Mise au point d'analyse peptidique de cellules tumorales HT29 par spectrométrie de masse à haute résolution

Les peptides sont impliqués dans la régulation de nombreux processus physiologiques. Ils représentent, ainsi, des cibles de choix pour la recherche pharmacologique et médicale. L objectif de ce travail est de mettre au point une méthode d analyse des peptides dans des cellules cancéreuses HT29. La peptidomique, basée sur des techniques modernes de spectrométrie de masse, est un domaine de recherche en plein essor. Ainsi, la mesure des masses moléculaires des peptides en mélange a été réalisée par MALDI/TOF (désorption/ionisation laser assistée par matrice) et l identification de quatre peptides a pu être obtenue par spectrométrie de masse à très haute résolution à l aide d un instrument hybride Qh-FT-ICR (résonance cyclotronique ionique à transformée de Fourier), équipé d une source electrospray. Cette source est couplée en amont à une chromatographie liquide. Une des perspectives de ce travail est de détecter des peptides impliqués dans des voies de signalisation menant à l apoptose.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Prise en compte de trois risques d'origine externe pour les installations du CEA Cadarache.

Les installations du CEA Cadarache peuvent être soumises, comme toutes les autres, aux risques d'origine externe tels que les séismes, les chutes d'avions, les événements climatiques extrêmes, etc. Le présent document fait le point sur trois risques auxquels il a fallu faire face dans un passé relativement récent : les incendies de forêt, la foudre et la neige

The presence of the anti-inflammatory protein MAM, from Faecalibacterium prausnitzii, in the intestinal ecosystem.

International audienceFaecalibacterium prausnitzii strain A2-165 was previously reported to have anti-inflammatory properties and prevent colitis in a TNBS model. We compared the immunomodulatory properties of strain A2-165 to four different F. prausnitzii isolates and eight abundant intestinal commensals using human dendritic cells (DCs) and mouse BMDCs in vitro. Principal component analysis revealed that the cytokine response to F. prausnitzii A2-165 is distinct from the other strains in eliciting high amounts of IL-10 secretion. The mouse DNBS model of relapsing IBD was used to compare the protective effects of F. prausnitzii A2-165 and Clostridium hathewayi, a low secretor of IL-10, on the Th1-driven inflammatory response to DNBS; attenuation of disease parameters was only observed with F. prausnitzii. In an in vivo mouse model of nasal tolerance to ovalbumin, F. prausnitzii A2-165 enhanced ovalbumin-specific T cell proliferation and reduced the proportion of IFN-γ(+) T cells in CLNs. Similarly, in vitro F. prausnitzii A2-165 stimulated BMDCs increased ovalbumin-specific T cell proliferation and reduced the number of IFN-γ(+) T cells. These mechanisms may contribute to the anti-inflammatory effects of F. prausnitzii in colitis and support the notion that this abundant bacterium might contribute to immune homeostasis in the intestine via its anti-inflammatory properties

High-resolution mass spectrometry and partial de novo sequencing constitute a useful approach for determining the profile of chemokine secretion following the stimulation of human intestinal epithelial cells.

International audienc

Acyl Chains of Phospholipase D Transphosphatidylation Products in Arabidopsis Cells: A Study Using Multiple Reaction Monitoring Mass Spectrometry

<div><h3>Background</h3><p>Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor.</p> <h3>Methodology/Principal findings</h3><p>Potential PLD substrates and products were studied in <em>Arabidopsis thaliana</em> suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of <em>n</em>-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in <em>in vitro</em> assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the <em>in vivo</em> products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates.</p> <h3>Conclusions</h3><p>MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products <em>per se</em> is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.</p> </div

Inter-kingdom effect on epithelial cells of the N-Acyl homoserine lactone 3-oxo-C12:2, a major quorum-sensing molecule from gut microbiota.

BACKGROUND AND AIMS:N-acyl homoserine lactones (AHLs), which are autoinducer quorum-sensing molecules involved in the bacterial communication network, also interact with eukaryotic cells. Searching for these molecules in the context of inflammatory bowel disease (IBD) is appealing. The aims of our study were to look for AHL molecules in faecal samples from healthy subjects (HS) and IBD patients to correlate AHL profiles with the microbiome and investigate the effect of AHLs of interest on epithelial cells. METHODS:Using mass spectrometry, we characterised AHL profiles in faecal samples from HS (n = 26) and IBD patients in remission (n = 24) and in flare (n = 25) and correlated the presence of AHLs of interest with gut microbiota composition obtained by real-time qPCR and 16S sequencing. We synthesised AHLs of interest to test the inflammatory response after IL1β stimulation and paracellular permeability on Caco-2 cells. RESULTS:We observed 14 different AHLs, among which one was prominent. This AHL corresponded to 3-oxo-C12:2 and was found significantly less frequently in IBD patients in flare (16%) and in remission (37.5%) versus HS (65.4%) (p = 0.001). The presence of 3-oxo-C12:2 was associated with significantly higher counts of Firmicutes, especially Faecalbacterium prausnitzii, and lower counts of Escherichia coli. In vitro, 3-oxo-C12:2 exerted an anti-inflammatory effect on Caco-2 cells. Interestingly, although 3-oxo-C12, the well-known AHL from Pseudomonas aeruginosa, increased paracellular permeability, 3-oxo-C12:2 did not. CONCLUSIONS:We identified AHLs in the human gut microbiota and discovered a new and prominent AHL, 3-oxo-C12:2, which correlates with normobiosis and exerts a protective effect on gut epithelial cells

Profiles of PBut produced <i>in vitro</i> by Arabidopsis microsomal PLDs.

<p>Microsomes were used in an enzymatic assay on lipid vesicles. The substrate used was PE. The reaction assay was defined as α-type, β/γ-type or δ-type. The reactions were performed at 37°C for 20 min in the presence of 0.6% (v/v) <i>n-</i>butanol. White bars, substrate; black bars, PBut.</p