Expression, purification, crystallization and preliminary X-ray analysis of the cathelicidin motif of the protegrin-3 precursor

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Therapeutic Approaches Using Host Defence Peptides to Tackle Herpes Virus Infections

One of the most common viral infections in humans is caused by herpes simplex virus (HSV). It can easily be treated with nucleoside analogues (e.g., acyclovir), but resistant strains are on the rise. Naturally occurring antimicrobial peptides have been demonstrated to possess antiviral activity against HSV. New evidence has also indicated that these host defence peptides are able to selectively stimulate the innate immune system to fight of infections. This review will focus on the anti-HSV activity of such peptides (both natural and synthetic), describe their mode of action and their clinical potential

DNA Display Selection of Peptide Ligands for a Full-Length Human G Protein-Coupled Receptor on CHO-K1 Cells

The G protein-coupled receptors (GPCRs), which form the largest group of transmembrane proteins involved in signal transduction, are major targets of currently available drugs. Thus, the search for cognate and surrogate peptide ligands for GPCRs is of both basic and therapeutic interest. Here we describe the application of an in vitro DNA display technology to screening libraries of peptide ligands for full-length GPCRs expressed on whole cells. We used human angiotensin II (Ang II) type-1 receptor (hAT1R) as a model GPCR. Under improved selection conditions using hAT1R-expressing Chinese hamster ovary (CHO)-K1 cells as bait, we confirmed that Ang II gene could be enriched more than 10,000-fold after four rounds of selection. Further, we successfully selected diverse Ang II-like peptides from randomized peptide libraries. The results provide more precise information on the sequence-function relationships of hAT1R ligands than can be obtained by conventional alanine-scanning mutagenesis. Completely in vitro DNA display can overcome the limitations of current display technologies and is expected to prove widely useful for screening diverse libraries of mutant peptide and protein ligands for receptors that can be expressed functionally on the surface of CHO-K1 cells

Binding of protegrin-1 to Pseudomonas aeruginosa and Burkholderia cepacia

BACKGROUND: Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia. METHODS: The PG-1 sensitivity/resistance and PG-1 binding properties of P. aeruginosa and B. cepacia were assessed using radial diffusion assays, radioiodinated PG-1, and surface plasmon resonance (BiaCore). RESULTS: The six P. aeruginosa strains examined were very sensitive to PG-1, exhibiting minimal active concentrations from 0.0625–0.5 μg/ml in radial diffusion assays. In contrast, all five B. cepacia strains examined were greater than 10-fold to 100-fold more resistant, with minimal active concentrations ranging from 6–10 μg/ml. When incubated with a radioiodinated variant of PG-1, a sensitive P. aeruginosa strain bound considerably more protegrin molecules per cell than a resistant B. cepacia strain. Binding/diffusion and surface plasmon resonance assays revealed that isolated lipopolysaccharide (LPS) and lipid A from the sensitive P. aeruginosa strains bound PG-1 more effectively than LPS and lipid A from resistant B. cepacia strains. CONCLUSION: These findings support the hypothesis that the relative resistance of B. cepacia to protegrin is due to a reduced number of PG-1 binding sites on the lipid A moiety of its LPS

A theoretical approach to spot active regions in antimicrobial proteins

Background: Much effort goes into identifying new antimicrobial compounds able to evade the increasing resistance of microorganisms to antibiotics. One strategy relies on antimicrobial peptides, either derived from fragments released by proteolytic cleavage of proteins or designed from known antimicrobial protein regions. Results: To identify these antimicrobial determinants, we developed a theoretical approach that predicts antimicrobial proteins from their amino acid sequence in addition to determining their antimicrobial regions. A bactericidal propensity index has been calculated for each amino acid, using the experimental data reported from a high-throughput screening assay as reference. Scanning profiles were performed for protein sequences and potentially active stretches were identified by the best selected threshold parameters. The method was corroborated against positive and negative datasets. This successful approach means that we can spot active sequences previously reported in the literature from experimental data for most of the antimicrobial proteins examined. Conclusion: The method presented can correctly identify antimicrobial proteins with an accuracy of 85% and a sensitivity of 90%. The method can also predict their key active regions, making this a tool for the design of new antimicrobial drugs

Stimuli of Sensory-Motor Nerves Terminate Arterial Contractile Effects of Endothelin-1 by CGRP and Dissociation of ET-1/ETA-Receptor Complexes

Endothelin-1 (ET-1), a long-acting paracrine mediator, is implicated in cardiovascular diseases but clinical trials with ET-receptor antagonists were not successful in some areas. We tested whether the quasi-irreversible receptor-binding of ET-1 (i) limits reversing effects of the antagonists and (ii) can be selectively dissociated by an endogenous counterbalancing mechanism.-receptor complexes.-receptors by ET-1 (i) occur at an antagonist-insensitive site of the receptor and (ii) are selectively terminated by endogenously released CGRP. Hence, natural stimuli of sensory-motor nerves that stimulate release of endogenous CGRP can be considered for therapy of diseases involving ET-1

Milling of pharmaceutical compounds : physical state changes and stability manipulation

Ce mémoire présente l'étude d'un principe actif pharmaceutique (PA) peu soluble dans l'eau. Sa formulation à l'état amorphe serait un moyen d'augmenter sa solubilité. Tout d'abord, nous avons montré que le polymorphisme du PA est plus complexe que les deux formes polymorphiques 1 (stable) et II (méta stable) jusqu'à présent connues et liées par une relation de monotropie. Deux nouvelles formes polymorphiques métastables ont été révélées par calorimétrie et diffraction des rayons X. Nous avons ensuite montré que le broyage de la forme 1 du PA conduit à son amorphisation. Le verre obtenu présente les mêmes caractéristiques structurales que le liquide trempé. Cependant, il n'est pas stable et cristallise, au réchauffage, peu après la transition vitreuse et en quelques jours lorsqu'il est stocké à température et humidité ambiantes. Nous avons cherché à stabiliser le PA amorphe par co-amorphisation avec un excipient polymérique amorphe (HPMC) et un excipient moléculaire cristallin (tréhalose anhydre). Des solutions solides et des alliages moléculaires amorphes homogènes, qui ne peuvent pas être formés par le procédé de fusion/trempe, ont été obtenus pour toute composition par co-broyage des mélanges excipient/PA(forme 1). La stabilité au réchauffage de ces solutions solides a été nettement améliorée. Au stockage, les solutions solides HPMC/PA présentent une meilleure stabilité que les alliages vitreux tréhalose/PA (stabilité de 2 ans à température et humidité ambiantes). Cette étude montre que la valeur de la température de transition vitreuse n'est pas un paramètre suffisant pour prédire la stabilité de composés amorphes. Un mécanisme d'amorphisation par broyage est proposé.Ln this thesis, a study of an active pharmaceutical ingredient (APl), weakly soluble in water, is reported. Its formulation in the amorphous state would be a means to increase its solubility. First, we showed that the polymorphism of this compound is more complex than the two forms, 1 (stable) and II (metastable), till now known and linked together by a monotropic relation. Two new metastable polymorphic forms were revealed by calorimetry and X-ray diffraction. Then, we showed that the milling of the APl stable form 1 induces its amorphization. The obtained glass has the same structural characteristics than the quenched liquid. However, it is not stable and crystallizes, on heating, just after the glass transition and in a few days when stored under ambient temperature and humidity. We tried to stabilize the amorphous APl by co-amorphization with an amorphous polymeric excipient (HPMC) and with a crystalline molecular excipient (anhydrous trehalose). Homogeneous amorphous solid solutions and molecular alloys, unreachable by the meltinglquenching process, were obtained by co-milling for any composition of the mixtures excipient/API(form 1). The stability on heating of these glass solutions was really enhanced. During storages, the HPMCIAPI glass solutions present a better stability than the trehalose/API vitreous alloys (stability of 2 years under ambient temperature and humidity). This investigation shows that the glass transition temperature value is not a sufficient parameter to predict the stability of amorphous compounds. A mechanism for the amorphization by milling is proposed

Bare and Shrouded vertical axis water turbine modelling : development of an experimental device and a numerical facility for the study of cavitation

Cette thèse s'inscrit dans le cadre des énergies renouvelables au sein du programme HARVEST centré sur le développement d'un concept d'hydrolienne dérivé des turbines Darrieus et Gorlov. L'ajout d'un dispositif appelé carénage à la turbine permet à celle-ci d'extraire une portion de l'énergie cinétique du courant plus grande. Toutefois ce dernier peut favoriser la cavitation qui nuit à la turbine. Parmi les différents axes du programme, les travaux de thèse se situent dans cette problématique. En régime subcavitant et cavitant, l'analyse de l'hydrolienne a été menée suivant une approche numérique et expérimentale. Pour ce faire deux outils ont été mis en place. Du coté expérimental, le tunnel hydrodynamique du LEGI a été équipé d'une balance qui donne la mesure instantanée des forces et du couple qui s'exercent sur la turbine. Du coté numérique, les efforts ont été orientés sur l'amélioration et le développement du code de calcul universitaire, CAVKA. L'utilisation intensive de ces deux moyens, couplée à des modèles théoriques, a permis de mettre en évidence d'une part le fonctionnement de la turbine libre ou carénée et, d'autre part, les limites de fonctionnement vis-à-vis de la cavitation.The general context of the present thesis is renewable energies within the HARVEST project, which consists in a water current turbine (WCT) development, inspired from the Darrieus and Gorlov geometries. The main advantage of the HARVEST WCT is the introduction of a channelling device, which allows extracting a bigger amount of the kinetic energy contained in the flowstream. However, the shrouding device can eventually increase cavitating risks, which generally damage the WCT itself and its performance. The main topic of this work is cavitation. The hydrodynamic behavior of the WCT is analyzed both numerically and experimentally, in non cavitating and cavitating conditions. For this analysis, two devices have been developed. On the one hand, the LEGI hydrodynamic channel is equipped with a measurement platform which provides the instantaneous and average measurements of two dimensional thrusts as well as the hydrodynamic torque. On the other hand, in the numerical domain, the work has been oriented to the improvement and the development of a CFD code, named Cavka. The intensive utilisation of these two devices, coupled to theoretical models, allow highlighting the functioning of the bare and shrouded WCTs and their limits in cavitating conditions