BBA - Molecular Basis of Disease (v.1690, #3)

Identification and characterisation of a novel KCNQ1 mutation in a family with Romano–Ward syndrome by J. Zehelein; D. Thomas; M. Khalil; A.-B. Wimmer; M. Koenen; M. Licka; K. Wu; J. Kiehn; K. Brockmeier; V.A.W. Kreye; C.A. Karle; H.A. Katus; H.E. Ulmer; W. Schoels (185-192).
Romano–Ward syndrome (RWS), the autosomal dominant form of the congenital long QT syndrome, is characterised by prolongation of the cardiac repolarisation process associated with ventricular tachyarrhythmias of the torsades de pointes type. Genetic studies have identified mutations in six ion channel genes, KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 and the accessory protein Ankyrin-B gene, to be responsible for this disorder. Single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequence analysis have identified a KCNQ1 mutation in a family that were clinically conspicuous due to several syncopes and prolonged QTc intervals in the ECG. The mutant subunit was expressed and functionally characterised in the Xenopus oocyte expression system. A novel heterozygous missense mutation with a C to T transition at the first position of codon 343 (CCA) of the KCNQ1 gene was identified in three concerned family members (QTc intervals: 500, 510 and 530 ms, respectively). As a result, proline 343 localised within the highly conserved transmembrane segment S6 of the KCNQ1 channel is replaced by a serine. Co-expression of mutant (KCNQ1-P343S) and wild-type (KCNQ1) cRNA in Xenopus oocytes produced potassium currents reduced by ≈92%, while I Ks reconstitution experiments with a combination of KCNQ1 mutant, wild-type and KCNE1 subunits yielded currents reduced by ≈60%. A novel mutation (P343S) identified in the KCNQ1 subunit gene of three members of a RWS family showed a dominant-negative effect on native I Ks currents leading to prolongation of the heart repolarisation and possibly increases the risk of malign arrhythmias with sudden cardiac death.
Keywords: I Ks current; KCNQ1 (KVLQT1); Long QT syndrome; Electrophysiology; Mutation;

Anti-amyloidogenic activity of tannic acid and its activity to destabilize Alzheimer's β-amyloid fibrils in vitro by Kenjiro Ono; Kazuhiro Hasegawa; Hironobu Naiki; Masahito Yamada (193-202).
Inhibition of the accumulation of amyloid β-peptide (Aβ) and the formation of β-amyloid fibrils (fAβ) from Aβ, as well as the destabilization of preformed fAβ in the CNS would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). We previously reported that nordihydroguaiaretic acid (NDGA) and wine-related polyphenols inhibit fAβ formation from Aβ(1–40) and Aβ(1–42) as well as destabilizing preformed fAβ(1–40) and fAβ(1–42) dose-dependently in vitro. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of polymeric polyphenol, tannic acid (TA) on the formation, extension, and destabilization of fAβ(1–40) and fAβ(1–42) at pH 7.5 at 37 °C in vitro. We next compared the anti-amyloidogenic activities of TA with myricetin, rifampicin, tetracycline, and NDGA. TA dose-dependently inhibited fAβ formation from Aβ(1–40) and Aβ(1–42), as well as their extension. Moreover, it dose-dependently destabilized preformed fAβs. The effective concentrations (EC50) of TA for the formation, extension and destabilization of fAβs were in the order of 0–0.1 μM. Although the mechanism by which TA inhibits fAβ formation from Aβ as well as destabilizes preformed fAβ in vitro is still unclear, it could be a key molecule for the development of therapeutics for AD.
Keywords: Alzheimer's disease; Tannic acid; β-Amyloid fibril; Thioflavin; Electron microscopy;

Significantly increased fractions of transformed to total α2-macroglobulin concentrations in plasma from patients with multiple sclerosis by Poul Erik H. Jensen; Signe Humle Jørgensen; Pameli Datta; Per Soelberg Sørensen (203-207).
We examined the proteinase inhibitor α2-macroglobulin (α2M) in plasma from patients with multiple sclerosis (MS); a neurological disease of the central nervous system. The plasma concentrations of native and transformed α2M were measured in 90 patients with clinically definite MS, 73 with relapsing–remitting and 17 with secondary progressive MS, and 132 healthy individuals. Significantly lower concentrations of native α2M and significantly higher concentrations of transformed α2M were found in MS patients. A significant correlation between the concentrations of native and transformed α2M was found. The fraction of transformed to total α2M in the MS patients was 36% higher than in the healthy individuals. The results suggest an important involvement of α2M in regulation of increased proteolytic activity occurring in MS disease.
Keywords: α2-Macroglobulin; Multiple sclerosis; Protease inhibitor; Plasma;

Restoration of copper metabolism and rescue of hepatic abnormalities in LEC rats, an animal model of Wilson disease, by expression of human ATP7B gene by Yan Meng; Ichiro Miyoshi; Masumi Hirabayashi; Mu Su; Yasumasa Mototani; Tadashi Okamura; Kunihiko Terada; Masatsugu Ueda; Katsuhiko Enomoto; Toshihiro Sugiyama; Noriyuki Kasai (208-219).
Hepatic abnormalities in Long–Evans Cinnamon (LEC) rats, an animal model of Wilson disease (WD), were restored by the expression of the human ATP7B cDNA under the control of CAG promoter. Expression of ATP7B transcript and protein in the liver of the transgenic rats resulted in the restoration of biosynthesis of holoceruloplasmin and biliary copper excretion. Meanwhile, transgenic rats showed striking improvements in their hepatic abnormalities, i.e., rescue from fulminant hepatitis, late onset of hepatic cholangiofibrosis, suppression of hepatocellular carcinoma and much improved survival rates. Moreover, dramatic decreases were noted both in the levels of hepatic copper and iron in transgenic rats before the occurrence of hepatitis. These results indicated that the human ATP7B product compensated for the deficiency of the endogenous rattus protein and did function in intrahepatic copper transport by secreting copper into the plasma via incorporation into ceruloplasmin and by the excretion of copper into the bile, and that ATP7B is critical to hepatic dysfunctions in WD. This first successful transgenic rescue has important implications for the gene therapy of WD.
Keywords: LEC rat; Wilson disease; ATP7B; Transgenic rat; Copper metabolism; Fulminant hepatitis;

Deamidation and cross-linking of gliadin peptides by transglutaminases and the relation to celiac disease by Hanne Skovbjerg; Claus Koch; Dorit Anthonsen; Hans Sjöström (220-230).
Activation of small intestinal gluten-reactive CD4+ T cells is a critical event in celiac disease. Such cells predominantly recognise gluten peptides in which specific glutamines are deamidated. Deamidation may be catalysed by intestinal tissue transglutaminase (TG2), a protein which is also the main autoantigen in celiac disease. Our aim was to study how the two main catalytic activities of transglutaminase—deamidation and transamidation (cross-linking) of an immunodominant gliadin epitope—are influenced by the presence of acceptor amines in the intestinal mucosa, and thereby contribute to further elucidation of the pathogenetic mechanisms in celiac disease. We prepared monoclonal antibodies, reacting specifically with the non-deamidated epitope QPFPQPQLPYPQPQ-amide and/or the deamidated epitope QPFPQPELPYPQPQ-amide. A solid phase immunoassay combined with gel filtration chromatography was used to analyse deamidation and cross-linking of these peptides to proteins. Our results show that QPFPQPQLPYPQPQ-amide was deamidated when incubated with purified TG2, with fresh mucosal sheets and with mucosal homogenates. Of other transglutaminases tested, only Streptoverticillium transglutaminase was able to generate the deamidated epitope. A fraction of the non-deamidated epitope was cross-linked to proteins, including TG2. The results suggest that intestinal TG2 is responsible for generation of the active deamidated epitope. As the epitope often occurs in a repeat structure, the result may be cross-linking of a deamidated, i.e., activated cell epitope. Alternatively, the deamidation may occur by reversal of the cross-linking reaction. The results provide a basis for the suggestion that binding of a peptide to a protein, in connection to its modification to a T cell epitope, might be a general explanation for the role of TG2 in celiac disease and a possible mechanism for the generation of autoantigens.
Keywords: Celiac disease; Transglutaminase; Gliadin peptide; Cross-linking; Deamidation; Mucosal protein;

Effects of chronic renal failure on caveolin-1, guanylate cyclase and AKT protein expression by Ram K. Sindhu; Ashkan Ehdaie; Nosratola D. Vaziri; Christian K. Roberts (231-237).
Chronic renal failure (CRF) has been documented to cause oxidative stress and alter nitric oxide (NO) metabolism. However, the effect of CRF on proteins related to NO bioactivity has not been investigated. The present study was designed to test the hypothesis that CRF would induce changes in caveolin-1 (Cav-1), soluble guanylate cyclase (sGC) and Akt, three proteins important in regulating NO synthase (NOS) functionality. Male Sprague–Dawley rats were randomized to CRF via 5/6 nephrectomy or sham-operated control groups. After 6 weeks, body weight, blood pressure, creatinine clearance, plasma creatinine, urinary cyclic guanosine monophosphate (cGMP) and immunodetectable levels of Cav-1, sGC and Akt were determined in the renal, aorta, heart and liver tissues from both groups. CRF resulted in marked decreases in body weight and creatinine clearance, and elevation of blood pressure and plasma creatinine. An apparent upregulation of sGC protein abundance in renal tissue was noted, with no change in aorta, heart and liver. This was accompanied by a reduction in urinary cGMP levels, indicative of sGC dysfunction. Cav-1 protein abundance was increased in aortic, liver and renal tissues. In contrast, CRF depressed Akt abundance in aorta, heart and liver tissues. These data document that CRF is characterized by alteration in the abundance of proteins regulating NO function in hepatic, vascular, cardiac and renal tissues, and a decrease in cGMP, which contributes to hypertension and changes in NO bioactivity previously noted in this model.
Keywords: CRF; Hypertension; Blood pressure; Nitric oxide; Guanylate cyclase; Caveolin-1; Akt;

A high proportion of polymorphisms in the promoters of brain expressed genes influences transcriptional activity by Paul R. Buckland; Bastiaan Hoogendoorn; Carol A. Guy; Sharon L. Coleman; S. Kaye Smith; Joseph D. Buxbaum; Vahram Haroutunian; Michael C. O'Donovan (238-249).
There is increasing interest in the possibility that polymorphisms affecting gene expression are responsible for a significant proportion of heritable human phenotypic variation, including human disease. We have sought to determine if polymorphisms in the promoters of brain expressed genes are commonly functional. We screened for polymorphism 56 genes previously reported to be differentially expressed in the brains of schizophrenics [Y. Hakak, J.R. Walker, C. Li, W.H. Wong, K.L. Davis, J.D. Buxbaum, V. Haroutunian, A.A. Fienberg, Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia. Proc. Natl. Acad. Sci. 98 (2001) 4746–4751.]. We found 60 variants distributed across 31 of the genes. A total of 77 haplotypes representing 28 different putative promoters were analyzed in a reporter gene assay in two cell lines. Of a total of 54 sequence variants represented in the haplotypes, 12 (or around 22%) were functional according to a highly conservative definition. These were found in the promoters of eight genes: NPY, PCSK1, NEFL, KIAA0513, LMO4, HSPA1B, TF and MDH1. We therefore estimate that around 20–25% of promoter polymorphisms in brain expressed genes are functional, and this is likely to be an underestimate. Our data therefore provide for the first time empirical evidence that promoter element polymorphisms, at least in brain expressed genes, should be afforded a high priority for molecular genetic studies.
Keywords: TF; NPY; PCSK1; NEFL; LMO4; HSPA1B;

Fabry disease is an inborn error of glycosphingolipid metabolism caused by the deficiency of lysosomal α-galactosidase A (α-Gal A). We have established transgenic mice that exclusively express human mutant α-Gal A (R301Q) in an α-Gal A knock-out background (TgM/KO mice). This serves as a biochemical model to study and evaluate active-site specific chaperone (ASSC) therapy for Fabry disease, which is specific for those missense mutations that cause misfolding of α-Gal A. The α-Gal A activities in the heart, kidney, spleen, and liver of homozygous TgM/KO mice were 52.6, 9.9, 29.6 and 44.4 unit/mg protein, respectively, corresponding to 16.4-, 0.8-, 0.6- and 1.4-fold of the endogenous enzyme activities in the same tissues of non-transgenic mice with a similar genetic background. Oral administration of 1-deoxygalactonojirimycin (DGJ), a competitive inhibitor of α-Gal A and an effective ASSC for Fabry disease, at 0.05 mM in the drinking water of the mice for 2 weeks resulted in 13.8-, 3.3-, 3.9-, and 2.6-fold increases in enzyme activities in the heart, kidney, spleen and liver, respectively. No accumulation of globotriaosylceramide, a natural substrate of α-Gal A, could be detected in the heart of TgM/KO mice after DGJ treatment, indicating that degradation of the glycolipid in the heart was not inhibited by DGJ at that dosage. The α-Gal A activity in homozygous or heterozygous fibroblasts established from TgM/KO mice (TMK cells) was approximately 39 and 20 unit/mg protein, respectively. These TgM/KO mice and TMK cells are useful tools for studying the mechanism of ASSC therapy, and for screening ASSCs for Fabry disease.
Keywords: Transgenic; α-Galactosidase A; Fabry disease; Active-site specific chaperone; 1-deoxygalactonojirimycin;

Oxidative stress due to activated neutrophils, macrophages and endothelial cells plays a crucial role in acute lung injury. This study compares the effects of the nonradical oxidants hypochlorite (HOCl) and hydrogen peroxide (H2O2) on pulmonary artery pressure [PAP (torr)], capillary filtration coefficient (K f,c), tissue lipid peroxidation (LPO) and reduced glutathione (GSH) depletion. HOCl, H2O2 (1000 nmol min−1) or buffer (control) is infused into isolated rabbit lungs. PAP, K f,c and lung weight were measured. Experiments were terminated after 105 min or when fluid retention exceeded 50 g. Lung tissue was analyzed for LPO products and GSH. The oxidants induced comparable maximum effects. However, the patterns of lung injury were distinct: H2O2 infusion evoked an early biphasic pressure response (ΔPAPmax 2.8±0.22/4.2±0.37 after 5.7±1.4/39±4.0 min) and a sixfold increase in K f,c after 90 min. HOCl application caused a late pressure response (ΔPAPmax 7.6±1.7 after 50.6±3.7 min) and a sevenfold increase in K f,c after 60 min. H2O2-induced effects were attenuated by desferal. This may suggest an involvement of transition metal catalysed hydroxyl radical formation. Different oxidants induced distinct patterns of changes in PAP and K f,c, which are accompanied by a comparable accumulation of LPO products and by a distinct degree of GSH depletion.
Keywords: Hypochlorite; Hydrogen peroxide; Acute lung injury; Oxidative stress;

Effects of adrenomedullin on aldosterone-induced cell proliferation in rat cardiac fibroblasts by Wei Jiang; Jing-Hui Yang; Shu-heng Wang; Chun-Shui Pan; Yong-Fen Qi; Jing Zhao; Chao-Shu Tang (265-275).
Aldosterone induces cardiac remodeling in cardiovascular diseases by stimulating the proliferation, production and secretion of collagen in fibroblasts. It also stimulates vascular smooth muscle cells to produce and secrete adrenomedullin (ADM), which has a cytoprotective effect against cardiovascular damage. We examined the effect of aldosterone on ADM production and secretion in rat cardiac fibroblasts, and the effect of ADM on aldosterone-stimulated fibroblast proliferation to observe the interaction between endogenous ADM and aldosterone. We detected ADM produced and secreted from cultured cardiac fibroblasts and the intracellular cAMP level by radioimmunoassay; evaluated cell proliferation by the level of [3H]-thymine incorporation; measured preproADM gene expression by reverse transcriptase polymerase chain reaction (RT-PCR); and monitored extracellular signal related kinase (ERK) activity by the phosphorylation of myelin basic protein in the presence of [γ-32P] ATP. Our results showed that aldosterone-stimulated secretion of ADM and its mRNA expression were concentration-dependent, which could be inhibited by the specific antagonist of mineralocorticoid receptor, spironolactone. In contrast, ADM inhibited aldosterone-induced fibroblast proliferation and ERK activity. Treatment with ADM24–50 (a new antagonist of specific ADM receptors) and calcitonin gene-related peptide (CGRP)8–37 (the antagonist of CGRP receptor type 1), to attenuate the action of endogenous ADM, reinforced the aldosterone-induced proliferation and inhibited the intracellular cAMP production stimulated by aldosterone. Thiorphan, an inhibitor of ADM degradation, inhibited the [3H]-thymine incorporation and reinforced the intracellular cAMP level induced by aldosterone. We reach the conclusion that aldosterone stimulates rat cardiac fibroblasts to produce and secrete ADM, which in turn regulates the proliferation-induced effects of aldosterone in these cells.
Keywords: Aldosterone; Adrenomedullin; Cardiac fibroblast; Proliferation; Cyclic adenosine monophosphate; Extracellular signal related kinase activity;

Methylthioadenosine phosphorylase gene expression is impaired in human liver cirrhosis and hepatocarcinoma by Carmen Berasain; Henar Hevia; Jokin Fernández-Irigoyen; Esther Larrea; Juan Caballería; José M. Mato; Jesús Prieto; Fernando J. Corrales; Elena R. García-Trevijano; Matías A. Avila (276-284).
Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine and adenine salvage pathways. In mammals, the liver plays a central role in methionine metabolism, and this essential function is lost in the progression from liver cirrhosis to hepatocarcinoma. Deficient MTAP gene expression has been recognized in many transformed cell lines and tissues. In the present work, we have studied the expression of MTAP in human and experimental liver cirrhosis and hepatocarcinoma. We observe that MTAP gene expression is significantly reduced in human hepatocarcinoma tissues and cell lines. Interestingly, MTAP gene expression was also impaired in the liver of CCl4-cirrhotic rats and cirrhotic patients. We provide evidence indicating that epigenetic mechanisms, involving DNA methylation and histone deacetylation, may play a role in the silencing of MTAP gene expression in hepatocarcinoma. Given the recently proposed tumor suppressor activity of MTAP, our observations can be relevant to the elucidation of the molecular mechanisms of multistep hepatocarcinogenesis.
Keywords: Liver; Methylthioadenosine phosphorylase; Cirrhosis; Hepatocarcinoma; Gene expression;

Author Index (285-287).

Cumulative Contents (288-289).