Liposome-Mediated Cellular Delivery of Active gp91phox

International audienceBACKGROUND: Gp91(phox) is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils. Lack of this protein causes chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms. METHODOLOGY: Here we optimize a prokaryotic cell-free expression system to produce integral mammalian membrane proteins. CONCLUSIONS: Using this system, we over-express truncated forms of the gp91(phox) protein under soluble form in the presence of detergents or lipids resulting in active proteins with a "native-like" conformation. All the proteins exhibit diaphorase activity in the presence of cytosolic factors (p67(phox), p47(phox), p40(phox) and Rac) and arachidonic acid. We also produce proteoliposomes containing gp91(phox) protein and demonstrate that these proteins exhibit activities similar to their cellular counterpart. The proteoliposomes induce rapid cellular delivery and relocation of recombinant gp91(phox) proteins to the plasma membrane. Our data support the concept of cell-free expression technology for producing recombinant proteoliposomes and their use for functional and structural studies or protein therapy by complementing deficient cells in gp91(phox) protein

Electrical, Thermal and Optical Diagnostics of an Atmospheric Plasma Jet System

Plasma diagnostics of atmospheric plasmas is a key tool in helping to understand processing performance issues. This paper presents an electrical, optical and thermographic imaging study of the PlasmaStream atmospheric plasma jet system. The system was found to exhibit three operating modes; one constricted/localized plasma and two extended volume plasmas. At low power and helium flows the plasma is localized at the electrodes and has the electrical properties of a corona/filamentary discharge with electrical chaotic temporal structure. With increasing discharge power and helium flow the plasma expands into the volume of the tube, becoming regular and homogeneous in appearance. Emission spectra show evidence of atomic oxygen, nitric oxide and the hydroxyl radical production. Plasma activated gas temperature deduced from the rotational temperature of nitrogen molecules was found to be of order of 400 K: whereas thermographic imaging of the quartz tube yielded surface temperatures between 319 and 347 K.<br/

Etude de l'ATPase CA + du reticulum sarco/endoplasmique (mise au point d'une nouvelle méthode de purification de SERCA1A de lapin exprimée chez S. cerevisiae permettant sa cristallisation et applications au mutant E309Q. Etude d'une autre isoforme SERCA3A )

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Etude de l'ATPase Ca2+ du réticulum sarco/endoplasmique : Mise au point d'une nouvelle méthode de purification de SERCA1a de lapin exprimée chez S. cerevisiae permettant sa cristallisation et applications au mutant E309Q-Etude d'une autre isoforme, SERCA3a

We describe here a new purification method of the rabbit SERCA1a after its heterologous expression in the yeast S. cerevisiae . A “biotin acceptor domain” is fused at the C-terminal part of the protein and is biotinylated in vivo by the yeast. The purification procedure, based on the strong interaction between avidin and biotin, allows us to obtain a protein pure at 40-50% and well active. After an additional purification step by SEC-HPLC the protein is pure at about 70% and always well active. Crystals were obtained from SERCA1a purified by this method and they diffract X-rays at 3,1 Å.This new method was used with sucicess to purify the mutant E309Q. Small crystals of the mutant were obtained. SERCA3a could also be purified using this procedure, but the low yield of expression limits the amount that could be purified. In parallel, immunolocalization of SERCA3a was performed in several cell lines and in cryosections of skin.Dans cette thèse est présentée une nouvelle stratégie de purification de SERCA1a après son expression hétérologue chez S. cerevisiae.. La protéine est fusionnée à un domaine accepteur de biotine, biotinylé in vivo par la levure. La procédure de purification, basée sur la forte interaction entre avidine et biotine, permet d'obtenir une protéine active pure à 40-50%. Une étape supplémentaire de filtration sur gel en HPLC a permis d'augmenter la pureté d'environ 70%, tout en conservant une très bonne activité spécifique. Les cristaux obtenus de SERCA1a ainsi purifiée diffractent les rayons X à 3,1Å. Cette nouvelle méthode de purification a été appliquée avec succès au mutant SERCA1a-E309Q. De petits cristaux de ce mutant ont pu être isolés. Cette méthode a également permis de purifier SERCA3a, bien que le faible taux d'expression de la protéine de fusion chez S. cerevisiae limite la quantité purifiée. En parallèle, des essais d'immunolocalisation cellulaire de SERCA3a dans différentes lignées cellulaires et dans des coupes de peau ont été réalisé

Caractérisation électrique et couplages électro-thermiques des décharges à barrières diélectriques dans l'air à pression atmosphérique (faisabilité de l'électrofiltration d'aérosol)

Résumé françaisRésumé anglaisORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF