely, addition of H2O2 caused a dose-dependent stimulation of IL-8 release. These results provide evidence that reactive oxygen metabolites play an important role in the regulation of IL-8 production and suggest that reduction of IL-8 release may contribute to the beneficial effects of antioxidants in experimental models of inflammation and ischemia/reperfusion injury.
L E DeForge, J C Fantone, J S Kenney, D G Remick
Bilirubin is conjugated with glucuronic acid in hepatocytes and subsequently secreted in bile. The major conjugate is bilirubin diglucuronide. Using sealed vesicles which are primarily derived from the canalicular (CMV) and sinusoidal (SMV) membrane vesicle domains of the plasma membrane of hepatocytes, we demonstrated that bilirubin glucuronides are transported by CMV by both ATP- and membrane potential-dependent transport systems. In CMV from normal rats, these processes are additive. In CMV from TR- rats, which have an autosomal recessively inherited defect in biliary secretion of nonbile acid organic anions, ATP-dependent transport of bilirubin diglucuronide was absent whereas the membrane potential driven system was retained. Other canalicular ATP-dependent transport systems, which were previously described for organic cations and bile acids, are functionally retained in TR- rats. Our study indicates that bilirubin glucuronides are primarily secreted into the bile canaliculus by an ATP-dependent mechanism which is defective in an animal model of the human Dubin-Johnson syndrome.
T Nishida, Z Gatmaitan, J Roy-Chowdhry, I M Arias
The use of cyclosporin A (CsA) as an immunosuppressive agent has markedly improved the clinical outcome in solid organ transplantation. However, posttransplantation infection remains a significant problem and may contribute to subsequent organ rejection. In this study the effect of cytomegalovirus (CMV) immediate early (IE) gene products on interleukin 2 (IL-2) gene transcription in the absence and presence of CsA was investigated using a transient transfection system. Jurkat T cells were transfected with plasmids expressing the CMV IE gene products or with a control plasmid. The presence of the CMV IE2 gene product abolished the inhibitory effect of CsA on IL-2 promoter activation and gene transcription. This effect was noted regardless of the time of CsA addition relative to the time of stimulation and was independent of CsA concentration. CsA had no effect on the CMV or the IL-2 receptor promoters. These studies suggest that the CMV IE gene products may play a role in graft rejection after solid organ transplantation.
L J Geist, M M Monick, M F Stinski, G W Hunninghake