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Didier Fournier

Publications and source records attributed to Didier Fournier.

At least 19 recordsLinked to original sources
PubMed2006

Stable polymethacrylate nanocapsules from ultraviolet light-induced template radical polymerization of unilamellar liposomes.

We employed UV-induced template polymerization to create hollow nanometer-sized polymer capsules. Homogeneous, unilamellar liposomes served as a two-dimensional template for the cross-linking of either butyl methacrylate or hydroxyethyl methacrylate with the bifunctional ethyleneglycol dimethacrylate. Different molar ratios of lipid/hydrophobic monomer/bifunctional monomer/photoinitiator were tested and dynamic light scattering revealed negligible changes of size at a defined molar ratio of 2/1/10/20, respectively. Cryo-transmission electron microscopy provided clear evidence that incorporation of the methacrylate monomers into and polymerization in the hydrophobic bilayer phase does not disrupt vesicle integrity. Moreover, after solubilization of the lipids, the polymethacrylate nanocapsules were stable at conditions needed for negative staining and could be visualized by atomic force microscopy. In contrast to previous findings, the nanocapsule size and shape did not change considerably after removal of the template phase, and the size distribution remained strictly monomodal. The employed method is not only an advance to fortify liposomes, but the nanocapsules themselves can be functionalized.

Cryoelectron Microscopy↗
PubMed2006

Structural insights into substrate traffic and inhibition in acetylcholinesterase.

Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.

Acetylcholine↗
PubMed2006

The effect of engineered disulfide bonds on the stability of Drosophila melanogaster acetylcholinesterase.

BACKGROUND: Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use in biosensors for detection of these insecticides. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, its stability has to be improved for extensive utilization. RESULTS: To create a disulfide bond that could increase the stability of the Drosophila melanogaster acetylcholinesterase, we selected seven positions taking into account first the distance between Cbeta of two residues, in which newly introduced cysteines will form the new disulfide bond and second the conservation of the residues in the cholinesterase family. Most disulfide bonds tested did not increase and even decreased the stability of the protein. However, one engineered disulfide bridge, I327C/D375C showed significant stability increase toward denaturation by temperature (170 fold at 50 degrees C), urea, organic solvent and provided resistance to protease degradation. The new disulfide bridge links the N-terminal domain (first 356 aa) to the C-terminal domain. The quantities produced by this mutant were the same as in wild-type flies. CONCLUSION: Addition of a disulfide bridge may either stabilize or unstabilize proteins. One bond out of the 7 tested provided significant stabilisation.

Acetonitriles↗
PubMed2006

Cholinesterase from the common prawn (Palaemon serratus) eyes: catalytic properties and sensitivity to organophosphate and carbamate compounds.

The main purpose of this study was to describe the kinetic properties of the cholinesterase (ChE) enzyme present in the eyes of the prawn Palaemon serratus, an abundant, ecological and commercially relevant species of European coastal environments. The obtained results suggest that the studied enzyme is a ChE and not a non-specific esterase, due to its apparent affinity for choline esters and the high sensitivity to eserine sulphate. This ChE displays a distinct preference for the substrate acetylthiocholine, showing a triphasic behaviour, with activation at low concentrations and inhibition by excess of substrate. Moreover, irreversible ChE inhibition by several organophosphate and carbamate compounds was characterized. All the irreversible inhibitions were homogeneous following a second-order rate reaction. The bimolecular rate constant (k(i)) values of ChE inhibition by the tested pesticides were also estimated and compared with available data from other invertebrate and vertebrate species. In conclusion, the results of the present study showed that prawn eyes possess only one ChE with typical properties of acetylcholinesterase, which is highly sensitive to the tested anti-cholinesterase compounds.

Acetylthiocholine↗
PubMed2005

Protection of mammalian cell used in biosensors by coating with a polyelectrolyte shell.

In order to detect xenoestrogens which induce perturbations of mammalian cells, design of biosensor using a mammalian cell line enable to detect these compounds is necessary. MELN cell line is suitable to detect estrogen activity, since they are stably transfect with an estrogen regulated luciferase gene. To realize this biosensor, it appeared necessary to add a protection to the mamalian cell, which is devoided, of the wall protecting yeasts or plant cells. With this aim in view, MELN cells have been isolated with a polyelectrolyte shell using the layer-by-layer technique. Among several polyelectrolyte-couples, the best cell survival (>80%) was obtained by alternating the polycation poly-diallyldimethyl ammonium chloride layer and the negatively charged poly-styrene sulfonate. We observed that the composition of the buffer used for layer-deposition was crucial to preserving cell viability, e.g. potassium ions were preferred to sodium ions during the coating. Furthermore, viability was increased when cells were allowed to recover for 2 h between each bilayer deposition. The use of engineered mammalian cells that synthesize luciferase as a response to exposure to estradiol, demonstrated that coating not only permits cell survival, but also allows essential metabolic functions, such as RNA and protein synthesis to take place. Capsule formation allows free diffusion of small molecules, while it prevents internalization in the cells of proteins larger than 60 kDa.

Ammonium Chloride↗
PubMed2005

Determination of thermodynamic parameters of Xerocomus chrysenteron lectin interactions with N-acetylgalactosamine and Thomsen-Friedenreich antigen by isothermal titration calorimetry.

BACKGROUND: Lectins are carbohydrate-binding proteins which potentially bind to cell surface glycoconjugates. They are found in various organisms including fungi. A lectin from the mushroom Xerocomus chrysenteron (XCL) has been isolated recently. It shows insecticidal activity and has antiproliferative properties. RESULTS: As the monosaccharide binding specificity is an important determinant of lectin function, we determined the affinity of XCL for the galactose moiety. Isothermal titration calorimetry studies revealed a dissociation constant Kd of 5.2 microM for the XCL:N-acetylgalactosamine interaction at 27 degrees C. Higher affinities were observed at lower temperatures and higher osmotic pressures. The dissociation constant was five hundred times higher for the disaccharide beta-D-Gal(1-3)-D-GalNAc, Thomsen-Friedenreich (TF) antigen (Kd of 0.94 microM). By using fetuin and asialofetuin in interaction with the XCL, we revealed its ability to recognize the Thomsen-Friedenreich motif on glycoproteins. CONCLUSION: The XCL antiproliferative effect and the TF antigen specificity presented in this work suggest that XCL and ABL may have similar binding mechanisms. The recent structure determination of these two proteins lead us to analyse these interactions in the light of our thermodynamic data. The understanding of this type of interaction may be a useful tool for the regulation of cell proliferation.

Acetylgalactosamine↗
PubMed2005

Genetically engineered acetylcholinesterase-based biosensor for attomolar detection of dichlorvos.

The design of a biosensor for the detection of dichlorvos at attomolar levels is described based on a highly sensitive double mutant (E69Y Y71D) of the Drosophila melanogaster acetylcholinesterase (Dm. AChE). This enzyme has a k(i) for dichlorvos equal to 487 microM(-1)min(-1), which is 300 and 20,000 times higher than that of the wild type Dm. AChE and the Electrophorus electricus AChE (E.el. AChE), respectively. The enzyme is immobilized into microporous-activated conductive carbon, and is used as such for the development of an inhibitor electrochemical biosensor. This E69Y Y71D mutant enables the decrease in the detection limit of the biosensor down to 10(-17) M, which is five orders of magnitude lower compared to the Electropharus electricus-based biosensor and eight orders of magnitude lower than the biosensors described so far.

Acetylcholinesterase↗
PubMed2005

Liposome retention in size exclusion chromatography.

BACKGROUND: Size exclusion chromatography is the method of choice for separating free from liposome-encapsulated molecules. However, if the column is not presaturated with lipids this type of chromatography causes a significant loss of lipid material. To date, the mechanism of lipid retention is poorly understood. It has been speculated that lipid binds to the column material or the entire liposome is entrapped inside the void. RESULTS: Here we show that intact liposomes and their contents are retained in the exclusion gel. Retention depends on the pore size, the smaller the pores, the higher the retention. Retained liposomes are not tightly fixed to the beads and are slowly released from the gels upon direct or inverted eluent flow, long washing steps or column repacking. Further addition of free liposomes leads to the elution of part of the gel-trapped liposomes, showing that the retention is transitory. Trapping reversibility should be related to a mechanism of partitioning of the liposomes between the stationary phase, water-swelled polymeric gel, and the mobile aqueous phase. CONCLUSION: Retention of liposomes by size exclusion gels is a dynamic and reversible process, which should be accounted for to control lipid loss and sample contamination during chromatography.

Acetylcholinesterase↗
PubMed2005

Hybrid nanocapsules: interactions of ABA block copolymers with liposomes.

Amphiphilic ABA triblock copolymers, such as poly(2-methyloxazoline)-block-poly(dimethylsiloxan)-block-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA), form vesicular structures. Here, the interaction of these ABA molecules with lipids is investigated by electron microscopy, fluorescence spectroscopy, light scattering, and differential scanning calorimetry. Our observations suggest the formation of homogeneous mixed polymer-lipid composites, independent of preparation method, i.e. film hydration, dispersion, or detergent removal. When ABA polymersomes and liposomes are mixed, we observed monomer exchanges on a time scale of minutes. The possibility of forming mixed structures and the exchanges between preformed structures allow the combination of the properties of lipids and polymers such as stability and loading encapsulation capacity.

Calorimetry, Differential Scanning↗
PubMed2005

Novel immobilized liposomal glucose oxidase system using the channel protein OmpF and catalase.

The reactivity of immobilized glucose oxidase-containing liposomes (IGOL) prepared in our previous work (Wang et al. [2003] Biotechnol Bioeng 83:444-453) was considerably improved here by incorporating the channel protein OmpF from Escherichia coli into the liposome membrane as well as by entrapping inside the liposome's aqueous interior not only glucose oxidase (GO), but also catalase (CA), both from Aspergillus niger. CA was used for decomposing the hydrogen peroxide produced in the glucose oxidation reaction inside the liposomes. The presence of OmpF enhanced the transport of glucose molecules from the exterior of the liposomes to the interior. In a first step of the work, liposomes containing GO and CA (GOCAL) were prepared and characterized. A remarkable protection effect of the liposome membrane on CA inside the liposomes at 40 degrees C was found; the remaining CA activity at 72 h incubation was more than 60% for GOCAL, while less than 20% for free CA. In a second step, OmpF was incorporated into GOCAL membranes, leading to the formation of OmpF-embedded GOCAL (abbreviated GOCAL-OmpF). The activity of GO inside GOCAL-OmpF increased up to 17 times in comparison with that inside GOCAL due to an increased glucose permeation across the liposome bilayer, without any leakage of GO or CA from the liposomes. The optimal system was estimated to contain on average five OmpF molecules per liposome. Finally, GOCAL-OmpF were covalently immobilized into chitosan gel beads. The performance of this novel biocatalyst (IGOCAL-OmpF) was examined by following the change in glucose conversion, as well as by following the remaining GO activity in successive 15-h air oxidations for repeated use at 40 degrees C in an airlift bioreactor. IGOCAL-OmpF showed higher reactivity and reusability than IGOL, as well as IGOL containing OmpF (IGOL-OmpF). The IGOCAL-OmpF gave about 80% of glucose conversion even when the catalyst was used repeatedly four times, while the corresponding conversions were about 60% and 20% for the IGOL and IGOL-OmpF, respectively. Due to the absence of CA, IGOL-OmpF was less stable and resulted in drastically inhibited GO.

Bioreactors↗
PubMed2005

Slow internal release of bioactive compounds under the effect of skin enzymes.

A new strategy for the skin delivery of bioactive compounds has been developed, using enzymes involved in the maintenance of the epidermal barrier function and the enzymatic transformation of corresponding precursors. This new strategy has been tested with regard to two enzymatic activities of the skin barrier: extracellular glucosidase and esterase/lipase. An analysis of the requirements for the glycosidic bond hydrolysis of any glycoconjugate by beta-glucocerebrosidase indicates that the release of the moiety linked to the glucose unit is obtained as long as the glycosidic bond being broken is not hindered, and as long as the leaving group property of the released moiety is good enough. This strategy was first applied to the release of the antioxidant delta-tocopherol. It was then extended to retinoic acid by introducing a spacer between the glucose unit and the bioactive moiety. This spacer was either a good leaving group such as hydroquinone, or a structure akin to a ceramide, namely glycerol. In these conditions, beta-glucocerebrosidase releases the complex spacer-active compound that is cleaved by an esterase. One of the advantages of this strategy lies in the slow release of the bioactive compound, extending in time its effect and most likely its tolerance, as is the case for retinoic acid.

Antioxidants↗
PubMed2004

A new lectin family with structure similarity to actinoporins revealed by the crystal structure of Xerocomus chrysenteron lectin XCL.

A newly defined family of fungal lectins displays no significant sequence similarity to any protein in the databases. These proteins, made of about 140 amino acid residues, have sequence identities ranging from 38% to 65% and share binding specificity to N-acetyl galactosamine. One member of this family, the lectin XCL from Xerocomus chrysenteron, induces drastic changes in the actin cytoskeleton after sugar binding at the cell surface and internalization, and has potent insecticidal activity. The crystal structure of XCL to 1.4 A resolution reveals the architecture of this new lectin family. The fold of the protein is not related to any of the several lectin folds documented so far. Unexpectedly, the structure similarity is significant with actinoporins, a family of pore-forming toxins. The specific structural features and sequence signatures in each protein family suggest a potential sugar binding site in XCL and a possible evolutionary relationship between these proteins. Finally, the tetrameric assembly of XCL reveals a complex network of protomer-protomer interfaces and generates a large, hydrated cavity of 1000 A3, which may become accessible to larger solutes after a small conformational change of the protein.

Amino Acid Sequence↗
PubMed2004

Fluorescence detection of enzymatic activity within a liposome based nano-biosensor.

The encapsulation of enzymes in microenvironments and especially in liposomes, has proven to greatly improve enzyme stabilization against unfolding, denaturation and dilution effects. Combining this stabilization effect, with the fact that liposomes are optically translucent, we have designed nano-sized spherical biosensors. In this work liposome-based biosensors are prepared by encapsulating the enzyme acetylcholinesterase (AChE) in L-a phosphatidylcholine liposomes resulting in spherical optical biosensors with an average diameter of 300+/-4 nm. Porins are embedded into the lipid membrane, allowing for the free substrate transport, but not that of the enzyme due to size limitations. The enzyme activity within the liposome is monitored using pyranine, a fluorescent pH indicator. The response of the liposome biosensor to the substrate acetylthiocholine chloride is relatively fast and reproducible, while the system is stable as has been shown by immobilization within sol-gel.

Acetylcholinesterase↗
PubMed2004

Rational polynomial equation as an unbiased approach for the kinetic studies of Drosophila melanogaster acetylcholinesterase reaction mechanism.

The hydrolysis of substrates by cholinesterases does not follow the Michaelis-Menten reaction mechanism. The well-known inhibition by excess substrate is often accompanied by an unexpectedly high activity at low substrate concentrations. It appears that these peculiarities are the consequence of an unusual architecture of the active site, which conducts the substrate molecule over many stages before it is cleaved and released. Structural and kinetic data also suggest that two substrate molecules can attach at the same time to the free, as well as to the acetylated, enzyme. We present a procedure which provides an unbiased framework for mathematical modelling of such complex reaction mechanisms. It is based on regression analysis of a rational polynomial using classical initial rate data. The determination of polynomial degree reveals the number of independent parameters that can be evaluated from the available information. Once determined, these parameters can substantially facilitate the construction and evaluation of a kinetic model reflecting the expected molecular events in an enzymic reaction. We also present practical suggestions for testing the postulated kinetic model, using an original thermodynamic approach and an isolated effect in a specifically mutated enzyme.

Acetylcholinesterase↗
PubMed2004

Sorting out molecules reacting with acetylcholinesterase by enzyme encapsulation in liposome.

Enzymes are considered as providential molecules for biosensor design because of their sensitivity and the high specificity of the reactions they catalyse. However, their active sites often display low selectivity, a lot of molecules may enter and interfere with catalysis. These molecules may be either competitive inhibitors, activators or molecules which change the physico-chemical environment of the enzyme (pH, ionic strength). They produce the "matrix effect" that lowers the reliability of biosensors. We show here that encapsulation of enzymes in liposomes inserts a barrier between the enzyme and the external environment and protects the enzyme in a stable nano-environment for an optimal activity. This barrier sorts out the molecules that could react with the enzyme according to their hydrophobicity. Acetylcholinesterase is used to detect organophosphorous and carbamate insecticide residues but several molecules (reversible inhibitors, pH and ionic strength modifiers) generate matrix effects in free conditions. These perturbations were completely ineffective following enzyme encapsulation.

Acetylcholinesterase↗
PubMed2004

Molecular recognition of cocaine by acetylcholinesterases for affinity purification and bio-sensing.

Cholinesterases can be used as sensitive biorecognition elements for widely used agricultural pesticides. This requires highly purified and inhibitor-free enzyme preparations. In the present work the cocaine derivative benzoylecgonine was for the first time used as the molecular recognition element for the purification of acetylcholinesterase from Electrophorus electricus by affinity chromatography. The preparation of enriched enzyme without the contamination by an inhibitor, which is traditionally used for eluting the "affinity" bound protein, was achieved. The specific activity was 2.2-fold increased to 3100 Umg(-1). The same cocaine derivative was immobilized on the surface of a piezoelectric crystal in order to analyze the binding of acetylcholinesterases from two different species, E. electricus and Drosophila melanogaster, to the immobilized inhibitor. Evaluation of the binding curves allowed the analysis of the binding kinetics. These experiments are fundamental for the development of a (competitive) biosensor for inhibitors of cholinesterase.

Acetylcholinesterase↗
PubMed2004

Mutation of exposed hydrophobic amino acids to arginine to increase protein stability.

BACKGROUND: One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface. RESULTS: In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion. CONCLUSION: Although the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions of nonpolar residues with water or from addition of new hydrogen bonds with the solvent. Other mechanisms may also contribute to the increased stability observed with some mutants. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface.

Acetylcholinesterase↗