Étude de l'hydrolyse d'oligopeptides dérivés de l'exon 24 et du polypeptide EP20-24-24-24-24 de la tropoélastine humaine par des élastases de mammifères et les protéases de Pseudomonas aeruginosa

Au cours de certaines pathologies, l'élastine, protéine responsable de l'élasticité de nombreux tissus peut être dégradée par des protéases spécifiques, les élastases. Parmi les peptides libérés, les matrikines, qui renferment la séquence GXXPG, sont connues pour réguler l'activité cellulaire. L'hydrolyse de peptides de type (VGVAPG)n et du polypeptide EP20-24-24-24-24, modèle de la tropoélastine humaine alternant des séquences de pontage et des régions hydrophobes riches en séquences VGVAPG a été étudiée. Les sites et produits d'hydrolyse de l'élastase pancréatique porcine, de l'élastase et de la protéinase 3 leucocytaires humaines et des protéases de Pseudomonas aeruginoa (élastase, protéase alcaline et LasA) ont été identifiés par spectrométrie de masse (ESI et MALDI-TOF) et par CLHP. L'élastase pancréatique de porc et la protéinase 3 libèrent des hexapeptides actifs. Nous avons également comparé l'adsorption des élastases sur l'élastine insoluble, première étape de l'élastolyseLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Interferon in chronic myeloid leukaemia: past and future.

International audienceImatinib has revolutionized the therapy of chronic myeloid leukaemia. However the complete eradication of leukaemic stem cells is still a matter of discussion. Interferon (IFN) has been used in the past with success. However the proportion of patients who achieved sustained complete cytogenetic response was small. Recently, in addition to its direct antineoplastic effect and immunomodulatory activity, IFN has been shown to stimulate the quiescent leukaemic stem cells. Thus there is now a rational for combining Imatinib and IFN. Large prospective phase III trials are in good progress to demonstrate in humans the usefullness of a combination therapy using Imatinib and IFN

Highly sensitive conductometric biosensors for total lactate, d- and l-lactate determination in dairy products

International audienceAn original method combining two conductometric biosensors is proposed for the determination of total lactate, l- and d-lactate in dairy products. The biosensors were prepared through cross-linking of l-lactate oxidase from Pediococcus sp. (LODP) or a combination of LODP and horseradish peroxidase (HRP) at the surface of gold interdigitated microelectrodes using glutaraldehyde (GA) vapors. LODP is reported to catalyze specifically l-lactate oxidation into pyruvate and hydrogen peroxide in solution. In this work, we showed that LODP chiral selectivity was lost following enzyme cross-linking, rendering LODP sensor suitable for total lactate determination. Biosensor sensitivity towards both stereoisomers was 1.16 ± 0.04 μS μM−1. The addition of HRP significantly improved the linear range and stability of LODP biosensor, but also increased its sensitivity, the effect being more pronounced for d-lactate (+381%) than for l-lactate (+260%). This result was attributed to a modification in LODP chiral selectivity combined with the production of additional ions (acetate, H+ and HCO3−) via the HRP-catalyzed oxidation of pyruvate. A method, taking advantage of the difference in d- and l-lactate behaviours and combining the monoenzymatic and bienzymatic biosensors calibration data, was proposed for the calculation of their concentrations in unknown samples. Different parameters (HRP/LODP ratio, exposure time to GA vapors, pH and concentration of measurement medium) were optimized in order to achieve the best compromise between sensitivity and stability of the biosensors. The best limit of detection for l- and d-lactate, calculated as three times the signal to noise ratio was achieved with the bi-enzymatic sensor and was equal to 0.05 μM. LODP/HRP biosensor response towards l-lactate was not significantly affected by glucose, fructose and lactose. The proposed biosensors were finally applied to the determination of total lactate, d- and l-lactate concentrations in yogurt samples. Results were in good agreement with those obtained using a reference colorimetric enzymatic method

Caractérisation électrochimique des propriétés conductrices d’hybrides nanoparticulesd’or/polyéthylèneimine/alcool polyvinylique/thermolysine. Application au développement d’un biocapteurenzymatique.

International audienceCaractérisation électrochimique des propriétés conductrices d’hybrides nanoparticulesd’or/polyéthylèneimine/alcool polyvinylique/thermolysine. Application au développement d’un biocapteurenzymatiqu

Biomimetic vesicles for electrochemical sensing

International audienceBiomimetic vesicles, mainly composed of self-assembled bilayers of phospholipids, have attracted great attention for applications in the biosensor field over a number of decades, as a means to amplify the signal through encapsulated signal probes. In this review paper the most important developments in biomimetic vesicles for electrochemical biosensing within the last 2 years are presented, with a focus on the format of bioassays, their inclusion in microfluidic chip devices and their use in mimicking cell membranes. Key issues and the remaining challenges for future commercialization are analyzed

Thermolysin entrapped in a gold nanoparticles/polymer composite for direct and sensitive conductometric biosensing of ochratoxin A in olive oil

International audienceAn original biosensor was developed for the direct conductometric detection of ochratoxin A (OTA) in olive oil samples. The biosensor is based on thermolysin (TLN) immobilization into a polyvinyl alcohol (PVA)/polyethylenimine (PEI) matrix containing gold nanoparticles (AuNPs) and cross-linked at the surface of gold interdigitated microelectrodes using glutaraldehyde. Under optimal conditions (35 min cross-linking time, working pH of 7 and temperature of 25 degrees C), the biosensor response was linear up to 60 nM OTA, with a sensitivity of 597 mu S mu M-1 and a limit of detection of 1 nM. This value was 700 times lower than the detection limit obtained using the more classical method based on enzyme cross-linking in the presence of bovine serum albumin (BSA). PVA/PEI hydrogel creates a very favorable aqueous environment for the enzyme. In addition, interactions between protonated amino groups of PEI and negative charges of both citrated AuNPs and thermolysin improve their dispersion in the polymer blend, favoring enzyme stabilization and accessibility of the substrate. No conductometric signal was observed after OTA injection in the absence of AuNPs, in agreement with the insulating properties of the cross-linked PVA/PEI hydrogel film. Incorporation of AuNPs into the TLN/BSA biomembrane helped improving the sensitivity by 5.3 but this latter remained 140 times lower than the sensitivity of TLN/AuNPs/(PVA/PEI) biosensor. The study of enzyme kinetics showed that V-i vs [OTA] plot exhibited a non-hyperbolic trend, indicating that kinetics does not display a Michaelis-Menten behavior. Biosensor response times are longer (7-48 min) comparatively to TLN/BSA biosensor with a maximal value of 25 min. This difference is due to the diffusion phenomenon through the pores of the polymer membrane. The proposed OTA biosensor was very reproducible with a relative standard deviations (RSDs) in the 3-15% range and stable over a 30-days period when stored at 4 degrees C in 20 mM phosphate buffer between two measurements. The method was further evaluated using commercial doped olive oil samples. No pretreatment of the sample was needed for testing and no matrix effect was observed. Recovery values were close to 100% demonstrating the suitability of the proposed method for OTA screening in olive oil