Comparison of the RAGE, ALCAM, BCAM and MCAM protein sequences.

<p>The multiple alignment was performed with CLUSTALW. Exons encoding a portion of the corresponding protein are highlighted alternatively in yellow and grey. Asterisks indicate conserved exon-intron boundaries; if conservation is among all four proteins, the asterisks are red, if conservation is between RAGE and at least one of the others the colour is blue. RAGE (receptor for advanced glycation endproducts); ALCAM (activated leukocyte cell adhesion molecule); BCAM (basal cell adhesion molecule); MCAM (melanoma cell adhesion molecule).</p

RAGE expression enhances cell spreading.

<p>Cell spreading assay. (<b>A</b>) Spreading of R3/1-pLXSN or R3/1-FL-RAGE cells onto culture dishes coated with 10 µg/ml of ECM proteins (Coll I, FN, or Lam) or PBS was assessed at 90 minutes after seeding. Photographs were taken in phase contrast at 40× magnification. Bar corresponds to 20 µm. (<b>B</b>) Quantification of cell spreading based on cell surface area. Results are displayed as means±SEM (ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001).</p

Phylogenetic analysis of the <i>Agpat1</i>–<i>Gpsm3</i> locus including <i>Ager</i>.

<p>(<b>A</b>) The panel represents the structures of the <i>Agpat1</i>-<i>Rnf5</i>-<i>Ager</i>-<i>Pbx2</i>-<i>Gpsm3</i> genes. The locus spans more than 1 Mbp in human chr 6. <i>Ager</i> (receptor for advanced glycation endproducts); <i>Agpa1</i> (1-acylglycerol-3-phosphate O-acyltransferase 1); <i>Gpsm3</i> (G-protein signaling modulator 3); <i>Pbx2</i> (pre-B-cell leukemia homeobox 2); <i>Rnf5</i> (ring finger protein 5). (<b>B</b>) A schematic phylogenetic tree with the main nodes. The presence of a gene is depicted by a green box, while the absence of the box indicates that the gene is not found in the specific phylogenetic group.</p

Phylogenetic tree derived from 3D structural alignments.

<p>The phylogenetic tree is based on the analysis of the closest 500 matches resulting from a search of the protein structure database using the structure of RAGE Ig domains V-C1 as a query. After filtering the results for a minimum length of alignment and removal of duplicates 27 different proteins were retrieved. Twenty four out of these 27 are cell adhesion molecules, only 2 belong to the T-cell receptor family and one protein represents the light chain of an antibody. RAGE V-C1 Ig domains group very closely with cell adhesion molecules BCAM and CD80. OCAM (olfactory cell adhesion molecule; pdb code 2jll); CEACAM-1 (Carcinoembryonic antigen-related cell adhesion molecule 1, CD 66a; pdb code 3R4D); PD-L1 (programmed death1 inhibitory receptor; pdb code 3BIS); CD80 (cluster of differentiation 80; pdb code 1DR9); RAGE (receptor for advanced glycation endproducts; pdb code 3CJJ); BCAM (Lutheran glycoprotein; basal cell adhesion molecule; pdb code 2PET); NECTIN-1 (pdb code 4FMF); NECTIN-2 (pdb code 4FMK); NECTIN-4 (pdb code 4FRW); CD155 (cluster of differentiation 155, poliovirus receptor; pdb code 3URO); CD2 (cluster of differentiation 2; pdb code 1HNG); MADCAM-1 (mucosal vascular addressin cell adhesion molecule 1; pdb code 1GSM); VCAM (vascular cell adhesion molecule; pdb code 1VSC); ICAM-1 (intercellular adhesion protein 1, pdb code 1IC1); ICAM-2 (intercellular adhesion protein 2; pdb code 1ZXQ); TCR-alpha (T-cell receptor a chain; pdb code 1NFD); TCR-gamma (T-cell receptor g chain; pdb code 1HXM); Fab-LC (antibody Fab fragment light chain; pdb code 3QNX); Neuroplastin (pdb code 2WV3); MUSK (pdb code 2IEP); NCAM-Ig-1–2 (neural cell adhesion molecule Ig domains 1 and 2; pdb code 1EPF); NCAM-Ig-2–3 (neural cell adhesion molecule Ig domains 1 and 2; pdb code 1QZ1); DSCAM (Down syndrom cell adhesion molecule; pdb code 3DMK); Hemolin (pdb code 1BIH); ROBO (Roundabout; pdb code 2VRA); ROBO-1 (Roundabout homolog 1; pdb code 2V9R); Contactin (protein tyrosine phosphatase z (PTPRZ); pdb code 3JXA); TAG-1 (axonin, pdb code 1CS6).</p

Comparison of RAGE V-C1 structure with homophilic cell adhesion molecules BCAM, ALCAM and MCAM.

<p>(<b>A</b>) The Ig domains V and C1 of RAGE which reside most distal from the cytoplasmic membrane (shown in blue) adopt a slightly bent structure. This spatial arrangement is very well conserved in the Ig domains 1 and 2 of its close homologue BCAM (red). Structural models of the corresponding Ig domains of ALCAM (green) and MCAM (magenta) suggest that these adopt as well a very similar structure that might be required for homophilic interaction. RAGE (receptor for advanced glycation endproducts); ALCAM (activated leukocyte cell adhesion molecule); BCAM (basal cell adhesion molecule); MCAM (melanoma cell adhesion molecule). (<b>B</b>) Model for RAGE-RAGE homophilic interaction mediating cell adhesion (derived from pdb code 4LP5) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086903#pone.0086903-Yatime1" target="_blank">[44]</a>. Left hand side: as observed in the crystal structure of sRAGE the extracellular domain adopts an extended conformation; alongside a cartoon representation is shown. Right hand side: in the crystal the V-domains (dark blue) form a large contact in trans orientation. This mode of interaction is conserved among different crystal structures suggesting that RAGE homophlic interaction occurs via the V-domain.</p

Additional file 2: of The arrhythmogenic cardiomyopathy-specific coding and non-coding transcriptome in human cardiac stromal cells

Supplementary information. Contains supplementary information, tables and figures. (PDF 1224 kb

Hypoxia/Reoxygenation Cardiac Injury and Regeneration in Zebrafish Adult Heart

<div><p>Aims</p><p>the adult zebrafish heart regenerates spontaneously after injury and has been used to study the mechanisms of cardiac repair. However, no zebrafish model is available that mimics ischemic injury in mammalian heart. We developed and characterized zebrafish cardiac injury induced by hypoxia/reoxygenation (H/R) and the regeneration that followed it.</p><p>Methods and Results</p><p>adult zebrafish were kept either in hypoxic (H) or normoxic control (C) water for 15 min; thereafter fishes were returned to C water. Within 2–6 hours (h) after reoxygenation there was evidence of cardiac oxidative stress by dihydroethidium fluorescence and protein nitrosylation, as well as of inflammation. We used Tg(cmlc2:nucDsRed) transgenic zebrafish to identify myocardial cell nuclei. Cardiomyocyte apoptosis and necrosis were evidenced by TUNEL and Acridine Orange (AO) staining, respectively; 18 h after H/R, 9.9±2.6% of myocardial cell nuclei were TUNEL⁺ and 15.0±2.5% were AO⁺. At the 30-day (d) time point myocardial cell death was back to baseline (n = 3 at each time point). We evaluated cardiomyocyte proliferation by Phospho Histone H3 (pHH3) or Proliferating Cell Nuclear Antigen (PCNA) expression. Cardiomyocyte proliferation was apparent 18–24 h after H/R, it achieved its peak 3–7d later, and was back to baseline at 30d. 7d after H/R 17.4±2.3% of all cardiomyocytes were pHH3⁺ and 7.4±0.6% were PCNA⁺ (n = 3 at each time point). Cardiac function was assessed by 2D-echocardiography and Ventricular Diastolic and Systolic Areas were used to compute Fractional Area Change (FAC). FAC decreased from 29.3±2.0% in normoxia to 16.4±1.8% at 18 h after H/R; one month later ventricular function was back to baseline (n = 12 at each time point).</p><p>Conclusions</p><p>zebrafish exposed to H/R exhibit evidence of cardiac oxidative stress and inflammation, myocardial cell death and proliferation. The initial decrease in ventricular function is followed by full recovery. This model more closely mimics reperfusion injury in mammals than other cardiac injury models.</p></div

Direct RAGE-RAGE binding.

<p>Surface plasmon resonance analysis of homophilic interaction between soluble sRAGE and sRAGE immobilized on a Ni-NTA sensor chip. Soluble sRAGE was injected over the sensor chip at a flow rate of 10 µl/min. The arrows indicate the start and end of injections.</p

RAGE expression enhances cell-matrix adhesion.

<p>(<b>A</b>) Western blot analysis on rat lung lysate and R3/1 cells using three different antibodies recognizing RAGE extracellular (anti-RAGE N-term1 and anti-RAGE N-term2) or intracellular (anti-RAGE C-term) domains. Asterisk (*) indicates nonspecific bands. Forty µg of cell lysate were loaded and detection of GAPDH was used as loading control<b>.</b> (<b>B</b>) Western blot analysis on R3/1-pLXSN and R3/1-FL-RAGE cells using anti-RAGE N-term1 antibody. Forty µg of cell lysate were loaded and detection of GAPDH was used as loading control<b>.</b> (<b>C, D</b>) Cell-matrix adhesion assay. Adhesion of R3/1-pLXSN or R3/1-FL-RAGE cells onto culture dishes coated with 10 µg/ml ECM proteins. Adhesion to PBS, collagen I (Coll I), Fibronectin (FN), or Laminin (Lam) was assayed for 15 minutes (C) or 45 minutes (D). One representative experiment out of three is shown. Results from triplicate wells are displayed as means±SEM (ns, not significant; *, P<0.05; **, P<0.01).</p

Detection of HIF-1α-dependent genes expression in whole hearts after H/R <i>in vivo.</i>

<p>Graphs show selected HIF-1α-dependent genes expression in whole hearts in control (C) and at different time points (3 h, 6 h, and 9 h) after H/R. (a) <i>Hmox1</i> mRNA expression exhibited a progressive increase and, at the 9 h time point, <i>hmox1</i> was ∼8-fold higher than in C. (b) <i>Vegfaa</i> mRNA increased and achieved its peak 6 h after H/R. (c) <i>Epo</i> mRNA exhibited a peak increase at 3 h which was ∼1.7-fold higher than in C but failed to achieve statistical significance. (n = 6; * <i>p</i><0.05 and *** <i>p</i><0.001 <i>vs.</i> C).</p