Non-Metabolic Membrane Tubulation and Permeability Induced by Bioactive Peptides

BACKGROUND: Basic cell-penetrating peptides are potential vectors for therapeutic molecules and display antimicrobial activity. The peptide-membrane contact is the first step of the sequential processes leading to peptide internalization and cell activity. However, the molecular mechanisms involved in peptide-membrane interaction are not well understood and are frequently controversial. Herein, we compared the membrane activities of six basic peptides with different size, charge density and amphipaticity: Two cell-penetrating peptides (penetratin and R9), three amphipathic peptides and the neuromodulator substance P. METHODOLOGY/PRINCIPAL FINDINGS: Experiments of X ray diffraction, video-microscopy of giant vesicles, fluorescence spectroscopy, turbidimetry and calcein leakage from large vesicles are reported. Permeability and toxicity experiments were performed on cultured cells. The peptides showed differences in bilayer thickness perturbations, vesicles aggregation and local bending properties which form lipidic tubular structures. These structures invade the vesicle lumen in the absence of exogenous energy. CONCLUSIONS/SIGNIFICANCE: We showed that the degree of membrane permeabilization with amphipathic peptides is dependent on both peptide size and hydrophobic nature of the residues. We propose a model for peptide-induced membrane perturbations that explains the differences in peptide membrane activities and suggests the existence of a facilitated “physical endocytosis,” which represents a new pathway for peptide cellular internalization

Development and Validation of an Internationally-Standardized, High-Resolution Capillary Gel-Based Electrophoresis PCR-Ribotyping Protocol for Clostridium difficile

PCR-ribotyping has been adopted in many laboratories as the method of choice for C. difficile typing and surveillance. However, issues with the conventional agarose gel-based technique, including inter-laboratory variation and interpretation of banding patterns have impeded progress. The method has recently been adapted to incorporate high-resolution capillary gel-based electrophoresis (CE-ribotyping), so improving discrimination, accuracy and reproducibility. However, reports to date have all represented single-centre studies and inter-laboratory variability has not been formally measured or assessed. Here, we achieved in a multi-centre setting a high level of reproducibility, accuracy and portability associated with a consensus CE-ribotyping protocol. Local databases were built at four participating laboratories using a distributed set of 70 known PCR-ribotypes. A panel of 50 isolates and 60 electronic profiles (blinded and randomized) were distributed to each testing centre for PCR-ribotype identification based on local databases generated using the standard set of 70 PCR-ribotypes, and the performance of the consensus protocol assessed. A maximum standard deviation of only ±3.8bp was recorded in individual fragment sizes, and PCR-ribotypes from 98.2% of anonymised strains were successfully discriminated across four ribotyping centres spanning Europe and North America (98.8% after analysing discrepancies). Consensus CE-ribotyping increases comparability of typing data between centres and thereby facilitates the rapid and accurate transfer of standardized typing data to support future national and international C. difficile surveillance programs

Dynamique à basses températures d’une sonde adicalaire nitroxyde en milieux poreux

La dynamique du radical nitroxyde TEMPO dans l'ethanol adsorbé dans des matériaux poreux est étudiée par RPE en bande X entre 100 et 250 K. Au dessus du point de transition vitreuse de l'éthanol, le radical se répartit entre les parois des pores et le solvant libre. On a déterminé les proportions de ces sites et leur vitesse d'échange qui se situe entre 106 et 108s-1 lorsqu'il n'y a pas de liaison hydrogène entre le radical et les parois. Lorsque celle-ci existe, dans la silice par exemple, l'échange n'est plus observé. Ce comportement est corrélé avec le couplage hyperfin de l'azote qui augmente avec la polarité locale. Des expériences analogues sur des résines de polymère, confirment une large distribution de tailles de pores mise en évidence par relaxation nucléaire

Simulations de spectres de résonance paramagnétique électronique avec ajustement automatique des paramètres spectroscopiques et du temps de corrélation de réorientation

La détermination précise du tenseur de couplage hyperfin A et du tenseur g peut être réalisée par ajustement automatique des paramètres recherchés, en minimisant la somme des carrés des écarts entre les spectres de RPE expérimental et calculé, au moyen de l'algorithme de Levenberg-Marquardt. Cette démarche est illustrée par l'exemple d'un gel de poly 4-vinylepyridine réticulé par des ions vanadyles, rigide à basses températures.Les mouvements de réorientation de la sonde paramagnétique modifient la forme des spectres comme on peut le constater pour ce gel au-dessus de 260 K. Le temps de corrélation τR pour un modèle de réorientation brownienne isotrope est pris comme paramètre ajustable des simulations spectrales, en utilisant l'algorithme de minimisation de la suite de Fibonacci

NMR investigation of the micellar properties of monoalkylphoshpates

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Some recent applications of computer simulations of ESR spectra to the dynamics of spin probes in gels and membranes

The ESR of S=1/2 paramagnetic species is one of the most convenient methods for probing microheterogeneous structures in gels, lyotropic liquid crystals or membranes. The interpretation of ESR spectra in term of dynamics of the spin probes generally requires computer simulations whose principles are briefly described. Some examples are given of simulations applied to the determination of the flexibility of the tridimensional network in polymer gels crosslinked by Cu2+ and VO2+ as well as to the dynamics of nitroxide spin probes in gels and in phospholipid model membranes

Radicaux libres formés par action d’atomes d’hydrogène produits en phase gazeuse sur quelques composés organiques à 77°K

Des composés organiques insaturés, refroidis à 77°K ont été soumis à l’action d’atomes d’hydrogène, produits en phase gazeuse par décharge hyperfréquence, afin d’étudier par Résonance Paramagnétique Électronique les radicaux formés par addition sur les doubles liaisons C = C et C = O. Dans quelques expériences le deutérium a été substitué à l’hydrogène pour faciliter l’interprétation des spectres. La validité de ces interprétations a été vérifiée chaque fois que cela était possible par la méthode du 2e moment. Les monomères vinyliques donnent de préférence des radicaux du type CH3 — [math]H — X ou CH3 — [math](CH3) — X et il semble que ces espèces ne réagissent pas à 77°K avec d’autres molécules de monomère. Dans le cas ou X = CN, nous avons pu évaluer l’interaction de l’électron célibataire avec l’azote. Les composés carbonylés donnent les radicaux R2 = [math] — OH avec une faible proportion de radicaux R2 = CH — O•. L’addition de H• ne se produit pas sur la double liaison C = C de l’alcool allylique et l’on obtient le radical CH2 = CH — [math]HOH par arrachement d’un hydrogène. Ce résultat nous a conduit à étudier les réactions d’arrachement produits par les atomes H•, sur quelques alcools et composés halogénés à 77°K

NMR and ESR study of the conformations and dynamical properties of poly(L-lysine) in aqueous solutions

International audienceThe conformations and dynamical behavior of poly(L-lysine) (PLL) in aqueous solutions have been investigated by and l8C NMR as well as by ESR on the end-chain spin-labeled polymer. The ESR allowed the motion of the macromolecular chain to be studied up to pH 13, showing that the random coil-*-helix transition at pH 11 gives rise to a twofold increase in the correlation time, with evidence of an anisotropic reorientation. In the random coil state at pH 7, where the segmental motion of the backbone is quasi-isotropic, the correlation time given by ESR is compared to that obtained by the relaxation of the methyl protons of the reduced Tempo radical residue and of the a carbons. The different methods yield an activation energy of 6.5 kcal mol"1 for this motion whereas the frequency dependence of the Ca relaxation may be interpreted by a Cole-Cole distribution of correlation times with a width parameter = 0.7. The rotational isomerism and temperature dependences of interconversion rates of the aminobutyl side chains have been analyzed from the proton vicinal couplings and the 13C and relaxation at different frequencies, assuming that the methylene groups undergo 120°j umps among three sites, two of them being equiprobable. These two kinds of information concur to show that the PLL side chains are less flexible than a hydrocarbon chain of same length, possibly because of the hydration of the NH3+ terminal group

NMR and nuclear fuel reprocessing

31P NMR is useful for the study of the hydrolytic and radiolytic degradation of tributylphosphate (TBP), extractant used for nuclear fuel reprocessing. Each degradation product can be easily identified and measured. NMR can also be used to study the behaviour of technetium, (99Tc), a fission product which able to cause problems in the implementation of the process. N,N- dialkylamides are alternative extractants to TBP for fuel reprocessing. Their extracting properties can also be studied using NMR. An NMR conformational analysis helps us to understand the behaviour of these new molecules