The human paraoxonase gene (HUMPONA) is codominantly expressed as alleles A and G. The A allele codes for glutamine (A genotype) and the G allele for arginine (B genotype) at position 191 of the paraoxonase enzyme. This genetic polymorphism has been suggested to be associated with the predisposition to coronary artery disease (CAD). We investigated the frequency of paraoxonase A and G alleles in 380 well-characterized CAD patients and in 169 controls. The most common genotype in both the patients with CAD (211/380) and in healthy Finnish individuals (87/169) was AA (Gln/Gln). The heterozygous AM (Gln/Arg) genotype was present in 140 of the patients and in 75 controls. The frequency of the A allele was 0.74 in both patients and controls. The genotype distribution between the two groups did not differ (P = 0.12, chi2 test). The genotype distributions were also similar to those reported earlier in other caucasoid populations. In conclusion, we found no association between the Gln-Arg 191 polymorphism of the human paraoxonase gene and coronary artery disease in Finns.
M Antikainen, S Murtomäki, M Syvänne, R Pahlman, E Tahvanainen, M Jauhiainen, M H Frick, C Ehnholm
Endothelial cell proliferation is inhibited by the establishment of cell to cell contacts. Adhesive molecules at junctions could therefore play a role in transferring negative growth signals. The transmembrane protein VE-cadherin (vascular endothelial cadherin/cadherin-S) is selectively expressed at intercellular clefts in the endothelium. The intracellular domain interacts with cytoplasmic proteins called catenins that transmit the adhesion signal and contribute to the anchorage of the protein to the actin cytoskeleton. Transfection of VE-cadherin in both Chinese hamster ovary (CHO) and L929 cells confers inhibition of cell growth. Truncation of VE-cadherin cytoplasmic region, responsible for linking catenins, does not affect VE-cadherin adhesive properties but abolishes its effect on cell growth. Seeding human umbilical vein endothelial cells or VE-cadherin transfectants on a recombinant VE-cadherin amino-terminal fragment inhibited their proliferation. These data show that VE-cadherin homotypic engagement at junctions participates in density dependent inhibition of cell growth. This effect requires both the extracellular adhesive domain and the intracellular catenin binding region of the molecule.
L Caveda, I Martin-Padura, P Navarro, F Breviario, M Corada, D Gulino, M G Lampugnani, E Dejana
Hypertension is associated with insulin-resistant states such as diabetes and obesity. Nitric oxide (NO) contributes to regulation of blood pressure. To gain insight into potential mechanisms linking hypertension with insulin resistance we directly measured and characterized NO production from human umbilical vein endothelial cells (HUVEC) in response to insulin using an amperometric NO-selective electrode. Insulin stimulation of HUVEC resulted in rapid, dose-dependent production of NO with a maximal response of approximately 100 nM NO (200,000 cells in 2 ml media; ED50 approximately 500 nM insulin). Although HUVEC have many more IGF-1 receptors than insulin receptors (approximately 400,000, and approximately 40,000 per cell respectively), a maximally stimulating dose of IGF-1 generated a smaller response than insulin (40 nM NO; ED50 approximately 100 nM IGF-1). Stimulation of HUVEC with PDGF did not result in measurable NO production. The effects of insulin and IGF-1 were completely blocked by inhibitors of either tyrosine kinase (genestein) or nitric oxide synthase (L-NAME). Wortmannin (an inhibitor of phosphatidylinositol 3-kinase [PI 3-kinase]) inhibited insulin-stimulated production of NO by approximately 50%. Since PI 3-kinase activity is required for insulin-stimulated glucose transport, our data suggest that NO is a novel effector of insulin signaling pathways that are also involved with glucose metabolism.
G Zeng, M J Quon
Aging is an important determinant of vascular disease. Endothelium-derived nitric oxide (NO) is protective as a vasodilator and inhibitor of platelet function. This study was designed to directly measure effects of prolonged aging on endotheliai NO release in isolated blood vessels and to delineate differences between the systemic and pulmonary circulation. Aortas and pulmonary arteries from 5-6-mo-old (young), 18-19-mo-old (middle-aged), and 32-33-mo-old (old) normotensive female rats were used. Blood pressure and plasma estradiol-17beta (E2) remained unchanged. In isolated blood vessels, NO release was induced by the receptor-independent agonist calcium ionophore A23187 (10 micromol/liter) and measured in situ on the endothelial surface of vessels using a porphyrinic microsensor. In vessels suspended in organ chambers isometric tension was recorded. In the aorta, the initial rate of NO release and peak NO concentration were reduced in middle-aged and old rats (P < 0.0006 vs. young rats, n = 6). Furthermore, endothelium-dependent relaxations to calcium ionophore and acetylcholine (both 10(-10) - 10(-5) mol/liter) were also reduced in aortas from old as compared with young rats (n = 6, P < 0.05). The initial rate of NO release and peak NO concentration significantly correlated with maximal relaxation to calcium ionophore A23187 (correlation coefficients r - 0.916, P < 0.0018 and r = 0.961, P < 0.0001, respectively, n = 7). In pulmonary arteries, however, the initial rate of NO release as well as peak NO concentration did not decrease with age (n = 6 for each age group, NS). In both blood vessels, the NO release was unaffected by superoxide dismutase in all age groups (n = 6, NS). Thus, aging specifically reduces initial rate and peak concentrations of endothelial NO release from aorta but not pulmonary artery indicating reduced NO production. As arterial pressure did not change with aging, the chronic exposure of the aorta to higher pressure and/or pulsatility than in the pulmonary artery may be the cause. This appears important as NO plays a protective role by preventing vasoconstriction, thrombosis and atherosclerosis.
M R Tschudi, M Barton, N A Bersinger, P Moreau, F Cosentino, G Noll, T Malinski, T F Lüscher
To evaluate the possible role of a putative vesicle-targeting protein, syntaxin-4, in vasopressin-regulated trafficking of aquaporin-2 water channel vesicles to the apical plasma membrane of renal collecting duct cells, we have carried out immunoblotting, immunocytochemistry, and reverse transcription (RT)-PCR experiments in rat kidney. Immunochemical studies used an affinity-purified, peptide-directed polyclonal antibody to rat syntaxin-4. Immunoblots using membrane fractions from inner medullary collecting duct (IMCD) cell suspensions revealed a solitary protein of 36 kD, the expected molecular mass of syntaxin-4. This protein was enriched in a plasma membrane-enriched membrane fraction from IMCD cells. Immunoperoxidase immunocytochemistry in 0.85-microm cryosections from rat inner medulla revealed discrete labeling of the apical plasma membrane of IMCD cells. RT-PCR demonstrated the presence of syntaxin-4 mRNA in microdissected IMCD segments, confirmed by direct sequencing of the PCR product. In addition, RT-PCR experiments demonstrated syntaxin-4 mRNA in glomeruli, vasa recta, connecting tubules, and thin descending limbs of Henle's loops. The demonstrated localization of syntaxin-4 in the apical plasma membrane of collecting duct principal cells, coupled with previous demonstration of syntaxin-4's putative cognate receptor VAMP2 in aquaporin-2-containing vesicles, supports the view that these proteins could play a role of aquaporin-2 vesicle targeting to the apical plasma membrane.
B Mandon, C L Chou, S Nielsen, M A Knepper
The A/Japan/57 influenza hemagglutinin (HA) was expressed in BALB/c mice under the transcriptional control of the surfactant protein C (SP-C) promoter, resulting in expression of HA in type II alveolar epithelial cells, as well as low level variable expression in other tissues, including the thymus in some of the founder lines. Transgenic animals were able to recover from infection with A/Japan/57 influenza, and they were able to mount antibody responses to A/Japan/57 HA in titers similar to wild type. We therefore tested their CD4+ T lymphocyte responses to HA and found them to be similar to wild type responses. However, CD8+ T cells from A/Japan/57-infected transgenic animals were unable to express cytolytic activity against target cells expressing the A/Japan/57 HA. The CD8+ T cell tolerance was also extremely specific, since transgenics immunized with an influenza strain containing a single amino acid substitution in a dominant HA epitope were able to mount full cytolytic responses to that epitope, but not the wild-type epitope. Adoptive transfer of CD8+ T cell clones into transgenic animals resulted extensive interstitial pneumonitis that was antigen-specific and associated with significant morbidity and mortality. We conclude that a lung-specific transgene may lead to specific CD8+ T cell tolerance, with CD4+ T cell and B cell reactivity to the antigen, and that CD4+ T cell reactivity may remain intact to an antigen expressed in the thymus, even when CD8+ T cell tolerance exists. This observation may have profound implications concerning immune-mediated lung diseases, particularly those mediated by CD4+ T cells.
R I Enelow, M H Stoler, A Srikiatkhachorn, C Kerlakian, S Agersborg, J A Whitsett, T J Braciale
Several clinical studies have suggested that excess hepatic iron accumulation is a progressive factor in some liver diseases including chronic viral hepatitis and hemochromatosis. However, it is not known whether iron-induced hepatotoxicity may be directly involved in hepatitis, cirrhosis, and liver cancer. The Long-Evans Cinnamon (LEC) rat, which accumulates excess copper in the liver as in patients with Wilson's disease, is of a mutant strain displaying spontaneous hemolysis, hepatitis, and liver cancer. We found previously that LEC rats harbored an additional abnormality: accumulation of as much iron as copper in the liver. In the present study, we compared the occurrence of hepatitis and liver cancer in LEC rats fed an iron-deficient diet (ID) with those in rats fed a regular diet (RD). The RD group showed rapid increments of hepatic iron concentrations as the result of hemolysis, characteristics of fulminant hepatitis showing apoptosis, and a 53% mortality rate. However, no rats in the ID group died of fulminant hepatitis. Hepatic iron, especially "free" iron concentration and the extent of hepatic fibrosis in the ID group were far less than those of the RD group. At week 65, all rats in the RD group developed liver cancer, whereas none did in the ID group. These results suggest that the accumulation of iron, possibly by virtue of synergistic radical formation with copper, plays an essential role in the development of fulminant hepatitis, hepatic fibrosis, and subsequent hepatocarcinogenesis in LEC rats.
J Kato, M Kobune, Y Kohgo, N Sugawara, H Hisai, T Nakamura, S Sakamaki, N Sawada, Y Niitsu
The hexosamine biosynthetic pathway has been hypothesized to be involved in mediating some of the toxic effects of hyperglycemia. Glutamine:fructose-6-phosphate amidotransferase (GFA), the first and rate limiting enzyme of the hexosamine biosynthetic pathway, was overexpressed in skeletal muscle and adipose tissue of transgenic mice. A 2.4-fold increase of GFA activity in muscle of the transgenic mice led to weight-dependent hyperinsulinemia in random-fed mice. The hyperinsulinemic-euglycemic clamp technique confirmed that transgenic mice develop insulin resistance, with a glucose disposal rate of 68.5 +/- 3.5 compared with 129.4 +/- 9.4 mg/kg per min (P < 0.001) for littermate controls. The decrease in the glucose disposal rate of the transgenic mice is accompanied by decreased protein but not mRNA levels of the insulin-stimulated glucose transporter (GLUT4). These data support the hypothesis that excessive flux through the hexosamine biosynthesis pathway mediates adverse regulatory and metabolic effects of hyperglycemia, specifically insulin resistance of glucose disposal. These mice can serve as a model system to study the mechanism for the regulation of glucose homeostasis by hexosamines.
L F Hebert Jr, M C Daniels, J Zhou, E D Crook, R L Turner, S T Simmons, J L Neidigh, J S Zhu, A D Baron, D A McClain
1-O-Octadecyl-2-O-methyl-glycerophosphocholine (ET18-OCH3) is an ether lipid with selective antiproliferative properties whose mechanism of action is still unresolved. We hypothesized that since ET18-OCH3 affects a wide variety of cells, its mechanism of action was likely to involve the inhibition of a common widely used pathway for transducing growth signals such as the mitogen-activated protein kinase (MAPK) cascade. To test this, we established conditions whereby quiescent MCF-7 cells took up ET18-OCH3 in sufficient quantities that inhibited cell proliferation subsequent to the addition of growth medium and examined the activation of components of the MAPK cascade under these conditions. ET18-OCH3 inhibited the sustained phosphorylation of MAPK resulting in a decrease in the magnitude and duration of activation of MAPK in cells stimulated with serum or EGF. ET18-OCH3 had no effect on the binding of EGF to its receptors, their activation, or p21ras activation. However, an interference in the association of Raf-1 with membranes and a resultant decrease in Raf-1 kinase activity in membranes of ET18-OCH3-treated cells was observed. ET18-OCH3 had no direct effect on MAPK or Raf-1 kinase activity. A direct correlation between ET18-OCH3 accumulation, inhibition of cell proliferation, Raf association with the membrane, and MAPK activation was also established. These results suggest that inhibition of the MAPK cascade by ET18-OCH3 as a result of its effect on Raf-1 activation may be an important mechanism by which ET18-OCH3 inhibits cell proliferation.
X Zhou, X Lu, C Richard, W Xiong, D W Litchfield, R Bittman, G Arthur
Genetic and environmental factors are important in the pathogenesis of clinical and experimental chronic intestinal inflammation. We investigated the influence of normal luminal bacteria and several groups of selected bacterial strains on spontaneous gastrointestinal and systemic inflammation in HLA-B27 transgenic rats. Rats maintained germfree for 3-9 mo were compared with littermates conventionalized with specific pathogen-free bacteria. Subsequently, germfree transgenic rats were colonized with groups of five to eight bacteria that were either facultative or strictly anaerobic. Transgenic germfree rats had no gastroduodenitis, colitis, or arthritis, but developed epididymitis and dermatitis to the same degree as conventionalized rats. Colonic proinflammatory cytokine expression was increased in transgenic conventionalized rats but was undetectable in germfree and nontransgenic rats. Colitis progressively increased over the first 4 wk of bacterial exposure, then plateaued. Only transgenic rats colonized with defined bacterial cocktails which contained Bacteroides spp. had colitis and gastritis. Normal luminal bacteria predictably and uniformly induce chronic colonic, gastric and systemic inflammation in B27 transgenic F344 rats, but all bacterial species do not have equal activities.
H C Rath, H H Herfarth, J S Ikeda, W B Grenther, T E Hamm Jr, E Balish, J D Taurog, R E Hammer, K H Wilson, R B Sartor
Besides a prominent mononuclear cell infiltration of the islets of Langerhans, nonobese diabetic (NOD) mice also show massive cellular infiltrates of the submandibular and lacrimal glands concomitant with histological signs of tissue damage. To obtain insights into the mechanisms operative during the initiation and progression of tissue damage, we followed by in situ hybridization the appearance of cells containing mRNA of the gene encoding the proinflammatory cytokine TNF-alpha in the cellular infiltrates. Cells expressing TNF-alpha are mainly located in infiltrates, are absent in nonaffected glands, and are preferentially found among CD4 T cells. Secretion of TNF-alpha by gland-infiltrating cells was confirmed by an ELISPOT procedure. Direct evidence for an instrumental role of TNF-alpha in initiation and progression of submandibular and lacrimal gland infiltration is provided by the observed significant reduction in the extent of infiltration in nonobese diabetic mice transgenic for a soluble TNF receptor p55 fused to the Fc part of human IgG3. This protection from infiltration is paralleled by decreased expression of the adhesion molecules ICAM-1 and VCAM-1 in submandibular and lacrimal glands. These data suggest a central role of TNF-alpha in the initiation and progression of autoimmune tissue destruction of salivary glands and indicate beneficial effects of soluble TNF receptors in the treatment of organ-specific autoimmune diseases.
R E Hunger, S Müller, J A Laissue, M W Hess, C Carnaud, I Garcia, C Mueller
Type 2 iodothyronine deiodinase (D2) is a recently cloned selenodeiodinase thought to provide intracellular 3,5,3' triiodothyronine (T3) to a restricted group of tissues. We report here the presence of D2 mRNA in human thyroid at levels 50-150-fold higher than in placenta. Surprisingly, while type 1 deiodinase (D1) is known to be present in human thyroid, D2 has not been evaluated previously. D2 mRNA was especially high in thyroids from Graves' patients and in follicular adenomas. Stimulated thyroids had higher D2 to D1 mRNA ratios than normal or multinodular glands suggesting differential regulation of D1 and D2 expression. Microsomes from normal, Graves', and TSH-stimulated thyroids contained low Km D2 activity resistant to propylthiouracil (1 mM) or to inactivation by N-bromoacetyl T3, agents which block or inactivate D1. At 2 nM thyroxine (T4), 100 times the physiological-free T4 levels, 60-80% of T4 to T3 conversion in stimulated, but only 27% of that in normal thyroids, is catalyzed by D2. We conclude that intrathyroidal T4 to T3 conversion by D2 may contribute significantly to the relative increase in thyroidal T3 production in patients with Graves' disease, toxic adenomas, and, perhaps, iodine deficiency.
D Salvatore, H Tu, J W Harney, P R Larsen
Atrial natriuretic peptide (ANP) binds to natriuretic peptide receptor-A (NPR-A), a membrane guanylyl cyclase, and to natriuretic peptide receptor-C (NPR-C), which plays a role in peptide clearance. Rat ANP (rANP) mutants that bind rat NPR-A selectively over rat NPR-C were isolated from randomized libraries of rANP-display phage by differential panning. One variant was identified with reduced NPR-C binding; rANP (G16R, A17E, Q18A) [rANP(REA18)]. Synthetic rANP(REA18) was equipotent with rANP in stimulating cGMP production from cloned rat NPR-A (ED50 = 1.8 nM) and was reduced in NPR-C binding by approximately 200-fold. When infused into conscious rats at 0.325 microg/min for 30 min rANP elicited an identical decrease in blood pressure compared with 0.25 microg/min of rANP(REA18), however the natriuretic (P < 0.05) and diuretic (P = 0.07) responses to rANP(REA18) were greater. These data are consistent with a role for NPR-C as a local decoy receptor attenuating NPR-A effects in the kidney, where these receptors are coexpressed. Improved NPR-A specificity could provide more effective natriuretic peptides for treatment of acute renal failure or heart failure.
H Jin, B Li, B Cunningham, J Tom, R Yang, P Sehl, G R Thomas, A Ko, D Oare, D G Lowe
The NADPH-dependent respiratory burst oxidase of human neutrophils catalyzes the reduction of oxygen to superoxide using NADPH as the electron donor and is essential for normal host defenses. To gain insight into the function of the various oxidase subunits that are required for the full expression of catalytic activity, we studied the interactions between the 2',3'-dialdehyde derivative of NADPH (NADPH dialdehyde) and neutrophil cytosol. NADPH dialdehyde treatment of cytosol resulted in the loss of the ability of the cytosol to participate in cell-free oxidase activation; this inactivation was blocked by NADPH but not by NAD, NADP, or GTP. Partial purification of neutrophil cytosol yielded a single peak which could restore the activity lost in cytosol treated with NADPH dialdehyde. This peak contained p67phox but not p47phox or Rac2. Purified recombinant p67phox was similarly able to restore the activity lost in NADPH dialdehyde-treated cytosol and bound [32P]NADPH dialdehyde in a specific fashion. The activity of recombinant p67phox in cell-free oxidase assays was lost on treatment with NADPH dialdehyde. Together, these data suggest p67phox contains the catalytic NADPH-binding site of the leukocyte NADPH oxidase.
R M Smith, J A Connor, L M Chen, B M Babior
The scavenger receptor, class B, type I (SR-BI) binds HDL and mediates the selective transfer of cholesteryl esters from HDL to cultured cells. The tissue distribution of SR-BI in mice suggests that this receptor may deliver HDL-cholesterol to the liver and to nonplacental steroidogenic tissues. To examine the role of SR-BI in vivo, we determined its tissue and cell type-specific expression pattern and regulation in rats. High levels of immunodetectable SR-BI were present in the adrenal gland, ovary, and liver. In pregnant animals, the mammary gland also expressed high levels of the protein. SR-BI was localized by immunofluorescence to the surfaces of steroidogenic cells in the zona fasciculata and zona reticularis of the adrenal gland and to the corpus luteal cells of the ovary. High-dose estrogen treatment dramatically reduced SR-BI in the liver and increased SR-BI in the adrenal gland and corpus luteal cells of the ovary. These estrogen-induced increases in SR-BI in the adrenal gland and ovary were accompanied by enhanced in vivo uptake of fluorescent lipid from HDL. The administration of human chorionic gonadotropin induced a dramatic increase in SR-BI in the steroidogenic Leydig cells of the testes. These findings suggest that SR-BI mediates physiologically relevant uptake of cholesterol from HDL to nonplacental steroidogenic tissues in vivo.
K T Landschulz, R K Pathak, A Rigotti, M Krieger, H H Hobbs
Very little is known about the turnover of extracellular matrix in the human intervertebral disc. We measured concentrations of specific molecules reflecting matrix synthesis and degradation in predetermined regions of 121 human lumbar intervertebral discs and correlated them with ageing and Thompson grade of degeneration. Synthesis in intervertebral discs, measured by immunoassay of the content of a putative aggrecan biosynthesis marker (846) and the content of types I and II procollagen markers, is highest in the neonatal and 2-5-yr age groups. The contents of these epitopes/molecules progressively diminished with increasing age. However, in the oldest age group (60-80 yr) and in highly degenerated discs, the type I procollagen epitope level increased significantly. The percentage of denatured type II collagen, assessed by the presence of an epitope that is exposed with cleavage of type II collagen, increased twofold from the neonatal discs to the young 2-5-yr age group. Thereafter, the percentage progressively decreased with increasing age; however, it increased significantly in the oldest group and in highly degenerate discs. We identified three matrix turnover phases. Phase I (growth) is characterized by active synthesis of matrix molecules and active denaturation of type II collagen. Phase II (maturation and ageing) is distinguished by a progressive drop in synthetic activity and a progressive reduction in denaturation of type 11 collagen. Phase III (degeneration and fibrotic) is illustrated by evidence for a lack of increased synthesis of aggrecan and type II procollagen, but also by an increase in collagen type II denaturation and type I procollagen synthesis, both dependent on age and grade of tissue degeneration.
J Antoniou, T Steffen, F Nelson, N Winterbottom, A P Hollander, R A Poole, M Aebi, M Alini
The ob gene product, leptin, is a signaling factor regulating body weight and energy balance. ob gene expression in rodents is increased in obesity and is regulated by feeding patterns and hormones, such as insulin and glucocorticoids. In humans with gross obesity, ob mRNA levels are higher, but other modulators of human ob expression are unknown. In view of the importance of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipocyte differentiation, we analyzed whether ob gene expression is subject to regulation by factors activating PPARs. Treatment of rats with the PPARalpha activator fenofibrate did not change adipose tissue and body weight and had no significant effect on ob mRNA levels. However, administration of the thiazolidinedione BRL49653, a PPARgamma ligand, increased food intake and adipose tissue weight while reducing ob mRNA levels in rats in a dose-dependent manner. The inhibitory action of the thiazolidinedione BRL49653 on ob mRNA levels was also observed in vitro. Thiazolidinediones reduced the expression of the human ob promoter in primary adipocytes, however, in undifferentiated 3T3-L1 preadipocytes lacking endogenous PPARgamma, cotransfection of PPARgamma was required to observe the decrease. In conclusion, these data suggest that PPARgamma activators reduce ob mRNA levels through an effect of PPARgamma on the ob promoter.
P De Vos, A M Lefebvre, S G Miller, M Guerre-Millo, K Wong, R Saladin, L G Hamann, B Staels, M R Briggs, J Auwerx
We have characterized the progressive stages of chronic intestinal inflammation that develops spontaneously in specific pathogen-free (SPF) mice with a targeted disruption in the IL-10 gene (IL-10-/-). Our longitudinal studies showed that inflammatory changes first appear in the cecum, ascending and transverse colon of 3-wk-old mutants. As the disease progressed, lesions appeared in the remainder of the colon and in the rectum. Some aged IL-10-/- mice also developed inflammation in the small intestine. Prolonged disease with transmural lesions and a high incidence of colorectal adenocarcinomas (60%) was observed in 6-mo-old mutants. Mechanistic studies have associated uncontrolled cytokine production by activated macrophages and CD4+ Th1-like T cells with the enterocolitis exhibited by IL-10-/- mice. A major role for a pathogenic Th1 response was further suggested by showing that anti-IFNgamma antibody (Ab) treatment significantly attenuated intestinal inflammation in young IL-10-/- mice. When weanlings were treated with IL-10, they failed to develop any signs of intestinal inflammation. Interestingly, IL-10 treatment of adults was not curative but did ameliorate disease progression. Our studies have also shown that inheritable factors strongly influence the disease susceptibility of IL-10-/- mice. In 3-mo-old mutants, intestinal lesions were most severe in IL-10-/- 129/SvEv and IL-10-/- BALB/c strains, of intermediate severity in the IL-10-/- 129 x C57BL/6J outbreds, and least severe in the IL-10-/- C57BL/6J strain.
D J Berg, N Davidson, R Kühn, W Müller, S Menon, G Holland, L Thompson-Snipes, M W Leach, D Rennick
Individuals deficient in hepatic methionine adenosyltransferase (MAT) activity (MAT I/III deficiency) have been demonstrated to contain mutations in the gene (MATA1) that encodes the major hepatic forms, MAT I and III. MAT I/III deficiency is characterized by isolated persistent hypermethioninemia and, in some cases, unusual breath odor. Most individuals with isolated hypermethioninemia have been free of major clinical difficulties. Therefore a definitive diagnosis of MAT I/III deficiency, which requires hepatic biopsy, is not routinely made. However, two individuals with isolated hypermethioninemia have developed abnormal neurological problems, including brain demyelination, suggesting that MAT I/III deficiency can be deleterious. In the present study we have examined the MATA1 gene of eight hypermethioninemic individuals, including the two with demyelination of the brain. Mutations that abolish or reduce the MAT activity were detected in the MATA1 gene of all eight individuals. Both patients with demyelination are homozygous for mutations that alter the reading frame of the encoded protein such that the predicted MATalpha1 subunits are truncated and enzymatically inactive. The product of MAT, S-adenosylmethionine (AdoMet), is the major methyl donor for a large number of biologically important compounds including the two major myelin phospholipids, phosphatidylcholine and sphingomyelin. Both are synthesized primarily in the liver. Our findings demonstrate that isolated persistent hypermethioninemia is a marker of MAT I/III deficiency, and that complete lack of MAT I/III activity can lead to neurological abnormalities. Therefore, a DNA-based diagnosis should be performed for individuals with isolated hypermethioninemia to assess if therapy aimed at the prevention of neurological manifestations is warranted.
M E Chamberlin, T Ubagai, S H Mudd, W G Wilson, J V Leonard, J Y Chou
Mitochondrial trifunctional protein (MTP) is a recently identified enzyme involved in mitochondrial beta-oxidation, harboring long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and long-chain 3-ketothiolase activity. A deficiency of this protein is associated with impaired oxidation of long-chain fatty acids which can lead to sudden infant death. Furthermore, it is clear that this inborn error of fatty acid oxidation is very frequent, second to medium chain acyl-CoA dehydrogenase deficiency. In most patients only the LCHAD activity of MTP is deficient with near normal activity of the two other enzyme activities of the complex. We recently described the occurrence of a frequent G1528C mutation in the cDNA coding for the a subunit of MTP. Using S. cerevisiae for expression of wild type and mutant protein we show that the G1528C mutation is directly responsible for the loss of LCHAD activity. Furthermore, we describe a newly developed method allowing identification of the G1528C mutation in genomic DNA. The finding of an 87% allele frequency of the G1528C mutation in 34 LCHAD deficient patients makes this a valuable test for prenatal diagnosis. Finally, we show that the gene encoding the alpha subunit of MTP is located on chromosome 2p24.1-23.3.
L IJlst, J P Ruiter, J M Hoovers, M E Jakobs, R J Wanders
Keratins 8 and 18 (K8/18) are intermediate filament phosphoglycoproteins that are expressed preferentially in simple-type epithelia. We recently described transgenic mice that express point-mutant human K18 (Ku, N.-O., S. Michie, R.G. Oshima, and M.B. Omary. 1995. J. Cell Biol. 131:1303-1314) and develop chronic hepatitis and hepatocyte fragility in association with hepatocyte keratin filament disruption. Here we show that mutant K18 expressing transgenic mice are highly susceptible to hepatotoxicity after acute administration of acetaminophen (400 mg/Kg) or chronic ingestion of griseofulvin (1.25% wt/wt of diet). The predisposition to hepatotoxicity results directly from the keratin mutation since nontransgenic or transgenic mice that express normal human K18 are more resistant. Hepatotoxicity was manifested by a significant difference in lethality, liver histopathology, and biochemical serum testing. Keratin glycosylation decreased in all griseofulvin-fed mice, whereas keratin phosphorylation increased dramatically preferentially in mice expressing normal K18. The phosphorylation increase in normal K18 after griseofulvin feeding appears to involve sites that are different to those that increase after partial hepatectomy. Our results indicate that hepatocyte intermediate filament disruption renders mice highly susceptible to hepatotoxicity, and raises the possibility that K18 mutations may predispose to drug hepatotoxicity. The dramatic phosphorylation increase in nonmutant keratins could provide survival advantage to hepatocytes.
N O Ku, S A Michie, R M Soetikno, E Z Resurreccion, R L Broome, R G Oshima, M B Omary
Since recent studies suggest an imbalance between cathepsin B and its tissue protease inhibitors (PI) in the pathogenesis of acute and chronic diseases, we tested the hypothesis that release of activated cysteine proteases (P) such as cathepsins B, H, and L might play a role in the pathogenesis of gastric hemorrhagic mucosal lesions (HML) induced by ethanol (E) or ammonia (A). Anesthetized rats received 1 ml of 50% E or 1% A solution intragastrically for 1 min during in situ gastric luminal perfusion. Rapid activation and release of cathepsins B, L, and H into the luminal perfusate preceded the formation of HML quantified by planimetry. Mucosal presence and activity of cysteine PI and cathepsin B have also been investigated in the pathogenesis of chemically induced HML. We extracted and partially isolated acid and thermostable inhibitors of cathepsin B in the gastric mucosa, and found rapid inactivation of PI and activation of cathepsin B in the early phase of E- or A-induced HML. Negative correlations were found between P and PI activities by E or A solutions. Both the activation of cathepsins B, L, and H and the development of E-induced HML were prevented by pretreatment with the sulfhydryl alkylator N-ethylmaleimide. These results suggest that cysteine P may be activated in the rat stomach after E or A exposure, and cysteine P may have a role in the pathogenesis of E- or A-induced gastric HML. Endogenous PI may also participate in the mechanisms of gastric mucosal lesions and gastroprotection.
L Nagy, S Kusstatscher, P V Hauschka, S Szabo
Adrenal hypoplasia congenita (AHC) is an X-linked disorder that typically presents with adrenal insufficiency during infancy. Hypogonadotropic hypogonadism (HHG) has been identified as a component of this disorder in affected individuals who survive into childhood. Recently, AHC was shown to be caused by mutations in DAX-1, a protein that is structurally similar in its carboxyterminal region to orphan nuclear receptors. We studied two kindreds with clinical features of AHC and HHG. DAX-1 mutations were identified in both families. In the JW kindred, a single base deletion at nucleotide 1219 was accompanied by an additional base substitution that resulted in a frameshift mutation at codon 329 followed by premature termination. In the MH kindred, a GGAT duplication at codon 418 caused a frameshift that also resulted in truncation of DAX-1. Baseline luteinizing hormone (LIT), follicle-stimulating hormone (FSH), and free-alpha-subunit (FAS) levels were determined during 24 h of frequent (q10 min) venous sampling. In patient MH, baseline LH levels were low, but FAS levels were within the normal range. In contrast, in patient JW, the mean LH and FSH were within the normal range during baseline sampling, but LH secretion was erratic rather than showing typical pulses. FAS was apulsatile for much of the day, but a surge was seen over a 3-4-h period. Pulsatile gonadotropin releasing hormone (GnRH) (25 ng/kg) was administered every 2 h for 7 d to assess pituitary responsiveness to exogenous GnRH. MH did not exhibit a gonadotropin response to pulsatile GnRH. JW exhibited a normal response to the first pulse of GnRH, but there was no increase in FAS. In contrast to the priming effect of GnRH in GnRH-deficient patients with Kallmann syndrome, GnRH pulses caused minimal secretory responses of LH and no FAS responses in patient JW. The initial LH response in patient JW implies a deficiency in hypothalamic GnRH. On the other hand, the failure to respond to pulsatile GnRH is consistent with a pituitary defect in gonadotropin production. These two cases exemplify the phenotypic heterogeneity of AHC/HHG, and suggest that DAX-1 mutations impair gonadotropin production by acting at both the hypothalamic and pituitary levels.
R L Habiby, P Boepple, L Nachtigall, P M Sluss, W F Crowley Jr, J L Jameson
The renin-angiotensin-aldosterone system (RAAS) participates in the injury sustained by the remnant kidney. Our studies assessed the importance of aldosterone in that model and the response of aldosterone to drugs interfering with the RAAS. Initially, four groups of rats were studied: SHAM-operated rats, untreated remnant rats (REM), REM rats treated with losartan and enalapril (REM AIIA), and REM AIIA rats infused with exogenous aldosterone (REM AIIA + ALDO). The last group was maintained with aldosterone levels comparable to those in untreated REM rats by constant infusion of exogenous aldosterone. REM rats had larger adrenal glands and a > 10-fold elevation in plasma aldosterone compared to SHAM. REM AIIA rats demonstrated significant suppression of the hyperaldosteronism as well as marked attenuation of proteinuria, hypertension, and glomerulosclerosis compared to REM. REM AIIA + ALDO rats manifested greater proteinuria, hypertension, and glomerulosclerosis than REM AIIA rats. Indeed, by 4 wk of observation all of these features of the experimental disease were similar in magnitude in REM AIIA + ALDO and untreated REM. In separate REM rats spironolactone administration did not reduce glomerular sclerosis but did transiently reduce proteinuria, lowered arterial pressure, and lessened cardiac hypertrophy. In summary, aldosterone contributes to hypertension and renal injury in the remnant kidney model.
E L Greene, S Kren, T H Hostetter