BBA - Molecular Basis of Disease (v.1772, #1)

A word of farewell and a word of welcome by Dennis Vance; Peter van der Vliet (1-2).

BBA in the year 2007 by Dennis E. Vance (3-4).

One of the major conceptual advances in the understanding of the pathogenesis of heart failure has been the insight that myocardial dysfunction and heart failure may progress as the result of the sustained over-expression of nitric oxide (NO) metabolites locally and in blood modulated by inducible nitric oxide synthase (iNOS). This by virtue of their deleterious effects is sufficient to contribute to disease progression by provoking left ventricular (LV) remodeling, hypertrophy and progressive LV dysfunction. Recently, tumor necrosis factor-alpha (TNF-α) has also been identified in this setting of heart failure. Analogous to the situation with NO, the over-expression of TNF-α is sufficient to contribute to disease progression in heart failure phenotype. Although important interactions between TNF-α and the NO have been recognized in the cardiovascular system for over a decade, the nature and importance of the interactions between these biologically active molecules in cardiac hypertrophy has become apparent only in the recent times. Therefore, we focused on the prevailing updated evidence which suggests that there is a functionally significant cross-regulation between NO and TNF-α signaling in blood thus playing a part in cardiac hypertrophy and failure. The discussions presented here will have a bearing on the therapeutic potential via inhibitors of these pathways in reducing cardiomyocyte hypertrophy and the LV dysfunction.
Keywords: TNF-α; Proinflammatory cytokines; Nitric oxide; Cardiomyopathy; Left ventricular function;

Quantification of chloride channel 2 (CLCN2) gene isoforms in normal versus lesion- and epilepsy-associated brain tissue by Matteo Bertelli; Stefano Cecchin; Cristina Lapucci; Paola de Gemmis; Daniela Danieli; Emanuele S.G. d'Amore; Luciano Buttolo; Filippo Giunta; Pietro Mortini; Massimo Pandolfo (15-20).
The chloride channel 2 (CLCN2) gene codes for a protein organized in N- and C-terminal regions with regulatory functions and a transmembrane region which forms the ring of the pore. Mutations in the gene have previously been described in patients with idiopathic familial epilepsy. In this study we looked for new isoforms of CLCN2 and we estimated expression levels by real time PCR in brain tissue containing epileptic foci. Samples used in this study were first analyzed and selected to exclude mutations in the coding region of the gene. Four isoforms (skipping exons 3, 16, 22 and 6/7) were identified and quantified by Real Time PCR and compared with total expression of the gene. Expression of the region common to all CLCN2 isoforms was 50% less in epilepsy-associated brain tissue than in controls. The ratio of the various isoforms was slightly greater in epileptic than control tissue. The greatest difference was recorded in the temporal lobe for the isoform with skipped exon 22. Analysis of these isoforms in brain tissue containing epileptic foci suggests that CLCN2 could be implicated in epilepsy, even in the absence of mutations.
Keywords: Epilepsy; Chloride Channel 2; Alternative splicing; Real Time PCR;

Preliminary observation of elevated levels of nanocrystalline iron oxide in the basal ganglia of neuroferritinopathy patients by Dimitri Hautot; Quentin A. Pankhurst; Chris M. Morris; Andrew Curtis; John Burn; Jon Dobson (21-25).
Magnetometry analysis of brain tissue sub-samples from two neuroferritinopathy patients provides a preliminary indication that the amount of magnetic iron compounds associated with this rare disease is significantly larger than in age/sex-matched controls. The primary iron compounds contributing to the remnant magnetization of the tissue above 50 K and at body temperature are both blocked and superparamagnetic (SPM) biogenic magnetite (Fe3O4) and/or maghemite (γ-Fe2O3). The concentration of SPM magnetite is significant and appears to be proportional to the concentration of ferritin, which varies with progression of the disease. The mutated ferritin protein appears to be responsible for the presence of iron oxide nano-particules, which in turn could be responsible for extensive damage in the brain.
Keywords: Neuroferritinopathy; Magnetite; Verwey transition; SQUID magnetometry; Iron; Nanoparticle; Biomineralization; Neurodegenerative disease;

Up-regulation of NPY gene expression in hypothalamus of rats with experimental chronic renal failure by Elzbieta Sucajtys-Szulc; Joanna Karbowska; Zdzislaw Kochan; Wojciech Wolyniec; Michal Chmielewski; Boleslaw Rutkowski; Julian Swierczynski (26-31).
Anorexia is possibly one of the most important causes of malnutrition in uremic patients. The cause of this abnormality is still unknown. Considering that: (a) NPY is one of the most important stimulants of food intake; (b) eating is a central nervous system regulated process and (c) NPY is expressed in hypothalamus, we hypothesized that the decrease of NPY gene expression in the hypothalamus could be an important factor contributing to anorexia associated with uremic state. In contrast to the prediction, the results presented in this paper indicate that the NPY gene expression in the hypothalamus of chronic renal failure (CRF) rats was significantly higher than in the hypothalamus of control (pair-fed) rats. Moreover, we found that serum NPY concentration in CRF rats was higher than in control (pair-fed) animals. The increase of plasma NPY concentration in CRF rats may be due to the greater synthesis of the neuropeptide in liver, since higher level of NPY mRNA was found in liver of CRF rats. The results obtained revealed that experimental chronic renal failure is associated with the increase of NPY gene expression in hypothalamus and liver of rats.
Keywords: NPY; Hypothalamus; Liver; Chronic renal failure;

Lipopolysaccharide attenuates mRNA levels of several adenylyl cyclase isoforms in vivo by Petter Kirkeby Risøe; Yunyong Wang; Jon Fredrik Stuestøl; Ansgar O. Aasen; Jacob E. Wang; Maria K. Dahle (32-39).
Signals that elevate intracellular levels of cyclic adenosine monophosphate (cAMP) are among the factors that control lipopolysaccharide (LPS)-mediated inflammatory mediator production by macrophages. cAMP signaling is also involved in maintaining body functions that are commonly impaired in sepsis, including the endothelial cell barrier function and heart function. Several agents successfully used for sepsis intervention target cAMP signaling, and it was recently shown that liver and lung may be protected from inflammation injury by cAMP-elevating phosphodiesterase inhibitors. Here, we show that LPS attenuates adenylyl cyclase (AC) mRNA levels in liver, lung, heart, spleen and kidney in an animal model of endotoxemia, and in macrophages from liver and lung. In particular, AC5, AC6, AC7 and AC9 mRNA were reduced in most tissues examined and in tissue macrophages. In Kupffer cells, prostaglandin E2-mediated cAMP production was inhibited by LPS treatment. The reduction in AC mRNA by LPS would be expected to lead to a lowered potential for cAMP production in most organs, and in particular, changes in AC6 mRNA may affect endothelial cell barrier function and heart function. In contrast, AC4 mRNA was elevated in heart and lung. The present work indicates a possible mechanism for LPS-mediated alteration of cAMP signaling in vivo.
Keywords: Adenylyl cyclase; cAMP; Lipopolysaccharide; Endotoxemia; Inflammation;

Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53 by Pedro M. Borralho; Isabel B. Moreira da Silva; Márcia M. Aranha; Cristina Albuquerque; Carlos Nobre Leitão; Clifford J. Steer; Cecília M.P. Rodrigues (40-47).
Drug resistance to 5-fluorouracil (5-FU) is still a major limitation to its clinical use. In addition, the clinical value of p53 as a predictive marker for 5-FU-based chemotherapy remains a matter of debate. Here, we used HCT116 human colorectal cancer cells expressing wild-type p53 and investigated whether inhibition of Fas expression by interference RNA modulates 5-FU-induced apoptosis. Cells were treated with 5-FU (1, 4 or 8 μM) for 8–48 h. Cell viability was evaluated by trypan blue dye exclusion. Apoptosis was assessed by changes in nuclear morphology and caspase activity. The interference RNA technology was used to silence Fas expression. Caspase activation, p53, Fas, cytochrome c, and Bcl-2 family protein expression was evaluated by immunoblotting. 5-FU was cytotoxic in HCT116 cells (p  < 0.001). Nuclear fragmentation and caspase-3, -8 and -9 activities were also markedly increased in HCT116 cells after 5-FU (p  < 0.001). In addition, wild-type p53 and Fas expression were 25- and 4-fold increased (p  < 0.05). Notably, when interference RNA was used to inhibit Fas, 5-FU-mediated nuclear fragmentation and caspase activity were markedly reduced in HCT116 cells. Finally, western blot analysis of mitochondrial extracts from HCT116 cells exposed to 5-FU showed a 6-fold increase in Bax, together with a 3-fold decrease in cytochrome c (p  < 0.001). In conclusion, 5-FU exerts its cytotoxic effects, in part, through a p53/Fas-dependent apoptotic pathway that involves Bax translocation and mitochondrial permeabilization.
Keywords: Apoptosis; Fas; 5-fluorouracil; Mitochondria; p53; siRNA;

Carcinogen-induced early molecular events and its implication in the initiation of chemical hepatocarcinogenesis in rats: Chemopreventive role of vanadium on this process by Tridib Chakraborty; Amrita Chatterjee; Ajay Rana; Duraisami Dhachinamoorthi; Ashok Kumar P; Malay Chatterjee (48-59).
Carcinogen-induced formation of DNA adducts and other types of DNA lesions are the critical molecular events in the initiation of chemical carcinogenesis and modulation of such events by chemopreventive agents could be an important step in limiting neoplastic transformation in vivo. Vanadium, a dietary micronutrient has been found to be effective in several types of cancers both in vivo and in vitro and also possesses profound anticarcinogenicity against rat models of mammary, colon and hepatocarcinogenesis. Presently, we report the chemopreventive potential of vanadium on diethylnitrosamine (DEN)-induced early DNA damages in rat liver. Hepatocarcinogenesis was induced in male Sprague–Dawley rats with a single, necrogenic, intraperitoneal (i.p.) injection of DEN (200 mg/kg body weight) at week 4. There was a significant induction of tissue-specific ethylguanines, steady elevation of modified DNA bases 8-hydroxy-2′-deoxyguanosines (8-OHdGs) (P  < 0.0001; 89.93%) along with substantial increment of the extent of single-strand breaks (SSBs) (P  < 0.0001) following DEN exposure. Supplementation of 0.5 ppm of vanadium throughout the experiment abated the formations of O6-ethylguanines and 7-ethylguanines (P  < 0.0001; 48.71% and 67.54% respectively), 8-OHdGs (P  < 0.0001; 81.37%), length:width (L:W) of DNA mass (P  < 0.01; 62.12%) and the mean frequency of tailed DNA (P  < 0.001; 53.58%), and hepatic nodulogenesis in preneoplastic rat liver. The study indicates that 0.5 ppm vanadium is potentially and optimally effective, as derived from dose–response studies, in limiting early molecular events and preneoplastic lesions, thereby modulating the initiation stage of hepatocarcinogenesis. Vanadium is chemopreventive against DEN-induced genotoxicity and resulting hepatocellular transformation in rats.
Keywords: Vanadium; Hepatocarcinogenesis; DNA adduct; Ethylguanines; 8-OHdGs; DNA strand-break;

Characterization of the molecular mechanisms involved in the increased insulin secretion in rats with acute liver failure by Masashi Kuwahata; Yuka Tomoe; Nagakatsu Harada; Saki Amano; Hiroko Segawa; Sawako Tatsumi; Mikiko Ito; Tatsuzo Oka; Ken-ichi Miyamoto (60-65).
To investigate the mechanism of hyperinsulinaemia in rats with acute liver failure induced by the administration of d-galactosamine (GalN), we focused on the role of polyprimidine tract-binding protein (PTB) in islet insulin synthesis. Recent reports indicate that PTB binds and stabilizes mRNA encoding insulin and insulin secretory granule proteins, including islet cell autoantigen 512 (ICA512), prohormone convertase 1/3 (PC1/3), and PC2. In the present study, glucose-stimulated insulin secretion was significantly increased in GalN-treated rats compared to controls. Levels of mRNA encoding insulin 1, ICA512, and PC1/3 were increased in the pancreatic islets of GalN-treated rats. This mRNA level elevation was not prevented by pretreatment with actinomycin D. When the PTB-binding site in insulin 1 mRNA was incubated with the islet cytosolic fraction, the RNA–protein complex level was increased in the cytosolic fraction obtained from GalN-treated rats compared to the level in control rats. The cytosolic fraction obtained from pancreatic islets obtained from GalN-treated rats had an increased PTB level compared to the levels obtained from the pancreatic islets of control rats. These findings suggest that, in rats with acute liver failure, cytosolic PTB binds and stabilizes mRNA encoding insulin and its secretory granule proteins.
Keywords: Insulin mRNA; Insulin secretory granule protein mRNAs; Polypyrimidine tract-binding protein; Hyperinsulinaemia; Acute liver failure;

Effects of retinoic acid on the development of liver fibrosis produced by carbon tetrachloride in mice by Lan Wang; James J. Potter; Lynda Rennie-Tankersley; Gennadiy Novitskiy; Jennifer Sipes; Esteban Mezey (66-71).
The role of retinoic acid (RA) in liver fibrogenesis was previously studied in cultured hepatic stellate cells (HSCs). RA suppresses the expression of α2(I) collagen by means of the activities of specific nuclear receptors RARα, RXRβ and their coregulators. In this study, the effects of RA in fibrogenesis were examined in carbon tetrachloride (CCl4) induced liver fibrosis in mice. Mice were treated with CCl4 or RA and CCl4, along side control groups, for 12 weeks. RA reduced the amount of histologically detectable fibrosis produced by CCl4. This was accompanied by a attenuation of the CCl4 induced increase in α2(I) collagen mRNA and a lower (2-fold versus 3-fold) increase in liver hydroxyproline. Furthermore, RA reduced the levels of 3-nitrotyrosine (3-NT) protein adducts and thiobarbituric acid (TBA) reactive substance (TBARS) in the liver, which are formed as results of oxidative stress induced by CCl4 treatment. These in vivo findings support our previous in vitro studies in cultured HSC of the inhibitory effect of RA on type I collagen expression. The data also provide evidence that RA reduces CCl4 induced oxidative stress in liver, suggesting that the anti-fibrotic role of RA is not limited to the inhibition of type I collagen expression.
Keywords: Liver fibrosis; Collagen; Retinoic acid; Carbon tetrachloride; Oxidative stress;

Epigenetic changes in estrogen receptor β gene in atherosclerotic cardiovascular tissues and in-vitro vascular senescence by Jei Kim; Jee Yeon Kim; Kyu Sang Song; Young Ho Lee; Joong Seok Seo; Jaroslav Jelinek; Pascal J. Goldschmidt-Clermont; Jean-Pierre J. Issa (72-80).
Epigenetic changes marked by DNA methylation have been proposed to play a role in age-related disease. We investigated DNA methylation changes in cardiovascular atherosclerotic tissues and in-vitro vascular senescence in the promoter of estrogen receptor β gene, which has essential roles in vascular function. Coronary atherosclerotic tissues showed higher methylation levels (28.7%) than normal appearing arterial (6.7%–10.1%) and venous tissues (18.2%). In comparing estrogen receptor β methylation between plaque and non-plaque regions in ascending aorta, common carotid artery, and femoral artery of two patients, the plaque lesions showed consistently higher methylation levels than non-plaque regions. Passage-dependent increased estrogen receptor β methylation was observed in three of six human aortic endothelial or smooth muscle cell lines cultured in-vitro to vascular senescence. Estrogen receptor β expression in these vascular cell lines was significantly activated by DNA-methyltransferase inhibition. This activity was augmented by histone deacetylase inhibition. These findings provide evidence of epigenetic dysregulation of estrogen receptor β in atherosclerosis and vascular aging. We suggest that focal epigenetic changes in estrogen receptor β contribute to the development of atherosclerosis and vascular aging.
Keywords: Aging; Atherosclerosis; DNA methylation; Epigenetic dysregulation; Estrogen receptor beta; In-vitro senescence;

Haplotype analyses of the APOA5 gene in patients with familial combined hyperlipidemia by Gerly M. van der Vleuten; Aaron Isaacs; Wu-Wei Zeng; Ewoud ter Avest; Philippa J. Talmud; Geesje M. Dallinga-Thie; Cornelia M. van Duijn; Anton F.H. Stalenhoef; Jacqueline de Graaf (81-88).
Background: Familial combined hyperlipidemia (FCH) is the most common genetic lipid disorder with an undefined genetic etiology. Apolipoprotein A5 gene (APOA5) variants were previously shown to contribute to FCH. The aim of the present study was to evaluate the association of APOA5 variants with FCH and its related phenotypes in Dutch FCH patients. Furthermore, the effects of variants in the APOA5 gene on carotid intima-media thickness (IMT) and cardiovascular disease (CVD) were examined. Materials and methods: The study population consisted of 36 Dutch families, including 157 FCH patients. Two polymorphisms in the APOA5 gene (− 1131T > C and S19W) were genotyped. Results: Haplotype analysis of APOA5 showed an association with FCH (p  = 0.029), total cholesterol (p  = 0.031), triglycerides (p  < 0.001), apolipoprotein B (p  = 0.011), HDL-cholesterol (p  = 0.013), small dense LDL (p  = 0.010) and remnant-like particle cholesterol (p  = 0.001). Compared to S19 homozygotes, 19W carriers had an increased risk of FCH (OR  =  1.6 [1.0–2.6]; p  = 0.026) and a more atherogenic lipid profile, reflected by higher triglyceride (+ 22%) and apolipoprotein B levels (+ 5%), decreased HDL-cholesterol levels (− 7%) and an increased prevalence of small dense LDL (16% vs. 26%). In carriers of the − 1131C allele, small dense LDL was more prevalent than in − 1131T homozygotes (29% vs. 16%). No association of the APOA5 gene with IMT and CVD was evident. Conclusion: In Dutch FCH families, variants in the APOA5 gene are associated with FCH and an atherogenic lipid profile.
Keywords: Familial combined hyperlipidemia; APOA5 gene; Haplotype; Triglyceride level; Intima-media thickness; Remnant-like particle;

Ovol1 encodes a zinc finger transcriptional repressor that is downstream of the LEF1/β-catenin complex, nuclear effectors of canonical Wnt signaling. Previous gene knockout studies performed in a 129Sv × C57BL/6 mixed genetic background revealed that Ovol1-deficient mice survive to adulthood but display multiple tissue defects. In this study, we describe a C57BL/6 strain-specific reduction in perinatal survival of Ovol1 mutant mice. The perinatal lethality is accompanied by kidney epithelial cysts of embryonic onset and delayed skin barrier acquisition. Genetic analysis suggests a partial functional compensation by Ovol2 for the loss of Ovol1. The expression of Ovol2 was up-regulated in Ovol1-deficient epidermis, and Ovol1 represses the activity of Ovol2 promoter in a DNA binding-dependent manner. Collectively, these studies uncover novel functions of Ovol1 in mouse development and identify Ovol2 as a downstream target of Ovol1.
Keywords: Ovol1; Ovol2; Perinatal lethality; Skin barrier; Cystic kidney; Transcriptional repression;

The ITPA c.94C > A and g.IVS2 + 21A > C sequence variants contribute to missplicing of the ITPA gene by Monica Arenas; John Duley; Satoshi Sumi; Jeremy Sanderson; Anthony Marinaki (96-102).
Inosine triphosphate pyrophosphatase (ITPase) catalyzes the conversion of inosine triphosphate (ITP) to the correspondent monophosphate. The ITPA c.94C > A and g.IVS2 + 21A > C allelic variants are associated with decreased red cell enzyme activity. The ITPA c.94C > A [P32T] sequence variant is associated with an increased risk of adverse drug reactions in patients treated with the thiopurine drug azathioprine. The aim of this study was to explore the molecular mechanisms of ITPase deficiency. ITPA mRNA was extracted from peripheral blood leukocytes (PBL), Epstein–Barr virus transformed lymphoblast cell cultures, reticulocytes, and cultured fibroblast from patients with known ITPA genotypes. ITPA mRNA was reversed transcribed, sequenced and the relative amounts of misspliced transcripts quantitated from three independent experiments. The ITPA g.IVS2 + 21A > C sequence variant resulted in missplicing of exon 3. The ITPA c.94C > A allelic variant resulted in missplicing of exons 2 and 3 representing, in PBL samples, 61% of the total mRNA expressed in ITPA c.94C > A homozygotes. We proposed that the ITPA c.94C > A allelic variant destroys an exonic splicing silencing (ESS) element in exon 2, resulting in the activation of two nearby upstream 5′ splice sites and missplicing of the exons 2 and 3 cassette causing structural changes to the enzyme and contributing to ITPase deficiency.
Keywords: ITPA; ITPase, Inosine triphosphate pyrophosphatase; Azathioprine; ADR, Adverse drug reaction; Pharmacogenetic; Cryptic 5′ splice site; Missplicing;

Acknowledgment (103-105).