Entrepreneur's profile of health staff (HS): a SEM approach

This study aims at identifying the entrepreneurial spirit of a specific professional group: health staff (HS). In this context, the psychological and cognitive structure of the HS was compared with the non entrepreneurial HS. The study used primary data collected through a face to face and an online survey, using industry organisations and institutions, and labour unions in order to get access. The results regarding the entrepreneur’s psychological and cognitive profile supported the hypothesis that HS who have created a firm have psychological and cognitive characteristics that support entrepreneurial activities

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

High glucose induced intracellular accumulation of methylglyoxal, which in turn led to decreased levels of HIF-1α.

<p>(A) ARPE-19 cells were grown in 15 mM (basal DMEM: F12 medium) or 40 mM of D-glucose during 10 days. During the last 61 hours of incubation, cells were exposed to hypoxia (2% O₂). After the treatments, the proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed against HIF-1α and actin. The results represent the mean ± SD of at least three independent experiments. * p<0.05, significantly different from control (t test). (B) ARPE-19 cells were grown in DMEM: F12 medium containing 15 mM or 40 mM of D-glucose during 10 days. Cells were then lysed in acetic acid (0.1 M) and the intracellular levels of MGO were determined by HPLC analysis after derivatization with DDB. The results represent the mean ± SD of at least three independent experiments. * p<0.05, significantly different from control (t test). (C and D) ARPE-19 cells were exposed to hypoxia (2% O₂) for 6 hours and MGO (1 mM or 3 mM) was added for 30 minutes or 3 hours. The cell lysates were analyzed by immunoblot using antibodies for HIF-1α and actin. (E) ARPE-19 cells were treated with different MGO concentrations (100 µM to 3 mM) for 2 hours. The cell lysates were analyzed by western blot using antibodies for Nrf-2 and actin. (F) ARPE-19 cells were treated with 3 mM of MGO for 30 minutes or 3 hours. Cells were then lysed in acetic acid (0.1 M) 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 and ** p<0.01, significantly different from control (one-way ANOVA with the Dunnet's comparison test). (G) After metabolic labeling with L-[³⁵S] methionine/cysteine under hypoxia, ARPE-19 cells were maintained under hypoxia, either in the absence or the presence of MGO (3 mM). Cells were harvested at 0, 10, 30, 60 and 90 minutes in 0.5% NP-40 lysis buffer. HIF-1α was immunoprecipitated and radiolabeled HIF-1α protein was assessed by SDS-PAGE and autoradiography.</p

Proposed model for MGO-dependent degradation of HIF-1α.

<p>MGO induces modifications on HIF-1α protein (as for example, formation of MG-H1 adducts) and promotes increased association of Hsp40/70 to HIF-1α. This association leads to recruitment of CHIP, which promotes polyubiquitination and proteasomal degradation of HIF-1α. MGO-induced degradation of HIF-1α is activated under high glucose and is inhibited by overexpression of glyoxalase I.</p

Methylglyoxal modified HIF-1α and induced its polyubiquitination.

<p>(A) ARPE-19 cells were subjected to hypoxia (2% O₂) for 6 hours and then incubated with MGO (3 mM) for 40 and 70 minutes. HIF-1α was immunoprecipitated and the immunoprecipitates were probed against CML and MG-H1. (B and C) ARPE-19 cells were treated with MG132 (20 µM) for 4 hours or subjected to hypoxia (2% O₂) for 6 hours and then incubated with MGO (3 mM) for 30 minutes, 90 minutes (B) or 3 hours (C). The cell lysates were analyzed by immunoblot against HIF-1α and the PVDF membranes were overexposed to reveal higher molecular weight bands; * HIF-1α of 120 kDa; ** posttranslationally modified HIF-1α (with higher molecular weights). (D) ARPE-19 cells were treated with MG132 (20 µM) for 4 hours either in the presence or absence of MGO (3 mM) for the last 90 minutes. HIF-1α was immunoprecipitated and immunoprecipitates were probed against HIF-1α and ubiquitin (P4D1). IP controls were carried out both with no antibody and with an irrelevant mouse IgG1 antibody (anti-GFP). (E) ARPE-19 cells cultured in coverslips were transfected with HA tagged HIF-1α. Subsequently, cells were treated with MG132 (20 µM) for 4 hours in the absence or presence of MGO (3 mM) for 90 minutes. Cells were fixed with 4% PFA for 10 minutes and used for immunocytochemistry using specific antibodies directed against HA and ubiquitin (FK1). (F) ARPE-19 cells were incubated with L-[³⁵S] methionine/cysteine under hypoxia. After metabolic labeling, cells were maintained under hypoxia, in the presence of 3 mM of MGO and either in the absence or presence of MG132 (20 µM). Cells were harvested at 0, 10, 40 and 70 minutes in 0.5% NP-40 lysis buffer. HIF-1α was immunoprecipitated and radiolabeled HIF-1α protein was assessed by SDS-PAGE and autoradiography. (G) ARPE-19 cells were treated with the proteasome inhibitor MG132 (20 µM) for 4 hours, either in the absence or presence of MGO (3 mM for 3 hours). The 20S proteasome activities were determined by <i>in vitro</i> fluorogenic assays, using specific substrates for each activity: Suc-LLVY-MCA (chymotrypsin-like activity), Z-LLE-MCA (caspase-like activity) and Boc-LRR-MCA (trypsin-like activity). The values reported in the graph correspond to measurements at 30 minutes of activity.</p

Destabilization of HIF-1α induced by methylglyoxal was independent on VHL.

<p>(A and B) ARPE-19 cells were transiently transfected with HIF-1α wt-V5 or HIF-1α (P402A/P564A)-V5 plasmids. Cells were subsequently subjected to hypoxia (2% O₂) for 6 hours (A) or treated with MG132 (20 µM) for 4 hours (B), in the absence or presence of MGO (3 mM for 3 hours). The cell lysates were immunoblotted against HIF-1α and V5. (C) RCC4 VHL^-/- cells were treated with MGO (3 mM for 3 hours) and cell lysates were analyzed by western blot for HIF-1α and actin.</p

Glyoxalase I overexpression significantly stabilized HIF-1α under high glucose or methylglyoxal treatment.

<p>(A) ARPE-19 cells were infected with pAD hGLOI adenovirus for 48 hours. By the end of infection, cells were treated with 3 mM of MGO for 3 hours and were, subsequently, lysed in acetic acid (0.1 M). 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<0.001, significantly different from Mock; ### p<0.001, significantly different from Mock + MGO3h (one-way ANOVA with Tukey's multiple comparison test). (B) 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 hGLOI adenovirus for 48 hours. During the last 6 hours of incubation, cells were subjected to hypoxia and subsequently lysed. Proteins were separated by SDS-PAGE, transferred to PVDF membranes and analyzed by immunoblot against HIF-1α and actin. (C) ARPE-19 cells were infected with pAD hGLOI adenovirus for 48 hours. During the last 6 hours of incubation, cells were subjected to hypoxia in the presence of MGO (3 mM for 3 hours). Subsequently, cells were lysed and the proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed against HIF-1α and actin. 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

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

Additional file 1: of Health conditions and occupational risks in a novel group: waste pickers in the largest open garbage dump in Latin America

Questionnaire “Water, the Environment and Health: impact on the living conditions of waste pickers”. (PDF 639 kb