DNA demethylation-dependent enhancement of toll-like receptor-2 gene expression in cystic fibrosis epithelial cells involves SP1-activated transcription-2

PGL3-T2P (-120), SP1 binding site-mutant pGL3-T2P (-120) SPm, pGL3-T2P (-12), or pGL3-null plasmids were each transiently transfected into HeLa cells. Luciferase activity is expressed as the percent activity of the pGL3-T2P (-120) construct. (B) Assessment of the effect of mutating the ETS binding site within the TLR2 -120/+110 promoter region. The pGL3-T2P (-120), ETS binding site-mutant pGL3-T2P (-120) ETSm, pGL3-T2P (-12), or pGL3-null plasmids were each transiently transfected into HeLa cells. Luciferase activity is expressed as the percent activity of the pGL3-T2P (-120) construct. (C) Assessment of the effect of mutating the ETS binding site within TLR2 -60/+110 promoter region. The pGL3-T2P (-60), mutant ETS binding site- pGL3-T2P (-60) ETSm construct, the pGL3-T2P (-12) construct, or the pGL3-null plasmid were transiently transfected into HeLa cells. Luciferase activity is expressed as the percent activity of the pGL3-T2P (-60) construct. Data are presented as the mean ± S.E.M. from three independent experiments performed in triplicate. n.s. = not significant as assessed by Student's test.<p><b>Copyright information:</b></p><p>Taken from "DNA demethylation-dependent enhancement of toll-like receptor-2 gene expression in cystic fibrosis epithelial cells involves SP1-activated transcription"</p><p>http://www.biomedcentral.com/1471-2199/9/39</p><p>BMC Molecular Biology 2008;9():39-39.</p><p>Published online 21 Apr 2008</p><p>PMCID:PMC2387165.</p><p></p

Acta de Renovación de Convenio de cooperación académica y científica entre la Università della Calabria (UNICAL, Italia) y la Universidad Nacional de la Plata, (UNLP, Argentina)

La Università della Calabria (UNICAL, Italia) y la Universidad Nacional de la Plata, (UNLP, Argentina) acuerdan renovar en todos sus términos, y por un período de vigencia de cinco (5) años, el Convenio de Colaboración Científica y Cultural suscripto entre las partes el 1 del mes de septiembre de 2008 en el cual convinieron en programar y ejecutar conjuntamente acciones de cooperación en los campos de la Docencia, Extensión, Investigación y Postgrado, así como el intercambio de información, de recursos humanos y materiales, a través de proyectos comunes de asistencia técnica.Universidad Nacional de La Plat

DNA demethylation-dependent enhancement of toll-like receptor-2 gene expression in cystic fibrosis epithelial cells involves SP1-activated transcription-0

D as +1.<p><b>Copyright information:</b></p><p>Taken from "DNA demethylation-dependent enhancement of toll-like receptor-2 gene expression in cystic fibrosis epithelial cells involves SP1-activated transcription"</p><p>http://www.biomedcentral.com/1471-2199/9/39</p><p>BMC Molecular Biology 2008;9():39-39.</p><p>Published online 21 Apr 2008</p><p>PMCID:PMC2387165.</p><p></p

DNA demethylation-dependent enhancement of toll-like receptor-2 gene expression in cystic fibrosis epithelial cells involves SP1-activated transcription-4

Cted with pGL3-null and pcDNA3.1 as control. Luciferase activity is expressed as the percent activity of the pGL3-T2P (-120) plasmid in pcDNA3.1-co-transfected, methylase-untreated cells. Data are presented as the mean ± S.E.M. from three independent experiments performed in triplicate. *p < 0.0001, assessed by Student's -test.<p><b>Copyright information:</b></p><p>Taken from "DNA demethylation-dependent enhancement of toll-like receptor-2 gene expression in cystic fibrosis epithelial cells involves SP1-activated transcription"</p><p>http://www.biomedcentral.com/1471-2199/9/39</p><p>BMC Molecular Biology 2008;9():39-39.</p><p>Published online 21 Apr 2008</p><p>PMCID:PMC2387165.</p><p></p

Mild Electrical Stimulation and Heat Shock Ameliorates Progressive Proteinuria and Renal Inflammation in Mouse Model of Alport Syndrome

<div><p>Alport syndrome is a hereditary glomerulopathy with proteinuria and nephritis caused by defects in genes encoding type IV collagen in the glomerular basement membrane. All male and most female patients develop end-stage renal disease. Effective treatment to stop or decelerate the progression of proteinuria and nephritis is still under investigation. Here we showed that combination treatment of mild electrical stress (MES) and heat stress (HS) ameliorated progressive proteinuria and renal injury in mouse model of Alport syndrome. The expressions of kidney injury marker neutrophil gelatinase-associated lipocalin and pro-inflammatory cytokines interleukin-6, tumor necrosis factor-α and interleukin-1β were suppressed by MES+HS treatment. The anti-proteinuric effect of MES+HS treatment is mediated by podocytic activation of phosphatidylinositol 3-OH kinase (PI3K)-Akt and heat shock protein 72 (Hsp72)-dependent pathways <em>in vitro</em> and <em>in vivo</em>. The anti-inflammatory effect of MES+HS was mediated by glomerular activation of c-jun NH₂-terminal kinase 1/2 (JNK1/2) and p38-dependent pathways <em>ex vivo</em>. Collectively, our studies show that combination treatment of MES and HS confers anti-proteinuric and anti-inflammatory effects on Alport mice likely through the activation of multiple signaling pathways including PI3K-Akt, Hsp72, JNK1/2, and p38 pathways, providing a novel candidate therapeutic strategy to decelerate the progression of patho-phenotypes in Alport syndrome.</p> </div

MES+HS suppresses the expressions of pro-inflammatory cytokines in kidneys of Alport mice in vivo.

<p>(<i>a–j</i>) Forty-eight hours after the last treatment, total RNA was isolated from kidneys (<i>a</i>–<i>f</i>) or glomeruli (<i>g</i>–<i>j</i>) of Alport mice sham-treated (CON) or treated with MES+HS for 8 weeks. Q-PCR analysis was performed to assess the expression of the indicated cytokines (<i>a</i>–<i>e, g</i>–<i>j</i>) and renal injury marker NGAL (<i>f</i>). Values are the mean ± S.E. (n = 6). *<i>P</i><0.05, *** <i>P</i><0.001 assessed by unpaired <i>t</i>-test. n.s., not significant.</p

MES+HS directly suppresses the expressions of pro-inflammatory cytokines via PI3K-Akt or Hsp72-independent pathway in isolated glomeruli of Alport mice ex vivo.

<p>(<i>a</i>) Bright field image indicates isolated glomeruli from Alport mice in culture medium. (<i>b, c</i>) Glomeruli were isolated from Alport mice by mesh sieving followed by <i>ex vivo</i> MES+HS treatment for 10 min. Four hours after treatment, total RNA was extracted from glomeruli and subjected to Q-PCR analysis to assess the cytokines mRNA expression. (<i>d</i>–<i>f</i>) Isolated glomeruli from Alport mice were pre-treated with LY294002 (20 µM) or quercetin (200 µM) for 2 hr, and treated with MES+HS for 10 min. Thirty minutes after the treatment, proteins were isolated, and immunoblotted with p-Akt and total Akt antibodies (<i>d</i>). Five hours after the treatment, protein lysates were analyzed for Hsp72 expression (<i>e</i>), or total RNA was isolated and subjected to Q-PCR analysis to assess the expression of IL-6 (<i>f</i>). Values are the mean ± S.E. (n = 3 for b, <i>c</i> & <i>f</i>). *<i>P</i><0.05, **<i>P</i><0.01 assessed by unpaired <i>t</i>-test. n.s., not significant.</p

MES+HS activates Akt and induces Hsp72 expression in podocytes of Alport mice in vivo.

<p>(<i>a</i>, <i>c & e</i>) One hour after the final treatment, whole kidney protein lysates or glomeruli protein lysates were isolated from 8 weeks-MES+HS-treated and control Alport mice, and immunoblotted to detect p-Akt and total Akt proteins. Actin was used as loading control (<i>a, c</i>). Renal cryosections were subjected to immunohistochemical analysis to assess phosphorylated Akt. Arrowheads indicate phosphorylated Akt in podocytes. Cryosections were counterstained with DAPI (<i>e</i>). (<i>b, d</i> & <i>f</i>) Eight hours (<i>b</i> & <i>f</i>) or five hours (<i>d</i>) after the final treatment, whole kidney or glomeruli protein lysates were isolated from MES+HS-treated (8 weeks treated for <i>b</i> & <i>f</i>, or 4 weeks treated for <i>d</i>) and control Alport mice, and immunoblotted to assess Hsp72 and HSC70 expression. α-Tubulin or actin was used as loading control (<i>b, d</i>). Renal cryosections were subjected to immunohistochemical analysis to assess Hsp72. Arrowheads indicate up-regulated Hsp72 in podocytes. Cryosections were counterstained with DAPI (<i>f</i>). (<i>e</i> & <i>f</i>) Representative glomeruli images of three independent experiments are shown. Renal sections were immunostained with anti-synaptopodin antibody to assess podocyte localization (<i>lower panels</i>). Sections were counterstained with DAPI. White bars; 10 µm.</p

Primer sequences for Real-time quantitative RT-PCR.

<p>Primer sequences for Real-time quantitative RT-PCR.</p

Bromide supplementation exacerbated the renal dysfunction, injury and fibrosis in a mouse model of Alport syndrome

<div><p>A seminal study recently demonstrated that bromide (Br^-) has a critical function in the assembly of type IV collagen in basement membrane (BM), and suggested that Br^- supplementation has therapeutic potential for BM diseases. Because salts of bromide (KBr and NaBr) have been used as antiepileptic drugs for several decades, repositioning of Br^- for BM diseases is probable. However, the effects of Br^- on glomerular basement membrane (GBM) disease such as Alport syndrome (AS) and its impact on the kidney are still unknown. In this study, we administered daily for 16 weeks 75 mg/kg or 250 mg/kg (within clinical dosage) NaBr or NaCl (control) via drinking water to 6-week-old AS mice (mouse model of X-linked AS). Treatment with 75 mg/kg NaBr had no effect on AS progression. Surprisingly, compared with 250 mg/kg NaCl, 250 mg/kg NaBr exacerbated the progressive proteinuria and increased the serum creatinine and blood urea nitrogen in AS mice. Histological analysis revealed that glomerular injury, renal inflammation and fibrosis were exacerbated in mice treated with 250 mg/kg NaBr compared with NaCl. The expressions of renal injury markers (<i>Lcn2</i>, <i>Lysozyme</i>), matrix metalloproteinase (<i>Mmp-12</i>), pro-inflammatory cytokines (<i>Il-6</i>, <i>Il-8</i>, <i>Tnf-α</i>, <i>Il-1β</i>) and pro-fibrotic genes (<i>Tgf-β</i>, <i>Col1a1</i>, <i>α-Sma</i>) were also exacerbated by 250 mg/kg NaBr treatment. Notably, the exacerbating effects of Br^- were not observed in wild-type mice. These findings suggest that Br^- supplementation needs to be carefully evaluated for real positive health benefits and for the absence of adverse side effects especially in GBM diseases such as AS.</p></div