Cyrille Richard

Helical Crystallization of Proteins on Carbon Nanotubes: A First Step towards the Development of New Biosensors**

have made them very promisingnanomaterials for new applications in chemistry and physics,particularly for the development of new nanotechnologies.However, few applications have so far been reported inbiology. Small proteins have been introduced into the insidehollow of opened nanotubes thus forming natural nano testtubes.

Anionic pH-sensitive pegylated lipoplexes to deliver DNA to tumors

Anionic pegylated lipoplexes have been prepared from the combined formulation of cationic lipoplexes and pegylated anionic liposomes. To this end, two original (bis- and tetra-) carboxylated cholesterol derivatives have been synthesised. Titration of the particle surface charge was realised to determine the ratio between anionic and cationic lipids that would give pH-sensitive complexes. This ratio has been optimised to form particles sensitive to pH change in the range 5.5-6.5. Compaction of DNA into these newly formed anionic complexes was checked by DNA accessibility to picogreen and DNA electrophoresis on an agarose gel. Gene expression of the formulated gene was similar for the cationic formulation taken as a control and the anionic formulations prepared. The pH-sensitive properties of these formulations was shown in vitro using bafilomycin, a vacuolar H(+)ATPase inhibitor. The efficiency of the new formulations to deliver DNA to the tumor was compared with cholesteryl hemisuccinate (CHEMS) formulations. The tetracarboxylated compound gave the most efficient formulations for tumor delivery in vivo.

Helicale Kristallisation von Proteinen auf Kohlenstoffnanoröhren: ein erster Schritt zur Entwicklung neuer Biosensoren

Zwei ganz unterschiedliche Proteine, Streptavidin und HupR, binden an mehrwandige Kohlenstoffnanorohren und bilden dort unter geeigneten Bedingungen regelmasige helicale Anordnungen (siehe Bild). Die Beschichtung der auseren Oberflache dieser Nanostrukturen mit biologischen Makromolekulen wurde mit Elektronenmikroskopie untersucht.

Trifluoromethyl ketones derived from squalene: inhibition of the cholesterol biosynthesis in HepG2 cells

Trifluoromethyl ketones 1 and 2, have been prepared from trisnorsqualene aldehyde 3. These compounds and their corresponding alcohols 4 and 7 have been found to be good inhibitors of the biosynthesis of cholesterol in HepG2 cells. The inhibition is correlated with a decrease of the HMG-CoA reductase activity, probably resulting from the transformation of the fluorinated compounds into their corresponding oxysterols which could act as repressors of that enzyme.

Silicates doped with luminescent ions: useful tools for optical imaging applications

Fluorescence is increasingly used for in vivo imaging and has provided remarkable results. Howerver this technique presents several limitations, especially due to tissue autofluorescence under external illumination and weak tissue penetration of low wavelength excitation light. We have developed an alternative optical imaging technique using persistent luminescent nanoparticles suitable for small animal imaging. These nanoparticles can be excited before the injection, and their in vivo distribution can be followed in real-time for several hours. Chemical modifications of their surface is possible leading to lung or liver targeting, or to long-lasting blood circulation.

Persistent Luminescence Nanoparticles for Bioimaging

Optical imaging is a rapidly developing field of research aimed at noninvasive monitoring of disease progression, evaluating the effects and pharmacokinetic of a drug, or identifying pathological biomarkers . To this end, it requires the development of targeting and highly specific contrast agents . In fluorescence imaging, an external light of appropriate wavelength is used to excite the fluorescent molecule, followed almost immediately by the release of longer wavelength, lower energy light for imaging. Fluorescence is increasingly used for imaging and has provided remarkable results. However this technique presents several limitations, especially due to tissue autofluorescence under external illumination and weak tissue penetration of low wavelength excitation light. To overcome these drawbacks, we have developed an innovative technique using persistent luminescence nanoparticles (PLNP) for optical imaging in small animal. Such nanoparticles can be excited before systemic injection, and their biodistribution monitored in real-time for dozen of minutes without the need for any external illumination source. This review article will focus on recent works undertaken in our laboratory on the synthesis of PLNP, their surface modifications and applications for bioimaging.

Helical organisation of soluble macromolecules on carbon nanotubes

s Trinoculaire ‘98 des Microscopies, Strasbourg-lllkirch, France, l-3 July 1998 283 Electron microscopy localisation of beet western ye#tows hrteovlrus (BWW) particles in the aphid vector Myzus persicae Catherine Reinbold, Frederick E. Gildow’, VBronique Brault Christophe Ritzenthaler’ l , Kim Findlaq, Brian Wells2, and Etienne Herrbach Keith Roberts2, Lothaire Pinck3 and Andrew J. Maule’ INRA, 28 rue de Herrlisheim, 68021 Co/mar, France ‘Department of Plant Pathology, 319 Buckhout Lab, Pennsylvania State University, University Park, PA 16802, USA (11 Department of Virus Research and 121 Department of Cell Biology. John lnnes Centre, Norwich Research Park. Norwich NR4 7LJH. lJr&erJ Kingdom. ‘Present address: f3J lnstitut de Bidogie M&u/aire des Plantes. CNRS UP, 12 rue du G&&al i’immer F67084 Strasbourg. France. Like all Luteoviruses, beet western yellows virus is obligately transmitted from plant to plant by aphid vectors, according to a persistant-circulative manner. In this type of interaction, virions must circulate inside the vector’s body before being inoculated. Inside the vector, virions are transported across two epithelial barriers: first, through the intestine from the P;ut lumen to the haemolymph,’ and se&ndly, through the accessoyy salivary glands (ASG) from the haemolymph to the salivary duct. Both steps rely on an endocytosis-exocytosis mechanism, involving the presence of specific virus receptors (Gildow F.E. (1987). Curr. Top. Vector Res. 4, 93-120). In order to identify viral proteins determining the specific transport of virions across the intestine and ASG barriers, we studied the localisation within aphids of a nontransmissible mutant, BWYV 6.4, lacking the readthrough domain (Brault V. et al. (1995). EMBO 1. 14, 650-659), as compared to the localisation of the wildtype (WT) virus. Plasmodesmata are believed to play very important roles in the process of transport, pathogenesis, communication and development in plants. These complex intercellular channels, present within the plant cell wal1, allow cytoplasmic and endomembrane continuity between neigbouring cells. Structural and functional analysis of plasmodesmata are generally difficult and time consuming due to their small size and inacessibility within the cell wall. We have investigated the detection of plasmodesmata in cell wall preparations from leaves of Nicotialm clevelandii and N . benthamiana. using aniline blue, a specific marker of callose, and cationic amphiphile fluorescent probes widely used for the generalpurpose of membrane structure and dynamic studies. Punctate fluorescent staining was readily detected within pits fields with both aniline blue and the cationic probes. Pit fields are small depressions representing primary wall not recovered by secondary wall, which are known to be rich in plasmodesmata. Scanning electron microscopy was used to demonstrate that the punctate staining was specific of plasmodesmata. Purified cell wall fractions submitted to different biochemical treatments to dissolve lipids or proteins, were assayed for plasmodesmata labelling. Results concerning the effects of these treatments on the composition of plasmodesmata will be presented. Using electron microscopy on aphids fed on, or microinjected with, virus preparations, BWYV 6.4 virions could not be observed so far inside the cells of intestine and ASG, whereas WT virions were frequently observed in both cells and basal lamina of these organs. These preliminary observations may be interpreted as follows: either the mutant is not recognised by specific virus receptors of the vector, or the mutant virions are instable in the gut lumen and/or in the haemolymph. Helical organisation of soluble macromolecules on carbon nanotubes Fabrice Balavoinez Cyrille Richard? Charles Mioskowskij and Patrick Schultz1 Thomas W. Ebbeser?, 1LG.B.M.C. 1, rue Laurent Fries, F 67404 llfkirch Cedex, %ervice des mo/&ules maraukes, Centre d’Etudes de Saclay, F 91191 Gif sur Yvette Cedex, 3&J&C Research Institute, 4 ffldepend&ce Way, Princeton, New Jersey 08540, USA Analysis of self-organized arrays using electron microsco P y and image processing has become a powerful method or the determination of macromolecular structures. Helical symmetries are of particular interest for since several orientations of the object can be recorded in a single untilted view thus allowing the direct determination of a three-dimensional model. We investi ated the use of carbon nanotubes as a general sup CF” a rt to promote t e hehcal organisation of proteins. Since their lscovery in 1991, carbon nanotubes have attracted considerable interest because of their opposite side exhibit a specific recognition fonction. As a model system we used the streptavidin/biotm system for the high affinity and specifici 2 molecules dl of the interaction. To our surprise the streptavidin not only strongly interact with the carbon surface but did sometimes spontaneously oreanise into regular arrays showing helical symmetr resolution of 1 1.8 run-l as evidences by optical diffraction. This Y . Negatively stained helices where organised to a constitutes the first example of helical crystallisation of fy-2 molecules on carbon nanotubes and was reproduced wt distinct protein systems. Although the interaction of the protein molecules with the carbon tubes is presently poorly understood, these results demonstrate the feasibility of the approach. Studies on plasmodesmata composition from purified cell walls by immunofluorescence Specific interaction and two-dimensional crystal-Ii&ion of histidine tagged yeast RNA polymerase I on Nickel-chelating lipids Nicolas Bischlerl, Fabrice Balavoine? Phili Herbert Tschochners, Charles Mioskowskt *!! p Milkereitj, and Patrick Schultz1 ~l.G.6.M.C. 1, rue Laurent Fries, F-67404 fflkiich, -%ervice des mol&ules rnarqu6es, C.E.N. da Sacb . 04 F 91191 Gif SW Yvette Cedex, %ZH Im Neuenheimer FeM 328. 9120 Heidelberg, Germany Two-dimensional (2D) crystallisation of soluble macromolecules is a widely used method for structure determination by electron microscopy. Amphiphillic molecules are often used to ta 2x Interface. To attract the specimen to the into et the specimen to a plane e specific interactions are used when a l&and of the specimen is grafted on the lipid molecule &mctionalized lipid). In this case it is necessary to chemically synthesise a different lipid molecule for each system to be studied. We developed a more general method where the lipid molecule is modified by the nickelchelating moiety Nitrilo-Tri-Acetate @ITA). This chemical function was shown to interact specifically with the irnidazole ring of histidine and is used to puriQ bistidinetagged proteins. Nickel-chelating lipid monolayers were used to generate Z-D crystals from yeast RNA polymerase I which was histidine-tagged on one of its subunits. The interactton of the enzyme with the spread lipid layers was found to be imidazole dependent and the formation of 2-D crystals required small amounts of imidazole probably to select the specific interaction of the engineered tag with the nickel. Two distinct preparations of RNA polymerase I tagged on different subunits yielded two different crystal forms indicating that the position of the tag determines the crystallisation process. The orientation of the enzyme in both crystal forms is correlated with the location of the tagged subunits in a 3-D model which shows that the tagged subunits are in contact with the lipid layer. These results indicate that the 2-D cry&all&&ion is mediated by the interaction of the histidme tag with the nickel chelating lipid film. This type of lipid molecules is thus a general tool for the orientation and 2D crystallisation of histidine-tagged macromolecular assemblies.

Physico-chemical characterizations of Cr doped persistent luminescence nanoparticles

Persistent luminescence nanoparticles have recently been proposed as innovative optical probes for small animal in vivo imaging. The main advantage of such probes is their ability to emit light for a long time after the end of their excitation, allowing in vivo imaging with low background. This work reports new information on the physico-chemical characterizations of Cr doped ZnGa2O4 nanoprobes in terms of synthetic procedure, luminescence properties as well as colloidal stabilities in different aqueous media and over the time.