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Immobilized proteins or peptides are of critical importance for applications such as biosensing or cell culture. We analyze the structure of layers of a large variety of proteins and peptides, grafted on silicon substrates by different routes differing in the nature of the intermediate layer linking the biomolecules to the substrate, either a silane monolayer, or a polyelectrolyte multilayer made from synthetic or natural polymers. The structural analysis is essentially performed by X-ray reflectometry, which proves to be an efficient methodology not requiring the use of tagged biomolecules, capable of evaluating consistently the amount of grafted biomolecules per surface area with estimated precisions ranging from 10 to 20%. The study provides a quantitative basis for selecting one among a series of well-proofed and sturdy grafting methodologies and underlines the potential of XRR for assessing the amount of grafted biomacromolecules without requiring the expensive tagging of molecules. Our results also show that, for the coupling route resting on synthetic polyelectrolytes, the grafting density is significantly lower than for direct coupling over a silane layer. In contrast, when performed over a cushion based on polysaccharides, the grafting density is well above the values found for a dense layer grafted on a silane monolayer, indicating partial penetration and swelling of the polysaccharide cushion.

Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) was used to investigate the mucoadhesive properties of nanoparticles decorated with low, medium and high molar mass chitosan (CS). Uncoated and chitosan-coated polycaprolactone (PCL) nanoparticles loaded with curcumin were prepared by nanoprecipitation method and characterized in terms of size, surface charge and drug content. The interactions between nanoparticles and mucin layer were monitored after the treatment of SAM-functionalized gold-coated quartz crystals with bovine submaxillary gland mucin (BSM). The results show that all investigated chitosan-coated nanoparticles adsorb onto the BSM layer, and the mass uptake was found to be independent of the chitosan molar mass. Uncoated nanoparticles showed, however, no affinity with BSM layer, confirming that the adsorption of colloidal systems occurs due to their decoration with chitosan. The adhesion is mainly attributed to electrostatic interactions between protonated amino groups of mucoadhesive chitosan and negatively charged groups of mucin. The results suggest that chitosan-coated nanoparticles are promising carriers for hydrophobic drugs delivery in the buccal mucosa.

On procede a une revue critique des methodes d'ingenierie utilisees dans le processus de conception des centrales nucleaires en examinant les aspects geotechniques et structurels. Le cas de la centrale de Cruas, construite sur isolation sismique, est presente dans les grandes lignes. On decrit ensuite les methodes disponibles en situation de reevaluation sismique, avant de terminer par les perspectives d'evolution de la pratique d'ingenierie et les actions de R&D d'accompagnement.

A biosensor based on the immobilization of Lactate oxidase in laponite–organosilasesquioxane films on glassy carbon electrode for the quantification of L-lactate in wine and dairy products is presented. The bioelectrode showed a very high sensitivity (0.33±0.01) A cm−2 M−1 and a short time response (10 s) for less than 1 U of enzyme. No significant interferences, including ascorbic acid, were detected. For red wine, matrix effects assigned to polyphenols and anthocyanins were observed, which ware easily overcome by sample dilution. Our L-lactate determinations were in good agreement with those of two standard methods.

Deoxyuridine 5'-triphosphate pyrophosphatases (dUTPases) are ubiquitous enzymes essential for hydrolysis of dUTP, thus preventing its incorporation into DNA. Although Epstein-Barr virus (EBV) dUTPase is monomeric, it has a high degree of similarity with the more frequent trimeric form of the enzyme. In both cases, the active site is composed of five conserved sequence motifs. Structural and functional studies of mutants based on the structure of EBV dUTPase gave new insight into the mechanism of the enzyme. A first mutant allowed us to exclude a role in enzymatic activity for the disulfide bridge involving the beginning of the disordered C terminus. Sequence alignments revealed two groups of dUTPases, based on the position in sequence of a conserved aspartic acid residue close to the active site. Single mutants of this residue in EBV dUTPase showed a highly impaired catalytic activity, which could be partially restored by a second mutation, making EBV dUTPase more similar to the second group of enzymes. Deletion of the flexible C-terminal tail carrying motif V resulted in a protein completely devoid of enzymatic activity, crystallizing with unhydrolyzed Mg(2+)-dUTP complex in the active site. Point mutations inside motif V highlighted the essential role of lid residue Phe(273). Magnesium appears to play a role mainly in substrate binding, since in absence of Mg(2+), the K(m) of the enzyme is reduced, whereas the k(cat) is less affected.

Biologically programmed molecular recognition provides the basis of all natural systems and supplies evolution-optimized functional materials from self-assembly of a limited number of molecular building blocks. Biomolecules such as peptides, nucleic acids and carbohydrates represent a diverse supply of structural building blocks for the chemist to design and fabricate new functional nanostructured architectures. In this context, we review here the chemistry we have developed to conjugate peptides with nucleic acids, carbohydrates, and organic molecules, as well as combinations thereof using a template-assembled approach. With this methodology, we have prepared new integrated functional systems exhibiting designed properties in the field of nanovectors, biosensors as well as controlled peptide self-assembly. Thus this molecular engineering approach allows for the rational design of systems with integrated tailor-made properties and paves the way to more elaborate applications by bottom-up design in the domain of nanobiosciences.

The purpose of this paper is to present a stochastical approach, which analyse the torsional ground motion, induced by the spatial variability of seismic motions. For this purpose, a torsional ground motion analytical model is proposed and a normalised differential motion parameter is introduced. The approach regards the seismic motion as the combination of a travelling wave on the site (coherent component) and a zero mean randomizing factor that introduces a loss of correlation effect. The soil parameters as fundamental frequency and damping coefficient are integrating by modeling the coherent component with the commonly used Kanai-Tajimi power spectral density. The parametric analysis of the model shows an increase of the induced torsion with both the soil frequency and the motion scattering parameter, and a decrease with the separation distance, the apparent wave velocity and the correlation length. Finally, in order to test the proposed torsional ground motion model prediction, it is compared to the experimental results recorded by the EPRI LSST array in Lotung, Taiwan (Laouami and Labbe, 2002). The comparison leads to the identification of the model parameters for the Lotung soft site.

We have investigated the structure of (3-mercaptopropyl)sulfonate (MPS) monolayer self-assembled onto gold surfaces by quartz crystal microbalance with energy dissipation monitoring (QCM-D) and various electrochemical methods. QCM-D experiments show that the MPS monolayer behaves as a thin rigid film with a surfacic mass of 166 ng cm(-2). Interfacial capacitance measurements demonstrate that the MPS monolayer is a rather open structure that can be penetrated by the ionic species of the phosphate buffer electrolyte. From MPS reductive desorption experiments, MPS surface concentration corresponds to 4.6 x 10(-10) mol cm(-2), which represents 60% of the coverage reported for a densely packed thiol monolayer. Despite this low packing density, oxidation of catechol is strongly inhibited leading to voltammograms that are free ofdiffusional contribution. This unique behavior has been exploited to show that the MPS monolayer covers the entire gold surface with a surface coverage at least equal to theta = 0.9981, which means a very low number of MPS-free pinholes and/or defects. Kinetics of electron transfer toward soluble redox species has been studied using catechol as a neutral hydrophilic probe, but also ferrocyanide as hydrophilic anion and ferrocenemethanol as neutral hydrophobic molecule. It is proposed that the MPS monolayer provides a high kinetic barrier toward permeation of these species and that electron transfer mainly occurs by electron tunneling through the MPS monolayer.

We describe a new supramolecular architecture obtained by deposition of double‐stranded deoxyribonucleic acid (dsDNA) and chitosan derivatives on a thiolated gold surface. Surface plasmon resonance was used to monitor, in real time, the construction of the supramolecular architecture. Three chitosan derivatives were used, a quaternized N‐substituted by three methyl groups in a 40.0 mol%, and two others N‐substituted with an octyl chain in a 5.0 and 25.0 mol%. The multilayer formation depends on the degree of substitution of chitosan as well as on the nature of the substituents. The adsorption of dsDNA is slower than that of chitosan. The best immobilization of dsDNA is obtained by using a quaternized chitosan and dsDNA prepared in 0.050 M acetate buffer pH 5.00. The system is stable in buffer solution independently of the nature of the chitosan derivative. The effect of the nature of the electrolyte and ionic strength is also discussed.

Coproporphyrinogen oxidase (CPO) is an essential enzyme that catalyzes the sixth step of the heme biosynthetic pathway. Unusually for heme biosynthetic enzymes, CPO exists in two evolutionarily and mechanistically distinct families, with eukaryotes and some prokaryotes employing members of the highly conserved oxygen-dependent CPO family. Here, we report the crystal structure of the oxygen-dependent CPO from Saccharomyces cerevisiae (Hem13p), which was determined by optimized sulfur anomalous scattering and refined to a resolution of 2.0 A. The protein adopts a novel structure that is quite different from predicted models and features a central flat seven-stranded anti-parallel sheet that is flanked by helices. The dimeric assembly, which is seen in different crystal forms, is formed by packing of helices and a short isolated strand that forms a beta-ladder with its counterpart in the partner subunit. The deep active-site cleft is lined by conserved residues and has been captured in open and closed conformations in two different crystal forms. A substratesized cavity is completely buried in the closed conformation by the approximately 8-A movement of a helix that forms a lid over the active site. The structure therefore suggests residues that likely play critical roles in catalysis and explains the deleterious effect of many of the mutations associated with the disease hereditary coproporphyria.

Resume Les facettes articulaires posterieures participent a chaque articulation intervertebrale. Leurs caracteristiques anatomiques et biomecaniques expliquent leur role potentiel dans la genese de lombalgies chroniques, realisant le classique « syndrome des facettes ». Il n’existe cependant aucune donnee consensuelle, ni sur le plan diagnostique, ni sur le plan therapeutique, l’existence meme de ce syndrome etant controversee. Les donnees de la litterature permettent notamment de preciser l’interet des infiltrations intra-articulaires et leur realisation technique. Cependant les resultats specifiques d’un tel geste restent difficiles a interpreter, puisqu’il ne peut se concevoir qu’au sein d’un arsenal therapeutique plus large. En effet, le syndrome des facettes n’est qu’un des nombreux facteurs a l’origine de la lombalgie chronique, douleur-maladie qui necessite une prise en charge multidimensionnelle.

Build-up of enzyme–polyelectrolyte multilayer onto glassy carbon (GC) surfaces by electrostatic self-assembling method has been investigated. In order to functionalize GC surface by a starting negatively charged layer, two approaches have been carried out: (i) covalent linkage of phenyl acetic acid through electroreduction of 4-phenylacetic diazonium salt (GC A surface), and (ii) formation of a glucose oxidase (GOD) monolayer through an affinity reaction between a GOD conjugated antibody and a previously adsorbed antigen monolayer (GC B surface). GC A and GC B surfaces have been modified by a precursor film (PF) composed of one layer of poly(styrenesulfonate) (PSS) sandwiched between two layers of poly(dimethyldiallylammonium) (PDDA), which improves the further assembling of enzymes. GOD, used as a model enzyme, has been self-assembled with PDDA onto these GC/PF surfaces. Enzymatic activity of immobilized GOD has been electrochemically assessed layer-by-layer during the build-up of (GOD–PDDA) n multilayers. Cyclic voltammetry (CV) performed in the presence of excess glucose and using ferrocene derivatives as artificial redox mediators allowed to quantify the amount of electrically wired enzyme on the basis of kinetic models reported in literature. By using three mediators bearing different electrical charges we conclude that electrostatic interactions between the redox mediator and enzyme microenvironment play a key role in determining the rate of enzyme active site regeneration.

Beta-dystroglycan is expressed in a wide variety of tissues and has generally been reported with an Mr of 43 kDa, sometimes accompanied with a 31 kDa protein assumed to be a truncated product. This molecule was recently identified as the anomalous beta-dystroglycan expressed in various carcinoma cell lines. We produced and characterized a G5 polyclonal antibody specific to beta-dystroglycan that is directed against the C-terminal portion of the molecule. We provide evidence that beta-dystroglycan may vary in size and properties by studying different Xenopus tissues. Besides normal beta-dystroglycan with an Mr of 43 kDa in smooth and cardiac muscle and sciatic nerve extracts, we found it in skeletal muscle and brain proteins with an Mr of 38 and 65 kDa, respectively. Glycosylation properties and proteolytic susceptibilities of these different beta-dystroglycans are analysed and compared in this work. Crosslinking experiments with various beta-dystroglycan preparations obtained from skeletal and cardiac muscles and brain gave rise to specific new covalent products with Mr of 125 kDa (doublet band), or 120 and 130 kDa, or 140 and 240 kDa, respectively. We provide evidence, using various similar beta-dystroglycan preparations, that the immunoprecipitation procedure with G5 specific polyclonal antibody allows consistent pelleting of various dystrophin-family isoforms. Skeletal muscles from Xenopus reveals the presence of two distinct beta-dystroglycan complexes, one with dystrophin and another one which involves alpha-dystrobrevin. Cardiac muscle and brain from Xenopus are shown to contain three beta-dystroglycan complexes related to various dystrophin-family isoforms. Dystrophin or alpha-dystrobrevin or Dp71 were found in cardiac muscle and dystrophin or Dp180 or Up71 in brain. This variability in the relationship between beta-dystroglycan and dystrophin-family isoforms suggests that each protein – currently known as dystrophin associated protein – could not be present in each of these complexes.

The purpose of this study was to perform an experimental analysis of the amplitude of full-scale spatial variability of seismic motions with regard to earthquake engineering. The LSST-Lotung array in Taiwan provides a good set of records for this type of study. Of interest are the free-field torsional seismic components induced by the spatial variability of seismic motions. In this study, three events have been considered: LSST-06, LSST-07 and LSST-16. In time domain analysis, the experimental results obtained show that when the separation distance increases, the induced torsion decreases, and the normalized differential motion parameter increases. Also, the results show that the stronger the event, in terms of maximum PGA, the larger the induced torsional amplitude and the smaller the induced normalized differential motion parameter.

International audience; We demonstrate the quartz crystal microbalance as a novel method to quantify the reaction yields and stability of the terminal conjugation of chemically complex molecules. Oxime ligation is identified as a facile, broadly applicable method for the reducing-end conjugation of glycosaminoglycans that overcomes the limited stability and yield of popular hydrazone ligation

In this paper, a stochastical approach for the investigation and the comprehension of the spatial variation of the seismic motions is presented. For this purpose, a coherency function analytical model is proposed. The approach regards the seismic motion as the combination of a traveling wave on the site (coherent component) and a zero mean randomizing factor that introduces an incoherence effect. The soil parameters as fundamental frequency and damping coefficient are integrating by modeling the coherent component with the commonly used Kanai-Tajimi power spectral density. The parametric analysis of the model shows an increase of the coherency with the increase of (i) the apparent propagation velocity of the motions on the ground surface, and (ii) the correlation length. On the other hand, the coherency decrease with the increase of: (i) the frequency content of the motion, (ii) the separation distance between stations and (iii) the scattering parameter which is the randomizing factor standard deviation. Finally, in order to test the proposed model prediction, its coherency function is compared to empirical results obtained on the EPRI LSST array in Lotung, Taiwan (Abrahamson NA, Shneider JF, Stepp JC. Earthquake Spectra 1991; 7:1–28). The comparison shows that the proposed model fits the spatial variability of seismic motions on Lotung site. In the absence of spatially recorded seismic data on a site, the proposed analytical model can be utilised for the description of the spatial variability of the seismic motions for the seismic response analysis of structures with large dimensions as pipes, bridges, tunnels…etc.

The amperometric response toward phenol of PPO-based rotating disk bioelectrodes is analyzed on the basis of a kinetic model taking into account internal and external mass transport effects and a CEC' electroenzymatic mechanism. Monophenolase activity of PPO catalyses the oxidation of phenol to o-quinone (step C). o-Quinone can then enter an amplification recycling process involving electrochemical reduction (step E) and enzymatic reoxidation (step C': catecholase activity). The rate-limiting steps such as monophenolase activity, catecholase recycling, permeability of the membrane, and activity and accessibility of the catalytic enzyme sites are theoretically considered and experimentally demonstrated for different electrode configurations including PPO immobilized in Laponite hydrogels and layer-by-layer self-assembled multilayers of PPO and poly(diallyldimethylammonium).

A feasibility study in view of a future industrial development of a phenol biosensor is presented. The biosensor construction is based on the electropolymerization of a pyrrole amphiphilic monomer–tyrosinase (EC.1.14.18.1) mixture previously adsorbed on a glassy carbon electrode surface. The optimized biosensor provides a low detection limit (10 nM) and a linear response up to 10 μM of phenol. The procedure allows the construction of biosensors exhibiting very reproducible characteristics. These bioelectrodes are characterized by exceptional long-term stability since they could be stored for 1 year without significant loss of their electroenzymatic activity.

The association of an inorganic/organic composite matrix with a permselective poly(4-vinylpyridine-co-styrene) membrane allows to obtain a selective glucose biosensor, allowing a sensitivity of 30 mA M−1 cm−2, for a linear dynamic range from 10−5 M to 1.5 mM. Moreover, the combined use of such permselective membranes with a bienzymatic system, glucose oxidase/polyphenol oxidase, has demonstrated that the effect of electroactive interferent species such as ascorbate, urate and p-acetaminophen, can be entirely removed.

Amperometric catechol biosensors can be constructed by drying onto the surface of a glassy carbon rotating-disk electrode an aqueous sol of synthetic laponite clay containing controlled amounts of polyphenol oxidase (PPO) and polycationic oligosilasesquioxane additive. The procedure allows the electrode surface to be coated with composite enzyme–laponite clay films exhibiting improved adhesion, enhanced mechanical strength and high enzymatic activity. Electrodes prepared in this manner can be used to detect catechol in the range 0.5 nM to 10 μM. The low detection limit of 0.5 nM results from an efficient signal amplification as a consequence of the electrochemical recycling of catechol substrate. An intrinsic amplification factor of 3.35 has been measured. The observed responses from such an electrode as a function of applied potential, enzyme activity and electrode rotation rate are in excellent agreement with theory. From a comparison of the experimental results with theory, we are able to characterize diffusion and enzyme kinetics in the enzymatic layer. The results are consistent with a microporous structure of the enzymatic layer in which microchannels are distributed. Diffusion of catechol substrate and orthoquinone product occurs within the microchannels filled by electrolyte and can be described using a pinhole model. The study shows that only a fraction of PPO, the one which is entrapped in open micropores with interconnected microchannels, is accessible to catechol substrate while keeping its full enzymatic activity. The other fraction should correspond to inaccessible proteins enclosed within the walls of microchannels.

Electrode surface modification by organo-inorganic layered coatings can be achieved readily by drying a completely delaminated laponite clay sol mixed with polycationic silasesquioxane oligomers. Oligosilsesquioxanes were synthesized by hydrolytic polycondensation of trialkoxysilanes bearing an alkylamino or alkyltrimethylammonium function. Films of excellent quality with exceptional adhesion and mechanical properties can be obtained. The intercalation of organosiloxane oligomers is accompanied by the expansion of the film and by the existence of a mesoporosity as shown by XRD measurements and N 2 adsorption experiments. The ion-exchange properties of the resulting coatings as well as permeation of neutral molecules were studied in aqueous and non-aqueous electrolytes as a function of the oligomer loading. For oligomer loadings higher than the cation-exchange capacity (cec) of laponite, the coatings behave as anion-exchangers which allows the binding a wide range of redox anions. Incorporated anions remain electroactive not only in aqueous but also in non aqueous electrolytes as a consequence of a fixed pore size and permanent interlayer spacing of oligomer-expanded laponite. On the other hand intercalation of oligomers allows us to modulate the permeability of the coating as shown by permeation experiments using neutral electroactive probes in non aqueous electrolytes. In the field of electroanalysis, amperometric biosensors made by the entrapment of glucose oxidase inside the hybrid material have been investigated successfully. Such enzymatic films exhibit enhanced analytical performances as compared with those obtained using native sodic laponite. The potential applications of this new hybrid material in the field of electrocatalysis have been exemplified by the electroprecipitation of catalytic nanoparticles such as Pt(0) obtained from the incorporation of the anionic precursor PtCl 4 2− .

A new mixed membrane material for amperometric glucose biosensors, based on immobilization of glucose oxidase (GOD) in inorganic laponite gels followed by an enzyme crosslinking through glutaraldehyde (GA) is investigated. This method gives the possibility to modulate the enzyme loading in the biomembrane, the corresponding sensitivities obtained being directly proportional to the enzyme content in the membrane. Thus, with a GOD/laponite ratio of 3.3, sensitivities reach up to 132 mA M −1 cm −2 for a linear dymamic range from 10 −2 to 20 mM. Effects of oxygen and temperature have been investigated. The analysis of the bioelectrode characteristics shows a good permeability of this inorganic/organic enzymatic membrane. A specific design, combining a Nafion membrane and incorporation of polyphenol oxydase, allows to reduce the effects of interfering species such as ascorbate, urate and acetaminophen by a factor 4.

This paper presents an analysis of impedimetric measurements and their variations due to interaction with biomolecules on platinum electrodes functionalized with polymeric layers containing antibodies. The behavior of electrodes functionalized either with aminopolysiloxanes deposited by a dip coating process or with an electropolymerized polypyrrole is compared. Best results were obtained using polypyrrole film based electrodes; these are discussed in terms of better permeability for the analyte.

A new bienzymatic electrode for glucose sensing has been constructed with the aid of the sol−gel process. This mediatorless sensor was made by the simultaneous immobilization of graphite microparticles and two enzymes (horseradish peroxidase (HRP) and glucose oxidase (GOD)) in a silica gel matrix. The amperometric detection of glucose was assayed by potentiostating the biosensor at 0 V vs SCE in order to reduce the enzymically generated H2O2. The active graphite particles evenly dispersed within the silica gel matrix establish an efficient electrical communication between the electrode surface and the HRP active center. The effect of graphite and HRP amounts have been investigated in order to improve the analytical characteristics of the amperometric H2O2 biosensor. For determination of glucose, a bilayer structure where HRP and GOD were immobilized in two separated silica films exhibited the higher analytical performances.

The self-aggregation properties of a pyrrole-substituted cationic surfactant have been studied using different techniques, including conductivity and surface tension measurements, potentiometry with a surfactant-selective electrode and cyclic voltammetry. Potentiometry gave good results for the determination of its critical micelle concentration (CMC) as well in pure water as in electrolytic media. Furthermore, in spite of the highly irreversible oxidation of the pyrrole group contained in the surfactant, cyclic voltammetry afforded a fast and valuable estimation of the CMC from peak current and peak potential variations.

A kinetic model is presented for the electroenzymatic processes occurring at a polyphenol oxidase rotating-disk-bioelectrode for low concentrations of catechol. The work presented here is based on the same formalism as that previously used by Bartlett and Whitaker (J. Electroanal. Chem., 224 (1987) 27) for a glucose sensor. Our model allows one to analyze the effect of enzyme loading, film thickness, permeability and electrode rotation rate. It also unambiguously demonstrates for the first time that the high sensitivity and low detection limit observed for such an enzymatic electrode is the consequence of the electrochemical recycling of substrate which occurs upon transduction.

The feasibility of an amperometric glucose biosensor based on immobilization of glucose oxidase (GOD) in inorganic gels (laponite) is investigated. The gels modified with enzyme were deposited onto platinum electrodes and then were exposed to glutaraldehyde vapor. The effects of different parameters (glutaraldehyde crosslinking and time of drying) on the performances of the biosensor have been studied. For optimum conditions, remarkably good characteristics were obtained: larger dynamic range (0.1–4 mM of glucose), higher i max , higher sensitivity (threefold better than this obtained through immobilization of enzymes in laponite films). The main advantage of the biosensor reported here is its long stability. The biosensor was kept for several weeks in a buffer solution at 4°C. About 450 measurements were done without any loss of the linearity in the range (0.1–4 mM). The possibility of an increase in the amount of enzyme in the biofilm which has been demonstrated, allows us to obtain more sensitive devices.

Protoporphyrinogen oxidase, which catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX, is the molecular target of diphenyl ether type herbicides. The structural gene for the yeast protoporphyrinogen oxidase, HEM14, was isolated by functional complementation of a hem14-1 protoporphyrinogen oxidase-deficient yeast mutant, using a novel one-step colored screening procedure to identify heme-synthesizing cells. The hem14-1 mutation was genetically linked to URA3, a marker on chromosome V, and HEM14 was physically mapped on the right arm of this chromosome, between PRP22 and FAA2. Disruption of the HEM14 gene leads to protoporphyrinogen oxidase deficiency in vivo (heme deficiency and accumulation of heme precursors), and in vitro (lack of immunodetectable protein or enzyme activity). The HEM14 gene encodes a 539-amino acid protein (59,665 Da; pI 9.3) containing an ADP- beta alpha beta-binding fold similar to those of several other flavoproteins. Yeast protoporphyrinogen oxidase was somewhat similar to the HemY gene product of Bacillus subtilis and to the human and mouse protoporphyrinogen oxidases. Studies on protoporphyrinogen oxidase overexpressed in yeast and purified as wild-type enzyme showed that (i) the NH2-terminal mitochondrial targeting sequence of protoporphyrinogen oxidase is not cleaved during importation; (ii) the enzyme, as purified, had a typical flavin semiquinone absorption spectrum; and (iii) the enzyme was strongly inhibited by diphenyl ether-type herbicides and readily photolabeled by a diazoketone derivative of tritiated acifluorfen. The mutant allele hem14-1 contains two mutations, L422P and K424E, responsible for the inactive enzyme. Both mutations introduced independently in the wild-type HEM14 gene completely inactivated the protein when analyzed in an Escherichia coli expression system.

Electrode surface modification by organo-inorganic layered coatings can be readily achieved by drying a completely delaminated laponite clay sol mixed with neutral octa(3-aminopropylsilasesquioxane) (OAPS) cubane-like octameric pillar precursors. Films of excellent quality with exceptional adhesion and mechanical properties could be obtained. Under mild acidic conditions, protonation of the octamer amino groups occurs. The ion-exchange properties of the resulting coatings were studied in aqueous electrolyte as a function of the octamer loading using the cationic Ru(NH 3 ) 3+ 6 and the anionic Mo(CN) 4− 8 electroactive probes. For octamer loadings higher than the cation exchange capacity (cec) of laponite, the octamer-laponite coating behaves as an anion exchanger which allows the efficient incorporation of a wide range of redox anions, e.g. RuO 2− 4 , PtCl 2− 4 , PdCl 2− 4 , Fe(CN) 3− 6 , SiW 12 O 4− 40 polyoxometallate, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) disodium salt (ABTS) or anthraquinone sulfonate (AQS). The apparent diffusion coefficient of Mo(CN) 4− 8 varies from 4 × 10 −10 to 8 × 10 −10 depending on the amount of intercalated octamer. The original use of such a modified electrode to operate in organic medium has been examined with RuO 2− 4 , ABTS and AQS as electroactive immobilized species.

Summary: Saccharomyces cerevisiae was more resistant to cadmium when the growth medium contained excess iron. Cadmium reduced the amount of iron taken up by cells during growth, and the cell ferrireductase activity was also strongly inhibited. These effects depended on the YAP1 and CAD1/YAP2 gene dosage. The growth rate of cells in iron-deficient conditions and their ferrireductase activity in the absence of added cadmium were also strongly affected by the dosage of YAP1 and CAD1/YAP2 genes. Our results suggest an indirect influence of these genes on iron metabolism, possibly via modification of the cell redox status.

A polyphenol oxidase (PPO) enzyme electrode constructed by the electropolymerization of a pyrrole amphiphilic monomer-PPO mixture, previously adsorbed on a glassy carbon electrode, is applied for the direct amperometric response of phenol, 3-chlorophenol and 4-chlorophenol. Furthermore, the detection of 2-chlorophenol, several polychlorophenols and pentachlorophenol is carried out by an inhibition process of the bioelectrode functioning. The mechanism of the bioelectrode inhibition is investigated in terms of enzymatic inhibition and polymer fouling. The capacities of this system for the resolution of mixtures of phenolic pollutants are also explored on the basis of the kinetic behavior of the bioelectrode response.

Multilayers of poly(diallyldimethylammonium chloride) (PDDA) and citrate capped Au nanoparticles (AuNPs) anchored on sodium 3-mercapto-1-propanesulfonate modified gold electrode by electrostatic layer-by-layer assembly (LbL) technique are shown to be an excellent architecture for the direct electrochemical oxidation of As(III) species. The growth of successive layers in the proposed LbL architecture is followed by atomic force microscopy, UV–vis spectroscopy, quartz crystal microbalance with energy dissipation, and electrochemistry. The first bilayer is found to show rather different physico-chemical characteristics as compared to the subsequent bilayers, and this is attributed to the difference in the adsorption environments. The analytical utility of the architecture with five bilayers is exploited for arsenic sensing via the direct electrocatalytic oxidation of As(III), and the detection limit is found to be well below the WHO guidelines of 10 ppb. When the non-redox active PDDA is replaced by the redox-active Os(2,2′-bipyridine)2Cl-poly(4-vinylpyridine) polyelectrolyte (PVPOs) in the LbL assembly, the performance is found to be inferior, demonstrating that the redox activity of the polyelectrolyte is futile as far as the direct electro-oxidation of As(III) is concerned.

Protoporphyrinogen oxidase, the molecular target of diphenylether-type herbicides, was purified to homogeneity from yeast mitochondrial membranes and found to be a 55-kDa polypeptide with a pI of 8.5 and a specific activity of 40,000 nmol of protoporphyrin/h/mg of protein at 30 degrees C. The Michaelis constant (Km) for protoporphyrinogen IX was 0.1 microM. Due to the high affinity of the enzyme toward oxygen, the Km for oxygen could only be approximated to 0.5-1.5 microM. The purified enzyme contained a flavin as cofactor. Studies with rabbit antibodies to yeast protoporphyrinogen oxidase showed that the enzyme is synthesized as a high molecular weight precursor (58 kDa) that is rapidly converted in vivo to the mature (55 kDa) membrane-bound form. Protoporphyrinogen oxidase activity was found only in purified yeast mitochondrial inner membrane (not in the outer membrane). Acifluorfen-methyl, a potent diphenylether-type herbicide, competitively inhibited the purified enzyme (Ki = 10 nM). The mixed inhibition by acifluorfen-methyl previously reported for the membrane-bound protoporphyrinogen oxidase (Camadro, J.M., Matringe, M., Scalla, R., and Labbe, P. (1991) Biochem. J. 277, 17-21) was shown to be related to partial proteolysis of the enzyme.

International audience

A wide range of assays has been used to characterize protein-carbohydrates interactions allowing access to various quantitative information including thermodynamic ones such as stoichiometry of binding, binding constant, enthalpy of binding as well as kinetics and mechanistic ones. An understanding of each technique is essential in order to propose and validate appropriate interaction models that rationalize experimental data. Due to the complexity of carbohydrate-lectins interactions as an inherent consequence of multivalent interactions, a careful control of experimental conditions is necessary in order to avoid misinterpretation. This article will briefly present the various techniques that are commonly used to characterize sugar-lectin interaction: inhibition of hemagglutination assay (HIA), enzyme-linked lectin assays (ELLA), isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). Recently introduced, force spectroscopy by atomic force microscopy (AFM) is a new approach for measuring carbohydrate-protein interaction at the level of a single protein.

Soft attachment of streptavidin to β-cyclodextrin-modified pegylated SAMs was efficiently performed in a reversible and repetitive way via orthogonal bifunctional linkers involving streptavidin–biotin recognition and redox-driven multivalent host–guest (β-cyclodextrin–ferrocene) interactions.

The new mixed-valence ammonium vanadophosphate (NH 4 ) 2 (VO)(VP)O 7 (diammonium phosphorus divanadium octaoxide) is isostructural with K 2 VO(X 2 O 7 ) (X = P, V). Its structure consists of [V 2 PO 8 ]∞layers of corner-sharing VO 5 pyramids and VPO 7 groups, interleaved with NH 4 + ions. The unusual features of this structure are the existence of the VPO 7 groups involving VO 4 and PO 4 tetrahedra, and the simultaneous presence of V IV O 5 and V V O 4 polyhedra, observed here for the first time in this class of compounds. The VPO 7 groups are statistically distributed in the framework. Magnetic measurements and bond-valence calculations confirm the mixed-valence character of this phase and show that the VO 5 pyramids and VO 4 tetrahedra are occupied by V IV and V V , respectively.

The quartz crystal microbalance technique with dissipation monitoring and the complementary optical microscopy technique were used for monitoring cell adhesion on an RGD-functionalized supported lipid bilayer. A critical interligand RGD spacing of nearly 80 nm was estimated for cell adhesion when an RGD spacing of 10 nm was necessary to observe flattened cells on a fluid surface.

The ferri-reductase activity of whole cells of Saccharomyces cerevisiae (washed free from the growth medium) was markedly increased 3 to 6 h after transferring the cells from a complete growth medium (preculture) to an iron-deficient growth medium (culture). This increase was prevented by the presence of iron, copper, excess oxygen, or other oxidative agents in the culture medium. The cells with increased ferri-reductase activity had a higher reduced glutathione content and a higher capacity to expose exofacial sulfhydryl groups. Plasma membranes purified from those cells exhibited a higher reduced nicotinamide adenine phosphate (NADPH)-dependent ferri-reductase specific activity. However, the intracellular levels of NADPH, NADH, and certain organic acids of the tricarboxylic acids cycle were unchanged, and the activity of NADPH-generating enzymes was not increased. Addition of Fe(III)-EDTA to iron-deprived and iron-rich cells in resting suspension resulted in a decrease in intracellular reduced glutathione in the case of iron-deprived cells and in an increase in organic acids and a sudden oxidation of NADH in both types of cells. The depolarizing effect of Fe3+ was more pronounced in iron-rich cells. The metabolic pathways that may be involved in regulating the trans-plasma membrane electron transfer in yeast are discussed.

We have used the yeast Saccharomyces cerevisiae as a model organism to study the role of ferric iron reduction in eucaryotic iron uptake. S. cerevisiae is able to utilize ferric chelates as an iron source by reducing the ferric iron to the ferrous form, which is subsequently internalized by the cells. A gene (FRE1) was identified which encodes a protein required for both ferric iron reduction and efficient ferric iron assimilation, thus linking these two activities. The predicted FRE1 protein appears to be a membrane protein and shows homology to the β-subunit of the human respiratory burst oxidase. These data suggest that FRE1 is a structural component of the ferric reductase. Subcellular fractionation studies showed that the ferric reductase activity of isolated plasma membranes did not reflect the activity of the intact cells, implying that cellular integrity was necessary for function of the major S. cerevisiae ferric reductase. An NADPH-dependent plasma membrane ferric reductase was partially purified from plasma membranes. Preliminary evidence suggests that the cell surface ferric reductase may, in addition to mediating cellular iron uptake, help modulate the intracellular redox potential of the yeast cell.

Plant protoporphyrinogen oxidase is of particular interest since it is the last enzyme of the common branch for chlorophyll and heme biosynthetic pathways. In addition, it is the target enzyme for diphenyl ether-type herbicides, such as acifluorfen. Two distinct methods were used to investigate the localization of this enzyme within Percoll-purified spinach chloroplasts. We first assayed the enzymatic activity by spectrofluorimetry and we analyzed the specific binding of the herbicide acifluorfen, using highly purified chloroplast fractions. The results obtained give clear evidence that chloroplast protoporphyrinogen oxidase activity is membrane-bound and is associated with both chloroplast membranes, i.e. envelope and thylakoids. Protoporphyrinogen oxidase specific activity was 7-8 times higher in envelope membranes than in thylakoids, in good agreement with the number of [3H]acifluorfen binding sites in each membrane system: 21 and 3 pmol/mg protein, respectively, in envelope membranes and thylakoids. On a total activity basis, 25% of protoporphyrinogen oxidase activity were associated with envelope membranes. The presence of protoporphyrinogen oxidase in chloroplast envelope membranes provides further evidence for a role of this membrane system in chlorophyll biosynthesis. In contrast, the physiological significance of the enzyme associated with thylakoids is still unknown, but it is possible that thylakoid protoporphyrinogen oxidase could be involved in heme biosynthesis.

Adsorption of dodecyl sulfate by an aqueous flocculated suspension of laponite-RD clay which has been cation-exchanged by cetylpyridinium(CP + ) or dioctadecyldimethylammonium (DODMA + ) leads to the formation of a cationic/anionic mixed surfactant bilayer at the clay-water interface which, in deionized water, redisperses the flocs.

Diphenyl ethers (DPEs) and related herbicides are powerful inhibitors of protoporphyrinogen oxidase, an enzyme involved in the biosynthesis of haems and chlorophylls. The inhibition kinetics of protoporphyrinogen oxidase of various origins by four DPEs, (methyl)-5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid (acifluorfen and its methyl ester, acifluorfen-methyl), methyl-5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-chlorobenzoate (LS 820340) and methyl-5-[2-chloro-5-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid (RH 5348), were studied. The inhibitions of the enzymes from maize (Zea mays) mitochondrial and etiochloroplastic membranes and mouse liver mitochondrial membranes were competitive with respect to the substrate, protoporphyrinogen IX, for all four molecules. The relative efficiencies of the inhibitors were: acifluorfen-methyl greater than LS 820340 much greater than RH 5348 greater than or equal to acifluorfen. The four molecules showed mixed-competitive type inhibition of the enzyme from yeast mitochondria where acifluorfen, a carboxylic acid, had the same inhibitory activity as its methyl ester, acifluorfen-methyl, and both were much greater than that of LS 820340 and RH 5348.

Guanine-rich DNA sequences are known to form highly ordered structures called G quadruplexes. These structures play an important role in many relevant biological processes, such as telomere stabilization, oncogene activation, and the regulation of the immunoglobulin switch region. The G-quadruplex motif is based on the association of planar G quartets of four guanine residues that are held together by eight Hoogsteen-type hydrogen bonds (Figure 1A). The G-quadruplex motif requires monovalent cations, such as Na and K , for stabilization. A wide variety of topologies can be adopted depending on the number of strands involved in the structure, the strand direction, as well as variations in loop size and sequence (Figure 1). The structure of parallel-stranded as well as antiparallel-stranded quadruplexes have been extensively studied by using different methods, such as NMR spectroscopy, X-ray diffraction and circular dichroism, but the exact conformation present in vivo is still under discussion. The design of small molecules that can bind to G quadruplexes has thus received attention because these nucleic acid motifs represent valuable pharmaceutical targets. For this purpose, a large number of small molecules has been evaluated for their binding with these particular DNA structures. However, as mentioned, the G quadruplex can adopt different topologies that can confuse the study of recognition phenomena. The design of a system that is able to mimic a well-defined conformation of G quadruplex is thus of great interest to precisely study the molecular interactions that can occur with small organic molecules. In 1985, Mutter proposed the TASP concept (template-assembled synthetic proteins) for the design of folded proteins. These pioneering works described the use of a cyclodecapeptide that allows the preparation of artificial proteins with a predetermined three-dimensional structure. Despite a large number of examples that use this template, to our knowledge, it has not been applied for the design of a specific folded structure of nucleic acid. With this in mind, we investigated the use of a peptidic scaffold as a topological template that directs the intramolecular assembly of covalently attached oligonucleotides into a single characteristic folding topology of G quadruplex. We anticipated that the scaffold should permit the preorganization of the DNA strands and the stabilization of the quadruplex structure. We report herein the synthesis and characterization of the novel water-soluble peptidic scaffold–oligonucleotide conjugate 1 that mimics the parallel-stranded conformation of G quadruplex (Scheme 1). We demonstrate that the use of the scaffold allows the precise control of the conformation of the quadruplex and dramatically increases the stability of the motif—all the more so as the formation of the quadruplex motif is possible even without the addition of any monovalent cations, such as K . We also show that mimic 1 can be used for surface functionalization, and this permits the study of the molecular interaction with G-quadruplex ligands by using surface plasmon resonance (SPR). The scaffold used for the synthesis of mimic 1 is a cyclic decapeptide with two independently functionalizable faces, which are due to the orientation of the lysine side-chains. On one side, the four oligonucleotides derived from the human telomeric sequence d(TTAGGGT) were anchored by using oxime bond formation, and a biotin residue was incorporated on the other side for attachment to streptavidin-immobilized surfaces. Earlier work from our laboratory has demonstrated that the oxime coupling strategy allows the efficient preparation of peptide–oligonucleotide conjugates. Figure 1. G-quartet motif and possible folded structures of the G quadruplex. A) G quartet; B) intermolecular parallel form; C) intramolecular parallel form; D) intramolecular antiparallel form.

Abstract The benefits of synthetic fiber ropes for deepwater station keeping are now well established and their use is expanding. Nearly all current applications use a single grade of polyester fiber, but for different supports and environments this may not be the optimal choice. Properties of polyester fibers can be modified by adjusting processing parameters and there are other fibers available such as PEN, which offer higher stiffness. This study examines the benefits of intermediate stiffness fibres, stiffer than standard polyester but less stiff than the high performance fibers. The results indicate that there is scope for improving mooring line performance and reducing line weight by careful evaluation of material options. Introduction Polyester fiber ropes are finding increasing applications in offshore mooring systems as production moves to deeper water. Following successful installations offshore Brazil in the late 1990's [Pellegrin 1999] the first Gulf of Mexico mooring was for the Mad Dog spar [Bugg 2004] in 2004, which employed 1200 tons of polyester down to 1670 meters water depth. The recently installed Independence Hub platform also used polyester moorings, in 2440 meters water depth [Paganie 2007]. Different rope constructions have been used but these mooring lines were all composed of similar high tenacity polyester fibers. The Red Hawk spar [Haslum 2005], also installed in 2004, used a modified polyester fiber with a higher initial stiffness to facilitate installation, and this raised the question of whether a higher fiber stiffness might be beneficial for other supports and allow rope diameter to be reduced. Previous work within the French Mooring line project [Davies et al 2002] studied high performance fibres such as aramids and HMPE and concluded that their very high stiffness, while allowing much smaller rope diameter and weight, did not improve durability as it would result in high fatigue loading of the metallic components of the mooring line. However, there is an intermediate stiffness region, shown in Figure 1, situated between the currently used fibres, with initial tensile modulus around 10-15 GPa, and the high performance fibers (> 60 GPa) which has not been explored previously for deepwater mooring applications.