Plectin is a widely expressed cytomatrix component involved in the attachment of the cytoskeleton to the plasma membrane. We have recently reported that the skin and muscles of three patients affected by epidermolysis bullosa simplex with muscular dystrophy (MD-EBS), a genetic disorder characterized by skin blistering associated with muscle involvement, are not reactive with antibodies specific to plectin. We demonstrated that in the skin, lack of plectin leads to failure of keratin filaments to connect to the plasma membrane via the hemidesmosomes, whereas in the muscle the deficient expression of the molecule correlates with an aberrant localization of desmin in the muscle fibers. In this study we demonstrate that in a MD-EBS kindred with two affected members, the disease results from a homozygous nonsense mutation in the plectin (PLEC1) gene leading to a premature stop codon (CGA to TGA) and decay of the aberrant plectin messenger RNA. The segregation of the mutated allele implicates the mutation in the pathology of the disorder. These results confirm the critical role of plectin in providing cell resistance to mechanical stresses both in the skin and the muscle.
S Chavanas, L Pulkkinen, Y Gache, F J Smith, W H McLean, J Uitto, J P Ortonne, G Meneguzzi
Oxidative modification of LDL increases its atherogenicity, and 15-lipoxygenase (15-LO) has been implicated in the process. To address this issue, we generated transgenic rabbits that expressed 15-LO in a macrophage-specific manner and studied their susceptibility to atherosclerosis development when they were fed a high-fat, high-cholesterol (HFHC) diet (Teklad 0533 rabbit diet 7009 with 10% corn oil and 0.25% cholesterol) for 13.5 wk. Transgenic and nontransgenic rabbits developed similar degrees of hypercholesterolemia and had similar levels of triglyceride, VLDL, LDL, and HDL. Quantitative morphometric analysis of the aortic atherosclerosis indicated that the transgenic animals (n = 19) had significantly smaller lesion areas (9.8+/-6.5%, mean+/-SD) than their littermate controls (n = 14, 17.8+/-15.0%) (P < 0.05). In a subgroup (n = 9) of transgenic rabbits that received the HFHC diet plus the antioxidant N',N '-diphenyl-phenylenediamine (1%), the extent of lesion involvement (9.8+/-7.5%) did not differ from the subgroup (n = 10) that received the regular HFHC diet (9.7+/-5.9%). Since the results were unexpected, we repeated the experiments. Again, we found that the nontransgenic littermates (n = 12) had more extensive lesions (11.6+/-10.6%) than the transgenic rabbits (n = 13; 9.5+/-7.8%), although the difference was not significant. In a third set of experiments, we crossed 15-LO transgenic rabbits with Watanabe heritable hyperlipidemic (WHHL) rabbits and found that the lesion area in the 15-LO transgenic/heterozygous WHHL rabbits (n = 14) was only about one third (7.7+/-5.7%) that found in nontransgenic heterozygous WHHL littermate controls (n = 11, 20.7+/-19.4%) (P < 0.05). These data suggest that overexpression of 15-LO in monocytes/macrophages protects against lipid deposition in the vessel wall during early atherogenesis in these rabbit models of atherosclerosis.
J Shen, E Herderick, J F Cornhill, E Zsigmond, H S Kim, H Kühn, N V Guevara, L Chan
Myocardial infarcts heal by scar formation because there are no stem cells in myocardium, and because adult myocytes cannot divide and repopulate the wound. We sought to redirect the heart to form skeletal muscle instead of scar by transferring the myogenic determination gene, MyoD, into cardiac granulation (wound repair) tissue. A replication-defective adenovirus was constructed containing MyoD under transcriptional control of the Rous sarcoma virus long terminal repeat. The virus converted cultured cardiac fibroblasts to skeletal muscle, indicated by expression of myogenin and skeletal myosin heavy chains (MHCs). To determine if MyoD could induce muscle differentiation in vivo, we injected 2 x 10(9) or 10(10) pfu of either the MyoD or a control beta-galactosidase adenovirus into healing rat hearts, injured 1 wk previously by freeze-thaw. After receiving the lower viral dose, cardiac granulation tissue expressed MyoD mRNA and protein, but did not express myogenin or skeletal MHC. When the higher dose of virus was administered, double immunostaining showed that cells in reparative tissue expressed both myogenin and embryonic skeletal MHC. No muscle differentiation occurred after beta-galactosidase transfection. Thus, MyoD gene transfer can induce skeletal muscle differentiation in healing heart lesions. Modifications of this strategy might eventually provide new contractile tissue to repair myocardial infarcts.
C E Murry, M A Kay, T Bartosek, S D Hauschka, S M Schwartz
Angiotensin II (AII) is a critical factor in cardiac remodeling which involves hypertrophy, fibroblast proliferation, and extracellular matrix production. However, little is known about the mechanism by which AII accelerates these responses. Osteopontin is an acidic phosphoprotein with RGD (arginine-glycine-aspartate) sequences that are involved in the vascular smooth muscle cell remodeling process. We identified the presence of osteopontin mRNA and protein in cultured rat cardiac fibroblasts and its prominent regulation by AII (10(-11) M). Osteopontin message levels were increased fourfold (P < 0.01) and protein fivefold (P < 0.05) at 24 h after addition of AII (10(-7) M). This response was inhibited by the AT1 receptor blocker, losartan. Osteopontin mRNA levels were increased in hypertrophied ventricles from animals with renovascular hypertension (1.6-fold, P < 0.05) and aortic banding (2.9-fold, P < 0.05). To examine the function of osteopontin, we determined its effects on (a) the ability of cardiac fibroblasts to contract three-dimensional collagen gels and (b) cardiac fibroblast growth. A monoclonal antibody against osteopontin partially blocked AII-induced three-dimensional collagen gel contraction by cardiac fibroblasts (64+/-4 vs. 86+/-5% in the presence of antibody, P < 0.05), while osteopontin itself promoted contraction of the gels by fibroblasts (71+/-5%, P < 0.05 compared with control). Either a monoclonal antibody against beta3 integrin which is a ligand for osteopontin or the RGD peptide blocked both AII and osteopontin-induced collagen gel contraction. Thus, the osteopontin RGD sequence binds to beta3 integrins on the fibroblast to promote fibroblast binding to collagen. All induced a threefold increase in DNA synthesis of cardiac fibroblasts, which was completely blocked by antibodies against osteopontin and beta3 integrin, or by RGD peptide, but not by controls. Thus, All-induced growth of cardiac fibroblasts also requires osteopontin engagement of the beta3 integrin. Taken together, these results provide the first evidence that osteopontin is a potentially important mediator of AII regulation of cardiac fibroblast behavior in the cardiac remodeling process.
N Ashizawa, K Graf, Y S Do, T Nunohiro, C M Giachelli, W P Meehan, T L Tuan, W A Hsueh
Thymic size and density were studied in 23 untreated patients with Graves' disease and 38 control subjects using computed tomography. Both thymic size and density were higher in untreated patients with Graves' disease than in control subjects in the age-matched group. After treatment with antithyroid drugs, both thymic size and density were significantly reduced, with a concomitant decrease in thyrotropin receptor antibodies. PCR of human thymic cDNA using primers for human thyrotropin receptor amplified a fragment in a size expected for the receptor, and its nucleotide sequence was identical to human thyrotropin receptor cDNA in the thyroid. Northern blot analysis of human thymic poly(A)+ RNA demonstrated the presence of the full length form of thyrotropin receptor mRNA. Western blot analysis of human thymic membrane using anti-thyrotropin receptor peptide antibodies demonstrated a band of 100 kD that was also observed in the thyroid membrane. Immunohistochemistry of thymic tissue using mouse antihuman thyrotropin receptor monoclonal antibodies demonstrated the immunostaining of epithelial cells. These results indicate that thymic hyperplasia is apparently associated with Graves' disease and suggest that thymic thyrotropin receptor may act as an autoantigen that may be involved in the pathophysiology of development of Graves' disease.
M Murakami, Y Hosoi, T Negishi, Y Kamiya, K Miyashita, M Yamada, T Iriuchijima, H Yokoo, I Yoshida, Y Tsushima, M Mori
Although the switch process is frequently associated with affinity maturation, the constant region is not assumed to play a role in Ag-Ab binding. In the present work, we demonstrate that two clonally related human monoclonal Igs sharing identical V(H) and V(L) sequences, but expressing different isotypes (IgA1kappa(PER) and IgG1kappa(PER)), bind tubulin with significantly different affinities. This difference was mainly accounted for by a disparity in the association rate constants. These results suggest that affinity maturation of this clone could be achieved through class switching in the absence of further somatic mutations. Since the differences observed were found at the Fab level, they also suggest a role for the C(H)1 domain in structuring the Ag-binding site into a more kinetically competent form.
O Pritsch, G Hudry-Clergeon, M Buckle, Y Petillot, J P Bouvet, J Gagnon, G Dighiero
We tested the hypothesis that glucose plus insulin determine the rate of fat oxidation in humans by controlling the rate of fatty acid entrance into the mitochondria. We gave constant infusions of [1-13C]oleate, a long-chain fatty acid, and [1-14C]octanoate, a medium-chain fatty acid, for 3 h in seven volunteers (basal). Immediately after the basal period, a hyperinsulinemic (insulin infusion = 120 mU x m(-2) min(-1)), hyperglycemic (plasma glucose = 140 mg/dl) clamp was started and continued for 5 h. During the last 3 h of the clamp, the infusions of [1-13C]oleate and [1-14C]octanoate were repeated. Intracellular acylcarnitine concentrations were measured in muscle biopsies obtained before and after the clamp. Plasma oleate enrichment and FFA concentration were kept constant by means of variable infusions of lipids and heparin. Oleate, but not octanoate, requires carnitine binding to gain access to the mitochondrial matrix; hence, if glucose and/or insulin limit long-chain fatty acid entrance into the mitochondria, then, during the clamp, long-chain acylcarnitine formation should be decreased, causing a decrease in oleate, but not octanoate, oxidation. Oleate oxidation decreased from the basal value of 0.7+/-0.1 to 0.4+/-0.1 micromol x kg(-1) x min(-1) (P < 0.05). In contrast, octanoate oxidation remained unchanged. Long-chain acylcarnitine concentration decreased from 855+/-271 in the basal state to 376+/-83 nmol/gram dry weight during the clamp (P < 0.05). We conclude that glucose and/or insulin determine fatty acid oxidation by controlling the rate of long-chain fatty acid entrance into the mitochondria.
L S Sidossis, C A Stuart, G I Shulman, G D Lopaschuk, R R Wolfe
The effects of glucagon (G) on proximal tubule reabsorption (PTR) and GFR seem to depend on a prior action of this hormone on the liver resulting in the liberation of a mediator and/or of a compound derived from amino acid metabolism. This study investigates in anesthetized rats the possible contribution of cAMP and urea, alone and in combination with a low dose of G, on phosphate excretion (known to depend mostly on PTR) and GFR. After a 60-min control period, cAMP (5 nmol/min x 100 grams of body weight [BW]) or urea (2.5 micromol/min x 100 grams BW) was infused intravenously for 200 min with or without G (1.2 ng/min x 100 grams BW, a physiological dose which, alone, does not influence PTR or GFR). cAMP increased markedly the excretion of phosphate and sodium (+303 and +221%, respectively, P < 0.01 for each) but did not alter GFR. Coinfusion of cAMP and G induced the same tubular effects but also induced a 20% rise in GFR (P < 0.05). Infusion of urea, with or without G, did not induce significant effects on PTR or GFR. After G infusion at increasing doses, the increase in fractional excretion of phosphate was correlated with a simultaneous rise in plasma cAMP concentration and reached a maximum for doubling of plasma cAMP. These results suggest that cAMP, normally released by the liver into the blood under the action of G, (a) is probably an essential hepatorenal link regulating the intensity of PTR, and (b) contributes, in conjunction with specific effects of G on the nephron, to the regulation of GFR.
M Ahloulay, M Déchaux, C Hassler, N Bouby, L Bankir
To study the role of apoC1 in lipoprotein metabolism, we have generated transgenic mice expressing the human APOC1 gene. On a sucrose-rich diet, male transgenic mice with high APOC1 expression in the liver showed elevated levels of serum cholesterol and triglyceride compared with control mice (5.7+/-0.7 and 3.3+/-2.1 vs. 2.7+/-0.1 and 0.4+/-0.1 mmol/liter, respectively). These elevated levels were mainly confined to the VLDL fraction. Female APOC1 transgenic mice showed less pronounced elevated serum lipid levels. In vivo VLDL turnover studies revealed that, in hyperlipidemic APOC1 transgenic mice, VLDL particles are cleared less efficiently from the circulation as compared with control mice. No differences were observed in the hepatic production and extrahepatic lipolysis of VLDL-triglyceride. Also, VLDL isolated from control and APOC1 transgenic mice were found to be equally good substrates for bovine lipoprotein lipase in vitro. These data indicate that the hyperlipidemia in APOC1 transgenic mice results primarily from impaired hepatic VLDL particle clearance, rather than a defect in the hydrolysis of VLDL-triglyceride. To investigate which hepatic receptor is involved in the apoC1-mediated inhibition of VLDL clearance, APOC1 transgenic mice were bred with an LDL receptor-deficient (LDLR(-/-)) background. In addition, control, LDLR(-/-), and LDLR(-/-)/APOC1 mice were transfected with adenovirus carrying the gene for the receptor-associated protein (Ad-RAP). Both serum cholesterol and triglyceride levels were strongly elevated in LDLR(-/-)/APOC1 mice compared with LDLR(-/-) mice (52+/-19 and 36+/-19 vs. 8.4+/-0.9 and 0.5+/-0.2 mmol/liter, respectively), indicating that apoC1 inhibits the alternative VLDL clearance pathway via the remnant receptor. Transfection of LDLR(-/-) mice with Ad-RAP strongly increased serum cholesterol and triglyceride levels, but to a lesser extent than those found in LDLR(-/-)/APOC1 mice (39+/-8 and 17+/-8 vs. 52+/-19 and 36+/-19 mmol/liter, respectively). However, in LDLR(-/-)/APOC1 mice the transfection with Ad-RAP did not further increase serum cholesterol and triglyceride levels (52+/-19 and 36+/-19 vs. 60+/-10 and 38+/-7 mmol/liter, respectively). From these studies we conclude that, in the absence of the LDLR, apoC1 inhibits the hepatic uptake of VLDL via a RAP-sensitive pathway.
M C Jong, V E Dahlmans, P J van Gorp, K W van Dijk, M L Breuer, M H Hofker, L M Havekes
Viral mutations have been implicated in alteration of the biological phenotype of hepatitis B virus (HBV). We recently cloned and sequenced the viral genome of an HBV strain associated with an outbreak of fulminant hepatitis (FH strain). The FH strain contained numerous mutations in all genomic regions and was functionally characterized by a more efficient encapsidation of pregenomic RNA leading to highly enhanced replication. To define the responsible mutation(s) for the enhanced replication, we introduced individual mutations of the FH strain into a wild-type construct by oligonucleotide-directed mutagenesis. Analysis of viral replication showed that two adjacent mutations in the HBV core promotor (C to T at nucleotide 1768 and T to A at nucleotide 1770) led to high level replication. Similar to the FH strain, this mutant displayed the phenotype of enhanced encapsidation of pregenomic RNA. Functional studies in an encapsidation assay demonstrated that the identified mutations resulted in a minor increase of pregenomic RNA transcription (two- to threefold) and a major transcription-independent enhancement (> 10-fold) of viral encapsidation. Our results demonstrate that the two adjacent mutations in the HBV core promotor region are responsible for the enhanced replication of the FH strain. These two mutations, outside the previously described encapsidation signal, core, and polymerase polypeptides, appeared to affect a novel genetic element involved in viral encapsidation.
T F Baumert, S A Rogers, K Hasegawa, T J Liang
Abnormal vascular smooth muscle cell (SMC) proliferation and migration contribute to the development of restenosis after percutaneous transluminal coronary angioplasty and accelerated arteriopathy after cardiac transplantation. Previously, we reported that the macrolide antibiotic rapamycin, but not the related compound FK506, inhibits both human and rat aortic SMC proliferation in vitro by inhibiting cell cycle-dependent kinases and delaying phosphorylation of retinoblastoma protein (Marx, S.O., T. Jayaraman, L.O. Go, and A.R. Marks. 1995. Circ. Res. 362:801). In the present study the effects of rapamycin on SMC migration were assayed in vitro using a modified Boyden chamber and in vivo using a porcine aortic SMC explant model. Pretreatment with rapamycin (2 ng/ml) for 48 h inhibited PDGF-induced migration (PDGF BB homodimer; 20 ng/ml) in cultured rat and human SMC (n = 10; P < 0.0001), whereas FK506 had no significant effect on migration. Rapamycin administered orally (1 mg/kg per d for 7 d) significantly inhibited porcine aortic SMC migration compared with control (n = 15; P < 0.0001). Thus, in addition to being a potent immunosuppressant and antiproliferative, rapamycin also inhibits SMC migration.
M Poon, S O Marx, R Gallo, J J Badimon, M B Taubman, A R Marks
Substance P (SP), neurokinin A (NKA), and calcitonin gene-related peptide (CGRP) have potent proinflammatory effects in the airways. They are released from sensory nerve endings originating in jugular and dorsal root ganglia. However, the major sensory supply to the airways originates from the nodose ganglion. In this study, we evaluated changes in neuropeptide biosynthesis in the sensory airway innervation of ovalbumin-sensitized and -challenged guinea pigs at the mRNA and peptide level. In the airways, a three- to fourfold increase of SP, NKA, and CGRP, was seen 24 h following allergen challenge. Whereas no evidence of local tachykinin biosynthesis was found 12 h after challenge, increased levels of preprotachykinin (PPT)-A mRNA (encoding SP and NKA) were found in nodose ganglia. Quantitative in situ hybridization indicated that this increase could be accounted for by de novo induction of PPT-A mRNA in nodose ganglion neurons. Quantitative immunohistochemistry showed that 24 h after challenge, the number of tachykinin-immunoreactive nodose ganglion neurons had increased by 25%. Their projection to the airways was shown. Changes in other sensory ganglia innervating the airways were not evident. These findings suggest that an induction of sensory neuropeptides in nodose ganglion neurons is crucially involved in the increase of airway hyperreactivity in the late response to allergen challenge.
A Fischer, G P McGregor, A Saria, B Philippin, W Kummer
Proteolytic degradation of aggrecan is a hallmark of the pathology of arthritis, yet the identity of the enzyme(s) in cartilage responsible for this degradation is unknown. Previous studies have suggested that the matrix metalloproteinases (MMPs) may be involved but there has been no definitive evidence for their direct action in the proteolysis of aggrecan in human arthritis. We now show unequivocally that aggrecan fragments derived from the specific action of MMPs can be detected in synovial fluids from patients with both inflammatory and noninflammatory arthritis, with a neoepitope monoclonal antibody AF-28 that detects the NH2-terminal sequence F342FGVG.... The synovial fluid MMP fragments were of low buoyant density and distributed exclusively at the top of cesium chloride density gradients, suggesting that these fragments lacked chondroitin sulfate chains. AF-28 immunoblotting of synovial fluid aggrecan fragments revealed a population of small AF-28 fragments of 30-50 kD. Based on their size relative to characterized products of an MMP-8 digest (Fosang, A.J., K. Last, P. Gardiner, D.C. Jackson, and L. Brown. 1995, Biochem. J. 310:337-343), these AF-28 fragments were derived from proteinase cleavage at, or near, the ...ITEGE373 / ARGSV... aggrecanase site. Immunodetection with polyclonal anti-ITEGE antiserum revealed that these fragments lacked the ...ITEGE374 COOH terminus and were not therefore products of aggrecanase action. The same fluid samples contained a broad 68-90-kD G1 fragment that contained the COOH-terminal ...ITEGE374 neoepitope. The results suggest that in some circumstances, despite extensive proteolysis of the core protein, aggrecan molecules may be cleaved by MMPs or aggrecanase in the interglobular domain, but not both.
A J Fosang, K Last, R A Maciewicz
We studied a patient with a severe spherocytic hemolytic anemia without family history of spherocytosis. Analysis of patient's erythrocyte membrane proteins revealed spectrin deficiency and a truncated alpha spectrin protein. We determined that the patient is a compound heterozygote with two mutations in alpha spectrin gene. Mutation in the paternal allele, designated alpha spectrin(PRAGUE), is a transition A to G in the penultimate position of intron 36 that leads to skipping of exon 37, frameshift, and production of the truncated alpha spectrin protein. The maternal allele, designated alpha spectrin(LEPRA), contains transition C-->T in position -99 of intron 30. This mutation enhances an alternative acceptor splice site 70 nucleotides upstream from the regular site. The alternative splicing causes a frameshift and premature termination of translation leading to a significant decrease in alpha spectrin production. The alpha(LEPRA) mutation is linked to a spectrin alphaIIa marker that was found to be associated with recessive or nondominant spectrin-deficient hereditary spherocytosis in approximately 50% of studied families. We conclude that the alpha(LEPRA) mutation combined in trans with the alpha(PRAGUE) mutation underlie the severe hemolytic anemia in the proband. We suggest that allele alpha spectrin(LEPRA) may be frequently involved in pathogenesis of recessive or nondominant spectrin-deficient hereditary spherocytosis.
H Wichterle, M Hanspal, J Palek, P Jarolim
Group B Streptococcus (GBS) is an important perinatal pathogen. Because transplacentally acquired maternal antibodies to the GBS capsular polysaccharides (CPS) confer protection, prevention of infant disease may be possible after immunization of women. Unfortunately, the purified CPS of GBS are only variably immunogenic in adults; therefore to enhance immunogenicity we have designed and developed a CPS-protein conjugate vaccine. The lability of a conformationally dependent epitope on the III CPS containing a critical sialic acid residue was important to consider in vaccine design. 100 women were randomized to receive GBS type III CPS-tetanus toxoid conjugate (III-TT) vaccine at one of three doses; unconjugated GBS type III CPS; or saline. Serum samples were obtained before immunization and 2, 4, 8, and 26 wk thereafter, and specific antibody to type III CPS was measured. Vaccines were well tolerated. In sera from recipients of the highest dose of III-TT, CPS-specific IgG levels rose from a geometric mean of 0.09 microg/ml before immunization to 4.53 microg/ml 8 wk later, whereas levels in recipients of unconjugated type III CPS rose from 0.21 microg/ml to 1.41 microg/ml. Lower doses resulted in lower antibody levels. A > or = 4-fold rise in antibody concentration was achieved in 90% of recipients of III-TT compared with 50% of those that received III CPS (P = 0.0015). Antibodies evoked by the conjugate vaccine recognized a conformationally dependent epitope of the III-CPS, promoted opsonophagocytosis and killing of GBS, and, after maternal immunization, protected neonatal mice from lethal challenge with type III GBS. We conclude that directed coupling of type III GBS polysaccharide to a carrier protein yielded a conjugate vaccine with preserved expression of a highly labile conformational epitope involving sialic acid and enhanced immunogenicity compared with uncoupled CPS.
D L Kasper, L C Paoletti, M R Wessels, H K Guttormsen, V J Carey, H J Jennings, C J Baker
The negative correlation between coronary heart disease and plasma levels of HDL has been attributed to the ability of HDL to take up cellular cholesterol. The HDL3-induced removal of cellular cholesterol was reported to be impaired in fibroblasts from patients with familial HDL deficiency (Tangier disease, TD). In addition, we have recently shown that HDL3 stimulates the hydrolysis of phosphatidylcholine (PC) in cholesterol-loaded fibroblasts. To investigate whether this cell signaling pathway is involved in cholesterol efflux mechanisms, we compared the HDL3-induced PC hydrolysis in normal fibroblasts and in fibroblasts from a TD kindred, in whom the HDL3- and apolipoprotein A-I (apo A-I)-induced mobilization of cellular cholesterol was found to be reduced by 50%. The HDL3-induced formation of phosphatidic acid (PA) via PC-specific phospholipase D (PC-PLD) was markedly reduced by 60-80% in these cells, whereas the formation of diacylglycerol (DG) via PC-specific phospholipase C (PC-PLC) was two- to threefold enhanced. Defective regulation of PC-PLC and PC-PLD was similarly observed in response to apo A-I and endothelin, but not in response to the receptor-independent stimulation of PC hydrolysis by PMA. A Tangier-like PA and DG formation pattern could be induced in normal cells after preincubation with pertussis toxin, suggesting the involvement of a G-protein. The impaired mobilization of radiolabeled cellular cholesterol in TD cells could completely be overcome by increasing the PA levels in the presence of the PA phosphohydrolase inhibitor propranolol. Conversely, the inhibition of PA formation in the presence of 0.3% butanol as well as the inhibition of DG formation in the presence of the PC-PLC inhibitor D 609 reduced the mobilization of cellular cholesterol both in normal and in TD cells. Our data indicate that the coordinate formation of PA and DG via PC-PLD and PC-PLC is essential for efficient cholesterol efflux. The molecular defect in this TD kindred appears to affect an upstream effector of protein kinase C responsible for the G-protein-dependent regulation of PC-specific phospholipases.
M Walter, H Reinecke, U Gerdes, J R Nofer, G Höbbel, U Seedorf, G Assmann
Two forms of intercalated cells are present in kidney collecting tubules, the alpha cell has apical endocytosis, apical H+-ATPase and basolateral band 3, while beta cells have reversed polarity of these proteins and no apical endocytosis. When a beta cell line was seeded at high density, it changed into the alpha form. We previously showed that a partially purified 230 kD extracellular matrix protein of high density cells was able to retarget band 3 from apical to basolateral domains and stimulated apical endocytosis in vitro (Van Adelsberg, J., J.C. Edwards, J. Takito, B. Kiss, and Q. Al-Awqati. 1994. Cell. 76:1053-1061). We now purify this protein, which was named hensin, to near homogeneity and find that it belongs to the macrophage scavenger receptor cysteine rich (SRCR) family. An antibody, generated against a fusion protein made from a partial cDNA recognized a 230-kD protein in rabbit kidney and in the intercalated cell line. In vitro, the hensin antibody inhibited expression of apical endocytosis. Hensin was secreted in a polarized manner and bound to the basolateral membrane and extracellular matrix. Immunohistochemistry of the kidney showed that it was expressed only in collecting tubules. Double immunofluorescence with hensin and peanut lectin, H+-ATPase, or band 3 showed many patterns; most alpha-cells had hensin staining while 50% of beta-cells did not. These results suggest that hensin may also be involved in the polarity reversal of intercalated cells in vivo.
J Takito, C Hikita, Q Al-Awqati
Eosinophil accumulation is a distinctive feature of lung allergic inflammation. Here, we have used a mouse model of OVA (ovalbumin)-induced pulmonary eosinophilia to study the cellular and molecular mechanisms for this selective recruitment of eosinophils to the airways. In this model there was an early accumulation of infiltrating monocytes/macrophages in the lung during the OVA treatment, whereas the increase in infiltrating T-lymphocytes paralleled the accumulation of eosinophils. The kinetics of accumulation of these three leukocyte subtypes correlated with the levels of mRNA expression of the chemokines monocyte chemotactic peptide-1/JE, eotaxin, and RANTES (regulated upon activation in normal T cells expressed and secreted), suggesting their involvement in the recruitment of these leukocytes. Furthermore, blockade of eotaxin with specific antibodies in vivo reduced the accumulation of eosinophils in the lung in response to OVA by half. Mature CD4+ T-lymphocytes were absolutely required for OVA-induced eosinophil accumulation since lung eosinophilia was prevented in CD4+-deficient mice. However, these cells were neither the main producers of the major eosinophilic chemokines eotaxin, RANTES, or MIP-1alpha, nor did they regulate the expression of these chemokines. Rather, the presence of CD4+ T cells was necessary for enhancement of VCAM-1 (vascular cell adhesion molecule-1) expression in the lung during allergic inflammation induced by the OVA treatment. In support of this, mice genetically deficient for VCAM-1 and intercellular adhesion molecule-1 failed to develop pulmonary eosinophilia. Selective eosinophilic recruitment during lung allergic inflammation results from a sequential accumulation of certain leukocyte types, particularly T cells, and relies on the presence of both eosinophilic chemoattractants and adhesion receptors.
J A Gonzalo, C M Lloyd, L Kremer, E Finger, C Martinez-A, M H Siegelman, M Cybulsky, J C Gutierrez-Ramos
Insulin resistance is a predictor of the development of noninsulin-dependent diabetes mellitus (NIDDM) in humans. It is unclear whether insulin resistance is a primary defect leading to NIDDM or the result of hyperinsulinemia and hyperglycemia. To determine if insulin resistance is the result of extrinsic factors such as hyperinsulinemia primary skeletal muscle cell cultures were established from muscle biopsies from Pima Indians with differing in vivo insulin sensitivities. These cell cultures expressed a variety of muscle-specific phenotypes including the proteins alpha-actinin and myosin, muscle-specific creatine kinase activity, and RNA encoding GLUT4, MYF5, MYOD1, and MYOGENIN. Labeled glucose was used to measure the insulin-stimulated conversion of glucose to glycogen in these cultures. The in vivo rates of insulin-stimulated glycogen production (insulin resistance) were correlated with in vitro measures of glycogen production (P = 0.007, r = 0.58). This defect in insulin action is stable in a uniform culture environment and is retained over time. The retention of insulin resistance in myoblast derived cell cultures is consistent with the expression of an underlying biochemical defect in insulin resistant skeletal muscle.
D B Thompson, R Pratley, V Ossowski
We used a model whereby mechanical stimulation induces bone formation in rat caudal vertebrae, to test the effect of estrogen on this osteogenic response. Unexpectedly, estrogen administered daily throughout the experiments (8-11 d) suppressed, and ovariectomy enhanced, mechanically induced osteogenesis. Osteogenesis was unaffected by the resorption-inhibitor pamidronate, suggesting that the suppression of bone formation caused by estrogen was not due to suppression of resorption. We found that estrogen did not significantly reduce the proportion of osteocytes that were induced by mechanical stimulation to express c-fos and IGF-I mRNA; and estrogen suppressed mechanically induced osteogenesis whether administration was started 24 h before or 24 h after loading. This suggests that estrogen acts primarily not on the strain-sensing mechanism itself, but on the osteogenic response to signals generated by strain-sensitive cells. We also found that when estrogen administration was started 3 d after mechanical stimulation, by which time osteogenesis is established, estrogen augmented the osteogenic response. This data is consistent with in vitro evidence for estrogen responsiveness in two phenotypically distinct bone cell types: stromal cells, whose functional activities are suppressed, and osteoblasts, which are stimulated, by estrogen.
C J Jagger, J W Chow, T J Chambers
Arthrogryposis multiplex congenita (AMC), characterized by multiple joint contractures developing in utero, results from lack of fetal movement. Some cases are genetically determined, but AMC occasionally complicates pregnancy in patients with myasthenia gravis (MG) suggesting involvement of circulating maternal antibodies. We previously demonstrated antibodies that inhibited the function of fetal acetylcholine receptor (AChR) in one healthy woman with an obstetric history of recurrent AMC. Here we study sera from this woman, from one other with a similar history, and from three (one asymptomatic) whose babies had neonatal MG and AMC. All five maternal sera had high titers of antibodies that inhibited alpha-Bungarotoxin (alpha-BuTx) binding to fetal AChR, and their sera markedly inhibited fetal AChR function with little effect on adult AChR function. Moreover, in a further survey, 3 of 20 sera from anti-AChR negative AMC mothers inhibited fetal AChR function significantly at 1:100 dilution. These results demonstrate the role of antibodies to fetal AChR and perhaps other muscle antigens in some cases of AMC. More generally, they suggest that placental transfer of antibodies directed at fetal antigens should be considered as a cause of other recurrent fetal or perinatal disorders.
S Riemersma, A Vincent, D Beeson, C Newland, S Hawke, B Vernet-der Garabedian, B Eymard, J Newsom-Davis
Endothelin-1 (ET-1) has been implicated in the regulation of vascular tone in various pathological conditions. To examine the effect of in vivo overexpression of the peptide in rats, we prepared recombinant adenovirus stocks encoding the human preproET-1 cDNA (Ad.ET-1) or Escherichia coli lacZ (Ad.betaGal), each driven by cytomegalovirus early promoter. Ad.ET-1 or Ad.betaGal was injected into the caudal vein of rats and the animals were studied under anesthesia 96 h later. Hepatic overexpression of the virus-derived human ET-1 mRNA was accompanied by a 13-fold elevation of liver ET-1 content in the Ad.ET-1 group. Circulating plasma ET-1 levels in the Ad.ET-1 group were sixfold higher than those in the Ad.betaGal group. Mean arterial blood pressure was increased by 28 mmHg in the Ad.ET-1 group as compared with the Ad.betaGal group. In the Ad.ET-1 group, intravenous infusion of the ET(A) receptor antagonist FR 139317 reduced the blood pressure to levels seen in the Ad.betaGal group, whereas the same antagonist did not significantly alter the blood pressure in the Ad.betaGal group. Intravenous infusion of the ET(B) receptor antagonist BQ-788 caused a small but significant increase in blood pressure in both groups. These findings demonstrate that endogenous overexpression of preproET-1, accompanied by an elevation of plasma ET-1 concentrations to the levels seen in pathophysiological states, can cause systemic hypertension through the activation of the ETA receptor.
V Niranjan, S Télémaque, D deWit, R D Gerard, M Yanagisawa
Human plasma phospholipid transfer protein (PLTP) circulates bound to high density lipoprotein (HDL) and mediates both net transfer and exchange of phospholipids between different lipoproteins. However, its overall function in lipoprotein metabolism is unknown. To assess the effects of increased plasma levels of PLTP, human PLTP transgenic mice were established using the human PLTP gene driven by its natural promoter. One line of PLTP transgenic mice with moderate expression of PLTP mRNA and protein was obtained. The order of human PLTP mRNA expression in tissues was: liver, kidney, brain, small intestine > lung > spleen > heart, adipose tissue. Western blotting using a human PLTP monoclonal antibody revealed authentic human PLTP (Mr 80 kD) in plasma. Plasma PLTP activity was increased by 29% in PLTP transgenic mice. However, plasma lipoprotein analysis, comparing PLTP transgenic mice to control littermates, revealed no significant changes in the plasma lipoprotein lipids or apolipoproteins. Since previous studies have shown that human cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase only function optimally in human apoAI transgenic mice, the human PLTP transgenic mice were cross-bred with human apoAI transgenic mice. In the human apoAI transgenic background, PLTP expression resulted in increased PLTP activity (47%), HDL phospholipid (26%), cholesteryl ester (24%), free cholesterol (37%), and apoAI (22%). There was a major increase of apoAI in prebeta-HDL (56%) and a small increase in alpha-HDL (14%). The size distribution of HDL particles within alpha- and prebeta-migrating species was not changed. The results suggest that PLTP increases the influx of phospholipid and secondarily cholesterol into HDL, leading to an increase in potentially antiatherogenic prebeta-HDL particles.
X Jiang, O L Francone, C Bruce, R Milne, J Mar, A Walsh, J L Breslow, A R Tall
Chronic metabolic acidosis increases proximal tubular citrate uptake and metabolism. The present study addressed the effect of chronic metabolic acidosis on a cytosolic enzyme of citrate metabolism, ATP citrate lyase. Chronic metabolic acidosis caused hypocitraturia in rats and increased renal cortical ATP citrate lyase activity by 67% after 7 d. Renal cortical ATP citrate lyase protein abundance increased by 29% after 3 d and by 141% after 7 d of acid diet. No significant change in mRNA abundance could be detected. Hypokalemia, which causes only intracellular acidosis, caused hypocitraturia and increased renal cortical ATP citrate lyase activity by 28%. Conversely, the hypercitraturia of chronic alkali feeding was associated with no change in ATP citrate lyase activity. Inhibition of ATP citrate lyase with the competitive inhibitor, 4S-hydroxycitrate, significantly abated hypocitraturia and increased urinary citrate excretion fourfold in chronic metabolic acidosis and threefold in K+-depletion. In summary, the hypocitraturia of chronic metabolic acidosis is associated with an increase in ATP citrate lyase activity and protein abundance, and is partly reversed by inhibition of this enzyme. These results suggest an important role for ATP citrate lyase in proximal tubular citrate metabolism.
J Z Melnick, P A Srere, N A Elshourbagy, O W Moe, P A Preisig, R J Alpern
Although a linkage between aerobic glycolysis and sodium-potassium transport has been demonstrated in diaphragm, vascular smooth muscle, and other cells, it is not known whether this linkage occurs in skeletal muscle generally. Metabolism of intact hind-leg muscles from young rats was studied in vitro under aerobic incubation conditions. When sodium influx into rat extensor digitorum longus (EDL) and soleus muscles was facilitated by the sodium ionophore monensin, muscle weight gain and production of lactate and alanine were markedly stimulated in a dose-dependent manner. Although lactate production rose in both muscles, it was more pronounced in EDL than in soleus. Monensin-induced lactate production was inhibited by ouabain or by incubation in sodium-free medium. Preincubation in potassium-free medium followed by potassium re-addition also stimulated ouabain-inhibitable lactate release. Replacement of glucose in the incubation medium with pyruvate abolished monensin-induced lactate production but exacerbated monensin-induced weight gain. Muscles from septic or endotoxin-treated rats exhibited an increased rate of lactate production in vitro that was partially inhibited by ouabain. Increases muscle lactate production in sepsis may reflect linked increases in activity of the Na+, K+-ATPase, consumption of ATP and stimulation of aerobic glycolysis.
J H James, C H Fang, S J Schrantz, P O Hasselgren, R J Paul, J E Fischer
Fructose, a naturally occurring monosaccharide, is increasingly used as an added sweetener in processed foods in the form of high fructose corn syrup. Increased fructose intake combined with the identification of children with clinical evidence of isolated fructose malabsorption (IFM) has stimulated interest in possible disorders of fructose absorption. The intestinal absorption of fructose is carried out by the facilitative hexose transporter, which has been designated as GLUT5. Functional properties and tissue distribution of GLUT5 suggest that IFM might be due to mutations in the GLUT5 gene. To test this hypothesis, we screened the GLUT5 gene for mutations in a group of eight patients with IFM and in one subject with global malabsorption, as compared with 15 healthy parents of subjects and up to 6 unrelated controls. No mutations were found in the protein coding region of this gene in any of the subjects. A single G to A substitution in the 5' untranslated region of exon 1 was identified in the subject with global malabsorption. This subject and her healthy mother were heterozygous for the variant sequence, suggesting that it was unlikely to be clinically significant. In addition, sequence analysis of each of the 12 GLUT5 exons was performed in the index case and confirmed the negative single-strand conformation polymorphism findings. These studies demonstrate that IFM does not result from the expression of mutant GLUT5 protein.
D Wasserman, J H Hoekstra, V Tolia, C J Taylor, B S Kirschner, J Takeda, G I Bell, R Taub, E B Rand
Hyaluronan (HA) is a glycosaminoglycan constituent of extracellular matrix. In its native form HA exists as a high molecular weight polymer, but during inflammation lower molecular weight fragments accumulate. We have identified a collection of inflammatory genes induced in macrophages by HA fragments but not by high molecular weight HA. These include several members of the chemokine gene family: macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, cytokine responsive gene-2, monocyte chemoattractant protein-1, and regulated on activation, normal T cell expressed and secreted. HA fragments as small as hexamers are capable of inducing expression of these genes in a mouse alveolar macrophage cell line, and monoclonal antibody to the HA receptor CD44 completely blocks binding of fluorescein-labeled HA to these cells and significantly inhibits HA-induced gene expression. We also investigated the ability of HA fragments to induce chemokine gene expression in human alveolar macrophages from patients with idiopathic pulmonary fibrosis and found that interleukin-8 mRNA is markedly induced. These data support the hypothesis that HA fragments generated during inflammation induce the expression of macrophage genes which are important in the development and maintenance of the inflammatory response.
C M McKee, M B Penno, M Cowman, M D Burdick, R M Strieter, C Bao, P W Noble
Apolipoprotein(a) [apo(a)] contains multiple kringle 4 repeats and circulates as part of lipoprotein(a) [Lp(a)]. Apo(a) is synthesized by the liver but its clearance mechanism is unknown. Previously, we showed that kringle 4-containing fragments of apo(a) are present in human urine. To probe their origin, human plasma was examined and a series of apo(a) immunoreactive peptides larger in size than urinary fragments was identified. The concentration of apo(a) fragments in plasma was directly related to the plasma level of Lp(a) and the 24-h urinary excretion of apo(a). Individuals with low (< 2 mg/dl) plasma levels of Lp(a) had proportionally more apo(a) circulating as fragments in their plasma. Similar apo(a) fragments were identified in baboon plasma but not in conditioned media from primary cultures of baboon hepatocytes, suggesting that the apo(a) fragments are generated from circulating apo(a) or Lp(a). When apo(a) fragments purified from human plasma were injected intravenously into mice, a species that does not produce apo(a), apo(a) fragments similar to those found in human urine were readily detected in mouse urine. Thus, we propose that apo(a) fragments in human plasma are derived from circulating apo(a)/Lp(a) and are the source of urinary apo(a).
V Mooser, S M Marcovina, A L White, H H Hobbs
Previous studies suggested that tyrosine kinase activation is an important signal transduction event in the IL-1 response of chondrocytes. The present study identifies the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK)-1 and ERK-2 as major tyrosine phosphorylated proteins in IL-1 stimulated chondrocytes. Kinase assays on immunoprecipitates with myelin basic protein as substrate showed that ERK-1 and ERK-2 activation was detectable within 5 min after IL-1 stimulation and decreased to baseline within 60 min. Analysis of other members of the MAP kinase family showed that chondrocytes also express c-Jun NH2 terminal kinase (JNK)-1, JNK-2, and p38 proteins. These kinases were time-dependently activated by IL-1. Among other chondrocyte activators tested, only TNF activated all three of the MAP kinase subgroups. JNK and p38 were not activated by any of the other cytokines and growth factors tested. However, ERK was also activated by PDGF, IGF-1, and IL-6. Phorbol 12-myristate 13-acetate, calcium ionophore, and cAMP analogues only increased ERK activity but had no significant effects on JNK or p38. These results suggest differential activation of MAP kinase subgroups by extracellular stimuli. ERK is activated in response to qualitatively diverse extracellular stimuli and various second messenger agonists. In contrast, JNK and p38 are only activated by IL-1 or TNF, suggesting that these kinases participate in the induction of the catabolic program in cartilage.
Y Geng, J Valbracht, M Lotz
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