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Accueil > Publications > Recherche par années > Années 2010 > 2011

2011

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A substrate-based approach to convert SerpinB1 into a specific inhibitor of proteinase 3, the Wegener’s granulomatosis autoantigen.

The physiological and pathological functions of proteinase 3 (PR3) are not well understood due to its close similarity to human neutrophil elastase (HNE) and the lack of a specific inhibitor. Based on structural analysis of the active sites of PR3 and HNE, we generated mutants derived from the polyvalent inhibitor SerpinB1 (monocyte/neutrophil elastase inhibitor) that specifically inhibit PR3 and that differ from wt-SerpinB1 by only 3 or 4 residues in the reactive center loop. The rate constant of association between the best SerpinB1 mutant and PR3 is 1.4 x 10(7) M(-1) . s(-1), which is similar to 100-fold higher than that observed with wt-SerpinB1 and compares with that of alpha 1-protease inhibitor (alpha 1-PI) toward HNE. SerpinB1(S/DAR) is cleaved by HNE, but due to differences in rate, inhibition of PR3 by SerpinB1(S/DAR) is only minimally affected by the presence of HNE even when the latter is in excess. SerpinB1(S/DAR) inhibits soluble PR3 and also membrane-bound PR3 at the surface of activated neutrophils. Moreover, SerpinB1(S/DAR) clears induced PR3 from the surface of activated neutrophils. Overall, these specific inhibitors of PR3 will be valuable for defining biological functions of the protease and may prove useful as therapeutics for PR3-related inflammatory diseases, such as Wegener’s granulomatosis.-Jegot, G., Derache, C., Castella, S., Lahouassa, H., Pitois, E., Jourdan, M. L., Remold-O’Donnell, E., Kellenberger, C., Gauthier, F., Korkmaz, B. A substrate-based approach to convert SerpinB1 into a specific inhibitor of proteinase 3, the Wegener’s granulomatosis autoantigen.

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An alternative flexible conformation of the E. coli HUβ(2) protein : structural, dynamics, and functional aspects.

The histone-like HU protein is the major nucleoid-associated protein involved in the dynamics and structure of the bacterial chromosome. Under physiological conditions, the three possible dimeric forms of the E. coli HU protein (EcHUα₂, EcHUβ₂, and EcHUαβ) are in thermal equilibrium between two dimeric conformations (N₂ ↔ I₂) varying in their secondary structure content. High-temperature molecular dynamics simulations combined with NMR experiments provide information about structural and dynamics features at the atomic level for the N₂ to I₂ thermal transition of the EcHUβ₂ homodimer. On the basis of these data, a realistic 3D model is proposed for the major I₂ conformation of EcHUβ₂. This model is in agreement with previous experimental data.

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Breather-mediated energy transfer in proteins

In this paper we investigate how energy is redistributed across protein structures, following localized kicks, within the framework of a nonlinear network model. We show that energy is directed most of the times to a few specific sites, systematically within the stiffest regions. This effect is sharpened as the energy of the kicks is increased, with fractions of transferred energy as high as 70% already for kicks above [20] kcal/mol. Remarkably, we show that such site-selective, high-yield transfers mark the spontaneous formation of spatially localized, time-periodic vibrations at the target sites, acting as efficient energy-collecting centers. A comparison of our simulations with a previously developed theory reveals that such energy-pinning modes are discrete breathers, able to carry energy across the structure in an quasi-coherent fashion by jumping from site to site.

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CD133 Positive Progenitor Endothelial Cell Lines from Human Cord Blood

Endothelial progenitor cells (EPCs) modulate postnatal vascularization and contribute to vessel regeneration in adults. Stem cells and progenitor cells were found in umbilical cord blood, bone marrow, and mobilized peripheral blood cells, from where they were isolated and cultured. However, the yield of progenitor cells is usually not sufficient for clinical application and the quality of progenitor cells varies. The aim of the study was the immortalization of early progenitor cells with high proliferative potential, capable to differentiate to EPCs and, further, toward endothelial cells. Two cell lines, namely HEPC-CB. 1 and HEPC-CB. 2 (human endothelial progenitor cells-cord blood) were isolated. As assessed by specific antibody labeling and flow cytometric analysis, they express a panel of stem cell markers : CD133, CD13, CD271, CD90 and also endothelial cell markers : CD202b, CD309 (VEGFR2), CD146, CD105, and CD143 but they do not present markers of finally differentiated endothelial cells : CD31, vWf, nor CD45 which is a specific hematopoietic cell marker. Using the multiplex Cytometric Bead Assay, the simultaneous production of proangiogenic cytokines IL8, angiogenin, and VEGF was demonstrated in normoxia and was shown to be increased by hypoxia. Both cell lines, similarly as mature endothelial cells, underwent in vitro pre-angiogenic process, formed pseudovessel structures and present an accelerated angiogenesis in hypoxic conditions. To date, these are the first CD133 positive established cell lines from human cord blood cells. (C) 2011 International Society for Advancement of Cytometry.

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Cetuximab pharmacokinetics influences progression-free survival of metastatic colorectal cancer patients.

PURPOSE :

An ancillary phase II study was conducted to study interindividual variability in cetuximab pharmacokinetics and its influence on progression-free survival (PFS) in metastatic colorectal cancer patients cotreated with irinotecan and 5-fluorouracil.
EXPERIMENTAL DESIGN :

Ninety-six patients received cetuximab as an infusion loading dose of 400 mg/m(2) followed by weekly infusions of 250 mg/m(2). Doses of irinotecan and 5-fluorouracil were adjusted individually. Cetuximab concentrations were measured by ELISA. Compartmental pharmacokinetic parameters were estimated by a population approach, and PFS was analyzed using a Cox model.
RESULTS :

Cetuximab pharmacokinetics was best described using a two-compartment model with both first-order and saturable (zero-order) elimination. Estimated pharmacokinetic parameters (% standard error) were as follows : central volume of distribution V(1) = 2.96 L (4%), peripheral volume of distribution V(2) = 4.65 L (6%), elimination clearance CL = 0.497 L/d (4%), distribution clearance Q = 0.836 L/d (8%), and zero-order elimination rate k(0) = 8.71 mg/d (10%). Body surface area influenced V(1), V(2), and k(0). Pretreatment serum albumin influenced CL. Risk of disease progression decreased with cetuximab global clearance (cumulative dose/cumulative area under the concentration versus time curve ; P = 0.00016). Median PFS of patients with a cetuximab residual concentration on day 14 below median value was 3.3 months as compared with 7.8 months for the other patients (P = 0.004).
CONCLUSIONS :

Cetuximab pharmacokinetics in colorectal cancer patients can be described using a model combining linear and nonlinear elimination rates. PFS is influenced by global clearance of cetuximab, a parameter that can be estimated using cetuximab residual concentration on day 14.

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Chemical inhibitors of monogalactosyldiacylglycerol synthases in Arabidopsis thaliana

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the main lipids in photosynthetic membranes in plant cells. They are synthesized in the envelope surrounding plastids by MGD and DGD galactosyltransferases. These galacto-lipids are critical for the biogenesis of photosynthetic membranes, and they act as a source of polyunsaturated fatty acids for the whole cell and as phospholipid surrogates in phosphate shortage. Based on a high-throughput chemical screen, we have characterized a new compound, galvestine-1, that inhibits MGDs in vitro by competing with diacylglycerol binding. Consistent effects of galvestine-1 on Arabidopsis thaliana include root uptake, circulation in the xylem and mesophyll, inhibition of MGDs in vivo causing a reduction of MGDG content and impairment of chloroplast development. The effects on pollen germination shed light on the contribution of galactolipids to pollen-tube elongation. The whole-genome transcriptional response of Arabidopsis points to the potential benefits of galvestine-1 as a unique tool to study lipid homeostasis in plants.

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