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BBA - Molecular Basis of Disease (v.1762, #7)

Editorial Board (pp. ii).

Gene expression profiling in vLINCL CLN6-deficient fibroblasts: Insights into pathobiology by C.A.F. Teixeira; S. Lin; M. Mangas; R. Quinta; C.J.P. Bessa; C. Ferreira; M.C. Sá Miranda; R-M.N. Boustany; M.G. Ribeiro (pp. 637-646).

The CLN6 vLINCL is caused by molecular defects in CLN6 gene coding for an ER resident transmembrane protein whose function is unknown. In the present study gene expression profiling of CLN6-deficient fibroblasts using cDNA microarray was undertaken in order to provide novel insights into the molecular mechanisms underlying this neurodegenerative fatal disease. Data were validated by qRT-PCR. Statistically significant alterations of expression were observed for 12 transcripts. The two most overexpressed genes, versican and tissue factor pathway inhibitor 2, are related to extracellular matrix (ECM), predicting changes in ECM-related proteins in CLN6-deficient cells. Transcript profiling also suggested alterations in signal transduction pathways, apoptosis and the immune/inflammatory response. Up-regulated genes related to steroidogenesis or signalling, and the relationship between cholesterol dynamics and glycosphingolipid sorting, led to investigation of free cholesterol and gangliosides in CLN6-deficient fibroblasts. Cholesterol accumulation in lysosomes suggests a homeostasis block as a result of CLN6p deficiency. The cholesterol imbalance may affect structure/function of caveolae and lipid rafts, disrupting signalling transduction pathways and sorting cell mechanisms. Alterations in protein/lipid intracellular trafficking would affect the composition and function of endocytic compartments, including lysosomes. Dysfunctional endosomal/lysosomal vesicles may act as one of the triggers for apoptosis and cell death, and for a secondary protective inflammatory response. In conclusion, the data reported provide novel clues into molecular pathophysiological mechanisms of CLN6-deficiency, and may also help in developing disease biomarkers and therapies for this and other neurodegenerative diseases.

Keywords: Abbreviations; AKR1C3 (3α-HSD); aldo-keto reductase family I, member C3 (3-α-hydroxysteroid dehydrogenase, type II); BCHE; butyrylcholinesterase; CAV2; caveolin 2; CNS; central nervous system; CSPG2; chondroitin sulphate proteoglycan 2 (versican); C1R; complement component 1, r subcomponent; ECM; extracellular matrix; ER; endoplasmic reticulum; ERGIC; endoplasmic reticulum Golgi intermediate compartment; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; GARP; glycoprotein A repetitions predominant; HSPA1B7 (HSP70-2); Human HMC class III heat shock HSP70-2 gene (HLA), 70 kDa protein 1B; INCL; infantile NCL; JNCL; juvenile NCL; LDL; low-density lipoprotein; LINCL; late-infantile NCL; LOXL2; lysyl oxidase-like 2; MATP; membrane-associated transporter protein (AIM1, melanoma antigen AIM1/human non-lens beta gamma-crystallin like complex); NCL; neuronal ceroid lipofuscinosis; PP5; placental protein 5; QPCT; glutaminyl-peptide cyclotransferase (glutaminyl cyclase); qRT-PCR; quantitative real-time PCR; RAP1; GTP-GDP dissociation stimulator 1; RRAS2; Ras-like protein TC21; RTVP1; Glioma pathogenesis-related protein (RTVP-1 protein); TFPI-2; tissue factor pathway inhibitor 2; TRPM2/CLU; Testosterone-repressed message 2 (clusterin, complement lysis inhibitor, SP-40, sulfated glycoprotein 2, apolipoprotein J); vLINCL; variant late-infantile NCLNeuronal Ceroid Lipofuscinoses; CLN6; Genechip analysis; qRT-PCR analysis; Cholesterol

Inhibition of HER-2(neu/ErbB2) restores normal function and structure to polycystic kidney disease (PKD) epithelia by Samantha J. Wilson; Kurt Amsler; Deborah P. Hyink; Xiaohong Li; Weining Lu; Jing Zhou; Christopher R. Burrow; Patricia D. Wilson (pp. 647-655).

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a very common lethal monogenetic disease with significant morbidities and a high likelihood of progression to renal failure for which there is no proven disease-specific therapy currently available for clinical use. Human ADPKD cystic epithelia have proliferative abnormalities mediated by EGFR over-expression and mispolarization leading autocrine response to EGF family ligands. We now show that apical localization of EGFR complexes in normal fetal and ADPKD epithelia is associated with heterodimerization of EGFR(HER-1) with HER-2(neu/ErbB2), while basal membrane localization in normal adult renal epithelia is associated with EGFR(HER-1) homodimers. Since ADPKD epithelial cells have reduced migratory function, this was used as a bioassay to evaluate the ability of compounds to rescue the aberrant human ADPKD phenotype. General tyrosine kinase inhibition by herbimycin and specific inhibition of HER-2(neu/ErbB2) by AG825 or pretreatment with ErbB2 siRNA reversed the migration defect of ADPKD epithelia. Selective inhibition of EGFR(HER-1) showed partial rescue. Increased ADPKD cell migration after inhibition of p38MAP kinase but not of PI3-kinase implicated p38MAPK downstream of HER-2(neu/ErbB2) stimulation. Daily administration of AG825 to PKD1 null heterozygous mice significantly inhibited the development of renal cysts. These studies implicate HER2(neu/ErbB2) as an effector of apical EGFR complex mispolarization and that its inhibition should be considered a candidate for clinical therapy of ADPKD.

Keywords: EGF receptor; Polarity; Signal transduction; Renal development; Small molecule therapy; Tyrosine kinase; Dimerization; PKD null mouse; siRNA

Hyperhomocysteinemia induces liver injury in rat: Protective effect of folic acid supplementation by Connie W.H. Woo; Gamika A. Prathapasinghe; Yaw L. Siow; Karmin O (pp. 656-665).

Hyperhomocysteinemia, a condition of elevated blood homocysteine level, is an independent risk factor for cardiovascular diseases. Hyperhomocysteinemia is also found in patients with liver diseases. However, the direct effect of homocysteine on liver injury is not well known. Folic acid supplementation is a promising approach for improving endothelial function in patients with hyperhomocysteinemia. The aim of this study was to investigate the direct effect of hyperhomocysteinemia on liver injury and whether folic acid could offer any protective effect to the liver. Hyperhomocysteinemia was induced in rats fed a high-methionine diet for 4 weeks. There was a significant increase in the serum aspartate aminotransferase and alanine aminotransferase activities reflecting liver injury in hyperhomocysteinemic rats. Hepatic NAD(P)H oxidase was activated during hyperhomocysteinemia leading to increased superoxide anion production and peroxynitrite formation in the liver. As a consequence, the level of lipid peroxides was significantly elevated in livers of hyperhomocysteinemic rats. Folic acid supplementation effectively inhibited NAD(P)H oxidase-mediated superoxide anion production leading to reduced lipid peroxidation in the liver. Folic acid supplementation also alleviated hyperhomocysteinemia-induced liver injury. These results suggest that hyperhomocysteinemia can cause liver injury and supplementation of folic acid offers a hepatoprotective effect.

Keywords: Homocysteine; Folic acid; Oxidative stress; Liver function

Long-chain Acyl-CoA is not primarily increased in myotubes established from type 2 diabetic subjects by Malene Just; Nils J. Færgeman; Jens Knudsen; Henning Beck-Nielsen; Michael Gaster (pp. 666-672).

Accumulation of intramuscular long-chain acyl-CoA esters (LCACoA) has previously in animal and human models been suggested to play an important role in lipid induced insulin resistance. The aim of this study was to examine whether myotubes established from type 2 diabetic (T2D) subjects and lean controls express differences in long-chain acyl-CoA esters (LCACoA) precultured under physiological conditions and during chronic exposure to palmitate (PA) and oleic acids (OA) with/without acute insulin stimulation. No significant differences were found between diabetic and control myotubes, neither in the total amount nor among individual LCA–CoA species during basal and acute insulin stimulation. LCA–CoA accumulated during exposure to palmitic acid but not during exposure to oleic acid. During PA and OA exposure, only palmitoyl-CoA, oleoyl-CoA and total LCA–CoA change. PA exposure increased the palmitoyl-CoA, whereas oleoyl-CoA was reduced and vice versa during OA exposure. No differences were found in the LCA–CoA level between T2D and control subjects, neither in the total amount nor in the individual specific LCA–CoA species during fatty acid exposure. Chronic (24 h), high PA, but not OA exposure induced insulin resistance at the level of glycogen synthesis in control subjects. These results indicate that (1) no primary defects are responsible for LCA–CoA accumulation in diabetic subjects; (2) LCA–CoA changes in vivo are partly adaptive to changes in the PA level and possibly other saturated fatty acids; and (3) PA induced insulin resistance may be mediated through an increased level of palmitoyl-CoA.

Keywords: Abbreviations; T2D; type 2 diabetes; LCACoA; long-chain acyl-CoA esters; TAG; triacylglycerol; DAG; diacylglycerol; OA; oleic acid; PA; palmitic acid; HI; high insulin concentration; BSA; bovine serum albumin; FCS; fetal calf serum; GIR; glucose infusion rates; FFA; free fatty acid; HPLC; high pressure liquid chromatographyAcyl-CoA; Insulin resistance; Free fatty acid; Myotube; Type 2 diabetes; Oleic acid; Palmitic acid

Renal elimination of p-aminohippurate (PAH) in response to three days of biliary obstruction in the rat. The role of OAT1 and OAT3 by Anabel Brandoni; Naohiko Anzai; Yoshikatsu Kanai; Hitoshi Endou; Adriana Mónica Torres (pp. 673-682).

Pharmacokinetic studies of the drugs administered to subjects with mechanical cholestasis are scarce. The purpose of the present study was to examine the effects of bile duct ligation of 3 days (peak of elevation of serum bile acids and bilirubin) on the systemic and renal PAH clearance and on the expression of cortical renal OAT1 and OAT3 in a rat model. PAH is the prototypical substrate of the renal organic anion transport system. Male Wistar rats underwent a bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. BDL rats displayed a significantly lower systemic PAH clearance. Renal studies revealed a reduction in the renal clearance and in the excreted and secreted load of PAH in BDL rats. The OAT1 protein expression in kidney homogenates was not modified, but it decreased in the basolateral membranes from BDL rats. In contrast, OAT3 abundance in both kidney cortex homogenates and in basolateral membranes increased by 3 days after the ligation. Immunocytochemical studies (light microscopic and confocal immunofluorescence microscopic analyses) confirmed the changes in the renal expression of these transport proteins. The present study demonstrates the key role of OAT1 expression in the impaired elimination of PAH after 3 days of obstructive cholestasis.

Keywords: p-aminohippurate; Cholestasis; Renal depuration; OAT1; OAT3

In vitro tau fibrillization: Mapping protein regions by Ismael Santa-María; Mar Pérez; Félix Hernández; Victor Muñoz; Francisco J. Moreno; Jesús Avila (pp. 683-692).

We have investigated the propensity to form fibrillar aggregates of a variety of fragments and variants of the tau protein under the influence of a tau fibrillization inducer: coenzyme Q₀. To better identify fibrillization hotspots, we compare the polymerization propensity of tau fragments containing the sequence of putative hotspots with that of tau variants with that same sequence deleted. We also investigate the effects of biologically occurring modifications such as phosphorylation and deamidation. We found that residues 305 to 335 are essential for in vitro tau fibrillization. Residues 306 to 311 facilitate in vitro assembly, but are not sufficient to mimic the in vivo fibrillization of tau. Furthermore, the propensity of the 306–311 sequence to form fibrils is highly decreased by chemical modifications of tyrosine 310 that are commonly found in vivo.

Keywords: Tau; Polymerization; Coenzyme Q; ₀; Tau variant

Peptides derived from HIV-1, HIV-2, Ebola virus, SARS coronavirus and coronavirus 229E exhibit high affinity binding to the formyl peptide receptor by John S. Mills (pp. 693-703).

Peptides derived from the membrane proximal region of fusion proteins of human immunodeficiency viruses 1 and 2, Coronavirus 229 E, severe acute respiratory syndrome coronavirus and Ebola virus were all potent antagonists of the formyl peptide receptor expressed in Chinese hamster ovary cells. Binding of viral peptides was affected by the naturally occurring polymorphisms at residues 190 and 192, which are located at second extracellular loop–transmembrane helix 5 interface. Substitution of R190 with W190 enhanced the affinity for a severe acute respiratory syndrome coronavirus peptide 6 fold but reduced the affinity for N- formyl-Nle–Leu–Phe by 2.5 fold. A 12 mer peptide derived from coronavirus 229E (ETYIKPWWVWL) was the most potent antagonist of the formyl peptide receptor W190 with a K_i of 230 nM. Fluorescently labeled ETYIKPWWVWL was effectively internalized by all three variants with EC₅₀ of ∼25 nM. An HKU-1 coronavirus peptide, MYVKWPWYVWL, was a potent antagonist but N- formyl-MYVKWPWYVWL was a potent agonist. ETYIKPWWVWL did not stimulate GTPγS binding but inhibited the stimulation by formyl-NleLeuPhe. It also blocked β arrestin translocation and receptor downregulation induced by formyl-Nle–Leu–Phe. This indicates that formyl peptide receptor may be important in viral infections and that variations in its sequence among individuals may affect their likelihood of viral and bacterial infections.

Keywords: Abbreviations; FPR; formyl peptide receptor; CHO S; Chinese hamster ovary cells designed for suspension culture; HRSV; human respiratory syncytial virus; FIV; feline immunodeficiency virus; fMLF; N-formyl-methionyl-leucyl-phenylalanine; AIDS; Acquired Immunodeficiency Syndrome; SIV; Simian Immunodeficiency Virus; HIV; human immunodeficiency virus; SARS; severe acute respiratory syndrome; GP-41; 41 kilodalton glycoprotein; HR; Heptade Repeat; FITC; Fluorescein isothiocyanate; formyl-Nle–Leu–Phe–Nle–Tyr–Lys–FITC; formyl-Nle–Leu–Phe–Nle–Tyr–Lys labeled at the Lys residue with Fluorescein isothiocyanate; formyl-Nle–Leu–Phe–Nle–Tyr–Lys–Alexa Fluor; formyl-Nle–Leu–Phe–Nle–Tyr–Lys labeled at the Lys residue with Alexa Fluor N-hydroxy-succinimide; ETYIK-(Alexa Fluor)WPWWVWL; ETYIKWPWWVWL labeled with Alexa Fluor 488 N-hydroxy-succinimide; GTPγS; guanosine 5′-3-O-(thio)triphosphate; TMH; transmembrane helix; FPRL1; formyl peptide like receptor 1; FPRL2; formyl peptide receptor like 2Virus; Signal Transduction; G protein coupled receptor; Polymorphism

Identification and membrane localization of electrogenic sodium bicarbonate cotransporters in Calu-3 cells by James L. Kreindler; Kathryn W. Peters; Raymond A. Frizzell; Robert J. Bridges (pp. 704-710).

Cystic fibrosis (CF) is a severely life-shortening genetic disease resulting from mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR). Impaired bicarbonate secretion is a key component of CF-related pancreatic disease, but the role of impaired bicarbonate secretion in CF lung disease is less well understood. The submucosal glands of the conducting airways produce and secrete a complex airway surface liquid that lines the airway epithelium and plays a significant role in mucociliary clearance. The serous cell is the predominant cell type of the submucosal gland and a predominant site of CFTR expression. Calu-3 cells are a model of airway submucosal gland serous cells that demonstrates vectorial bicarbonate secretion in response to elevations in cAMP. Based on previously published measurements of unidirectional ion flux, pharmacological inhibition of short-circuit current and ion substitution studies, one can hypothesize the existence of an electrogenic sodium bicarbonate cotransporter (NBC) in the basolateral membrane of Calu-3 cells that mediates bicarbonate entry from the interstitium. To test this hypothesis, we performed reverse-transcriptase PCR, western blotting, and surface biotinylation to identify and localize electrogenic NBCs in Calu-3 cells. Our data demonstrate that both pNBC1 and NBC4 mRNAs can be identified and that their protein products are expressed at the basolateral membrane of polarized Calu-3 cells. These data suggest that these transporters contribute to regulated bicarbonate secretion across Calu-3 cells and perhaps human airway submucosal glands.

Keywords: Sodium bicarbonate cotransporter; Bicarbonate; Cystic fibrosis; Calu-3 cell

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BBA - Molecular Basis of Disease (v.1762, #7)