Oral administration of vanadate to diabetic streptozotocin-treated rats decreased the high blood glucose and D-3-hydroxybutyrate levels related to diabetes. The increase in the expression of the P-enolpyruvate carboxykinase (PEPCK) gene, the main regulatory enzyme of gluconeogenesis, was counteracted in the liver and the kidney after vanadate administration to diabetic rats. Vanadate also counteracted the induction in tyrosine aminotransferase gene expression due to diabetes and was able to increase the expression of the glucokinase gene to levels even higher than those found in healthy animals. Similarly, an induction in pyruvate kinase mRNA transcripts was observed in diabetic vanadate-treated rats. These effects were correlated with changes on glucokinase and pyruvate kinase activities. Vanadate treatment caused a decrease in the expression of the liver-specific glucose transporter, GLUT-2. Thus, vanadate was able to restore liver glucose utilization and block glucose production in diabetic rats. The increase in the expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCoAS) gene, the key regulatory enzyme in the ketone bodies production pathway, observed in diabetic rats was also blocked by vanadate. Furthermore, a similar pattern in the expression of PEPCK, GLUT-2, HMGCoAS, and the transcription factor CCAAT/enhancer-binding protein alpha genes has been observed. All of these results suggest that the regulation of the expression of genes involved in the glucose and ketone bodies metabolism could be a key step in the normalization process induced by vanadate administration to diabetic rats.
A Valera, J E Rodriguez-Gil, F Bosch
We have identified and characterized two mutations in the hormone binding domain of the vitamin D receptor (VDR) in patients with hereditary vitamin D-resistant rickets. One patient was found to have a premature stop mutation (CAG to TAG) in the hinge region affecting amino acid 149 (Q149X) and the other demonstrated a missense mutation (CGC to CTC) resulting in the substitution of arginine 271 by leucine (R271L) in the steroid binding domain. Eukaryotic expression analyses in CV-1 cells showed the inability of both patients' VDR to induce transcription from the osteocalcin hormone gene response element at 10(-7) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Normal transcription levels could, however, be elicited by the missense mutated VDR (R271L) in the presence of 1,000-fold higher 1,25-(OH)2D3 concentrations than needed for the wild-type receptor. This shows that Arg 271 directly affects the affinity of the VDR for its ligand and its conversion to leucine decreases its affinity for 1,25(OH)2D3 by a factor of 1,000. Arg 271 is located immediately 3-prime to a 30 amino acid segment (VDR amino acids 241-270) that is conserved among members of the steroid/thyroid/retinoid hormone receptor superfamily. These results represent the first missense mutation identified in the hormone binding domain of VDR and further define the structure-function relationship of 1,25(OH)2D3 ligand binding to its nuclear receptor.
K Kristjansson, A R Rut, M Hewison, J L O'Riordan, M R Hughes
Chenodeoxycholate is toxic to hepatocytes, and accumulation of chenodeoxycholate in the liver during cholestasis may potentiate hepatocellular injury. However, the mechanism of hepatocellular injury by chenodeoxycholate remains obscure. Our aim was to determine the mechanism of cytotoxicity by chenodeoxycholate in rat hepatocytes. At a concentration of 250 microM, glycochenodeoxycholate was more toxic than either chenodeoxycholate or taurochenodeoxycholate. Cellular ATP was 86% depleted within 30 min after addition of glycochenodeoxycholate. Fructose, a glycolytic substrate, maintained ATP concentrations at 50% of the initial value and protected against glycochenodeoxycholate cytotoxicity. ATP depletion in the absence of a glycolytic substrate suggested impairment of mitochondrial function. Indeed, glycochenodeoxycholate inhibited state 3 respiration in digitonin-permeabilized cells in a dose-dependent manner. After ATP depletion, a sustained rise in cytosolic free calcium (Cai2+) was observed. Removal of extracellular Ca2+ abolished the rise in Cai2+, decreased cellular proteolysis, and protected against cell killing by glycochenodeoxycholate. The results suggest that glycochenodeoxycholate cytotoxicity results from ATP depletion followed by a subsequent rise in Cai2+. The rise in Cai2+ leads to an increase in calcium-dependent degradative proteolysis and, ultimately, cell death. We conclude that glycochenodeoxycholate causes a bioenergetic form of lethal cell injury dependent on ATP depletion analogous to the lethal cell injury of anoxia.
J R Spivey, S F Bronk, G J Gores
To determine if an abnormality exists in the sympathetic nervous system of patients with accelerated hypertension, we recorded muscle sympathetic nerve activity (MSNA) from the tibial nerve by microneurography in eight benign essential hypertensives and seven accelerated essential hypertensives. Basal MSNA, plasma renin activity, and plasma angiotensin II levels were significantly higher in accelerated hypertensives than in benign hypertensives (P < 0.05). To clarify the relationship between the renin-angiotensin axis and sympathetic nervous system in the accelerated hypertensives, we measured the MSNA after 7 d of oral administration of captopril (75 mg/d) for antihypertensive treatment in the benign hypertensives and accelerated hypertensives. After administering captopril, the arterial pressure decreased significantly in the benign hypertensives and accelerated hypertensives with decreases in plasma angiotensin II levels, and the decreases in arterial pressure were greater in the accelerated hypertensive than in the benign hypertensives. After captopril administration, the MSNA decreased significantly in the accelerated hypertensives but did not change in the benign hypertensives. Thus, in accelerated hypertensives, sympathetic tone is elevated, and the elevated sympathetic tone is closely related to the activated renin-angiotensin axis tone.
T Matsukawa, T Mano, E Gotoh, M Ishii
Smooth muscle contraction is initiated primarily by an increase in intracellular Ca2+, Ca(2+)-dependent activation of myosin light chain kinase, and phosphorylation of myosin light chain. In this investigation, we identified pregnancy-associated alterations in myosin light chain phosphorylation, force of contraction, and content of contractile proteins in human myometrium. Steady-state levels of myosin light chain phosphorylation and contractile stress were correlated positively in both tissues, but the myometrial strips from pregnant women developed more stress at any given level of myosin light chain phosphorylation. During spontaneous contractions and during conditions that favor maximal generation of stress, the rate and extent of myosin light chain phosphorylation were attenuated in myometrial strips from pregnant women. The content of myosin and actin per milligram of protein and per tissue cross-sectional area was similar between myometrium of nonpregnant and pregnant women. Although cell size was significantly increased in tissues obtained from pregnant women, the amounts of contractile proteins per cellular cross-sectional area were similar. In addition, myosin light chain kinase and phosphatase activities were similar in the two tissues. The content of caldesmon was significantly increased in myometrium of pregnant women, whereas that of calponin (a smooth muscle-specific protein associated with the thin filaments) was not different. We conclude that adaptations of human myometrium during pregnancy include (a) cellular mechanisms that preclude the development of high levels of myosin light chain phosphorylation during contraction and (b) an increase in the stress generating capacity for any given level of myosin light chain phosphorylation.
R A Word, J T Stull, M L Casey, K E Kamm
Human antigen-specific CD4+ T cells become autoreactive after treatment with various DNA methylation inhibitors, including 5-azacytidine, procainamide, and hydralazine. This suggests a mechanism that could contribute to the development of some forms of autoimmunity. In this report we have asked whether T cells treated with DNA methylation inhibitors can induce autoimmunity. Murine CD4+ T cells were treated with 5-azacytidine or procainamide and were shown to respond to syngeneic antigen-presenting cells, similar to CD4+ human T cell clones treated with these drugs. Functional characterization demonstrated that cells treated with either drug spontaneously lysed syngeneic macrophages and secreted IL-4, IL-6, and IFN-gamma. Adoptive transfer of 5-azacytidine- or procainamide-treated cells into unirradiated syngeneic recipients induced an immune complex glomerulonephritis and IgG anti-DNA and antihistone antibodies. These experiments demonstrate that T cells treated with either of two distinct DNA methyltransferase inhibitors are sufficient to induce a lupus-like disease. It is possible that the lysis of macrophages, together with the release of cytokines promoting B cell differentiation, contributes to the autoantibody production and immune complex deposition. These results suggest that environmental agents that inhibit DNA methylation could interact with T cells in vivo to produce a lupus-like illness, a mechanism that could have relevance to drug-induced and idiopathic lupus.
J Quddus, K J Johnson, J Gavalchin, E P Amento, C E Chrisp, R L Yung, B C Richardson
We have developed two different models of tumor angiogenesis by human brain tumors: one being tube formation by bovine aortic endothelial (BAE) cells cocultured with tumor cells in vitro, and other being in vivo angiogenesis in mice when tumor cells are transplanted into the dorsal sac. We investigated whether tube formation could be induced in BAE cells in type I collagen gel when these cells were cocultured with seven human glioma cell lines. Four of the seven glioma cell lines, which had high levels of basic fibroblast growth factor (bFGF) mRNA, induced tube formation by BAE cells. The tube formation was blocked by coadministration of anti-bFGF antibody. In in vivo model system of tumor angiogenesis in mice, these four cell lines were highly angiogenic. In contrast, with the other three glioma cell lines, which had poor expression of bFGF, BAE cells showed no apparent tube formation. These three cell lines did not efficiently develop capillary networks in mice. The results demonstrated a correlative relationship in the tubulogenesis of BAE cells, bFGF mRNA levels and angiogenesis in mice. The present study with two model systems of tumor angiogenesis suggests that the angiogenesis of some human glioma cell lines is mediated by bFGF, possibly via paracrine control.
T Abe, K Okamura, M Ono, K Kohno, T Mori, S Hori, M Kuwano
The most common organ-specific autoimmune disease in humans involves the thyroid. Autoantibodies against thyroid peroxidase (TPO) are present in the sera of virtually all patients with active disease. We report the molecular cloning of the genes for 30 high-affinity, IgG-class human autoantibodies to TPO from thyroid-infiltrating B cells. Analysis of the putative germline genes used for the TPO human autoantibodies suggests the use of only five different H and L chain combinations involving four H chains and three L chains. In addition, the same combination of H and L chains was found in multiple patients. The F(ab) proteins expressed by these genes define two major, closely associated domains (A and B) in an immunodominant region on TPO. These A and B domains contain the binding sites of approximately 80% of IgG-class TPO autoantibodies in the sera of patients with autoimmune thyroid disease. The present information permits analysis, not previously possible, of the relationship between autoantibody H and L chain genes and the antigenic domains on an autoantigen. Our data, obtained using target organ-derived autoantibodies, indicate that there is restriction in H and L chain usage in relation to the interaction with specific antigenic domains in human, organ-specific autoimmune disease.
G D Chazenbalk, S Portolano, D Russo, J S Hutchison, B Rapoport, S McLachlan
Neutrophil (PMN) migration across intestinal epithelial barriers, such as occurs in many disease states, results in modifications in epithelial barrier. Here, we investigated the impact of lipoxin A4 (LXA4), an eicosanoid with counterregulatory inflammatory roles, on PMN migration across cultured monolayers of the human intestinal epithelial cell line T84. Transepithelial migration of PMN was assessed in the apical-to-basolateral direction and in the basolateral-to-apical direction. In the apical-to-basolateral direction, preexposure of PMN to LXA4 (10 nM, 15 min) stimulated an 87 +/- 5% increase in transepithelial migration of PMN as determined by a PMN myeloperoxidase assay. The LXA4-elicited effect on transmigration was present throughout the 2-h assay period and was not secondary to LXA4 effects on epithelial monolayer integrity as judged by measurement of transepithelial resistance. PMN migration in the basolateral-to-apical direction was modulated by LXA4 with a comparable time- and concentration-dependence to that in the apical-to-basolateral direction. However, qualitative differences in how LXA4 modulates transmigration in the two opposing directions were observed. In the basolateral-to-apical direction, preexposure of PMN to LXA4 (10 nM, 15 min) diminished PMN transepithelial migration by 33 +/- 4%. Structure-function studies revealed that LXA4 and 11-trans-LXA4 (50% of LXA4 effect), but not LXB4, inhibited basolateral-to-apical PMN transmigration. The action of LXA4 was not sensitive to inhibitors of cyclooxygenase or specific leukotriene biosynthesis, but was sensitive to staurosporine, a protein kinase C inhibitor. These results suggest that migration of PMN across epithelia in the physiological direction may be qualitatively different following PMN exposure to eicosanoids. We propose that such retention of PMN at this specific anatomic location may serve an important role in mucosal defense.
S P Colgan, C N Serhan, C A Parkos, C Delp-Archer, J L Madara
Protein plug obstruction of the pancreatic duct is one of the early events in chronic pancreatitis yet little is known about its pathogenesis. GP2, a protein in the exocrine pancreas, is a glycosyl phosphatidylinositol-anchored protein that is cleaved from the zymogen granule membrane and secreted into pancreatic juice. Since its homologue, uromodulin, is involved in renal cast formation, we asked the question whether GP2 might play a similar role in plug formation in chronic pancreatitis. The protein composition of intraductal plugs from patients with noncalcific chronic pancreatitis was examined. Plugs purified from pancreatic juice obtained by endoscopic cannulation were analyzed by SDS-PAGE. A 97-kD protein was found not only to be a reproducible constituent but also enriched within intraductal plugs. This protein was confirmed as GP2 by its localization to zymogen granule membranes, its isoelectric point, and by Western blotting. Although the pancreatic stone protein was identified in plugs, it was not a major reproducible component. These results demonstrate that GP2 is an integral component of plugs in pancreatic juice and suggest that GP2 may play a role in pancreatic plug formation that is analogous to the role played by uromodulin in the pathogenesis of renal casts.
S D Freedman, K Sakamoto, R P Venu
The mechanism by which FFA metabolism inhibits intracellular insulin-mediated muscle glucose metabolism in normal humans is unknown. We used the leg balance technique with muscle biopsies to determine how experimental maintenance of FFA during hyperinsulinemia alters muscle glucose uptake, oxidation, glycolysis, storage, pyruvate dehydrogenase (PDH), or glycogen synthase (GS). 10 healthy volunteers had two euglycemic insulin clamp experiments. On one occasion, FFA were maintained by lipid emulsion infusion; on the other, FFA were allowed to fall. Leg FFA uptake was monitored with [9,10-3H]-palmitate. Maintenance of FFA during hyperinsulinemia decreased muscle glucose uptake (1.57 +/- 0.31 vs 2.44 +/- 0.39 mumol/min per 100 ml tissue, P < 0.01), leg respiratory quotient (0.86 +/- 0.02 vs 0.93 +/- 0.02, P < 0.05), contribution of glucose to leg oxygen consumption (53 +/- 6 vs 76 +/- 8%, P < 0.05), and PDH activity (0.328 +/- 0.053 vs 0.662 +/- 0.176 nmol/min per mg, P < 0.05). Leg lactate balance was increased. The greatest effect of FFA replacement was reduced muscle glucose storage (0.36 +/- 0.20 vs 1.24 +/- 0.25 mumol/min per 100 ml, P < 0.01), accompanied by decreased GS fractional velocity (0.129 +/- 0.26 vs 0.169 +/- 0.033, P < 0.01). These results confirm in human skeletal muscle the existence of competition between glucose and FFA as oxidative fuels, mediated by suppression of PDH. Maintenance of FFA levels during hyperinsulinemia most strikingly inhibited leg muscle glucose storage, accompanied by decreased GS activity.
D E Kelley, M Mokan, J A Simoneau, L J Mandarino
The mechanisms by which hypoxia causes vasoconstriction in vivo are not known. Accumulating evidence implicates the endothelium as a key regulator of vascular tone. Hypoxia induces the expression and secretion of endothelin-1 (ET-1), a potent vasoconstrictor in cultured human endothelial cells. We report here that nitric oxide (NO), an endothelial-derived relaxing factor, modifies this induction of ET-1. Whereas low oxygen tension (PO2 = 20-30 Torr) increases ET-1 expression four- to eightfold above that seen at normal oxygen tension (PO2 = 150 Torr), sodium nitroprusside, which releases NO, suppresses this effect. This inhibition of hypoxia-induced ET-1 expression occurs within the first hour of exposure of cells to sodium nitroprusside. Moreover, when the endogenous constitutive levels of NO made by endothelial cells are suppressed using N-omega-nitro-L-arginine, a potent competitive inhibitor of NO synthase, the baseline levels of ET-1 produced in normoxic environments are increased three- to fourfold. The effects of hypoxia and the NO synthase inhibitor on ET-1 expression are additive. The regulation of ET-1 production by NO appears to be at the level of transcription. Similar effects of NO were observed on the expression of the PDGF-B chain gene. PDGF-B expression was suppressed by NO in a hypoxic environment and induced by N-omega-nitro-L-arginine in both normoxic and hypoxic environments. These findings suggest that in addition to its role as a vasodilator, NO may also influence vascular tone via the regulated reciprocal production of ET-1 and PDGF-B in the vasculature.
S Kourembanas, L P McQuillan, G K Leung, D V Faller
Reductions in dietary fat, saturated fat, and cholesterol have been recommended to reduce the risk of heart disease in our society. The effects of these modifications on human cytokine production and immune responses have not been well studied. 22 subjects > 40 yr of age were fed a diet approximating that of the current American (14.1% of calories as saturated fatty acids, [SFA], 14.5% monounsaturated fatty acids [MUFA], 6.1% [n-6] polyunsaturated fatty acids [PUFA], 0.8% [n-3] PUFA, and 147 mg cholesterol/1,000 calories) for 6 wk, after which time they consumed (11 in each group) one of the two low-fat, low-cholesterol, high-PUFA diets based on National Cholesterol Education Panel (NCEP) Step 2 recommendations (4.0-4.5% SFA, 10.8-11.6% MUFA, 10.3-10.5% PUFA, 45-61 mg cholesterol/1,000 calories) for 24 wk. One of the NCEP Step 2 diets was enriched in fish-derived (n-3) PUFA (low-fat, high-fish: 0.54% or 1.23 g/d eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA] [121-188 g fish/d]) and the other low in fish-derived (n-3) PUFA (low-fat, low-fish [0.13% or 0.27 g/d EPA and DHA] [33 g fish/d]). Measurements of in vivo and in vitro indexes of immune responses were taken after each dietary period. Long-term feeding of low-fat, low-fish diet enriched in plant-derived PUFA increased blood mononuclear cell mitogenic response to the T cell mitogen Con A, IL-1 beta, and TNF production and had no effect on delayed-type hypersensitivity skin response, IL-6, GM-CSF, or PGE2 production. In contrast, the low-fat, high-fish diet significantly decreased the percentage of helper T cells whereas the percentage of suppressor T cells increased. Mitogenic responses to Con A and delayed-type hypersensitivity skin response as well as the production of cytokines IL-1 beta, TNF, and IL-6 by mononuclear cells were significantly reduced after the consumption of the low-fat, high-fish diet (24, 40, 45, 35, and 34%, respectively; P < 0.05 by two-tailed Student's t test except for IL-1 beta and TNF, which is by one-tailed t test). Our data are consistent with the concept that the NCEP Step 2 diet that is high in fish significantly decreases various parameters of the immune response in contrast to this diet when it is low in fish. Such alterations may be beneficial for the prevention and treatment of atherosclerotic and inflammatory diseases but may be detrimental with regard to host defense against invading pathogens.
S N Meydani, A H Lichtenstein, S Cornwall, M Meydani, B R Goldin, H Rasmussen, C A Dinarello, E J Schaefer
A previous study suggested that muscles from hypocalorically fed rats were limited in their ability to rephosphorylate ADP. During muscle contraction hydrolysis of ATP results in an increase in phosphorus, free ADP, delta GATP, and a reduction in phosphocreatine levels that is reversed during rest by rephosphorylation of ADP to ATP and the resynthesis of phosphocreatine by ATP. We therefore hypothesized that these changes would be restored more slowly during postcontraction rest in hypocalorically fed rats as compared with controls. We compared controls fed ad lib to 2-d fasted and hypocalorically fed rats, losing 20% of their weight. We also compared hypocalorically fed rats that had been refed ad lib for 7 d with age-matched controls fed ad lib. The results showed that ATP, muscle pH, and total muscle creatine levels were not different in all groups. The raised phosphorus and delta GATP levels and lower phosphocreatine/phosphorus ratio at the end of contraction changed more slowly during rest in the hypocaloric rats. These abnormalities were partially corrected by refeeding. The data taken as a whole support the concept of impaired rephosphorylation of ADP in malnourished muscle that is not completely restored by refeeding in stimulated muscle.
A Mijan de la Torre, A Madapallimattam, A Cross, R L Armstrong, K N Jeejeebhoy
Myocardial propagation may contribute to fatal arrhythmias in patients with idiopathic dilated cardiomyopathy (IDC). We examined this property in 15 patients with IDC undergoing cardiac transplantation and in 14 control subjects. An 8 x 8 array with electrodes 2 mm apart was used to determine the electrical activation sequence over a small region of the left ventricular surface. Tissue from the area beneath the electrode array was examined in the patients with IDC. The patients with IDC could be divided into three groups. Group I (n = 7) had activation patterns and estimates of longitudinal (theta L = 0.84 +/- 0.09 m/s) and transverse (theta T = 0.23 +/- 0.05 m/s) conduction velocities that were no different from controls (theta L = 0.80 +/- 0.08 m/s, theta T = 0.23 +/- 0.03 m/s). Group II (n = 4) had fractionated electrograms and disturbed transverse conduction with normal longitudinal activation, features characteristic of nonuniform anisotropic properties. Two of the control patients also had this pattern. Group III (n = 4) had fractionated potentials and severely disturbed transverse and longitudinal propagation. The amount of myocardial fibrosis correlated with the severity of abnormal propagation. We conclude that (a) severe contractile dysfunction is not necessarily accompanied by changes in propagation, and (b) nonuniform anisotropic propagation is present in a large proportion of patients with IDC and could underlie ventricular arrhythmias in this disorder.
K P Anderson, R Walker, P Urie, P R Ershler, R L Lux, S V Karwandee
G M Reaven, Y D Chen, J Jeppesen, P Maheux, R M Krauss
Euglycemic hyperinsulinemia evokes both sympathetic activation and vasodilation in skeletal muscle, but the mechanism remains unknown. To determine whether insulin per se or insulin-induced stimulation of carbohydrate metabolism is the main excitatory stimulus, we performed, in six healthy lean subjects, simultaneous microneurographic recordings of muscle sympathetic nerve activity, plethysmographic measurements of calf blood flow, and calorimetric determinations of carbohydrate oxidation rate. Measurements were made during 2 h of: (a) insulin/glucose infusion (hyperinsulinemic [6 pmol/kg per min] euglycemic clamp), (b) exogenous glucose infusion at a rate matched to that attained during protocol a, and (c) exogenous fructose infusion at the same rate as for glucose infusion in protocol b. For a comparable rise in carbohydrate oxidation, insulin/glucose infusion that resulted in twofold greater increases in plasma insulin concentrations than did glucose infusion alone, evoked twofold greater increases in both muscle sympathetic nerve activity and calf blood flow. Fructose infusion, which increased carbohydrate oxidation comparably, but had only a minor effect on insulinemia, did not stimulate either muscle sympathetic nerve activity or calf blood flow. These observations suggest that in humans hyperinsulinemia per se, rather than insulin-induced stimulation of carbohydrate metabolism, is the main mechanism that triggers both sympathetic activation and vasodilation in skeletal muscle.
P Vollenweider, L Tappy, D Randin, P Schneiter, E Jéquier, P Nicod, U Scherrer
Aldose reductase (AR) is implicated in the pathogenesis of the diabetic complications and osmotic cataract. AR has been identified as an osmoregulatory protein, at least in the renal medulla. An outstanding question relates to the response of AR gene expression to diet-induced galactosemia in extrarenal tissues. This paper shows that AR gene expression in different tissues is regulated by a complex multifactorial mechanism. Galactose feeding in the rat is associated with a complex and, on occasions, multiphasic pattern of changes in AR mRNA levels in kidney, testis, skeletal muscle, and brain. These changes are not in synchrony with the temporal sequence of changes in tissue galactitol, galactose, and myoinositol concentrations. Moreover, galactose feeding results in changes in tissue AR activities that are not related, temporally or quantitatively, to the alterations in tissue AR mRNA or galactitol levels. It is concluded that AR gene expression and tissue AR activities are regulated by mechanisms that are not purely dependent on nonspecific alterations in intracellular metabolite concentrations. This conclusion is supported by the finding that chronic xylose feeding, despite being associated with intracellular xylitol accumulation, does not result in alterations in AR mRNA levels, at least in the kidney.
R R Wu, P A Lyons, A Wang, A J Sainsbury, S Chung, T N Palmer
To establish whether insulin resistance and/or postprandial fatty acid metabolism might contribute to familial combined hyperlipidemia (FCH) we have examined parameters of insulin resistance and lipid metabolism in six FCH kindreds. Probands and relatives (n = 56) were divided into three tertiles on the basis of fasting plasma triglycerides (TG). Individuals in the highest tertile (TG > 2.5 mM; n = 14) were older and had increased body mass index, systolic blood pressure, and fasting plasma insulin concentrations compared with individuals in the lowest tertile (n = 24). The former also presented with decreased HDL cholesterol and increased total plasma cholesterol, HDL-TG, and apoprotein B, E, and CIII concentrations. Insulin concentrations were positively correlated with plasma apo B, apo CIII, apo E, and TG, and inversely with HDL cholesterol. Fasting nonesterified fatty acids (NEFA) were elevated in FCH subjects compared to six unrelated controls and five subjects with familial hypertriglyceridemia. Prolonged and exaggerated postprandial plasma NEFA concentrations were found in five hypertriglyceridemic FCH probands. In FCH the X2 minor allele of the AI-CIII-AIV gene cluster was associated with increased fasting plasma TG, apo CIII, apo AI, and NEFA concentrations and decreased postheparin lipolytic activities. The clustering of risk factors associated with insulin resistance in FCH indicates a common metabolic basis for the FCH phenotype and the syndrome of insulin resistance probably mediated by an impaired fatty acid metabolism.
M Castro Cabezas, T W de Bruin, H W de Valk, C C Shoulders, H Jansen, D Willem Erkelens
Peroxisomal-deficient skin fibroblasts from patients with Zellweger's syndrome or infantile Refsum's disease produced fewer prostaglandins than normal skin fibroblasts. Radioimmunoassay indicated a 45-55% decrease in prostaglandin E2 (PGE2) production when Zellweger's fibroblasts were incubated with arachidonic acid. This deficiency was not overcome by pretreatment of the Zellweger's fibroblasts with media containing arachidonic acid, and it was not due to channeling of arachidonic acid into other eicosanoid products. Modifications in the peroxide tone of the Zellweger's fibroblasts by addition of H2O2 or catalase failed to increase PGE2 production. Using Northern analysis, we were unable to detect an mRNA transcript for PGH synthase in unstimulated Zellweger fibroblasts but identified a 4.2-kb mRNA transcript after treatment with phorbol myristate acetate (PMA). Treatment for 6 h with 10 nM PMA raised PGE2 production in normal and Zellweger fibroblasts to equivalent levels. These increases were prevented by addition of H-7, staurosporine, cycloheximide, or actinomycin D. Our findings suggest that the reduced PGE2 production in peroxisomal deficient fibroblasts is due to a decrease in PGH synthase mRNA. The reduction in PGH synthase can be overcome by treatment of the cells with agents which enhance gene expression.
J A Gordon, L J Warnock, A A Spector
We report here that a 92-kD gelatinolytic metalloproteinase is expressed as protein and mRNA in human osteoarthritic cartilage, but not in normal adult articular cartilage. Western immunoblotting demonstrated that the 92-kD gelatinolytic activity corresponded to 92-kD type IV collagenase/gelatinase (gelatinase B); mRNA for gelatinase B was identified by Northern blotting. Chondrocytes from normal cartilage also exhibited mRNA for 72-kD type IV collagenase/gelatinase (gelatinase A), tissue collagenase, and stromelysin-1, and these mRNAs were increased in osteoarthritic cartilage. Regional analysis of osteoarthritic cartilage samples from four individuals revealed that gelatinase B mRNA was expressed in grossly fibrillated areas; two of four nonfibrillated cartilage samples failed to exhibit the mRNA, but did have increased levels of mRNA for other neutral metalloproteinases. IL-1 alpha treatment of normal human cartilage explants or isolated chondrocytes induced increased levels of gelatinase B and increased mRNA for tissue collagenase and stromelysin-1. Under identical conditions, mRNA levels for gelatinase A were not increased indicating that regulation of this enzyme in human articular chondrocytes is distinct from that of other metalloproteinases. Our data showing expression of gelatinase B in fibrillated cartilage suggest that it is a marker of progressive articular cartilage degradation in osteoarthritis.
M Mohtai, R L Smith, D J Schurman, Y Tsuji, F M Torti, N I Hutchinson, W G Stetler-Stevenson, G I Goldberg
One of the salient pathological features of rheumatoid arthritis is synovial cell proliferation with bone erosion. Despite extensive investigation, the factors essential for synovial cell proliferation remain to be identified. Recent studies suggest that human T cell leukemia virus type I (HTLV-I) may play an important role in synovial overgrowth observed in patients with one type of chronic inflammatory synovitis. In order to confirm and extend these observations, we have established synovial cell clones (SCCs) from three HTLV-I carriers who demonstrated synovial overgrowth but were otherwise asymptomatic. HTLV-I proviral DNA randomly integrated into the cellular genome was present in 20-30% of SCCs. The SCCs carrying HTLV-I proviral DNA and expressing the tax gene exhibited high levels of proliferative potential. HTLV-I was found to function as a transcriptional trans-activator in these SCCs. Moreover, transfection of the tax expression plasmid into SCCs resulted in the same phenotype of increased proliferation and cytokine expression as exhibited by HTLV-I provirus-carrying and tax-expressing SCCs. These data suggest that tax plays a critical role not only in leukemogenesis but also in synovial overgrowth in humans.
T Nakajima, H Aono, T Hasunuma, K Yamamoto, I Maruyama, T Nosaka, M Hatanaka, K Nishioka
Synthetic oligodeoxynucleotides complementary to the break-point junction of bcr-abl transcripts selectively inhibit the proliferation of Philadelphia-positive leukemic cells, but residual leukemic cells persist in antisense oligodeoxynucleotides-treated cultures. Cyclophosphamide derivatives such as mafosfamide and 4-hydroperoxycyclophosphamide are used at high doses for purging of Philadelphia leukemic cells from marrows but such treatment can be associated with delayed engraftment and prolonged cytopenias. To develop a more effective procedure that might optimize the killing of leukemia cells and the sparing of normal hematopoietic progenitor cells, a 1:1 mixture of Philadelphia leukemic cells and normal bone marrow cells was exposed to a combination of a low dose of mafosfamide and bcr-abl antisense oligodeoxynucleotides and assayed for growth ability in clonogenic assays and in immunodeficient mice. Bcr-abl transcripts were not detected in residual colonies, and cytogenetic analysis of individual colonies revealed a normal karyotype. Normal but not leukemic hematopoietic colonies of human origin were also detected in marrows of immunodeficient mice 1 mo after injection of the treated cells. Our results indicate that a combination of a conventional chemotherapeutic agent and a tumor-specific antisense oligodeoxynucleotide is highly effective in killing leukemic cells and in sparing a much higher number of normal progenitor cells as compared with high-dose mafosfamide treatment. This offers the prospect of a novel and more selective ex vivo treatment of chronic myelogenous leukemia.
T Skorski, M Nieborowska-Skorska, C Barletta, L Malaguarnera, C Szcyzlik, S T Chen, B Lange, B Calabretta
A proband with chylomicronemia, pancreatitis, and non-insulin-dependent diabetes (NIDDM) bears two different mutations in exon 3 of the lipoprotein lipase (LPL) gene: a missense mutation, 75Arg-->Ser, inherited through the paternal line and a truncation, 73Tyr-->Ter, through the maternal line. NIDDM appeared to be independently segregating. The R75S mutant was studied in extracts and media from transfected COS-1 cells. Detectable amounts of catalytically competent R75S LPL suggested destabilization of the active homodimer as with exon 5 mutants (Hata et al. 1992. J. Biol. Chem. 267:20132-20139). Hydrolysis of a short-chain fatty acid ester indicated that R75S does not directly affect activation of LPL by apoC-II. Subjects with NIDDM and wild-type LPL, and nondiabetic middle-aged carriers of the 73Tyr-->Ter truncation had moderate hypertriglyceridemia (260-521 mg/dl) and reduced high density lipoprotein cholesterol. A maternal aunt with NIDDM carried the truncation. Her phenotype (triglycerides of 5,300 mg/dl, eruptive xanthomatosis, and recurrent pancreatitis) was as severe as that in homozygotes or compound heterozygotes. We conclude: (a) diabetic carriers of dysfunctional LPL alleles are at risk for severe lipemia; and (b) the physiologic defects in NIDDM may be additive or synergistic with heterozygous LPL deficiency.
D E Wilson, A Hata, L K Kwong, A Lingam, J Shuhua, D N Ridinger, C Yeager, K C Kaltenborn, P H Iverius, J M Lalouel
RATIONALE: Advanced glycosylation end products (AGEs) may play an important role in the development of diabetic vascular sequelae. An AGE cross-link, pentosidine, is a sensitive and specific marker for tissue levels of AGEs. OBJECTIVES: To evaluate the role of AGEs in the development of diabetic nephropathy and retinopathy, we studied pentosidine levels and the clinical characteristics of 48 subjects with insulin-dependent diabetes mellitus. Diabetic nephropathy was classified as normal, microalbuminuria, or gross proteinuria, and retinopathy was graded as none, background, or proliferative. NEWLY OBSERVED FINDINGS: Significant elevation of pentosidine (P = 0.025) was found in subjects with microalbuminuria or gross proteinuria (73.03 +/- 9.47 vs 76.46 +/- 6.37 pmol/mg col) when compared with normal (56.96 +/- 3.26 pmol/mg col). Multivariate analysis to correct for age, duration of diabetes, and gender did not modify the results. Elevated pentosidine levels were also found in those with proliferative when compared with those with background retinopathy (75.86 +/- 5.66 vs 60.42 +/- 5.98 pmol/mg col) (P < 0.05). CONCLUSIONS: Microalbuminuria is associated with elevated levels of pentosidine similar to those found in overt diabetic nephropathy suggesting that elevated AGE levels are already present during the earliest detectable phase of diabetic nephropathy.
P J Beisswenger, L L Moore, T Brinck-Johnsen, T J Curphey
Intracellular alkalinization is known to be associated with tumorigenic transformation. Besides phenotypical alterations alkali-transformed Madin-Darby canine kidney (MDCK) cells exhibit a spontaneously oscillating cell membrane potential (PD). Using single-channel patch clamp techniques, it was the aim of this study to identify the ion channel underlying the rhythmic hyperpolarizations of the PD. In the cell-attached patch configuration, we found that channel activity was oscillating. The frequency of channel oscillations is 1.1 +/- 0.1 min-1. At the peak of oscillatory channel activity, single-channel current was -2.7 +/- 0.05 pA, and in the resting state it was -1.95 +/- 0.05 pA. Given the single-channel conductance of 53 +/- 3 pS for inward (and of 27 +/- 5 pS for outward) current the difference of single-channel current amplitude corresponded to a hyperpolarization of approximately 14 mV. The channel is selective for K+ over Na+. Channel kinetics are characterized by one open and by three closed time constants. The channel is Ca2+ sensitive. Half maximal activation in the inside-out patch mode is achieved at a Ca2+ concentration of 10 mumol/liter. In addition, we also found a 13-pS K+ channel that shows no oscillatory activity in the cell-attached patch configuration and that was not Ca2+ sensitive. We conclude that the Ca(2+)-sensitive 53-pS K+ channel is underlying spontaneous oscillations of the PD. It has virtually identical biophysical properties as a Ca(2+)-sensitive K+ channel in nontransformed parent MDCK cells. Hence, alkali-induced transformation of MDCK cells did not affect the channel protein itself but its regulators thereby causing spontaneous fluctuations of the PD.
A Schwab, H J Westphale, L Wojnowski, S Wünsch, H Oberleithner
SR 49059, a new potent and selective orally active, nonpeptide vasopressin (AVP) antagonist has been characterized in several in vitro and in vivo models. SR 49059 showed high affinity for V1a receptors from rat liver (Ki = 1.6 +/- 0.2) and human platelets, adrenals, and myometrium (Ki ranging from 1.1 to 6.3 nM). The previously described nonpeptide V1 antagonist, OPC-21268, was almost inactive in human tissues at concentrations up to 100 microM. SR 49059 exhibited much lower affinity (two orders of magnitude or more) for AVP V2 (bovine and human), V1b (human), and oxytocin (rat and human) receptors and had no measurable affinity for a great number of other receptors. In vitro, AVP-induced contraction of rat caudal artery was competitively antagonized by SR 49059 (pA2 = 9.42). Furthermore, SR 49059 inhibited AVP-induced human platelet aggregation with an IC50 value of 3.7 +/- 0.4 nM, while OPC-21268 was inactive up to 20 microM. In vivo, SR 49059 inhibited the pressor response to exogenous AVP in pithed rats (intravenous) and in conscious normotensive rats (intravenous and per os) with a long duration of action (> 8 h at 10 mg/kg p.o). In all the biological assays used, SR 49059 was devoid of any intrinsic agonistic activity. Thus, SR 49059 is the most potent and selective nonpeptide AVP V1a antagonist described so far, with marked affinity, selectivity, and efficacy toward both animal and human receptors. With this original profile, SR 49059 constitutes a powerful tool for exploring the therapeutical usefulness of a selective V1a antagonist.
C Serradeil-Le Gal, J Wagnon, C Garcia, C Lacour, P Guiraudou, B Christophe, G Villanova, D Nisato, J P Maffrand, G Le Fur
The integrin VLA-2 mediates cell adhesion to collagen and laminin and also functions as a virus receptor, mediating cell surface attachment and infection by a human pathogen, echovirus 1. To determine whether extracellular matrix proteins and virus interact with VLA-2 in the same manner, we carried out a detailed comparison of these two functions and found that they differed markedly in six different respects. In contrast to the ECM/VLA-2 interaction, echovirus 1 binding did not discriminate between functional forms of VLA-2, showed a different pattern of inhibition by anti-beta1 and -alpha 2 antibodies, was not stimulated by phorbol esters, was not activated by beta 1 antibodies that stimulate ECM binding, was not inhibited by any particular divalent cation, and most notably was not inhibited by EDTA. These striking differences were found both with intact cells expressing VLA-2 and with solubilized VLA-2, suggesting that VLA-2 interacts with these different ligands by markedly different mechanisms, and probably at different functional sites. In addition, alterations in the alpha 2 cytoplasmic domain that had marked effects on cellular responses to collagen and laminin had no effect on virus internalization and cell killing. Thus VLA-2-mediated events that occur after receptor occupancy by extracellular matrix proteins also appear to be distinct from those that occur after receptor interaction with virus.
J M Bergelson, B M Chan, R W Finberg, M E Hemler
Patients with insulin-dependent diabetes (IDDM) possess antibodies to islet proteins of M(r)-64,000. Potential autoantigens of this M(r) include glutamate decarboxylase (GAD) and 65 kD heat shock protein. We have detected two distinct antibody specificities in IDDM that bind 50,000 M(r) or 37,000/40,000 M(r) proteolytic fragments of 64,000 M(r) proteins. In this study, we investigated relationships of these proteolytic fragments to GAD and heat shock proteins. Polyclonal antibodies to GAD bound 50,000 M(r) fragments of islet antigen. Recombinant GAD65, but not GAD67, blocked binding to this antigen, suggesting that 50,000 M(r) fragments are derived from islet GAD65. In contrast, GAD antibodies did not recognize 37,000/40,000 M(r) fragments, and neither GAD isoforms blocked autoantibody binding to precursors of these fragments. The 37,000/40,000 M(r) fragments, but not the 50,000 M(r) fragments, were detected after trypsin treatment of immunoprecipitates from insulinoma cells that lacked expression of major GAD isoforms. Antibodies in IDDM did not bind native or trypsinized islet heat shock proteins. Thus, IDDM patients possess antibodies to GAD, but also distinct antibodies to a 64,000 M(r) protein that is not related to known GAD isoforms or heat shock proteins.
M R Christie, J A Hollands, T J Brown, B K Michelsen, T L Delovitch
Several strategies have been used to obtain recombinant (r) human plasminogens (HPg) containing different oligosaccharide side chains on its sole N-linked glycosylation site, present at Asn289. The approaches included expression of the cDNA for HPg in insect cell lines under various conditions, addition of glycosidase inhibitors during expression, and purification of specific glycoforms of HPg using affinity chromatography on an insolubilized lectin column. The activation kinetics for urokinase (UK) of each of the purified HPgs, as well as their relative abilities to bind to the ligand, epsilon-aminocaproic acid (EACA), were then determined. Removal of both N- and O-linked oligosaccharide from HPg resulted in a slight increase in the Kcat/Km for its activation, while a glycoform containing tetrasialyl-tetra-antennary complex oligosaccharide on Asn289 was a slightly poorer substrate for UK than plasma HPg, which contains bisialyl-biantennary complex carbohydrate on Asn289. The most dramatic differences were observed for HPgs with high mannose-type glycans on Asn289. (Man9GlcNAc2)-HPg possessed only approximately 6% of the kcat/Km of plasma HPg, whereas (Glc3Man9GlcNAc2)-HPg did not undergo activation at a significant rate by UK. Differences were also found in the relative abilities of the HPg glycoforms to interact with EACA. The most effective interactions were observed with HPgs containing complex-type glycans, and the least effective binding was found for HPgs with high mannose-type oligosaccharides. The full range of the binding effects is represented by a fourfold difference between HPg containing tetrasialyl-tetra-antennary glycan and HPg with (Glc3Man9GlcNAc2) assembled on Asn289. These results clearly demonstrate that the nature of the N-linked glycan assembled on HPg dramatically influences its ability to be activated by UK and to bind to omega-amino acid effector molecules.
D J Davidson, F J Castellino
Upper body obesity (UB Ob) is associated with a reduced net free fatty acid (FFA) response to epinephrine compared with nonobese (Non Ob) and lower-body obese (LB Ob) women. Because catecholamines regulate some of the metabolic responses to exercise, we hypothesized that UB Ob would have a reduced net FFA response to exercise. Plasma FFA rate of appearance (Ra) ([1-14C]palmitate) and fatty acid oxidation (indirect calorimetry) were therefore measured during 2.5 h of stationary bicycle exercise (45% VO2 peak) in 13 UB Ob, 11 LB Ob, and 8 Non Ob premenopausal women. 10 UB Ob and 8 LB Ob women were retested after an approximately 8-kg weight loss. Results: During exercise Non Ob and LB Ob women had greater increments in FFA availability (51 +/- 7 and 53 +/- 8 mmol, respectively) than UB Ob women (27 +/- 4 mmol, P < 0.05). Total exercise FFA availability and fatty acid oxidation were not different between Non Ob, LB Ob, and UB Ob women, however. Following weight loss (approximately 8 kg), the FFA response to exercise increased (P < 0.01) and remained greater (P < 0.05) in LB Ob than in UB Ob women. In conclusion, the FFA response to exercise was reduced in UB Ob women before and after weight loss, but no effects on fatty acid oxidation were apparent.
J A Kanaley, P E Cryer, M D Jensen
Normal subjects demonstrate the presence of ultradian oscillations (period 80-150 min) in insulin secretion rate (ISR) tightly coupled to glucose oscillations of similar period. These oscillations appear to be a function of the feedback loop linking glucose and insulin. The present study was undertaken to determine whether the control by glucose of the ultradian oscillations in insulin secretion is altered in impaired glucose tolerance IGT and in non-insulin-dependent diabetes mellitus (NIDDM). Patients with NIDDM (n = 7), IGT (n = 4), and matched nondiabetic controls (n = 5) were studied under three separate protocols that involved administration of glucose at either a constant rate of 6 mg/kg per min for 28 h or in one of two oscillatory patterns at the same overall mean rate. The amplitude of the oscillations was 33% above and below the mean infusion rate, and their respective periods were 144 min (slow oscillatory infusion) or 96 min (rapid oscillatory infusion). Insulin, C-peptide, and glucose were sampled at 10-min intervals during the last 24 h of each study. ISRs were calculated by deconvolution of C-peptide levels. Analysis of the data showed that (a) the tight temporal coupling between glucose and ISR in the nondiabetic controls was impaired in the IGT and NIDDM groups as demonstrated by pulse analysis, cross-correlation analysis, and spectral analysis; (b) the absolute amplitude of the ISR pulses progressively declined with the transition from obesity to IGT to NIDDM; and (c) the absolute amplitude of the ISR oscillations failed to increase appropriately with increasing absolute amplitude of glucose oscillations in the IGT and NIDDM subjects compared with the control group. In conclusion, the present study demonstrates that important dynamic properties of the feedback loop linking insulin secretion and glucose are disrupted not only in established NIDDM but also in conditions where glucose tolerance is only minimally impaired. Further studies are needed to determine how early in the course of beta-cell dysfunction this lack of control by glucose of the ultradian oscillations in insulin secretion occurs and to define more precisely if this phenomenon plays a pathogenetic role in the onset of hyperglycemia in genetically susceptible individuals.
N M O'Meara, J Sturis, E Van Cauter, K S Polonsky
Insulin production was studied in transgenic mice expressing the human insulin gene under the control of its own promoter. Glucose homeostasis during a 48-h fast was similar in control and transgenic mice, with comparable levels of serum immunoreactive insulin. Northern blot and primer extension analyses indicated that more than twice as much insulin mRNA is present in pancreata from transgenic mice. Primer extension analysis using oligonucleotides specific for mouse insulins I and II or for human insulin, showed that the excess insulin mRNA was due solely to expression of the foreign, human insulin gene. The ratio of mRNA for mouse insulin I and II was unaffected by coexpression of human insulin. There were coordinate changes in the levels of all three mRNA during the 48-h fast, or after a 24-h fast followed by 24-h refeed. Despite the supraphysiologic levels of insulin mRNA in the transgenic mice, their pancreatic content of immunoreactive insulin was not significantly different from controls. The comparison of the relative levels of human and mouse insulin mRNAs with their peptide counterparts (separated by HPLC) indicates that the efficiency of insulin production from mouse insulin mRNA is greater than that from human, stressing the importance of posttranscriptional regulatory events in the overall maintenance of pancreatic insulin content.
B Schnetzler, G Murakawa, D Abalos, P Halban, R Selden
Histamine and IL-1 have been implicated in the pathogenesis of chronic inflammatory diseases, such as pulmonary allergic reactions and rheumatoid arthritis. We therefore investigated whether histamine modulated the synthesis of IL-1 beta. Human PBMC were stimulated with IL-1 alpha (10 ng/ml) in the absence or presence of histamine (10(-9)-10(-4) M). Histamine alone did not induce protein synthesis or mRNA accumulation for IL-1 beta. IL-1 alpha-induced IL-1 beta synthesis was enhanced two to threefold by histamine concentrations from 10(-6)-10(-4) M. Cimetidine, an H2 receptor antagonist, reversed the histamine (10(-5) M)-mediated increase in IL-1 alpha-induced IL-1 beta synthesis. Diphenhydramine, an H1 receptor antagonist, had no effect. Indomethacin, a cyclooxygenase inhibitor, significantly reduced IL-1 alpha-induced IL-1 beta synthesis, but had no effect on the histamine-mediated increase in IL-1 alpha-induced IL-1 beta synthesis. Histamine (10(-5) M) enhanced and sustained IL-1 beta mRNA levels in IL-1 alpha-stimulated PBMC. However, histamine reduced IL-1 beta mRNA half-life (2.4 vs 1.2 h), suggesting that histamine enhances IL-1 alpha-induced IL-1 beta synthesis at the level of transcriptional activation. On the other hand, histamine (10(-5) M) did not affect IL-1 alpha-induced synthesis of IL-1 receptor antagonist. These results suggest that mast cells may sustain chronic inflammatory processes by upregulating self-induction of IL-1 through histamine release.
E Vannier, C A Dinarello
The role of thrombospondin, a multifunctional matrix glycoprotein, in platelet adhesion is controversial: both adhesive and antiadhesive properties have been attributed to this molecule. Because shear flow has a significant influence on platelet adhesion, we have assessed thrombospondin-platelet interactions both under static and flow conditions. The capacity of thrombospondin to support platelet adhesion depended upon its conformation. In a Ca(2+)-depleted conformation, such as in citrated plasma, thrombospondin was nonadhesive or antiadhesive as it inhibited platelet adhesion to fibrinogen, fibronectin, laminin, and von Willebrand factor by 30-70%. In a Ca(2+)-replete conformation, however, thrombospondin effectively supported platelet adhesion. Shear rate influenced this adhesion; percent surface coverage on thrombospondin increased from 5.4 +/- 0.3 at 0 s-1 to 41.5 +/- 6.7 at 1,600 s-1. In contrast to the extensive platelet spreading observed on fibronectin at all shear rates, platelet spreading on thrombospondin occurred only sporadically and at high shear rates. GPIa-IIa, GPIIb-IIIa, GPIV, and the vitronectin receptor, which are all proposed platelet receptors for thrombospondin, were not solely responsible for platelet adhesion to thrombospondin. These results suggest that thrombospondin may play a dual role in adhesive processes in vivo: (a) it may function in conjunction with other adhesive proteins to maintain optimal platelet adhesion at various shear rates; and (b) it may serve as a modulator of cellular adhesive functions under specific microenvironmental conditions.
F R Agbanyo, J J Sixma, P G de Groot, L R Languino, E F Plow
Components of bacterial peptidoglycans have potent biological activities, including adjuvant effects, cytotoxicity, and induction of sleep. Mixtures of peptidoglycan components also induce inflammation in the lung, subarachnoid space, and joint, but the structural requirements for activity are unknown. Using a rabbit model for meningitis, we determined the biological activities of 14 individual muramyl peptides constituting > 90% of the peptidoglycan of the gram-negative pediatric pathogen Haemophilus influenzae. Upon intracisternal inoculation, most of the muropeptides induced leukocytosis in cerebrospinal fluid (CSF), influx of protein into CSF, or brain edema, alone or in combination. The disaccharide-tetrapeptide, the major component of all gram-negative peptidoglycans, induced CSF leukocytosis and protein influx at doses as low as 0.4 microgram (0.42 nM). Modification of the N-acetyl muramic acid or substitution of the alanine at position four in the peptide side chain decreased leukocytosis but enhanced brain edema. As the size of the muropeptide increased, the inflammatory activity decreased. Muropeptide carrying the diaminopimelyl-diaminopimelic acid cross-link specifically induced cytotoxic brain edema. These findings significantly expand the spectrum of biological activities of natural muramyl peptides and provide the basis for a structure-activity relationship for the inflammatory properties of bacterial muropeptides.
M Burroughs, E Rozdzinski, S Geelen, E Tuomanen
Rat skeletal muscle contains two enzymes which can make epinephrine: phenylethanolamine N-methyltransferase (PNMT) and nonspecific N-methyltransferase. We studied the time-course and mechanism by which the glucocorticoid dexamethasone increases muscle PNMT activity. We also examined the hypothesis that increased muscle E synthesis may contribute to glucocorticoid-induced insulin resistance. Dexamethasone (1 mg/kg s.c. for 12 d) increased muscle PNMT activity seven-fold but did not change NMT activity. Immunotitration with an anti-PNMT antibody indicated that the PNMT elevation was due to increased numbers of PNMT molecules. Dexamethasone rapidly increased PNMT activity and this elevation was largely maintained 6 d after glucocorticoid treatment stopped. Muscle epinephrine levels were transiently elevated by dexamethasone. Dexamethasone-treated rats had elevated insulin levels after a glucose load, and chronic administration of the PNMT inhibitor SKF 64139 reversed this increase. Chronic SKF 64139 improved glucose tolerance in normal rats. Dexamethasone induced muscle synthesis of the epinephrine-forming enzyme PNMT. A PNMT inhibitor lowered insulin levels in glucocorticoid-treated rats and glucose levels in untreated rats. These findings are compatible with antagonism of insulin-mediated glucose uptake by epinephrine synthesized in skeletal muscle.
B Kennedy, H Elayan, M G Ziegler
We studied phospholipid topology and transbilayer mobility in red cells during blood storage. The distribution of phospholipids was determined by measuring the reactivity of phosphatidylethanolamine with fluorescamine and the degradation of phospholipids by phospholipase A2 and sphingomyelinase C. Phospholipid mobility was measured by determining transbilayer movements of spin-labeled phospholipids. We were unable to detect a change in the distribution of endogenous membrane phospholipids in stored red cells even after 2-mo storage. The rate of inward movement of spin-labeled phosphatidylethanolamine and phosphatidylserine was progressively reduced, whereas that for phosphatidylcholine was increased. These changes in phospholipid translocation correlated with a fall in cellular ATP. However, following restoration of ATP, neither the rate of aminophospholipid translocation nor the transbilayer movement of phosphatidylcholine were completely corrected. Taken together, our findings demonstrate that red cell storage alters the kinetics of transbilayer mobility of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine, the activity of the aminophospholipid translocase, but not the asymmetric distribution of endogenous membrane phospholipids, at least at a level detectable with phospholipases. Thus, if phosphatidylserine appearance on the outer monolayer is a signal for red cell elimination, the amount that triggers macrophage recognition is below the level of detection upon using the phospholipase technique.
D Geldwerth, F A Kuypers, P Bütikofer, M Allary, B H Lubin, P F Devaux
We tested the hypothesis that hyperpnea-induced bronchoconstriction (HIB) and hyperpnea-induced bronchovascular hyperpermeability (HIBVH) are mediated through stimulation of NK-1 and NK-2 receptors in guinea pigs. We first established the efficacy and selectivity of (+/-) CP-96,345 (3 mg/kg i.v.) and of SR-48,968 (300 micrograms/kg i.v.) as NK-1 and NK-2 antagonists, respectively. (+/-) CP-96,345 substantially attenuated bronchoconstriction and systemic vascular leak caused by administration of Sar9,Met(O2)11-Substance P (a specific NK-1 agonist), but had no effect upon bronchoconstriction induced by selective NK-2 stimulation with Nle10-Neurokinin A[4-10]. Conversely, SR-48,968 antagonized the bronchoconstrictor response to Nle10-NKA[4-10], right-shifting the dose-response curve by 2 log units, but had no effect on Sar9, Met(O2)11-SP-induced bronchoconstriction. Anesthetized, tracheostomized, opened-chest male Hartley guinea pigs were pretreated with (+/-) CP-96,345 (3 mg/kg i.v.), SR-48,968 (300 micrograms/kg i.v.), or their respective vehicles, and Evans blue dye (30 mg/kg i.v.) to label circulating albumin. 10 min isocapnic dry gas hyperpnea (12 ml/kg, 150 breaths/min) provoked HIB and HIBVH in vehicle-treated animals. (+/-) CP-96,345 reduced the magnitude of HIB by one-half (peak posthyperpnea RL 7.8 +/- 1.9 [SE] times prehyperpnea baseline versus 16.1 +/- 2.6, vehicle-treated; P < or = 0.0001, ANOVA); SR-48,968 blocked HIB more completely (peak posthyperpnea RL 5.1 +/- 1.7 [SE] times prehyperpnea baseline versus 19.3 +/- 2.8, vehicle-treated; P < 0.0001, ANOVA). Neither drug reduced HIBVH. We conclude that dry gas hyperpnea causes bronchoconstriction in guinea pigs through activation of tachykinin receptors. The differential effects of neurokinin receptor blockade on HIB and HIBVH demonstrate that hyperpnea-induced airflow obstruction is not primarily a consequence of hyperpnea-induced bronchovascular leak.
J Solway, B M Kao, J E Jordan, B Gitter, I W Rodger, J J Howbert, L E Alger, J Necheles, A R Leff, A Garland
We have found that an important Th2 cytokine, IL-10, is produced by tissues from patients acutely infected with Leishmania donovani. In all individuals tested, IL-10 mRNA production was increased in lymph nodes taken during acute disease over that observed in postacute samples. In contrast, both pre- and posttreatment lymph nodes had readily detected mRNA for IFN-gamma and IL-2. A down-regulating effect of IL-10 on leishmania-induced proliferative responses was demonstrated when Hu rIL-10 was added to cultures of PBMC from clinically cured individuals. PBMC from individuals with acute visceral leishmaniasis responded to stimulation with leishmania lysate by producing IL-10 mRNA. Simultaneously cultured PBMC collected from the same patients after successful chemotherapy produced no detectable IL-10 mRNA after leishmania antigen stimulation. Neutralizing anti-IL-10 mAb added to PBMC from patients with acute visceral leishmaniasis markedly increased the proliferative response to leishmania lysate. Finally, we observed mRNA for IL-10 and IFN-gamma concurrently in a lesion from a patient with post-kala-azar dermal leishmaniasis (PKDL). These results indicate the production of IL-10 during L. donovani infection, and suggest a role for this cytokine in the regulation of immune responsiveness during visceral leishmaniasis.
H W Ghalib, M R Piuvezam, Y A Skeiky, M Siddig, F A Hashim, A M el-Hassan, D M Russo, S G Reed
A 120-kD glycoprotein antigen abundantly expressed on Blastomyces dermatitidis yeasts is a target of cellular and humoral immune responses in human infection. To investigate the antigen and immune response more carefully at the molecular level, we screened an expression library from B. dermatitidis to identify clones that encode this antigen, designated WI-1. A 942-bp cDNA was isolated by immunologic screening with polyclonal, rabbit anti-WI-1 antiserum. Northern hybridization analysis showed that the cDNA hybridized to yeast message approximately equal to 3.9 kb. DNA and deduced protein sequence analysis of the clone demonstrated a 25-amino acid repeat arrayed in tandem, present in 4.5 copies near the 5' end, and rich in predicted antigenic epitopes. Further analysis showed strong homology in these tandem repeats with invasin, an adhesin of Yersiniae. Cloned cDNA was used to express a 30-kD fusion protein strongly recognized in western blots by rabbit anti-WI-1 antiserum, and by sera from all 35 blastomycosis patients studied. The fusion protein product of subcloned cDNA encoding only the tandem repeat also was strongly recognized in western blots by sera from the 35 blastomycosis patients, but not by sera from 10 histoplasmosis and 5 coccidioidomycosis patients. An antigen-inhibition radioimmunoassay showed that the tandem repeat alone completely eliminated rabbit and human anti-WI-1 antibody binding to radiolabeled native WI-1. From these results, we conclude that the 25-amino acid repeat of WI-1 displays an immunodominant B cell epitope, and that the carboxyl-terminus of the molecule exhibits an architecture that may promote adhesion of Blastomyces yeasts to host cells or extracellular matrix proteins and ultimately provide a clearer picture of the molecular pathogenesis of blastomycosis.
B S Klein, L H Hogan, J M Jones
Healing baboon polytetrafluoroethylene grafts express PDGF mRNA in the neointima. Perfusates of graft segments also contain PDGF-like mitogenic activity. To extend these findings, we studied the expression and regional distribution of the PDGF protein isoforms and their receptors in this prosthetic graft model. By immunohistochemistry, as well as ELISA and Western blot analysis of tissue extracts, both PDGF-A and PDGF-B were identified in macrophages within the interstices of the synthetic material. In contrast, the neointima contained predominantly PDGF-A localized to the endothelial surface and the immediate subjacent smooth muscle cell layers. Tissue extracts of neointima and graft material were mitogenic for baboon aortic smooth muscle cells in culture; nearly all of this proliferative activity was blocked by a neutralizing anti-PDGF antibody. PDGF receptor beta-subunit mRNA and protein were easily detectable in the neointima and graft material. PDGF receptor alpha-subunit mRNA was also observed in the graft matrix and at lower levels in the neointima. This pattern of ligand and receptor expression further implicates locally produced PDGF as a regulator of neointimal smooth muscle cell growth in this model. The coexpression of ligand and receptor in the macrophage-rich matrix also suggests that PDGF may participate in the foreign body response.
L W Kraiss, E W Raines, J N Wilcox, R A Seifert, T B Barrett, T R Kirkman, C E Hart, D F Bowen-Pope, R Ross, A W Clowes
Hemostasis in the brain is of paramount importance because bleeding into the neural parenchyma can result in paralysis, coma, and death. Consistent with this sensitivity to hemorrhage, the brain contains large amounts of tissue factor (TF), the major cellular initiator of the coagulation protease cascades. However, to date, the cellular source for TF in the central nervous system has not been identified. In this study, analysis of murine brain sections by in situ hybridization demonstrated high levels of TF mRNA in cells that expressed glial fibrillary acidic protein, a specific marker for astrocytes. Furthermore, primary mouse astrocyte cultures and astrocyte cell lines from mouse, rat, and human constitutively expressed TF mRNA and functional protein. These data indicated that astrocytes are the primary source of TF in the central nervous system. We propose that astrocytes forming the glia limitans around the neural vasculature and deep to the meninges are intimately involved in controlling hemorrhage in the brain. Finally, we observed an increase in TF mRNA expression in the brains of scrapie-infected mice. This modulation of TF expression in the absence of hemorrhage suggested that TF may function in processes other than hemostasis by altering protease generation in normal and diseased brain.
M Eddleston, J C de la Torre, M B Oldstone, D J Loskutoff, T S Edgington, N Mackman
We have identified a novel 69-kD peptide autoantigen (ICA69) associated with insulin-dependent diabetes mellitus (IDDM) by screening a human islet lambda gt11 cDNA expression library with cytoplasmic islet cell antibody positive sera from relatives of IDDM patients who progressed to the overt disease. The deduced open reading frame of the ICA69 cDNA predicts a 483-amino acid protein. ICA69 shows no nucleotide or amino acid sequence relation to any known sequence in GenBank, except for two short regions of similarity with BSA. The ICA69 cDNA probe hybridizes with a 2-kb mRNA in poly(A+) RNA from human pancreas, brain, heart, thyroid, and kidney, but not with skeletal muscle, placenta, spleen, or ovary. Expression of ICA69 was also detected in beta cells and cell lines, as well as in tumoral tissue of islet cell origin. The native ICA69 molecule migrates to 69 kD in SDS-PAGE as detected with specific antibodies. Serum samples from relatives of IDDM patients specifically reacted with affinity-purified recombinant ICA69 on Western blotting. The structural gene for ICA69 was designated ICA1. A homologue in the mouse, designated Ica-1 was mapped to the proximal end of chromosome 6 (within 6 cM of the Met protooncogene). ICA69 adds a novel autoantigen to the family of identified islet target molecules, and by the manner of its identification and characterization large amounts of antigen are available for development of quantitative, convenient predictive assays for autoantibodies and analysis of the role of this molecule in diabetes autoimmunity, as well as its physiologic function.
M Pietropaolo, L Castaño, S Babu, R Buelow, Y L Kuo, S Martin, A Martin, A C Powers, M Prochazka, J Naggert
Recent studies have demonstrated the induced expression of endothelial adhesion molecules including E-selectin (also called endothelial leukocyte adhesion molecule-1), vascular cell adhesion molecule and intercellular adhesion molecule in actively involved mucosa of patients with ulcerative colitis and Crohn's disease. Similar induction has been demonstrated in the colon of the Cotton-top tamarin (CTT), a New World primate that experiences a spontaneous acute and chronic colitis resembling ulcerative colitis. To assess the potential importance of leukocyte adhesion as a necessary step in acute colitis, the effect of parenteral mAb directed against adhesion molecules on CTT colitis was evaluated in placebo-controlled blinded trials. Serial administration of either of two anti-E-selectin mAb designated BB11 and EH8 effectively coated endothelial surfaces expressing this vascular adhesion molecule. Although colitis activity was slightly diminished after the 10-d treatment period in CTT receiving either BB11 or EH8, this reduction was not significantly different than that seen in animals given a placebo control when assessed by a previously validated standardized scale of inflammatory activity: mean histologic activity grade 2.2 +/- 0.2 pretreatment vs 1.5 +/- 0.5 posttreatment in group receiving mAb and 2.1 +/- 0.1 pretreatment vs 1.3 +/- 0.5 posttreatment in the placebo group (P > 0.2). In contrast, administration of an anti-alpha 4 integrin mAb designated HP1/2 that binds VLA4 (alpha 4 beta 1) and presumably alpha 4 beta 7 integrins resulted in significant attenuation of acute colitis when compared to both pretreatment activity index (P = 0.005) and the placebo control group (P < 0.01): mean histologic activity grade 1.6 +/- 0.3 pretreatment vs 0.2 +/- 0.1 posttreatment in the group receiving HP1/2 and 1.8 +/- 0.5 pretreatment and 1.2 +/- 0.2 posttreatment in the placebo control group. These studies using a model of spontaneous colitis in the CTT demonstrate the feasibility of modulation of leukocyte-vascular adhesion and/or other integrin-mediated events possibly including T cell aggregation and T cell-stromal interactions, as well as lymphocyte homing. These results suggest both that these processes are important and possibly essential elements in sustaining acute colitis and that their disruption may result in therapeutic benefit.
D K Podolsky, R Lobb, N King, C D Benjamin, B Pepinsky, P Sehgal, M deBeaumont
Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has, for cogent technical reasons, largely been undertaken to date in neonatal ventricular myocytes. To circumvent expected limitations of other methods, the present study was initiated to determine whether replication-deficient adenovirus would enable efficient gene transfer to adult cardiac cells in culture. Adult rat ventricular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a cytomegalovirus immediate-early promoter-driven lacZ reporter gene and were assayed for the presence of beta-galactosidase 48 h after infection. The frequency of lacZ+ rod-shaped myocytes was half-maximal at 4 x 10(5) plaque-forming units (PFU) and approached 90% at 1 x 10(8) PFU. Uninfected cells and cells infected with lacZ- virus remained colorless. Beta-galactosidase activity concurred with the proportion of lacZ+ cells and was contingent on the exogenous lacZ gene. At 10(8) PFU/dish, cell number, morphology, and viability each were comparable to uninfected cells. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant adenovirus. The relative uniformity for gene transfer by adenovirus should facilitate tests to determine the impact of putative regulators upon the endogenous genes and gene products of virally modified adult ventricular muscle cells.
L A Kirshenbaum, W R MacLellan, W Mazur, B A French, M D Schneider
Repair after acute lung injury requires elimination of granulation tissue from the alveolar airspace. We hypothesized that during lung repair, signals capable of inducing the death of the two principal cellular elements of granulation tissue, fibroblasts and endothelial cells, would be present at the air-lung interface. Bronchoalveolar lavage fluid obtained from patients during lung repair induced both fibroblast and endothelial cell death, while fluid obtained at the time of injury or from patient controls did not. The mode of cell death for endothelial cells was apoptosis. Fibroblast death, while morphologically distinct from necrosis, also differed from typical apoptosis. Only proliferating cells were susceptible to the bioactivities in lavage fluid, which were trypsin sensitive and lipid insoluble. Histological examination of lung tissue from patients after lung injury revealed evidence of apoptotic cells within airspace granulation tissue. Our results suggest that cell death induced by peptide(s) present at the air-lung interface may participate in the remodeling process that accompanies tissue repair after injury.
V A Polunovsky, B Chen, C Henke, D Snover, C Wendt, D H Ingbar, P B Bitterman
To elucidate the cellular mechanism by which angiotensin II (ANG II) induces cardiac hypertrophy, we investigated the possible autocrine/paracrine role of endogenous endothelin-1 (ET-1) in ANG II-induced hypertrophy of neonatal rat cardiomyocytes by use of synthetic ET-1 receptor antagonist and antisense oligonucleotides to preproET-1 (ppET-1) mRNA. Northern blot analysis and in situ hybridization revealed that ppET-1 mRNA was expressed in cardiomyocytes, but, to a lesser extent, in nonmyocytes as well. ANG II upregulated ppET-1 mRNA level by threefold over control level as early as 30 min, and it stimulated release of immunoreactive ET-1 from cardiomyocytes in a dose- and time-dependent manner. ET-1 stimulated ppET-1 mRNA levels after 30 min in a similar fashion as ANG II. Tetradecanoylphorbol-acetate (10(-7) M) mimicked the effects of ANG II and ET-1 on induction of ppET-1 mRNA. ANG II-induced ppET-1 gene expression was completely blocked by protein kinase C inhibitor H-7 or by down-regulation of endogenous protein kinase C by pretreatment with phorbol ester. ET-1 and ANG II stimulated twofold increase [3H]leucine incorporation into cardiomyocytes, whose effects were similarly and dose dependently inhibited by endothelin A receptor antagonist (BQ123). Introduction of antisense sequence against coding region of ppET-1 mRNA into cardiomyocytes resulted in complete blockade with ppET-1 mRNA levels and [3H]leucine incorporation stimulated by ANG II. These results suggest that endogenous ET-1 locally generated and secreted by cardiomyocytes may contribute to ANG II-induced cardiac hypertrophy via an autocrine/paracrine fashion.
H Ito, Y Hirata, S Adachi, M Tanaka, M Tsujino, A Koike, A Nogami, F Murumo, M Hiroe
Hyperthermia causes changes in expression of TGF-beta mRNA and protein in cultured cardiac cells, as well as in the heart in vivo. 12 h after hyperthermia, primary cultures of neonatal rat cardiomyocytes show a two- to threefold decreased expression of TGF-beta mRNAs which returns to control levels by 48 h after heat shock. In cultures of cardiac fibroblasts, expression of TGF-beta mRNAs increases 5-25-fold, 12-48 h after heat shock, while fetal bovine heart endothelial cells show little change in TGF-beta expression after hyperthermia. In each case, mRNAs for TGF-beta s 1, 2, and 3 are regulated similarly. Hearts isolated from rats exposed to hyperthermia show an initial 20-fold decrease in TGF-beta 1 and 3 mRNA levels which return to control levels by 24 h and subsequently are elevated two- to threefold above normal 48-72 h after heat shock; there is little change in TGF-beta 2 mRNA. Expression of immunoreactive TGF-beta 1 and 3 protein, localized intracellularly in myocytes, follows the same pattern as the mRNA expression. By 72 h, some myocytes show hyperstaining for TGF-beta 1. Staining for extracellular TGF-beta 1/3 exhibits the opposite time course, being most intense 3-6 h after heat shock and returning to control levels by 48 h. The increase in TGF-beta s after hyperthermia could play a role in mediating the reported cardioprotective effects of heat shock.
K C Flanders, T S Winokur, M G Holder, M B Sporn
We have discovered a novel compound, NO-1886, which possesses a powerful lipoprotein lipase (LPL) activity-increasing action. Administration of NO-1886 increased LPL activity in the postheparin plasma, adipose tissue, and myocardium of rats, and produced a reduction in plasma triglyceride levels with concomitant elevation of HDL cholesterol levels. Administration of NO-1886 increased LPL enzyme mass in postheparin plasma and mRNA activity in epididymal adipose tissue, and it was concluded that the mode of action of this compound is stimulation of tissue LPL synthesis. We also conducted long-term studies to assess the impact of increases in LPL activity and HDL levels on the development of atherosclerotic lesions in rats. Administration of NO-1886 for as long as 90 d significantly decreased the degree of atherosclerotic changes in the coronary arteries of vitamin D2-treated, cholesterol-fed rats. Statistical analysis indicated that increased concentration of HDL is the factor contributing mostly to the prevention of coronary artery sclerosis. In summary, the results of our study indicate that compound NO-1886 increases LPL activity, causing an elevation in HDL levels, and that long-term administration of NO-1886 to rats with experimental atherosclerosis provides significant protection against the development of coronary artery lesions.
K Tsutsumi, Y Inoue, A Shima, K Iwasaki, M Kawamura, T Murase
To establish the mechanism(s) and site(s) of action of cholecystokinin (CCK) on pancreatic secretion under physiological conditions, we used an in vivo model using anesthetized rats with pancreaticobiliary cannulas. Infusion of CCK-8 (10-160 pmol/kg per h) produced a dose-dependent increase in plasma CCK levels. CCK-8 infusion at 40 pmol/kg per h produced a plasma CCK level of 7.9 +/- 1.5 pM and an 80% increase in pancreatic protein output over basal. This level was closely approximated by a postprandial peak plasma CCK level by 6.2 +/- 1.1 pM. Pretreatment with atropine or hexamethonium completely abolished pancreatic protein response to low doses of CCK-8 (10-40 pmol/kg per h) but had only partial effect on doses > 40 pmol/kg per h. Bilateral vagotomy also abolished the pancreatic responses to low doses of CCK-8. Similarly perivagal treatment with a sensory neurotoxin, capsaicin, caused a complete inhibition of pancreatic protein secretion in response to CCK-8 infusion. In contrast, pancreatic protein responses to bethanechol were similar in control and capsaicin-treated rats. In separate studies we demonstrated that gastroduodenal but not jejunal application of capsaicin for 30 min abolished pancreatic protein secretion in response to physiological doses of CCK-8. In conclusion, CCK at physiological levels stimulates pancreatic enzyme secretion via a capsaicin-sensitive afferent vagal pathway originating from the gastroduodenal mucosa.
Y Li, C Owyang
PDGF isoforms have been postulated to serve as mediators of fibroblast proliferation and chemotaxis during lung fibrogenesis induced by asbestos inhalation. We have studied the interaction of chrysotile asbestos fibers with rat lung fibroblasts (RLF) in vitro and the consequent changes in PDGF receptor mRNA expression, PDGF binding, and mitogenic activity of PDGF isoforms. Northern blot analysis revealed that mRNA for the PDGF-receptor alpha subtype (PDGF-R alpha) on RLF was upregulated after a 24-h exposure to asbestos in culture (0.5-15 micrograms fibers/cm2). [125I]PDGF-BB receptor assays showed that normal RLF possess mainly PDGF-R beta and a paucity of PDGF-R alpha. In agreement with the Northern data, saturation binding of [125I]PDGF-BB to RLF exposed to asbestos demonstrated an approximately 40% increase in binding sites accompanied by a twofold decrease in receptor affinity. Treating asbestos-exposed RLF with PDGF-AA, which binds only PDGF-R alpha, blocked the PDGF binding sites that were upregulated by fiber exposure. PDGF-AA had increased mitogenic potency for fiber-exposed RLF, but PDGF-BB was a less potent mitogen for these RLF. Nonfibrogenic carbonyl iron spheres induced similar changes in PDGF growth responses. These data show that inorganic particulates alter the PDGF-R alpha population on RLF without significant change in PDGF-R beta.
J C Bonner, A L Goodell, P G Coin, A R Brody
We have previously shown that forskolin and 3-isobutyl-1-methylxanthine (IBMX) increased accumulation of cisplatin (DDP) in DDP-sensitive 2008 human ovarian carcinoma cells in proportion to their ability to increase cAMP. Since the major function of cAMP is to activate protein kinase A, it was conjectured that the stimulation of DDP accumulation was mediated by a protein kinase A substrate. We now show that exposure of 2008 cells to forskolin resulted in phosphorylation of a prominent 52-kD membrane protein. Microsequencing of the band demonstrated it to be human beta-tubulin. Similarly, pretreatment of 2008 cells with the microtubule stabilizing drug taxol increased platinum accumulation in a dose-dependent manner. In 11-fold DDP-resistant 2008/C13*5.25 cells, decreased DDP accumulation was associated with enhanced spontaneous formation of microtubule bundles and decreased expression of beta-tubulin and the tubulin-associated p53 antioncogene relative to 2008 cells. 2008/C13*5.25 cells had altered sensitivity to tubulin-binding drugs, being hypersensitive to taxol and cross-resistant to colchicine. We conclude that pharmacologic alterations of tubulin enhance accumulation of DDP, and that the DDP-resistant phenotype in 2008/C13*5.25 cells is associated with tubulin abnormalities.
R D Christen, A P Jekunen, J A Jones, F Thiebaut, D R Shalinsky, S B Howell
A low ratio of whole-body 24-h fat/carbohydrate (CHO) oxidation has been shown to be a predictor of subsequent body weight gain. We tested the hypothesis that the variability of this ratio may be related to differences in skeletal muscle metabolism. Since lipoprotein lipase (LPL) plays a pivotal role in partitioning lipoprotein-borne triglycerides to adipose (storage) and skeletal muscle (mostly oxidation), we postulated that a low ratio of fat/CHO oxidation was associated with a low skeletal muscle LPL (SMLPL) activity. As an index of substrate oxidation, 24-h RQ was measured under sedentary and eucaloric conditions in 16 healthy nondiabetic Pima males. During a 6-h euglycemic, hyperinsulinemic clamp, muscle biopsies were obtained at baseline, 3, and 6 h. Heparin-elutable SMLPL activity was 2.92 +/- 0.56 nmol free fatty acids/g.min (mean +/- SD) at baseline, was unchanged (2.91 +/- 0.51) at the third hour, and increased significantly (P < 0.05) to 3.13 +/- 0.57 at the sixth hour of the clamp. The mean (of baseline and 3-h) SMLPL activity correlated inversely with 24-h RQ (r = 0.57, P < 0.03) but not with body size, body composition, or insulin-mediated glucose uptake. Since SMLPL activity is related to the ratio of whole body fat/CHO oxidation rate, a decreased muscle LPL activity may, therefore, predispose to obesity.
R T Ferraro, R H Eckel, D E Larson, A M Fontvieille, R Rising, D R Jensen, E Ravussin
Elevation of cytosolic calcium ([Ca2+]i) has been reported to induce apoptosis in a number of cell types. However, in the neutrophil, which undergoes apoptosis constitutively during aging in vitro, activation by inflammatory mediators elevates [Ca2+]i and prolongs lifespan via inhibition of apoptosis. To examine this paradox, we investigated the effects of modulation of [Ca2+]i upon apoptosis of neutrophils in vitro. Calcium ionophores (A23187, ionomycin) retarded apoptosis in neutrophil populations after 20 h (P < 0.001). Conversely, intracellular Ca(2+)-chelation, using bis-(o-aminophenoxy)-N,N,N'N'-tetraacetic acid (BAPTA) acetoxymethyl ester (AM) promoted apoptosis (P < 0.02). W-7 (an inhibitor of calmodulin) also promoted apoptosis (P < 0.05). Measurements of [Ca2+]i, using fura-2, showed (a) increased apoptosis in neutrophil populations was not associated with elevated [Ca2+]i, (b) neutrophils cultured with ionophore at concentrations inhibiting apoptosis exhibited transient (< 1 h) elevations of [Ca2+]i, to levels previously reported with receptor-mediated stimuli, and (c) BAPTA was able to prevent the elevation of [Ca2+]i and the inhibition of apoptosis produced by ionophore. Modulation of apoptosis occurred without alterations in intracellular pH. Thus, in the neutrophil, unlike lymphoid cells, elevation of [Ca2+]i exerts an inhibitory effect upon apoptosis. Furthermore, these data suggest that transient elevation of [Ca2+]i elicits signaling events leading to prolonged inhibition of apoptosis.
M K Whyte, S J Hardwick, L C Meagher, J S Savill, C Haslett
Oral administration to five postmenopausal women of dl-norgestrel (0.075 mg/d for 7 wk) reduced mean fasting plasma levels of triglycerides by 29% (P < 0.001), VLDL triglycerides by 39% (P < 0.01), and VLDL apo B by 26% (P < 0.05), while lowering mean total cholesterol by 7% (P < 0.06). To explain these observations the kinetics of VLDL and LDL apo B turnover were studied by injecting autologous 125I-labeled VLDL and 131I-labeled LDL under control conditions and again in the fourth week of a 7-wk course of dl-norgestrel. VLDL apo B pool size fell by an average of 27% (1.2 vs 1.7 mg/kg, P < 0.06) and production of apo B by 18% (18 vs 22 mg/kg per d, P < 0.05) with unchanged fractional catabolic rate. Production of LDL apo B increased 36% with dl-norgestrel (12 vs 9.4 mg/kg per d, P < 0.05), but this was compensated by a 36% increase in fractional catabolic rate of LDL apo B (0.33 vs 0.25 pools/d, P < 0.005), thereby maintaining pool size. Lipoprotein (a) fell by an average of 12% (16 vs 18 mg/dl, P < 0.06). dl-Norgestrel reduced VLDL triglycerides (40 vs 64 mg/dl, P < 0.05), intermediate density lipoprotein cholesterol (14 vs 19 mg/dl, P < 0.02), IDL apo B (5.3 vs 7.2 mg/dl, P < 0.05), and VLDL cholesterol (3.1 vs 5.1 mg/dl, 0.10 > P > 0.05), in parallel with the reductions in VLDL apo B production and pool size. dl-Norgestrel significantly lowered the production rate of VLDL apo B, thereby decreasing plasma VLDL and intermediate density lipoprotein concentrations.
B M Wolfe, M W Huff
In previous studies we showed that cultured human keratinocytes expressed the 55-kD TNF receptor (TNFR) and that its expression the important for TNF alpha-mediated upregulation of intercellular adhesion molecule-1 (ICAM-1) expression on keratinocytes. Because factors that either reduce or enhance TNFR expression are likely to have a major impact on the biological effects of TNF alpha on keratinocytes, these studies were conducted to determine the factors that regulate its expression on keratinocytes. Using reverse transcriptase polymerase chain reaction, human keratinocytes were shown to lack 75-kD TNFR expression, indicating that TNF responsiveness of human keratinocytes critically depended on regulation of 55-kD TNFR expression. Human keratinocyte 55-kD TNFR surface and mRNA expression was found to be regulated in vitro by recombinant human (rh) TNF alpha. Stimulation of keratinocytes with rhTNF alpha initially decreased, but later increased, 55-kD TNFR surface expression. This biphasic modulation of 55-kD TNFR surface expression was associated with concomitant changes in 55-kD TNFR mRNA expression. Ultraviolet B (UVB) radiation, a well-known inducer of synthesis and secretion of TNF alpha by human keratinocytes, was found to mimic TNF alpha-induced modulation of 55-kD TNFR surface and mRNA expression via a TNF alpha-mediated autocrine regulatory mechanism. Production of soluble 55-kD TNFR by human keratinocytes remained unaffected by TNF alpha stimulation or UVB irradiation. These studies provide clear evidence that membrane expression of the human 55-kD TNFR may be regulated in human keratinocytes by the ligand itself: TNF alpha. Since in previous studies UVB irradiation transiently inhibited TNF alpha-induced human keratinocyte ICAM-1 expression, it is proposed that UVB radiation-induced biphasic modulation of human keratinocyte 55-kD TNFR expression may affect the capacity of these cells to respond to TNF alpha.
U Trefzer, M Brockhaus, H Lötscher, F Parlow, A Budnik, M Grewe, H Christoph, A Kapp, E Schöpf, T A Luger
We have previously shown that minimally oxidized LDL (MM-LDL) activated endothelial cells to increase their interaction with monocytes but not neutrophils, inducing monocyte but not neutrophil binding and synthesis of monocyte chemotactic protein-1 and monocyte colony-stimulating factor (M-CSF). In the present studies we have examined the signaling pathways by which this monocyte-specific response is induced. Both induction of monocyte binding and mRNA levels for M-CSF by MM-LDL were not inhibited in protein kinase C-depleted endothelial cells. A number of our studies indicate that cAMP is the second messenger for the effects of MM-LDL cited above. Incubation of endothelial cells with MM-LDL caused a 173% increase in intracellular cAMP levels. Agents which increased cAMP levels, including cholera toxin, pertussis toxin, dibutyryl cAMP, and isoproterenol mimicked the actions of MM-LDL. Agents which elevated cAMP were also shown to activate NF kappa B, suggesting a role for this transcription factor in activation of monocyte-endothelial interactions. Although endothelial leukocyte adhesion molecule (ELAM) mRNA synthesis can be regulated by NF kappa B, ELAM was not expressed and ELAM mRNA was only slightly elevated in response to MM-LDL. We present evidence that induction of neutrophil binding by LPS is actually suppressed by agents that elevated cAMP levels.
F Parhami, Z T Fang, A M Fogelman, A Andalibi, M C Territo, J A Berliner
We have identified the molecular defect in two siblings presenting with classical clinical and biochemical features of Fish Eye disease (FED), including corneal opacities, HDL cholesterol < 10 mg/dl, normal plasma cholesteryl esters, and elevated triglycerides. In contrast to previously reported patients with FED who are unable to esterify HDL-associated cholesterol, our patients' plasma lecithin-cholesterol acetyltransferase (alpha-LCAT)-specific activities assayed using an HDL-like proteoliposome substrate were 12.7-25.7 nmol/micrograms (19.5 +/- 1.8 in controls). In addition, significant residual cholesterol esterification was present in VLDL/LDL-depleted plasma, confirming the presence of HDL-associated alpha-LCAT activity. DNA sequence analysis of the proband's LCAT gene identified deletion of the triplet coding for leu300, which resulted in the loss of a restriction site for MlnI. Digestion of PCR-amplified DNA using MlnI established that both siblings are homozygous for this defect. Expression of LCAT300-del. in human embryonic kidney-293 cells revealed normal mRNA and intracellular LCAT concentrations. However, reduced amounts of LCAT300-del., which had a normal specific alpha-LCAT activity, were present in the media. In summary, we report the first case of FED associated with a mutant enzyme that has a normal alpha-LCAT-specific activity. The functional significance of this LCAT gene defect has been established in an in vitro expression system, which demonstrates that very small amounts of this functional LCAT mutant enzyme accumulate in the media. Characterization of LCAT300-del. established that selective alpha-LCAT deficiency is not a prerequisite for the development of FED. On the basis of our combined results, we propose that the residual amounts of total plasma LCAT activity and not its distribution on lipoproteins primarily determines the heterogeneity in phenotypic expression observed in familial LCAT deficiency syndromes.
H G Klein, S Santamarina-Fojo, N Duverger, M Clerc, M F Dumon, J J Albers, S Marcovina, H B Brewer Jr
Insulin resistance for glucose metabolism in skeletal muscle is a key feature in non-insulin-dependent diabetes mellitus (NIDDM). Which cellular effectors of glucose metabolism are involved is still unknown. We investigated whether transmembrane glucose transport in vivo is impaired in skeletal muscle in nonobese NIDDM patients. We performed euglycemic insulin clamp studies in combination with the forearm balance technique (brachial artery and deep forearm vein catheterization) in six nonobese NIDDM patients and five age- and weight-matched controls. Unlabeled D-mannitol (a nontransportable molecule) and radioactive 3-O-methyl-D-glucose (the reference molecular probe to assess glucose transport activity) were simultaneously injected into the brachial artery, and the washout curves were measured in the deep venous effluent blood. In vivo transmembrane transport of 3-O-methyl-D-glucose in forearm muscle was determined by computerized analysis of the washout curves. At similar steady-state plasma concentrations of insulin (approximately 500 pmol/liter) and glucose (approximately 5.15 mmol/liter), transmembrane inward transport of 3-O-methyl-D-glucose in skeletal muscle was markedly reduced in the NIDDM patients (6.5 x 10(-2) +/- 0.56 x 10(-2).min-1) compared with controls (12.5 x 10(-2) +/- 1.5 x 10(-2).min-1, P < 0.005). Mean glucose uptake was also reduced in the diabetics both at the whole body level (9.25 +/- 1.84 vs. 28.3 +/- 2.44 mumol/min per kg, P < 0.02) and in the forearm tissues (5.84 +/- 1.51 vs. 37.5 +/- 7.95 mumol/min per kg, P < 0.02). When the latter rates were extrapolated to the whole body level, skeletal muscle accounted for approximately 80% of the defect in insulin action seen in NIDDM patients. We conclude that transmembrane glucose transport, when assessed in vivo in skeletal muscle, is insensitive to insulin in nonobese NIDDM patients, and plays a major role in determining whole body insulin resistance.
R C Bonadonna, S Del Prato, M P Saccomani, E Bonora, G Gulli, E Ferrannini, D Bier, C Cobelli, R A DeFronzo
Infection with HIV-1 occasionally results in a sicca syndrome, termed the diffuse infiltrative lymphocytosis syndrome, characterized by infiltration of the salivary glands with a predominance of CD8 T cells. This response is strongly associated with certain MHC class I and class II alleles. To define the salivary gland T cell receptor (TCR) repertoire, the primary structure of the TCR beta-chains was determined using in situ cDNA synthesis followed by the "anchored" polymerase chain reaction. The sequences of 59 beta-chains from five individuals with diffuse infiltrative lymphocytosis syndrome shared structural features suggesting antigenic clonal selection. Certain combinations of V beta J beta gene segments were selectively overrepresented in the repertoire sample, demonstrating a common restricted usage of certain V beta and J beta gene segments. The beta-chains derived from these overrepresented V beta J beta combinations revealed a preference for specific amino acids at position 97 in the third complementarity-determining region, a residue postulated to contact peptide antigen. Moreover, the nucleotides encoding this position were not germline in origin. TCR beta-chains in nonoverrepresented V beta J beta combinations did not exhibit preferential usage of selected somatically encoded residues. The pattern of TCR beta-chains expressed in the salivary gland of a control person with primary Sjögren's syndrome was considerably more heterogeneous and different from that found in diffuse infiltrative lymphocytosis syndrome.
E Dwyer, S Itescu, R Winchester
Expression of heat shock protein 70 (hsp70) is stimulated during ischemia, but its proposed cytoprotective function during metabolic stress has remained conjectural. We introduced a human hsp70 gene into mouse 10T1/2 cells and assessed the susceptibility of these cells to injury in response to conditions that mimic ischemia. Transiently transfected cells, in the absence of stress, expressed human hsp70 to levels equal to or greater than those induced by heat shock, as assessed by RNAse protection, immunoblot, and immunohistochemical analyses. By comparison to cells transfected with a control plasmid, cells expressing the human hsp70 transgene were resistant to injury induced by glucose deprivation and inhibition of mitochondrial respiration. These results provide direct evidence for a cytoprotective function of hsp70 during metabolic stress.
R S Williams, J A Thomas, M Fina, Z German, I J Benjamin
Measurement of beta-cell function is an important marker of progression to diabetes in individuals at risk for the disease. Although the peak incidence for the disease occurs before 17 years of age, normal values for insulin secretion were not available in this age group. We performed a simplified intravenous glucose tolerance test in 167 normal children, and in 98 islet cell antibody (ICA)-negative and 12 ICA-positive siblings of diabetic patients. Their age range was 1-16 yr. The first phase of insulin secretion, evaluated as the sum of plasma insulin concentrations at 1 and 3 min, increased with age and was significantly lower in ICA-negative siblings (86 +/- 6 microU/ml, P < 0.002) than in normal controls (115 +/- 6 microU/ml). This difference was not apparent before 8 yr of age. None of the ICA-negative siblings developed diabetes after an average of 4.5 yr. ICA-positive siblings at first study had a first phase insulin response similar to that of ICA negative siblings, but significantly lower than that of the normal controls (74 +/- 13 microU/ml, P < 0.02). The reason for the decreased insulin secretion in ICA-negative siblings is unknown, but could involve a defect in the growth of beta-cell mass or insulin secretion that could be part of the multifactorial pathogenesis of type 1 diabetes.
J C Carel, C Boitard, P F Bougnères
L K Olson, J B Redmon, H C Towle, R P Robertson
C Brugnara, L de Franceschi, S L Alper
The presence of receptors for the cytokine IL-2 was assessed in the IEC-6 cell line established from normal rat crypt epithelium and primary intestinal epithelial cells. 125I-IL-2 was found to specifically bind to subconfluent IEC-6 cells. Maximal binding was observed within 30 min after addition of the ligand; binding could be inhibited by excess unlabeled IL-2 or addition of antibody to the IL-2 receptor. Both intermediate and low affinity receptors with approximate Kd of 10 and 100 pM, respectively were present. Kinetic analysis were consistent with the results of Western blot analysis using an antisera to the 75-kD IL-2 receptor beta chain. IL-2 receptors appeared to be functional; addition of IL-2 led to modulation of proliferation with initial stimulation at 24 h followed by inhibition at 48 h. This effect could be blocked by addition of antibody to the IL-2 receptor beta chain. IL-2 treatment could be shown to enhance expression (range = 4- to 50-fold stimulation) of TGF-beta, as well as the lectin protein mac-2, in IEC-6 cells. The relevance of observations in the IEC-6 cell line to intestinal mucosa in vivo was supported by the demonstration of a gradient of expression of the IL-2 receptor in primary rat intestinal epithelial cells by Western blot analysis. In addition, mRNA for the IL-2 receptor-beta chain was demonstrated by Northern blot analysis using mRNA from primary rat intestinal epithelial cells depleted of detectable contaminating intraepithelial lymphocytes by two cycles of fractionation on Percoll gradients. Collectively, these observations suggest that the range of cellular targets of the putative lymphokine IL-2 is broader than appreciated, and IL-2 may serve to integrate epithelial and lymphocyte responses in the intestinal mucosa.
C Ciacci, Y R Mahida, A Dignass, M Koizumi, D K Podolsky