BBA - Molecular Basis of Disease (v.1689, #1)

The prion-like protein termed Doppel (Dpl) shows approx. 25% sequence identity with all known prion proteins (PrP). We recently showed that the cellular PrP is dimeric under native conditions, a finding which was confirmed by the investigation of its crystal structure. Human PrP further interacts with its cellular receptor, the 37 kDa/67 kDa laminin receptor (LRP/LR). Here we report that human Doppel fails to interact with (i) itself, (ii) the human 37 kDa/67 kDa LRP/LR, and (iii) the human cellular prion protein (huPrP) in the yeast two-hybrid system. Our findings suggest that Dpl and PrP are not related or are only marginally related with respect to their ligand binding behaviour.
Keywords: Doppel; Dpl; Prion; PrP; 37 kDa/67 kDa laminin receptor; LRP/LR; Yeast two-hybrid system; Scrapie; Ligand;

High concentrations of magnesium modulate vascular endothelial cell behaviour in vitro by Jeanette A.M Maier; Daniela Bernardini; Yves Rayssiguier; Andrzej Mazur (6-12).
Magnesium supplementation has been reported to prevent cardiovascular diseases through the decrease of plasma lipids and to improve endothelial function in patients with coronary artery disease. In the present work, we evaluated whether high magnesium concentrations can directly affect the function of cultured endothelial cells, which play a crucial role in maintaining the functional integrity of the vascular wall. We cultured human umbilical vein endothelial cells for various times in media containing different concentration of magnesium (range 2 to 10 mM) and compared them to the corresponding controls (1 mM Mg). High Mg concentrations stimulated endothelial proliferation, enhanced the motogenic response to angiogenic factors and attenuated the response to lipopolysaccharide (LPS). In addition, we demonstrate that high concentrations of magnesium did not modulate the levels of plasminogen activator inhibitor-1, but enhanced the synthesis of nitric oxide, in part through the up-regulation of endothelial nitric oxide synthase.Our results demonstrate a direct role of magnesium in maintaining endothelial function. We therefore anticipate that magnesium may have a protective effect against atherosclerosis and could play a role in promoting the growth of collateral vessels in chronic ischemia. Moreover, because it induces the synthesis of nitric oxide, this cation could be a helpful tool in hypertension as well as in preventing thrombosis.
Keywords: Endothelial cell; Magnesium; Atherosclerosis; Nitric oxide;

Low magnesium promotes endothelial cell dysfunction: implications for atherosclerosis, inflammation and thrombosis by Jeanette A.M Maier; Corinne Malpuech-Brugère; Wioletta Zimowska; Yves Rayssiguier; Andrzej Mazur (13-21).
Because (i) endothelial cells are important players in cardiovascular diseases and (ii) Mg deficiency promotes atherosclerosis, thrombosis and hypertension, we evaluated whether low concentrations of Mg could directly affect endothelial behavior. We found that low Mg concentrations reversibly inhibit endothelial proliferation, and this event correlates with a marked down-regulation of the levels of CDC25B. The inhibition of endothelial proliferation is due to an up-regulation of interleukin-1 (IL-1), since an antisense oligonucleotide against IL-1 could prevent the growth inhibition observed in cells exposed to low concentrations of the cation. We also report the up-regulation of Vascular Cell Adhesion Molecule-1 (VCAM) and Plasminogen Activator Inhibitor (PAI)-1 after Mg deficiency. VCAM is responsible, at least in part, of the increased adhesion of monocytoid U937 cells to the endothelial cells grown in low magnesium. In addition, endothelial migratory response is severely impaired. By cDNA array, we identified several transcripts modulated by exposure to low Mg, some of which—c-src, ezrin, CD9, cytohesin and zyxin—contribute to endothelial adhesion to substrates and migration.In conclusion, our results demonstrate a direct role of low magnesium in promoting endothelial dysfunction by generating a pro-inflammatory, pro-thrombotic and pro-atherogenic environment that could play a role in the pathogenesis cardiovascular disease.
Keywords: Endothelial cell; Magnesium; Atherosclerosis; Gene expression; Inflammation;

Pro-inflammatory cytokine release after shock is central in the development of subsequent multiple organ dysfunction syndrome. Some studies suggest that interleukin-10 (IL-10) is an immunosuppressive mediator after injury or sepsis, while others suggest that IL-10 is an important regulator of the pro-inflammatory response. We hypothesized that in a model of trauma and hemorrhagic shock (TH), IL-10 regulates pro-inflammatory cytokine activity via an autocrine effect on cytokine mRNA transcription in Kupffer cells early after TH. To study this, male C3H/HeN mice were sham-operated or subjected to TH. Plasma levels of TNF-α, IL-6 and PGE2 were elevated following TH. A sharp peak in IL-10 levels was observed at 2 h after the insult. Kupffer cell (KC) depletion prior to TH reduced plasma IL-6, IL-10 and TNF-α levels, whereas treatment with anti-IL-10 after TH increased IL-6 and TNF-α levels. Kupffer cell mRNA expression for IL-6, IL-10 and TNF-α was elevated in the TH group and further increased by anti-IL-10 treatment. These findings indicate that KC-dependent IL-10 regulates the early systemic inflammatory response after TH. Thus, while IL-10 is an important mediator of immunosuppression following traumatic injury, it also is beneficial with regard to its ability to counter-regulate the early inflammatory response under such conditions.
Keywords: Kupffer cell; Immune response; Cytokine; Anti-IL-10; GdCl2;

Phagocytosis of photoreceptor outer segments (OS) by retinal pigment epithelium (RPE) is essential for OS renewal and survival of photoreceptors. Internalized, oxidatively modified macromolecules perturb the lysosomal function of the RPE and can lead to impaired processing of photoreceptor outer segments. In this study, we sought to investigate the impact of intracellular accumulation of oxidatively damaged lipid–protein complexes on maturation and distribution of cathepsin D, the major lysosomal protease in the RPE. Primary cultures of human RPE cells were treated with copper-oxidized low density lipoprotein (LDL) and then challenged with serum-coated latex beads to stimulate phagocytosis. Three observations were noted to occur in this experimental system. First, immature forms of cathepsin D (52 and 46 kDa) were exclusively associated with latex-containing phagosomes. Second, maturation of cathepsin D was severely impaired in RPE cells loaded with oxidized LDL (oxLDL) prior to the phagocytic challenge. Third, pre-treatment with oxLDL caused sustained secretion of pro-cathepsin D and the latent form of gelatinase A into the extracellular space in a dose-dependent manner. These data stimulate the hypothesis that intracellular accumulation of poorly degradable, oxidized lipid–protein cross-links, may alter the turnover of cathepsin D, causing its mistargeting into the extracellular space together with the enhanced secretion of a gelatinase.
Keywords: Pigment epithelium of retina; Lipid peroxidation; Lysosome; Cathepsin D; Membrane transport; Phagocytosis;

H3 mRNA level as a new proliferative marker in astrocytomas by Joanna Orchel; Jerzy Slowinski; Urszula Mazurek; Tadeusz Wilczok (42-46).
Keywords: Histone gene; Proliferation; Astrocytoma; Prognosis;

Two novel missense mutations in ABCA1 result in altered trafficking and cause severe autosomal recessive HDL deficiency by Christiane Albrecht; Kevin Baynes; Alessandro Sardini; Silke Schepelmann; Emily R Eden; Simon W Davies; Christopher F Higgins; Michael D Feher; James S Owen; Anne K Soutar (47-57).
Extremely low concentrations of high density lipoprotein (HDL)-cholesterol and apolipoprotein (apo) AI are features of Tangier disease caused by autosomal recessive mutations in ATP-binding cassette transporter A1 (ABCA1). Less deleterious, but dominantly inherited mutations cause HDL deficiency. We investigated causes of severe HDL deficiency in a 42-year-old female with progressive coronary disease.ApoAI-mediated efflux of cholesterol from the proband's fibroblasts was less than 10% of normal and nucleotide sequencing revealed inheritance of two novel mutations in ABCAI, V1704D and L1379F. ABCA1 mRNA was approximately 3-fold higher in the proband's cells than in control cells; preincubation with cholesterol increased it 5-fold in control and 8-fold in the proband's cells, but similar amounts of ABCA1 protein were present in control and mutant cells. When transiently transfected into HEK293 cells, confocal microscopy revealed that both mutant proteins were retained in the endoplasmic reticulum, while wild-type ABCA1 was located at the plasma membrane.Severe HDL deficiency in the proband was caused by two novel autosomal recessive mutations in ABCA1, one (V1704D) predicted to lie in a transmembrane segment and the other (L1379F) in a large extracellular loop. Both mutations prevent normal trafficking of ABCA1, thereby explaining their inability to mediate apoA1-dependent lipid efflux.
Keywords: ABCA1; Protein trafficking; HDL deficiency; Apolipoprotein AI; Cholesterol efflux;

Keywords: Kidney allograft; Banff 97 schema; Protocol biopsy; Chronic allograft nephropathy (CAN); ser/thr protein kinase Par1/Emk1; Splicing alteration; Cryptic exon;

Identification of hepatic molecular mechanisms of action of alpha-tocopherol using global gene expression profile analysis in rats by Luca Barella; Patrick Y Muller; Manfred Schlachter; Willi Hunziker; Elisabeth Stöcklin; Volker Spitzer; Nina Meier; Sonia de Pascual-Teresa; Anne-Marie Minihane; Gerald Rimbach (66-74).
The recent discovery that vitamin E (VE) regulates gene activity at the transcriptional level indicates that VE may exert part of its biological effects by mechanisms which may be independent of its well-recognised antioxidant function. The objective of this study was the identification of hepatic vitamin E-sensitive genes and examination of the effects of VE on their corresponding biological endpoints. Two groups of male rats were randomly assigned to either a VE-sufficient diet or to a control diet deficient in VE for 290 days. High-density oligonucleotide microarrays comprising over 7000 genes were used to assess the transcriptional response of the liver. Differential gene expression was monitored over a period of 9 months, at four different time-points, and rats were individually profiled. This experimental strategy identified several VE-sensitive genes, which were chronically altered by dietary VE. VE supplementation down-regulated scavenger receptor CD36, coagulation factor IX and 5-α-steroid reductase type 1 mRNA levels while hepatic gamma glutamyl-cysteinyl synthetase was significantly up-regulated. Measurement of the corresponding biological endpoints such as activated partial thromboplastin time, plasma dihydrotestosterone and hepatic glutathione substantiated the gene chip data which indicated that dietary VE plays an important role in a range of metabolic processes within the liver.
Keywords: α-Tocopherol; Rat; Liver; Gene expression; DNA array;

Air pollutants increase gene expression of the vasoconstrictor endothelin-1 in the lungs by Errol Thomson; Patrick Goegan; Prem Kumarathasan; Renaud Vincent (75-82).
Inhalation of urban pollutants elevates the circulating levels of the vasoactive peptides endothelin (ET)-1 and ET-3 in rats. This effect could explain the association between episodic variations of urban pollutants and acute cardiopulmonary morbidity and mortality documented in epidemiological studies. Because the lungs are the primary source of circulating ET-1 and the main site of clearance from circulation, we investigated the response of endothelin system genes in the lungs of Fischer-344 rats after 4-h nose-only inhalation of 0.8 ppm ozone plus 49 mg/m3 EHC-93 (Ottawa particles). The mRNA levels for preproET-1, preproET-3, endothelin-converting enzyme (ECE)-1, and ET receptor subtypes A and B were determined at 2 h, and 1, 2, 3, 7, and 14 days after exposure. The pollutants induced preproET-1 and ECE-1 (P<0.05) after 2 h, consistent with the notion of increased synthesis and conversion of the peptide ET-1 in lung endothelial cells. PreproET-3 mRNA was down-regulated at 2 h post-exposure (P<0.05), and returned to control levels by 24 h, indicating that induction of ET-3 in the lungs is not responsible for the sustained elevation of ET-3 in plasma reported after inhalation of pollutants. Our results indicate that lung endothelin system genes respond rapidly and transiently to inhalation of urban pollutants, consistent with the dynamics of urban pollutant health effects in the human population.
Keywords: Air pollution; Endothelin-1 (ET-1); Endothelin-converting enzyme-1 (ECE-1); Ozone; Particulate matter; Cardiovascular disease;

Defects in an intracellular chloride channel CLC-5 cause Dent's disease, an inherited kidney stone disorder. Using a collecting duct model, mIMCD-3 cells, we show expression of dimeric mCLC-5. Transient transfection of antisense CLC-5 reduces CLC-5 protein expression. Binding of both calcium phosphate (hydroxyapatite) and calcium oxalate monohydrate (COM) crystals overlaid onto mIMCD-3 cultures was affected by altered CLC-5 expression. Calcium phosphate crystal agglomerations (>10 μm) were minimal in control (9%) and sense (13%) CLC-5-transfected cells, compared to 66% of antisense CLC-5-transfected cells (P<0.001). Small calcium phosphate crystals (<10 μm) were found associated with 45% of sense CLC-5-treated cells, of which the majority (11/14 cells) appeared to be internalised within the cell. Calcium oxalate agglomerations (>10 μm) were also largely absent for controls or sense mCLC-5 transfectants (11% and 9% of cells, respectively) with COM crystal agglomerates predominating in antisense CLC-5 transfectants (66%, P<0.0001). We conclude that collecting duct cells with reduced CLC-5 expression lead to a tendency to form calcium crystal agglomeration, which may help explain the nephrocalcinosis and nephrolithiasis seen in Dent's disease.
Keywords: Dent's disease; Chloride channel; CLC-5; Endocytosis; Collecting duct; Calcium phosphate; COM crystal;