Modulation allostérique de la fonction des récepteurs FP et PAF

Les récepteurs couplés aux protéines-G (RCPGs) constituent la première étape d’une série de cascades signalétiques menant à la régulation d’une multitude de processus physiologiques. Dans le modèle classique connu, la liaison du ligand induit un changement de conformation du récepteur qui mène à sa forme active. Une fois activés, les RCPGs vont réguler l’activité d’une protéine membranaire cible qui peut être tant une enzyme qu’un canal ionique. L’interaction entre le récepteur et la cible nécessite l’intermédiaire d’une protéine hétérotrimérique appelée « protéine G », qui est activée pour favoriser l’échange du GDP (guanosine diphosphate) pour un GTP (guanosine triphosphate) et assurer la transduction du signal du récepteur à l’effecteur. Les mécanismes moléculaires menant à l’activation des effecteurs spécifiques via l’activation des RCPGs par les protéines G hétérotrimériques sont encore plutôt méconnus. Dans notre étude nous nous sommes intéressés aux récepteurs FP et PAF, à leurs ligands naturels, la PGF2α et le Carbamyl-PAF respectivement, et à des ligands à action antagoniste sur ces récepteurs. Des ligands considérés comme agonistes, sont des molécules qui interagissent avec le récepteur et induisent les mêmes effets que le ligand naturel. Les antagonistes, par contre, sont des molécules qui interagissent avec le récepteur et bloquent l’action du ligand naturel en prévenant le changement conformationnel du complexe, et ils peuvent avoir une action compétitive ou non-compétitive. Nous avons étudié aussi des ligands orthostériques et allostériques du récepteur FP des prostaglandines et du récepteur PAF. Un ligand orthostérique peut se comporter comme agoniste ou antagoniste en se fixant au site de liaison du ligand (agoniste) naturel. Un ligand allostérique est un agoniste ou antagoniste se fixant à un site autre que celui du ligand naturel entraînant un changement de conformation ayant pour conséquence soit une augmentation (effecteur positif), soit une diminution (effecteur négatif) de l'activité du ligand naturel.

Le VEGF induit la synthèse du PAF par l’entremise de l’activation du VEGFR-2 : identification des phosphotyrosines impliquées

Notre laboratoire a démontré que la capacité proinflammatoire du vascular endothelial growth factor (VEGF-A165) implique la synthèse endothéliale du facteur d’activation plaquettaire (PAF) via l’activation du récepteur tyrosine kinase homodimérique VEGFR-2/R-2. La synthèse du PAF requiert l’activation de la p38 MAPK et p42/44 MAPK qui activent la phospholipase A2 secrétée de type V (sPLA2-V). Nous avons découvert que la synthèse aigue de prostacycline (PGI2) induite par le VEGF-A165 requiert l’activation des récepteurs hétérodimériques VEGFR-1/R-2. L’activation sélective des récepteurs du VEGF peut donc agir comme balance dans la synthèse de facteurs pro-(PAF) et anti-(PGI2) inflammatoire. Cependant, les tyrosines impliquées dans la transphosphorylation de VEGFR-2/R-2 menant à la synthèse du PAF sont inconnues. Par mutagenèse dirigée, nous avons effectué des transfections transitoires de cellules endothéliales avec des plasmides codant pour le VEGFR-2 dont les tyrosines ciblées ont été remplacées de façon séquentielle par une phénylalanine. Un vecteur vide pcDNA a été utilisé comme contrôle négatif. La stimulation des cellules endothéliales de l’aorte bovine (BAEC) transfectées avec le VEGF-A165 (1nM) pendant 15 minutes augmente la synthèse du PAF de 300%, laquelle était similaire dans les BAEC non transfectées. Dans les BAEC transfectées avec les vecteurs pcDNA codant pour les mutations Y801F, Y1059F, Y1175F et Y1214F, nous avons observé une réduction de 54, 73, 68, et 57% respectivement de la synthèse du PAF induite par le VEGF par rapport au pcDNA témoin. Nos résultats apportent un nouvel aperçu sur le mécanisme par lequel le VEGF induit la synthèse du PAF qui est connu pour sa contribution dans l’activité pro-inflammatoire du VEGF.

Staphylococcus aureus lipoteichoic acid inhibits platelet activation and thrombus formation via the Paf receptor

Impaired healing is common in wounds infected with the major human pathogen Staphylococcus aureus, although the underlying mechanisms are poorly understood. Here, we show that S.aureus lipoteichoic acid (LTA) inhibits platelet aggregation caused by physiological agonists and S. aureus and reduced platelet thrombus formation in vitro. The presence of D-alanine on LTA is necessary for the full inhibitory effect. Inhibition of aggregation was blocked using a monoclonal anti-platelet activating factor receptor (PafR) antibody and Ginkgolide B, a well-defined PafR antagonist, demonstrating that the LTA inhibitory signal occurs via PafR. Using a cyclic AMP (cAMP) assay and a western blot for phosphorylated VASP, we determined that cAMP levels increase upon platelet incubation with LTA, an effect which inhibits platelet activation. This was blocked when platelets were preincubated with Ginkgolide B. Furthermore, LTA reduced haemostasis in a mouse tail-bleed assay.

Platelet-activating factor receptor (PAF-R)-dependent pathways control tumour growth and tumour response to chemotherapy

Abstract Background Phagocytosis of apoptotic cells by macrophages induces a suppressor phenotype. Previous data from our group suggested that this occurs via Platelet-activating factor receptor (PAF-R)-mediated pathways. In the present study, we investigated the impact of apoptotic cell inoculation or induction by a chemotherapeutic agent (dacarbazine, DTIC) on tumour growth, microenvironmental parameters and survival, and the effect of treatment with a PAF-R antagonist (WEB2170). These studies were performed in murine tumours: Ehrlich Ascitis Tumour (EAT) and B16F10 melanoma. Methods Tumour growth was assessed by direct counting of EAT cells in the ascitis or by measuring the volume of the solid tumour. Parameters of the tumour microenvironment, such as the frequency of cells expressing cyclo-oxygenase-2 (COX-2), caspase-3 and galectin-3, and microvascular density, were determined by immunohistochemistry. Levels of vascular endothelium growth factor (VEGF) and prostaglandin E2 (PGE2) were determined by ELISA, and levels of nitric oxide (NO) by Griess reaction. PAF-R expression was analysed by immunohistochemistry and flow cytometry. Results Inoculation of apoptotic cells before EAT implantation stimulated tumour growth. This effect was reversed by in vivo pre-treatment with WEB2170. This treatment also reduced tumour growth and modified the microenvironment by reducing PGE2, VEGF and NO production. In B16F10 melanoma, WEB2170 alone or in association with DTIC significantly reduced tumour volume. Survival of the tumour-bearing mice was not affected by WEB2170 treatment but was significantly improved by the combination of DTIC with WEB2170. Tumour microenvironment elements were among the targets of the combination therapy since the relative frequency of COX-2 and galectin-3 positive cells and the microvascular density within the tumour mass were significantly reduced by treatment with WEB2170 or DTIC alone or in combination. Antibodies to PAF-R stained the cells from inside the tumour, but not the tumour cells grown in vitro. At the tissue level, a few cells (probably macrophages) stained positively with antibodies to PAF-R. Conclusions We suggest that PAF-R-dependent pathways are activated during experimental tumour growth, modifying the microenvironment and the phenotype of the tumour macrophages in such a way as to favour tumour growth. Combination therapy with a PAF-R antagonist and a chemotherapeutic drug may represent a new and promising strategy for the treatment of some tumours.

Activation of PAF-receptor induces regulatory dendritic cells through PGE2 and IL-10

Activation of the platelet-activating factor receptor (PAFR) in macrophages is associated with suppressor phenotype. Here, we investigated the PAFR in murine dendritic cells (DC). Bone marrow-derived dendritic cells (BALB/c) were cultured with GM-CSF and maturation was induced by LPS. The PAFR antagonists (WEB2086, WEB2170, PCA4248) and the prostaglandin (PG) synthesis inhibitors (indomethacin, nimesulide and NS-398) were added before LPS. Mature and immature DCs expressed PAFR. LPS increased MHCII, CD40, CD80, CD86, CCR7 and induced IL-10, IL-12, COX-2 and PGE2 expression. IL-10, COX-2 and PGE2 levels were reduced by PAFR antagonists and increased by cPAF. The IL-10 production was independent of PGs. Mature DCs induced antigen-specific lymphocyte proliferation. PAFR antagonists or PG-synthesis inhibitors significantly increased lymphocyte proliferation. It is proposed that PAF has a central role in regulatory DC differentiation through potentiation of IL-10 and PGE2 production.

The PCNA-associated factor KIAA0101/p15(PAF) binds the potential tumor suppressor product p33ING1b

The KIAA0101/p15(PAF)/OEATC-1 protein was initially isolated in a yeast two-hybrid screen for proliferating cell nuclear antigen (PCNA) binding partners, and was shown to bind PCNA competitively with the cell cycle regulator p21(WAF). PCNA is involved in DNA replication and damage repair. Using polyclonal antisera raised against a p15(PAF) fusion protein, we have shown that in a range of mammalian tumor and non-tumor cell lines the endogenous p15(PAF) protein localises to the nucleus and the mitochondria. Under normal conditions no co-localisation with PCNA could be detected, however following exposure to UV it was possible to co-immunoprecipitate p15(PAF) and PCNA from a number of cell lines, suggesting a UV-enhanced association of the two proteins. Overexpression of p15(PAF) in mammalian cells was also found to protect cells from UV-induced cell death. Based on similarities between the behaviour of p15(PAF) and the potential tumor suppressor product p33ING1b, we have further shown that these two proteins interact in the same complex in cell cultures. This suggests that p15(PAF) forms part of a larger protein complex potentially involved in the regulation of DNA repair, apoptosis and cell cycle progression. (c) 2005 Elsevier Inc. All rights reserved.

Rôle de la MSK1 dans la signalisation intracellulaire menant à la synthèse endothéliale de PAF induite par le VEGF

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Rôle de la MSK1 dans la signalisation intracellulaire menant à la synthèse endothéliale de PAF induite par le VEGF

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Influência do estímulo por PAF no perfil de abundância proteica em neutrófilos : uma abordagem proteômica

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2015.

Programa de acondicionamiento f?sico (paf) como propuesta para pacientes adultos con obesidad m?rbida pendientes de cirug?a bari?trica

La obesidad m?rbida (OM) es una enfermedad asociada a la obesidad donde el riesgo de morbimortalidad se acrecienta exponencialmente pudiendo llevar a consecuencias graves a los sujetos que la padecen. Los riesgos de ?sta enfermedad pueden evidenciar problemas f?sicos hasta consecuencias m?s alarmantes como enfermedades cardiorespiratorias, metab?licas cerebrovasculares, osteomusculares; como consecuencia de un desbalance energ?tico causado por m?ltiples factores en los cuales tienen implicaci?n el componente gen?tico, adem?s de conductas alimentarias y sedentarias a las cuales se suman las condiciones socioecon?micas, educativas y culturales de la poblaci?n. El inter?s de ?ste trabajo es presentar una propuesta de acondicionamiento f?sico (PAF) para obesos m?rbidos pendientes de cirug?a bari?trica, la metodolog?a empleada es la revisi?n a trav?s de bibliograf?a sobre documentos acerca del ejercicio y obesidad, programas de acondicionamiento para obesos. Se concluye que si bien existen estudios generales sobre obesidad y ejercicio, no se encuentran referenciados dise?os de programas de ejercicio espec?ficos para obesos m?rbidos, pendientes de cirug?a bari?trica.

Australian foundations and the downturn

Building on the investigation of the Charity Commission (2009) on the effects of the economic downturn on the largest trusts and foundation in the United Kingdom, the purpose of this research was to assess the extent to which Australian trusts and foundations were taking an actively strategic approach to their investments and pursuit of mission (including grant-making), and the relationship between the two in the context of the economic downturn. Focus was given to identifying the issues raised as a consequence of the economic downturn, rather than providing a generalised snapshot of the ‘average’ foundations response. In September 2009, semi-structured, in depth interviews were conducted with executives of 23 grant making trusts and foundations. The interviews for this research focused on the largest grant makers in terms of grant expenditure, however included foundations from different geographical locations and from across different cause areas. It is important to stress at the outset that this was not a representative sample of foundations; the study aimed to identify issues rather than to present a representative picture of the ‘average’ foundation’s response. It is also important to note that the study was undertaken in September 2009 at a time when many foundations were beginning to feel more optimistic about the longer term future, but aware of continuing and possibly worsening short term income problems. But whatever the financial future, some of the underlying issues, concerning investment and grant making management practices, raised in this report will be of continuing relevance worthy of wider discussion. If a crisis is too good to waste, it is also too good to forget. One other introductory point – as previously noted, interviews for this study were conducted in September 2009 – just one month prior to the introduction of the new Private Ancillary Fund (PAF) legislation which replaced the previous Prescribed Private Fund (PPF) arrangement1. References to PAFs and/or PPFs reflect that time.

Estimation on the mortality and disease burden attributed to selected risk factors in Shandong province [山东省主要危险因素的归因死亡和疾病负担分析]

Objective: To determine the major health related risk factors and provide evidence for policy-making,using health burden analysis on selected factors among general population from Shandong province. Methods: Based on data derived from the Third Death of Cause Sampling Survey in Shandong. Years of life lcrat(YLLs),yearS Iived with disability(YLDs)and disability-adjusted life years(DALYs) were calculated according to the GBD ethodology.Deaths and DALYs attributed to the selected risk factors were than estimated together with the PAF data from GBD 2001 study.The indirect method was employed to estimate the YLDs. Results: 51.09%of the total dearlls and 31.83%of the total DALYs from the Shandong population were resulted from the 19 selected risk factors.High blood pre.ure,smoking,low fruit and vegetable intake,aleohol consumption,indoor smoke from solid fuels,high cholesterol,urban air pollution, physical inactivity,overweight and obesity and unsafe injections in health care settings were identified as the top 10 risk faetors for mortality which together caused 50.21%of the total deaths.Alcohol use,smoking,high blood pressure,Low fruit and vegetable intake, indoor smoke from solid fuels, overweight and obesity,high cholesterol, physical inactivity,urban air pollution and iron-deficiency anemia were proved as the top 10 risk factors related to disease burden and were responsible for 29.04%of the total DALYs. Conclusion: Alcohol use.smoking and high blood pressure were determined as the major risk factors which influencing the health of residents in Shandong. The mortality and burden of disease could be reduced significantly if these major factors were effectively under control.