BBA - Molecular Basis of Disease (v.1688, #1)

Association of apolipoprotein A5 variants with LDL particle size and triglyceride in Japanese Americans by Melissa A. Austin; Philippa J. Talmud; Federico M. Farin; Deborah A. Nickerson; Karen L. Edwards; Donna Leonetti; Marguerite J. McNeely; Hannah-Malia Viernes; Steve E. Humphries; Wilfred Y. Fujimoto (1-9).
A new apolipoprotein (apo) gene, APOA5, was recently identified on chromosome 11q23, and common variants in the gene have been associated with plasma triglyceride (TG) levels in several studies. The purpose of the present study was to examine the association of five single nucleotide polymorphisms (SNPs) and haplotypes in the APOA5 gene with low-density lipoprotein (LDL) particle size using a community-based sample of Japanese American families, including examining whether the associations with LDL size are independent of, or primarily reflecting, TG levels. Genetic association analyses were performed using 154 unrelated individuals, quantitative transmission disequilibrium tests (TDT) in 238 nuclear families, a sample of 24 hypertriglyceridemic subjects with matched, normotriglyceridemic controls, and using haplotype analyses. There was a high degree of allelic association between several of the SNPs, with complete linkage disequilibrium (LD) between −1131C>T and the −3A>G SNP which alters a potential Kozak sequence. All approaches demonstrated associations between the −3A>G APOA5 variant and both decreased LDL size and increased TG levels. The frequency of the rare allele was higher than reported for Caucasian, Hispanic, and African Americans, but similar to that in Japan and China. Therefore, the haplotype containing the −1131C and −3G variants, and possibly specifically the −3A>G SNP in APOA5, may be a major genetic determinant of LDL particle size and TG levels among ethnic Asians.
Keywords: Apolipoprotein; Cardiovascular disease; Genetics; Low-density lipoprotein; Triglyceride;

Role of lipid interactions in autoimmune demyelination by Benjamin Ohler; Karlheinz Graf; Richard Bragg; Travis Lemons; Robert Coe; Claude Genain; Jacob Israelachvili; Cynthia Husted (10-17).
A morphological transformation involving loss of adhesion between myelin lamellae and formation of myelin vesicles has been described as a mechanism for demyelination in multiple sclerosis and marmoset experimental allergic encephalomyelitis (EAE). Although protein interactions are involved in maintaining normal myelin structure, we describe here how lipids contribute to myelin stability and how lipid changes in EAE, including increases in lipid polyunsaturation and negatively charged phosphatidylserine (PS), promote demyelination. Three physico-chemical techniques were used to identify these changes: (1) Langmuir monolayer isotherms indicated that EAE white matter lipids were significantly more ‘expanded’ (fluid) than controls. (2) NMR spectroscopy indicated that EAE myelin lipids were more polyunsaturated than controls. (3) High-performance liquid chromatography (HPLC) with an evaporative light scattering detector indicated increased PS in EAE compared to controls, while sphingomyelin (SM), sulfatides and phosphatidylcholine (PC) were decreased. We present a physical model considering electrostatic, van der Waals and undulation forces to quantify the effect of these changes on myelin adhesion at the extracellular interface. Taken together, the isotherm, NMR, HPLC and modeling results support a mechanism for autoimmune demyelination whereby the composition of myelin lipids is altered in a manner that increases myelin fluidity, decreases myelin adhesion, increases membrane curvature, and promotes vesiculation.
Keywords: Experimental allergic encephalomyelitis; Myelin; Membrane lipid fluidity and packing; Multiple sclerosis;

Many cardiac patients undergo exercise conditioning with or without medication. Therefore, we investigated the interaction of exercise training and chronic nitroglycerin treatment on blood pressure (BP), aortic nitric oxide (NO), oxidants and antioxidants in rats. Fisher 344 rats were divided into four groups and treated as follows: (1) sedentary control, (2) exercise training (ET) for 8 weeks, (3) nitroglycerin (15 mg/kg, s.c. for 8 weeks) and (4) ET+nitroglycerin. BP was monitored with tail-cuff method. The animals were sacrificed 24 h after the last treatments and thoracic aorta was isolated and analyzed. Exercise training on treadmill for 8 weeks significantly increased respiratory exchange ratio (RER), aortic NO levels, and endothelial nitric oxide synthase (eNOS) protein expression. Training significantly enhanced aortic glutathione (GSH), reduced to oxidized glutathione (GSH/GSSG) ratio, copper/zinc-superoxide dismutase (CuZn-SOD), Mn-SOD, catalase (CAT), glutathione peroxidase (GSH-Px) glutathione disulfide reductase (GR) activities and protein expressions. Training significantly depleted aortic malondialdehyde (MDA) and protein carbonyls without change in BP. Nitroglycerin administration for 8 weeks significantly increased aortic NO levels and eNOS protein expression. Nitroglycerin significantly enhanced aortic Mn-SOD, CAT, GR and glutathione-S-transferase (GST) activities and protein expressions with decreased MDA levels, protein carbonyls and BP. Interaction of training and nitroglycerin treatment significantly increased aortic NO levels, eNOS protein expression, GSH/GSSG ratio, antioxidant enzymes and normalized BP. The data suggest that the interaction of training and nitroglycerin maintained BP by up-regulating the aortic NO and antioxidants and reducing the oxidative stress in rats.
Keywords: Aorta; Antioxidant enzyme; Blood pressure; Exercise training; Lipid peroxidation; Nitric oxide; Nitroglycerin; Rat;

Evidence that oxidative stress is increased in patients with X-linked adrenoleukodystrophy by C.R Vargas; M Wajner; L.R Sirtori; L Goulart; M Chiochetta; D Coelho; A Latini; S Llesuy; A Bello-Klein; R Giugliani; M Deon; C.F Mello (26-32).
X-linked adrenoleukodystrophy (X-ALD) is a hereditary disorder of peroxisomal metabolism biochemically characterized by the accumulation of very long chain fatty acids (VLCFA), particularly hexacosanoic acid (C26:0) and tetracosanoic acid (C24:0) in different tissues and in biological fluids. The disease is clinically characterized by central and peripheral demyelination and adrenal insufficiency, which is closely related to the increased concentrations of these fatty acids. However, the mechanisms underlying the brain damage in X-ALD are poorly known. Considering that free radical generation is involved in various neurodegererative disorders, like Parkinson disease, multiple sclerosis and Alzheimer's disease, in the present study we evaluated various oxidative stress parameters, namely chemiluminescence, thiobarbituric acid reactive species (TBA-RS), total radical-trapping antioxidant potential (TRAP), and total antioxidant reactivity (TAR) in plasma of X-ALD patients, as well as the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in erythrocytes and fibroblasts from these patients. It was verified a significant increase of plasma chemiluminescence and TBA-RS, reflecting induction of lipid peroxidation, as well as a decrease of plasma TAR, indicating a deficient capacity to rapidly handle an increase of reactive species. We also observed a significant increase of erythrocytes GPx activity and of catalase and SOD activities in fibroblasts from the patients studied. It is therefore proposed that oxidative stress may be involved in pathophysiology of X-ALD.
Keywords: X-linked adrenoleukodystrophy; Oxidative stress; Free radical;

Copper-mediated LDL oxidation by homocysteine and related compounds depends largely on copper ligation by Emi Nakano; Mike P. Williamson; Nick H. Williams; Hilary J. Powers (33-42).
Oxidation of low-density lipoprotein (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. Elevated plasma homocysteine is an accepted risk factor for atherosclerosis, and may act through LDL oxidation, although this is controversial. In this study, homocysteine at physiological concentrations is shown to act as a pro-oxidant for three stages of copper-mediated LDL oxidation (initiation, conjugated diene formation and aldehyde formation), whereas at high concentration, it acts as an antioxidant. The affinity for copper of homocysteine and related copper ligands homocysteine, cystathionine and djenkolate was measured, showing that at high concentrations (100 μM) under our assay conditions, they bind essentially all of the copper present. This is used to rationalise the behaviour of these ligands, which stimulate LDL oxidation at low concentration but generally inhibit it at high concentration. Albumin strongly reduced the effect of homocystine on lag time for LDL oxidation, suggesting that the effects of homocystine are due to copper binding. In contrast, copper binding does not fully explain the pro-oxidant behaviour of low concentrations of homocysteine towards LDL, which appears in part at least to be due to stimulation of free radical production. The likely role of homocysteine in LDL oxidation in vivo is discussed in the light of these results.
Keywords: Homocysteine; Cardiovascular disease; Citrate; LDL; Pro-oxidant; Antioxidant;

Cell protection, resistance and invasiveness of two malignant mesotheliomas as assessed by 10K-microarray by Steve Mohr; Gérard Keith; Françoise Galateau-Salle; Philippe Icard; Bertrand H Rihn (43-60).
Malignant pleural mesothelioma (MPM) is an aggressive serosal tumor, strongly associated with former exposure to asbestos fibers and for which there is currently no effective treatment available. In human, MPM is characterized by a high local invasiveness, poor prognosis and therapeutic outcomes. In order to assess molecular changes that specify this phenotype, we performed a global gene expression profiling of human MPM. Using a 10,000-element microarray, we analyzed mRNA relative gene expression levels by comparing a mesothelioma cell line to either a pleural cell line or tumor specimens. To analyze these gene expression data, we used various bioinformatics softwares. Hierarchical clustering methods were used to group genes and samples with similar expression in an unsupervised mode. Genes of known function were further sorted by enzyme, function and pathway clusters using a supervised software (IncyteGenomics). Taken together, these data defined a molecular fingerprint of human MPM with more than 700 up- or down-regulated genes related to several traits of the malignant phenotype, specially associated with MPM invasiveness, protection and resistance to anticancer defenses. This portrait is meaningful in disease classification and management, and relevant in finding new specific markers of MPM. These molecular markers should improve the accuracy of mesothelioma diagnosis, prognosis and therapy.
Keywords: Malignant mesothelioma; Pleura; Asbestos; Microarray; Transcriptomics; Molecular marker;

Hypobetalipoproteinemia with an apparently recessive inheritance due to a “de novo” mutation of apolipoprotein B by Sandra Lancellotti; Enza Di Leo; Junia Y. Penacchioni; Fiorella Balli; Laura Viola; Stefano Bertolini; Sebastiano Calandra; Patrizia Tarugi (61-67).
Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder either linked or not linked to apolipoprotein (apo) B gene. Abetalipoproteinemia (ABL) is a recessive disorder due to mutations of microsomal triglyceride transfer protein (MTP) gene. We investigated a patient with apparently recessive hypobetalipoproteinemia consistent with symptomatic heterozygous FHBL or a “mild” form of ABL. The proband had fatty liver associated with LDL-cholesterol (LDL-C) and apo B levels <5th percentile but no truncated apo B forms detectable in plasma. MTP gene sequence revealed that he was a carrier of the I128T polymorphism and an unreported amino acid substitution (V168I) unlikely to be the cause of hypobetalipoproteinemia. Apo B gene sequence showed that he was heterozygous for two single base substitutions in exon 9 and 22 resulting in a nonsense (Q294X) and a missense (R1101H) mutation, respectively. Neither of his parents carried the Q294X; his father and paternal grandmother carried the R1101H mutation. Analysis of polymorphic genetic markers excluded non-paternity. In conclusion, the proband has a “de novo “mutation of apo B gene resulting in a short truncated apo B form (apo B-6.46). Sporadic cases of FHBL with an apparently recessive transmission may be caused by “de novo” mutations of apo B gene.
Keywords: Familial hypobetalipoproteinemia; “De novo” mutation; Truncated apolipoprotein B; Microsomal triglyceride transfer protein;

Characterisation of 11β-hydroxysteroid dehydrogenases in feline kidney and liver by L. Schipper; B. Spee; J. Rothuizen; F. Woutersen-van Nijnanten; J. Fink-Gremmels (68-77).
11β-Hydroxysteroid dehydrogenases type 1 and 2 (11β-HSD1 and 11β-HSD2) are microsomal enzymes responsible for the interconversion of cortisol into the inactive form cortisone and vice versa. 11β-HSD1 is mainly present in the liver, and has predominantly reductase activity although its function has not yet been elucidated. 11β-HSD2, present in mineralocorticoid target tissues such as the kidney, converts cortisol into cortisone. Reduced activity due to inhibition or mutations of 11β-HSD2 leads to hypertension and hypokalemia resulting in the Apparent Mineralocorticoid Excess Syndrome (AMES). Like humans, cats are highly susceptible for hypertension. As large species differences exist with respect to the kinetic parameters (K m and V max) and amino acid sequences of both enzymes, we determined these characteristics in the cat. Both enzyme types were found in the kidneys. 11β-HSD1 in the feline kidney showed bidirectional activity with predominantly dehydrogenase activity (dehydrogenase: K m 1959±797 nM, V max 766±88 pmol/mg*min; reductase: K m 778±136 nM, V max 112±4 pmol/mg*min). 11β-HSD2 represents a unidirectional dehydrogenase with a higher substrate affinity (K m 184±24 nM, V max 74±3 pmol/mg*min). In the liver, only 11β-HSD1 is detected exerting reductase activity (K m 10462 nM, V max 840 pmol/mg*min). Sequence analysis of conserved parts of 11β-HSD1 and 11β-HSD2 revealed the highest homology of the feline enzymes with the correspondent enzymes found in man. This suggests that the cat may serve as a suitable model species for studies directed to the pathogenesis and treatment of human diseases like AMES and hypertension.
Keywords: 11β-Hydroxysteroid dehydrogenase; Enzyme kinetics; Sequence; Cat; Apparent mineralocorticoid excess syndrome; Hypertension;

Wilson disease is an autosomal disorder of copper transport caused by mutations in the ATP7B gene encoding a copper-transporting P-type ATPase. The Long Evans Cinnamon (LEC) rat is an established animal model for Wilson disease. We have used structural homology modelling of the N-terminal copper-binding region of the rat atp7b protein (rCBD) to reveal the presence of a domain, the fourth domain (rD4), which was previously thought to be missing from rCBD. Although the CXXC motif is absent from rD4, the overall fold is preserved. Using a wide range of techniques, rCBD is shown to undergo metal-induced secondary and tertiary structural changes similar to WCBD. Competition 65Zn(II)-blot experiments with rCBD demonstrate a binding cooperativity unique to Cu(I). Far-UV circular dichroism (CD) spectra suggest significant secondary structural transformation occurring when 2–3 molar equivalents of Cu(I) is added. Near-UV CD spectra, which indicate tertiary structural transformations, show a proportional decrease in rCBD disulfide bonds upon the incremental addition of Cu(I), and a maximum 5:1 Cu(I) to protein ratio. The similarity of these results to those obtained for the Wilson disease N-terminal copper-binding region (WCBD), which has six copper-binding domains, suggests that the metal-dependent conformational changes observed in both proteins may be largely determined by the protein–protein interactions taking place between the heavy metal-associated (HMA) domains, and remain largely unaffected by the absence of one of the six CXXC copper-binding sites.
Keywords: Copper; Wilson disease; ATP7B; LEC rat; HMA domain; Copper transport; Copper binding;

Leptin and the control of respiratory gene expression in muscle by G.B McClelland; C.S Kraft; D Michaud; J.C Russell; C.R Mueller; C.D Moyes (86-93).
Leptin plays a central role in the regulation of fatty acid homeostasis, promoting lipid storage in adipose tissue and fatty acid oxidation in peripheral tissues. Loss of leptin signaling leads to accumulation of lipids in muscle and loss of insulin sensitivity secondary to obesity. In this study, we examined the direct and indirect effects of leptin signaling on mitochondrial enzymes including those essential for peripheral fatty acid oxidation. We assessed the impact of leptin using the JCR:LA-cp rat, which lacks functional leptin receptors. The activities of marker mitochondrial enzymes citrate synthase (CS) and cytochrome oxidase (COX) were similar between wild-type (+/?) and corpulent (cp/cp) rats. In contrast, several tissues showed variations in the fatty acid oxidizing enzymes carnitine palmitoyltransferase II (CPT II), long-chain acyl-CoA dehydrogenase (LCAD) and 3-hydroxyacyl-CoA dehydrogenase (HOAD). It was not clear if these changes were due to loss of leptin signaling or to insulin insensitivity. Consequently, we examined the effects of leptin on cultured C2C12 and Sol8 cells. Leptin (3 days at 0, 0.2, or 2.0 nM) had no direct effect on the activities of CS, COX, or fatty acid oxidizing enzymes. Leptin treatment did not affect luciferase-based reporter genes under the control of transcription factors involved in mitochondrial biogenesis (nuclear respiratory factor-1 (NRF-1), nuclear respiratory factor-2 (NRF-2)) or fatty acid enzyme expression (peroxisome proliferator-activated receptors (PPARs)). These studies suggest that leptin exerts only indirect effects on mitochondrial gene expression in muscle, possibly arising from insulin resistance.
Keywords: Leptin; Fatty acid; Muscle; Mitochondrion; Enzyme; JCR:LA rat;