Opfermitverantwortung beim Betrug

Isolation of the peptide fraction P1-RPC-C18 by reverse-phase chromatography in a C4-column (RPC-C4) and mass spectrometry profile showing the peptide ions. (PDF 66 kb

MGO induces formation of large aggregates containing ubiquitin, CHIP and Hsp40.

<p>(A) ARPE-19 cells were treated with 1 mM MGO or 3 mM MGO for 3 hours and the cell lysates were analyzed by western blotting using specific antibodies against CHIP and actin. (B) ARPE-19 cells were transfected with myc-CHIP wt and treated with 3 mM MGO for 15, 30, 50, 79 and 90 minutes. Proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed for c-myc. (C) ARPE-19 cells were treated with 1 mM MGO or 3 mM MGO for 3 hours. The cell lysates were used to immunoprecipitate Hsp40 and immunoblot the immunoprecipitates against ubiquitin. (D) ARPE-19 cells were transfected with myc-CHIP wt and treated with 3 mM MGO for 3 hours. c-myc (CHIP) was immunoprecipitated and the immunoprecipitates were probed using a monoclonal antibody against ubiquitin. (E) ARPE-19 cells were transfected with c-myc CHIP wt, c-myc CHIP K30A or c-myc CHIP H260Q. Cells were subsequently treated with 3 mM MGO for 3 hours and the cell lysates were immunoblotted for c-myc and actin.</p

MGO decreases the levels of Hsc70 and Hsp90.

<p>(A, B and C) ARPE-19 cells were treated with 1 mM MGO or 3 mM MGO for 3 hours and the cell lysates were analyzed by western blotting using anti-Hsc70 (A), anti-Hsp90 (B) and anti-actin antibodies. The quantified results represent the mean ± SD of at least three independent experiments. *** p<0.001, ^## p<0.01, ^### p<0.001, significantly different from control; one-way ANOVA (Dunnet's <i>post hoc</i> test) (C).</p

MGO-induced stress activates Hsf-1 through increased oligomerization and DNA-binding, eliciting a cell response to stress.

<p>(A) ARPE-19 cells were treated with 50 µM CHX together with 1 mM MGO for 1, 2, 3, 4 or 5 hours and the cell lysates were analyzed by western blot using specific antibodies against Hsf-1 and actin. * Monomeric Hsf-1 (∼80 kDa); ** Dimeric Hsf-1 (∼160 kDa); *** Trimeric Hsf-1 (∼230 kDa). (B) ARPE-19 cells were treated with 1 mM MGO for 1, 3 or 5 hours and the cell lysates were analyzed by western blot using specific antibodies against Hsf-1 and actin. (C) ARPE-19 cells were transiently transfected with the HSE wt (x4)-luciferase or HSE mut (x4)-luciferase vectors and were treated with 1 mM MGO for 3, 5 or 8 hours. Subsequently, the luciferase activity was determined by luminescence. The quantified results represent the mean ± SD of at least three independent experiments. ** p<0.01, significantly different from control; one-way ANOVA (Dunnet's <i>post hoc</i> test). (D) ARPE-19 cells were treated with 1 mM MGO for 3, 5 or 8 hours. Total RNA was used to synthesize cDNA, which, in turn, was used as template to quantify HspB1, HspB2, Hsc70, Hsp70 and Hsp90 mRNA and 18S rRNA through RT-PCR. The quantified results represent the mean ± SD of at least three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, significantly different from the respective control; ## p<0.01 and ### p<0.001, significantly different from the corresponding “MGO 3 h” condition; § p<0.05, §§ p<0.01, significantly different from the corresponding “MGO 5 h” condition; one-way ANOVA (Tukey's <i>post hoc</i> test). (E) ARPE-19 cells were treated with 1 mM MGO for 3, 5 or 8 hours and the cell lysates were analyzed by western blot using anti-Hsc70, -Hsp90 and -actin antibodies. The quantified results represent the mean ± SD of at least three independent experiments. * p<0.05, ** p<0.01 and *** p<0.001, significantly different from the respective control; one-way ANOVA (Tukey's <i>post hoc</i> test); ## p<0.01, significantly different from the corresponding “MGO 5 h” condition; one-way ANOVA (Tukey's <b><i>post hoc</i></b> test). (F) ARPE-19 cells were treated with 1 mM MGO for 1, 3, 5, 8 or 10 hours and the cell lysates were analyzed by western blot using anti-Hsp27, -Hsp70, -GAPDH and -actin antibodies. (G) ARPE-19 cells, transfected with c-myc tagged Hsf-1, were treated with 1 mM MGO for 1, 3, 5 or 8 hours. c-myc was immunoprecipitated and immunoprecipitates were probed against c-myc and Hsp90. (H) ARPE-19 cells were treated with 1 mM MGO for 3, 8 or 10 hours and used to assess cell viability through the MTT colorimetric assay. The quantified results represent the mean ± SD of at least three independent experiments. * p<0.05, significantly different from control; # p<0.05, significantly different from “1 mM MGO 8 h” condition; one-way ANOVA (Dunnet's <i>post hoc</i> test).</p

MGO decreases the 20S proteasome activity, depletes free ubiquitin and leads to accumulation of ubiquitin conjugates.

<p>(A) ARPE-19 cells were treated with 1 mM or 3 mM of MGO for 3 hours. Cells were then lysed and the intracellular levels of MGO were determined by HPLC after derivatization with DDB. The results represent the mean ± SD of at least three independent experiments. * p<0.05, significantly different from control (one-way ANOVA). (B) ARPE-19 cells were treated with 3 mM MGO for 3 hours and the cell extracts were used to measure the proteolytic activities of the 20S proteasome (chymotrypsin-like, caspase-like and trypsin-like activities) using specific fluorogenic substrates. The results correspond to the measurement at 30 minutes of activity and represent the mean ± SD of at least three independent experiments. ** p<0.01, significantly different from control; paired T test. (C) ARPE-19 cells were treated with 1 mM MGO, 3 mM MGO or 20 µM MG132 for 3 hours and the proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with monoclonal antibodies against ubiquitin and actin.</p

MGO induces accumulation of oxidized and argpyrimidine-modified proteins and decreases cell viability.

<p>(A) ARPE-19 cells were treated with 1 mM MGO or 3 mM MGO for 3 hours and the cell lysates were used to derivatize proteins with DNPH. Derivatized proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with an antibody against dinitrophenylhydrazone derivatives. (B) ARPE-19 cells were treated with 1 mM or 3 mM MGO for 3 hours and the cell lysates were immunoblotted for argpyrimidine and actin. (C) ARPE-19 cell were treated with 1 mM MGO or 3 mM MGO for 3 hours and used to assess cell viability through the MTT colorimetric assay. The quantified results represent the mean ± SD of at least three independent experiments. * p<0.05, significantly different from control; one-way ANOVA (Dunnet's <i>post hoc</i> test).</p

Out of Africa: did Emys orbicularis occidentaliscross the Strait of Gibraltar twice?

<p>The narrow Strait of Gibraltar has separated the African and European continents since the Miocene (5.3 Mya), with a different degree of permeability for Mediterranean taxa. Southern and northern regions of the Iberian Peninsula and Morocco, respectively, are key areas to evaluate the colonization dynamics and biogeographic history of taxa occurring at both sides of this strait. The Ibero-Maghrebian subspecies of the European pond turtle, <em>Emys orbicularis occidentalis</em>, is patchily distributed and threatened throughout most of the Iberian Peninsula and northern Morocco and its origin is thought to be in North Africa. Here we expand the geographic sampling across the Iberian Peninsula and Morocco, with special emphasis in the southern tip of the peninsula and northern Morocco, and analyze mtDNA sequences of 183 <em>E. o. occidentalis </em>to better understand the complex biogeographic history of this subspecies. We provide for the first time evidence for shared haplotypes of Iberian and North African pond turtles, with an additional haplotype in the southern Iberian Peninsula derived from Moroccan haplotypes. This supports the hypothesis that the Strait of Gibraltar constitutes no significant biogeographic barrier for <em>E. orbicularis</em>. However, the newly discovered shared, or extremely similar, haplotypes of European pond turtles from the southern Iberian Peninsula and Morocco suggest either that at least two independent natural colonization waves from Morocco have reached the Iberian Peninsula or that Moroccan turtles were accidentally or deliberately introduced there.</p

Silencing of CHIP stabilized HIF-1α protein in the presence of methylglyoxal in ARPE-19, Cos-7 and RCC4 cells.

<p>(A) ARPE-19 cells were infected with pAd shRNA-hCHIP for 48 hours. Cells were subsequently lysed and protein extracts were immunoblotted against endogenous CHIP and actin. (B) ARPE-19 cells were co-infected with pAd V5-hCHIP and pAd shRNA-hCHIP for 24 hours. The cell lysates were analyzed by western blotting using anti-V5 and anti-actin monoclonal antibodies. (C) ARPE-19 cells were infected with pAd shRNA-hCHIP for 48 hours and during the last 6 hours of incubation, cells were subjected to hypoxia in the presence of MGO (3 mM for the last 70 minutes). Cell lysates were used to immunoprecipitate HIF-1α and the immunoprecipitates were probed with antibodies against HIF-1α and ubiquitin (P4D1). The data in the graph represents the mean ± SD of at least three independent experiments. ** p<0.01, significantly different from control (one-way ANOVA with the Dunnet's comparison test). (D) RCC4 VHL^-/- cells, infected with pAd shRNA-hCHIP for 48 hours, were treated with MGO (3 mM for 70 minutes). Cell lysates were used to immunoprecipitate HIF-1α and the immunoprecipitates were blotted against HIF-1α and ubiquitin (P4D1). (E) Cos-7 cells were infected with pAd shRNA-hCHIP for 48 hours and, during the last 6 hours of incubation, cells were subjected to hypoxia and treated with MGO (3 mM for the last 70 minutes). Cell lysates were used to immunoprecipitate HIF-1α and the immunoprecipitates were probed for HIF-1α and ubiquitin (P4D1). IP controls were carried out both with no antibody and with an irrelevant mouse IgG1 antibody (for example anti-GFP). (F) ARPE-19 cells were grown in 15 mM (basal DMEM: F12 medium) or 40 mM of D-glucose during 10 days. After 8 days of incubation, cells were infected with pAd shRNA-hCHIP for 48 hours. During the last 6 hours of incubation, cells were subjected to hypoxia and subsequently lysed. Proteins were blotted against HIF-1α and actin. The word “Mock” in the figures refers to a control with a scrambled shRNA sequence.</p

CHIP polyubiquitinated HIF-1α in the presence of methylglyoxal.

<p>(A and B) ARPE-19 cells were transiently transfected with the CHIP wt c-myc plasmid and treated with MG132 (20 µM) for 4 hours in the absence or presence of MGO (3 mM for 40 or 70 minutes). HIF-1α (A) or c-myc (B) were immunoprecipitated and the immunoprecipitates were probed for c-myc and HIF-1α. (C) ARPE-19 cells were transfected with CHIP wt c-myc or with the dominant negatives CHIP K30A c-myc and CHIP H260Q c-myc, simultaneously or separately, and treated with MG132 (20 µM) for 41 hours in the presence or absence of MGO (3 mM for 70 minutes). HIF-1α was then immunoprecipitated and the immunoprecipitates were blotted against HIF-1α, c-myc and ubiquitin (P4D1). (D) ARPE-19 cells were transfected with CHIP wt c-myc or with the dominant negative mutants CHIP K30A c-myc and CHIP H260Q c-myc, simultaneously or separately, and subjected to hypoxia for 6 hours in the presence or absence of MGO (3 mM for 70 minutes). HIF-1α was then immunoprecipitated and the immunoprecipitates were blotted for HIF-1α and ubiquitin (P4D1). (E) ARPE-19 cells were transfected with CHIP wt c-myc, CHIP K30A c-myc or/and CHIP H260Q c-myc dominant negatives, either simultaneously or separately, and subjected to hypoxia for 6 hours in the presence or absence of MGO (3 mM for 70 minutes). Proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed for HIF-1α and actin. (F) ARPE-19 cells were treated with MG132 (20 µM for 4 hours) either in the presence or absence of MGO (3 mM for 40 or 70 minutes). HIF-1α was immunoprecipitated and the immunoprecipitates were probed using antibodies against HIF-1α, Hsp70 and Hsp40. (G) ARPE-19 cells were transfected simultaneously with CHIP-c-myc and V5-tagged Hsp40 and/or HA-tagged Hsp70. Cells were subsequently treated with MG132 (20 µM) for 4 hours and MGO (3 mM) for the last 70 minutes of incubation. HIF-1α was immunoprecipitated and the immunoprecipitates were blotted against HA, V5 and c-myc. IP controls were carried out both with no antibody and with an irrelevant mouse IgG1 antibody (for example anti-GFP). The word “Mock” in the figures refers to a control with an empty vector.</p

Methylglyoxal decreased the transcriptional activity of HIF-1 and the release of VEGF_121/165 into the medium.

<p>(A) ARPE-19 cells were transiently transfected with the pT81 HRE-luciferase vector and were subjected to hypoxia (2% O₂) for 6 hours in the absence or presence of MGO (1 mM for 4 hours). Subsequently, the luciferase activity was determined and the values were expressed as fold induction over control. (B) ARPE-19 cells were subjected to hypoxia (2% O₂) for 6 hours either in the absence or the presence of MGO (1 mM for 4 hours). Total RNA was used to synthesize cDNA, which, in turn, was used as template to quantify VEGF mRNA and 18S rRNA through RT-PCR. (C) ARPE-19 cells were subjected to hypoxia (2% O₂) for 6 hours either in the absence or in the presence of MGO (1 mM for 4 hours). The concentration of the diffusible VEGF₁₂₁ and VEGF₁₆₅ isoforms were determined by ELISA using a monoclonal antibody for human VEGF. The results represent the mean ± SD of at least three independent experiments. ** p<0.01 and *** p<0.001, significantly different from control; ## p<0.01 and ### p<0.001, significantly different from hypoxia condition (one-way ANOVA).</p