Detection of wheat allergens using 2D Western blot and mass spectrometry.

BACKGROUND: Wheat allergy is relatively common and the associated clinical manifestations depend on the involved molecular allergens as well as on the way of exposure. Different symptoms have been described: wheat-dependent exercise-induced anaphylaxis (WDEIA), atopic dermatitis (AD) and pollen rhinitis (PR). Traditional diagnostic methods do not allow accurate molecular identification of the allergens that are essential for risk assessment and for the choice of the most adapted treatment. METHODS: Standardized total protein extracts obtained from wheat seeds were separated by 2D electrophoresis. Twenty-one sera with high wheat-specific immunoglobulin E (sIgE) levels were classified into three patients groups based on their clinical profile. These sera were tested by Western blot on 2D separated standardized wheat protein extract and their sIgE sensitization profiles were compared. RESULTS: Specific sensitization profiles were identified for each phenotype group. For WDEIA, protein spots around 37 kDa (pH 6-9) and 37-50 kDa (pH 5-6) were identified. For AD, spots were observed around 50 kDa (pH 9), 10 kDa (pH 9) and 20 to 75 kDa (pH3). For PR, specific spots were located around 90 kDa (pH 9). The mass spectrometry (UHPLC-MS/MS) analysis of these identified spots pointed out several potential interesting allergens: Tri a 26, Tri a bA, Tri a 34, Tri a tritin. CONCLUSIONS: The present study allowed the identification of different protein areas specific to these studied groups. The protein spots of interest were identified by UHPLC-MS/MS. It has been possible to establish a link between a specific symptomatology and the newly identified responsible allergens.

[Hypophosphatasia]. / L'hypophosphatasie.

Hypophosphatasia (HPP) is a rare genetic disorder characterized by the diminution of the enzymatic activity of the alkaline phosphatase (ALP). The disease mainly involves multiple defects of the mineralization of the skeleton including bone fragilities. It will be expressed to varying degrees of severity and will allow to characterize different forms of HPP. Unfortunately, the prevalence of this pathology remains probably underestimated and its diagnosis must be multidisciplinary by taking into account the biochemical assays, the clinical history as well as the radiological imaging. So, in the approach of this diagnosis, a retrospective screening was carried out by the clinical chemistry department of the CHU of Liège. The aim of this study is to potentially identify the affected patients on the basis of their biochemical assays and their anamnesis in order to propose a genetic screening. Unfortunately, no case could be formally identified, which testifies the difficulty to establish a diagnosis of the slight forms encountered mainly in the adults.

L'hypophosphatasie (HPP) est une pathologie héréditaire rare caractérisée par une diminution de l'activité enzymatique de la phosphatase alcaline (PAL). La maladie entraîne, principalement, de multiples défauts de minéralisation du squelette conduisant à des fragilités osseuses, qui s'exprimeront à des degrés plus ou moins sévères et permettront de caractériser différentes formes d'HPP. Malheureusement, la prévalence de cette pathologie reste probablement sous-estimée et son diagnostic doit être pluridisciplinaire, en prenant en compte les dosages biochimiques, l'histoire clinique du patient ainsi que des iconographies radiologiques. C'est donc dans une optique de diagnostic que nous avons réalisé un screening rétrospectif au sein du laboratoire de chimie clinique du CHU de Liège. Le but de cette étude était d'identifier des patients potentiellement atteints par cette pathologie sur base de leurs dosages biochimiques et d'une clinique évocatrice afin de proposer un dépistage génétique. Malheureusement, aucun cas n'a pu être formellement identifié, ce qui témoigne de la difficulté d'établir un diagnostic des formes légères rencontrées et ce, principalement chez l'adulte.

Poly(acrylic acid)-coated iron oxide nanoparticles: quantitative evaluation of the coating properties and applications for the removal of a pollutant dye.

In this work, 6-12 nm iron oxide nanoparticles were synthesized and coated with poly(acrylic acid) chains of molecular weight 2100 g mol(-1). Based on a quantitative evaluation of the dispersions, the bare and coated particles were thoroughly characterized. The number densities of polymers adsorbed at the particle surface and of available chargeable groups were found to be 1.9±0.3 nm(-2) and 26±4 nm(-2), respectively. Occurring via a multi-site binding mechanism, the electrostatic coupling leads to a solid and resilient anchoring of the chains. To assess the efficacy of the particles for pollutant remediation, the adsorption isotherm of methylene blue molecules, a model of pollutant, was determined. The excellent agreement between the predicted and the measured amounts of adsorbed dyes suggests that most carboxylates participate to the complexation and adsorption mechanisms. An adsorption of 830 mg g(-1) was obtained. This quantity compares well with the highest values available for this dye.

The effects of aggregation and protein corona on the cellular internalization of iron oxide nanoparticles.

Engineered inorganic nanoparticles are essential components in the development of nanotechnologies. For applications in nanomedicine, particles need to be functionalized to ensure a good dispersibility in biological fluids. In many cases however, functionalization is not sufficient: the particles become either coated by a corona of serum proteins or precipitate out of the solvent. In the present paper, we show that by changing the coating of iron oxide nanoparticles from a low-molecular weight ligand (citrate ions) to small carboxylated polymers (poly(acrylic acid)), the colloidal stability of the dispersion is improved and the adsorption/internalization of iron toward living mammalian cells is profoundly affected. Citrate-coated particles are shown to destabilize in all fetal-calf-serum based physiological conditions tested, whereas the polymer coated particles exhibit an outstanding dispersibility as well as a structure devoid of protein corona. The interactions between nanoparticles and human lymphoblastoid cells are investigated by transmission electron microscopy and flow cytometry. Two types of nanoparticle/cell interactions are underlined. Iron oxides are found either adsorbed on the cellular membranes, or internalized into membrane-bound endocytosis compartments. For the precipitating citrate-coated particles, the kinetics of interactions reveal a massive and rapid adsorption of iron oxide on the cell surfaces. The quantification of the partition between adsorbed and internalized iron was performed from the cytometry data. The results highlight the importance of resilient adsorbed nanomaterials at the cytoplasmic membrane.

Probing oppositely charged surfactant and copolymer interactions by isothermal titration microcalorimetry.

The complexation between charge-neutral block copolymers and oppositely charged surfactants was investigated by light scattering experiments and by isothermal titration calorimetry (ITC). The copolymer was poly(sodium acrylate)-b-poly(acrylamide) and the surfactant dodecyltrimethylammonium bromide (DTAB). In a previous report, we had shown that the copolymers and the surfactants coassembled spontaneously into colloidal complexes. Depending on the charge ratio, the complexes were either single surfactant micelles decorated by copolymers or core-shell hierarchical structures. ITC was performed in order to investigate the thermodynamics of the complex formation. Titrations of copolymers by surfactants and of surfactants by copolymers revealed that the electrostatic coassembly was an endothermic reaction, suggesting a process dominated by the entropy of the counterions. Here, we found that the thermodynamic quantities associated with the reaction depended on the mixing order. When surfactants were added stepwise to copolymers, the titration was associated with the formation of single micelles decorated by a unique polymer. Above a critical charge ratio, the micelles rearranged themselves into 100 nm colloidal complexes in a collective process which displayed the following features: (i) the process was very slow as compared to the time scale of Brownian diffusion, (ii) the thermodynamic signature was a endothermic peak, and (iii) the stoichiometry between the positive and negative charges was modified from n = 0.48 (single micelles) to 0.75 (core-shell complexes). When copolymers were added stepwise to surfactants, the titration resulted in the formation of the core-shell aggregates only. In both experiments, the amount of polyelectrolytes needed for complex formation exceeded the number required to compensate the net micellar charge, confirming the evidence of overcharging in the complex formation.

Structural investigation of an exopolysaccharide substituted with a lactyl ether group produced by Raoultella terrigena Ez-555-6 isolated in the Chernobyl exclusion zone.

Raoultella terrigena strain Ez-555-6, isolated from a root nodule of Medicago sativa harvested in the Chernobyl exclusion zone, produces a non-referenced high-molecular-mass exopolysaccharide (EPS). The structure of this EPS was determined using a combination approach including monosaccharide composition (GLC-FID, HPAEC-PAD), determination of glycosylation sites (GLC-EIMS) and 1D/2D NMR ((1)H, (13)C) and ESIMS (HR, MS/MS) studies of oligosaccharides obtained from mild acid hydrolysis. The EPS was found to be a charged pentasaccharide with a repeating unit composed of D-galactose, D-glucose, D-mannose and D-glucuronic acid (1:2:1:1). Lactic acid and O-acetyl substituents were localized on galactose and glucose residues, respectively, as presented in the following structure:

Oligogalacturonides induce resistance in wheat against powdery mildew.

Biological activities, priming and protective effects of two oligogalacturonides fractions (OGAs) were assayed during a compatible wheat/Blumeria graminis f. sp. tritici interaction. These fractions were obtained from commercial polygalacturonic acid. They both consisted of oligogalacturonides with polymerisation degrees (DP) ranging from 2 to 25, and one of them was a 30% chemically acetylated fraction. A 5 g x L(-1) solution of each fraction was infiltrated in the first leave of ten-days-old plantlets, and activities of defence-related enzymes were measured 48H post-treatment. Among them, oxalate oxidase and peroxidase activities increased, suggesting an elicitation due to both fractions of oligogalacturonides. Some of the pre-treated plantlets were subsequently submitted to powdery mildew infection. As revealed by 3,3'-diaminobenzidine (DAB) staining, the accumulation of hydrogen peroxide (H2O2) at the penetration site of the fungus increased 21H after inoculation to the same extent in areas of plantlets infiltrated by both fractions. On the other hand, the intensity of fluorescence associated with papillae was higher when plantlets were pre-infiltrated with the acetylated fraction, whereas no difference was observed between control plantlets and those treated with the non-acetylated fraction. Moreover, microscopic assessment of the number of haustoria occurring 40H post-inoculation showed it was only reduced when acetylated fraction was used. Despite different modes of action of these molecules, a similar 45% protective effect occurred in both cases when the oligogalacturonides fractions were sprayed on ten-days-old plantlets.

Oligogalacturonans production by free radical depolymerization of polygalacturonan.

The large production of acidic oligosaccharides was investigated by non-enzymatic depolymerization of polygalacturonic acid (PGA) using free hydroxyl radical hydrolysis process from H2O2/copper (II) system. A large amount of oligogalacturonides (OGAs) with degrees of polymerization up to 6 were fractionated, and characterized by ESI-Q/TOF-MASS spectrometry and 1H NMR spectroscopy. This efficient production of uronic oligosaccharides from PGA constitutes an original process to produce bioactive compounds in large scale up.

Production of oligoglucuronans using a monolithic enzymatic microreactor.

A glucuronan lyase (EC 4.2.2.14) was immobilized on a monolithic Convective Interaction Media (CIM) disk. The immobilization yield was equal to 29% of the initial activity and 35% of the initial protein amount. Degradations of three glucuronans with various O-acetylation degrees were investigated and compared with degradations using free enzyme. The immobilized glucuronan lyase was inhibited by the O-acetylation degree like the free enzyme. (1)H NMR analyses were used to study the O-acetylation degree of oligoglucuronans and demonstrated that the average degrees of polymerization were inclusive between 4 and 13 after 24h of degradation. This first immobilization of a glucuronan lyase constitutes a new tool to produce oligoglucuronans.

Purification and characterization of a novel glucuronan lyase from Trichoderma sp. GL2.

The filamentous fungus Trichoderma sp. GL2 produces an extracellular glucuronan lyase (GL) when grown on glucuronan as the sole carbon source. In this paper, we report the purification to electrophoretical homogeneity of this polysaccharide lyase by size exclusion chromatography and anion exchange chromatography. The purified GL, classified as an endopolyglucuronate lyase, is a monomer with an apparent molecular weight of 27 kDa and an isoelectric point of 6.95. Despite an inhibition of the activity when polysaccharide substrates were substituted by acetates, the enzyme was active toward glucuronans (acetylated or not) and ulvan, leading to various (4,5)-unsaturated products as oligoglucuronans (acetylated or deacetylated), highly acetylated low-molecular-weight (LMW) glucuronans, and LMW ulvans.

Purification of oligouronides using hollow-fiber membrane functionalised with L-histidine.

The pseudobioaffinity chromatography with L-histidines immobilized onto poly(ethylene-vinyl alcohol) hollow-fiber membranes was studied for a selective purification of anionic oligosaccharides. Oligoglucuronans with different degrees of polymerization and acetylation, prepared by an enzymatic degradation of bacterial polyglucuronic acid, were used as models. The adsorption and elution parameters were studied to optimise the selective adsorption. A better understanding of the physico-chemical phenomena governing this selectivity was attempted and a few hypotheses on the mechanism of selectivity are proposed.

Contributions of intermolecular interactions between constitutive arabinoxylans to the flaxseeds mucilage properties.

The main fraction (about 75%) of the mucilage extracted from seeds of Linum usitatissimum which consists of arabino-xylans (AX) has been studied in dilute and semidilute regimes by SEC/MALLS analysis and rheology, respectively. It has been found that AX contains 3 populations of about 5 000 000 g mol(-1) (less than 10%), 1 000 000 g mol(-1) (about 40%), and 200 000 g mol(-1) (about 50%). We have also observed a great retention of polymer during the filtration procedure, which is much pronounced as the AX concentration increases. This evidences the presence of large aggregates in the solution. The retention can be greatly diminished if the filtration is conducted under higher temperature. Aggregation could result from the establishment of intermolecular associations via hydrogen bonds. This hypothesis seems to be confirmed by the two higher populations in molar masses which present a random coil conformation consistent with a low degree of branching. Rheological measurements, conducted at 20 g L(-1), have confirmed the association tendency leading to pseudo gels behavior. Viscoelastic properties have been evidenced by time-temperature master curves of dynamic spectra. Such master curves have also been established with addition of chaotropic (i.e., KSCN) and lyotropic (i.e., NaCl) salts. It has been shown that intermolecular associations are greatly diminished under chaotropic salts influence. This has been also confirmed by SEC/MALLS analysis. These results point out the role of hydrogen bonds in the organization of the AX system.

Production of glucuronan oligosaccharides using a new glucuronan lyase activity from a Trichoderma sp. strain.

Sinorhizobium meliloti M5N1CS synthesizes a homopolymer of glucuronic acids beta-(1,4) linked and variably C2 and/or C3O-acetylated. To obtain beta-Delta-(4,5)-unsaturated oligoglucuronans, various acetylated forms of this bacterial polymer were cleaved by a Trichoderma sp. GL2 glucuronan lyase. Oligomers with polymerization degrees up to 8 were then produced, purified by liquid chromatography (size exclusion and anions exchange) and characterized using 1H NMR and ESI-Q/TOF-MS. Finally, the production (in gram quantity) of pure unsaturated oligoglucuronans non-acetylated (di- and trisaccharide) was investigated thanks to the complete depolymerization of deacetylated glucuronan.

Structural investigations of the neutral polysaccharide of Linum usitatissimum L. seeds mucilage.

The heterogeneity of the purified water-soluble neutral fraction coming from the mucilage extract of the yellow flaxseed was investigated. After fractionation by size-exclusion chromatography, the analyse of the neutral monosaccharides composition showed a mixture of three major families of polymers. They were all identified as arabinoxylans with a constant A/X ratio of 0.24, but varying in their galactose and fucose residues in the side chains. Furthermore, the molecular weight (M(w)) analysis acquired by multi-angle laser light scattered, revealed the association of two high M(w) polymers [5.7x10(6) (11.1%) and 9.3x10(5) (42.4%) g mol(-1)] with a smaller one [3.2x10(5) g mol(-1) (45.3%)].

Flax (Linum usitatissimum) seed cake: a potential source of high molecular weight arabinoxylans?

Water-soluble polysaccharides were extracted from flaxseed cake and analyzed. Two groups were separated by anion-exchange chromatography. The first one (nonretained) was the major fraction (83%) and possessed a high molecular weight (HMW) arabinoxylan (56%) with an Ara/Xyl ratio of 0.32 and an M(w) of 846 000. This polymer was accompanied by a smaller galactoglucan (44%), with an M(w) of 6.5 x 10(4). The latter group (17%), retained by the gel, was further described as a HMW pectin heterogeneous group, with, respectively, 3.1 x 10(5) and 1.3 x 10(5). Despite the presence of HMW arabinoxylans, the investigation of rheological flow sweep at the concentration of 2% (w/v) has shown a slight shear thinning behavior with a small zero-rate viscosity at 9.6 Pa.s.

Methods for obtaining neutral and acid oligosaccharides from flax pectins.

Esterified acid soluble pectins from flax (Linun usitatissimum L.) were degraded either with HCl or pectin lyase. Centrifugation and 2-propanol precipitation led to the isolation of two low molecular weight polygalacturonates after acid hydrolysis of pectins. However, after pectin lyase digestion and purification by size-exclusion HPLC, (1)H NMR analyses indicated that acetylated hairy regions, large methylated and acetylated oligogalacturonides together with small unsubstituted oligogalacturonides were produced. Thus, in a few steps, a panel of substituted neutral and acidic oligosaccharides was produced from a raw plant material. Such oligosaccharides could be useful for further fractionations such as chemical saponification and enzymatic removal of neutral sugar chains from the hairy regions. The procedures used for pectin extraction, for degradation, and for the purification of fragments seem appropriate for large-scale production of biologically active oligosaccharides from flax.

A rapid method for determining the antimicrobial activity of novel natural molecules.

Preservatives used in the Agro-food industries may be of natural origin or obtained chemically. Because of the increasing interest of consumers in food products that contain only natural ingredients, studies on preservative molecules of natural origin, such as organic acids or peptides, have been reported in the past several years. Such studies, which require numerous assays, may be limited by the large amount of molecules required. Microscale assays provide an opportunity for testing natural components available in low quantity. This study examined a rapid method that used microplates for the evaluation of anti-microbial substances. The method was validated using five foodborne pathogens. It required a low amount of product and was convenient for the determination of correlations between the bacterial growth inhibition and concentration of the antimicrobial substance.

Microfiltration and ultrafiltration of polysaccharides produced by fermentation using a rotating disk dynamic filtration system.

The recovery of exopolysaccharides (EPS) produced by Sinorhizobium meliloti bacteria by dynamic microfiltration was investigated using a rotating disk device designed in our laboratory, equipped with a 0.2 microm nylon membrane. This system differs from commercially available systems by the presence of vanes on the disk which produce a very important increase in permeate flux while yielding excellent EPS transmission. For polymers produced under standard fermentation conditions (70 h at 30 degrees C), the mass flux rose to 650 g h(-1) m(-2) using a disk equipped with 2 mm vanes rotating at 2000 rpm against 380 g h(-1) m(-2) with a smooth disk at the same speed. The maximum flux observed was 1560 g h(-1) m(-2) with a 6-mm vanes disk rotating at 3000 rpm and a 36 degrees C broth. An interesting finding was that the permeate flux J(f) for various disks can be correlated by the same function of the mean shear stress at the membrane tau(wm) according to J(f) = 4.6 tau(wm) (0.717) for a 30 degrees C broth, showing that the effect of vanes is merely to increase the shear stress by raising the fluid core velocity between the membrane and the disk. With 6-mm vanes the core angular velocity was found to be 84% of disk velocity vs. 45% for a smooth disk. When the fermentation temperature was increased to 36 degrees C to produce a lower molecular weight polymer, the permeate flux rose by about 250%, much more than what could be expected from the reduction in permeate viscosity and followed the same power law with membrane shear stress as for 30 degrees C. The same device was equipped with a PES 50 kDa membrane to concentrate EPS by ultrafiltration. Permeate fluxes were of the order of 160 L h(-1) m(-2) at 2000 rpm and 30 degrees C with nearly complete EPS rejection. Finally, the net electrical power consumed by the disk was measured by subtracting the power consumed without fluid from the power during filtration at the same speed. This power increases with speed and with the presence of vanes, but since the gain provided by the vanes is very high, the specific energy per m(3) of permeate is minimal with the highest vanes tested (6 mm) and maximal for smooth disks.

Polysaccharide lyases: recent developments as biotechnological tools.

Polysaccharide lyases, which are polysaccharide cleavage enzymes, act mainly on anionic polysaccharides. Produced by prokaryote and eukaryote organisms, these enzymes degrade (1,4) glycosidic bond by a beta elimination mechanism and have unsaturated oligosaccharides as major products. New polysaccharides are cleaved only by their specific polysaccharide lyases. From anionic polysaccharides controlled degradations, various biotechnological applications were investigated. This review catalogues the degradation of bacterial, plant and animal polysaccharides (neutral and anionic) by this family of carbohydrate acting enzymes.