K. El Kirat

Sample preparation procedures for biological atomic force microscopy

Since the late 1980s, atomic force microscopy (AFM) has been increasingly used in biological sciences and it is now established as a versatile tool to address the structure, properties and functions of biological specimens. AFM is unique in that it provides three‐dimensional images of biological structures, including biomolecules, lipid films, 2D protein crystals and cells, under physiological conditions and with unprecedented resolution. A crucial prerequisite for successful, reliable biological AFM is that the samples need to be well attached to a solid substrate using appropriate, nondestructive methods. In this review, we discuss common techniques for immobilizing biological specimens for AFM studies.

Ennoblement of stainless steel in the presence of glucose oxidase: Nature and role of interfacial processes

The ennoblement of the free corrosion potential (E(corr)) of AISI 316L stainless steel which did not occur in synthetic fresh water (SFW), was observed after introduction of glucose oxidase (Gox) and glucose, or of hydrogen peroxide (H(2)O(2)). The composition of the surface was monitored using AFM and XPS, a detailed XPS analysis being based on the discrimination between oxygen of organic and inorganic nature proposed in a previous study. In H(2)O(2) medium, the main changes regarding the inorganic phase were the increase of the oxygen concentration in the passive film, the increase of the molar concentration ratio of oxidized species Fe(ox)/Cr(ox) and the growth of nanoparticles, presumably made of ferric oxide/hydroxide. In Gox medium, no significant changes were observed in both oxygen concentration and Fe(ox)/Cr(ox) ratio, but the density of colloidal particles decreased, indicating a dissolution of Fe oxide/hydroxide under the influence of gluconate. In contrast with H(2)O(2), in SFW and Gox the amount of organic compounds increased due to the accumulation of polysaccharides and proteins. The influence of glucose oxidase on the ennoblement of stainless steel is not due to indirect effects of H(2)O(2) through the change of surface composition. The E(corr) ennoblement seems to be directly due to the presence of H(2)O(2) and to the electrochemical behavior of H(2)O(2) and related oxygen species. This consideration is important for understanding and controlling microbial influenced corrosion.

Lipid-destabilizing properties of the hydrophobic helices H8 and H9 from colicin E1.

Colicins are toxic proteins produced by Escherichia coli that must cross the membrane to exert their activity. The lipid insertion of their pf domain is linked to a conformational change which enables the penetration of a hydrophobic hairpin. They provide useful models to more generally study insertion of proteins, channel formation and protein translocation in and across membranes. In this paper, we study the lipid-destabilizing properties of helices H8 and H9 forming the hydrophobic hairpin of colicin E1. Modelling analysis suggests that those fragments behave like tilted peptides. The latter are characterized by an asymmetric distribution of their hydrophobic residues when helical. They are able to interact with a hydrophobic/hydrophilic interface (such as a lipid membrane) and to destabilize the organized system into which they insert. Fluorescence techniques using labelled liposomes clearly show that H9, and H8 to a lesser extent, destabilize lipid particles, by inducing fusion and leakage. AFM assays clearly indicate that H8 and especially H9 induce membrane fragilization. Holes in the membrane are even observed in the presence of H9. This behaviour is close to what is seen with viral fusion peptides. Those results suggest that the peptides could be involved in the toroidal pore formation of colicin E1, notably by disturbing the lipids and facilitating the insertion of the other, more hydrophilic, helices that will form the pore. Since tilted, lipid-destabilizing fragments are also common to membrane proteins and to signal sequences, we suggest that tilted peptides should have an ubiquitous role in the mechanism of insertion of proteins into membranes.

Silanization with APTES for Controlling the Interactions Between Stainless Steel and Biocomponents: Reality vs Expectation

Jessem Landoulsi1, Michel J. Genet2, Karim El Kirat3, Caroline Richard4, Sylviane Pulvin5 and Paul G. Rouxhet2 1Laboratoire de Reactivite de Surface, Universite Pierre & Marie Curie -Paris VI, 2Institute of Condensed Matter and Nanosciences – Bio & Soft Matter, Universite Catholique de Louvain, 3Laboratoire de Biomecanique et Bioingenierie, 4Laboratoire Roberval, 5Genie Enzymatique et Cellulaire, Universite de Technologie de Compiegne, 1,3,4,5France 2Belgium

Modulation of cell behaviour by fibronectin or collagen adsorption on anti-adhesive biomaterials

Development of adhesive as well as anti-adhesive surfaces is necessary in various biomaterial applications. Cell behaviour on adhesive substrata is well documented, whereas very little is known about anti-adhesive surfaces. Our previous studies (Velzenberger et al. 2007) demonstrated the capability of two anti-adhesive surfaces to induce pre-osteoblast (MC-3T3) cell aggregation and death by apoptosis. Here, we combined biological and physico-chemical studies in order to progress in the understanding of cell/anti-adhesive surface interactions. Four model surfaces were used to investigate protein adsorption and subsequent MC-3T3 cell morphology and adhesion. For that purpose, fibronectin (Fn) or collagen adsorption was characterised on each surface. Then, we examined the effect of both proteins on MC-3T3 adhesion and spreading.