Gibberellin-regulated proteins: Emergent allergens.

About 10 years ago, a protein family was shown for the first time to contain allergenic members, gibberellin-regulated protein (GRP). The first reported member was from peach, Pru p 7. One can hypothesize that it was not detected before because its physicochemical characteristics overlap with those of lipid transfer protein (LTP), a well-known allergen, or because the exposure to GRP increased due to an increase in the gibberellin phythormone level in plant food, either exogenous or endogenous. Like LTPs, GRPs are small cationic proteins with disulfide bridges, are resistant to heat and proteolytic cleavage, and are involved in the defense of the plant. Besides peach, GRP allergens have been described in Japanese apricot (Pru m 7), sweet cherry (Pru av 7), orange (Cit s 7), pomegranate (Pun g 7), bell pepper (Cap a 7), strawberry (Fra a GRP), and also in pollen with a restriction to Cupressaceae tree family (Cup s 7, Cry j 7, and Jun a 7). IgE cross-reactivities were described between GRPs, and the reported peach/cypress and citrus/cypress syndromes may therefore be explained because of these GRP cross-reactivities. GRPs are clinically relevant, and severe adverse reactions may sometimes occur in association with cofactors. More than 60% and up to 95% sequence identities are calculated between various allergenic GRPs, and three-dimensional models show a cleft in the molecule and predict at least three epitopic regions. The structure of the protein and its properties and the matrix effect in the original allergenic source should be unraveled to understand why, despite the ubiquity of the protein family in plants, only a few members are able to sensitize patients.

Technical and economic analysis of real anaerobic digester centrate by means of partial nitrification and sustainable heterotrophic denitrification.

The reliability of partial nitrification coupled with heterotrophic denitrification for the treatment of real anaerobic digester centrate produced in a wastewater treatment plant was technically and economically assessed in two sequencing batch reactors. Removal efficiencies above 90% were consistently achieved at N-ammonium loads above 1.2 g N L⁻¹ d⁻¹. Ethanol, affluent from a waste water treatment plant (biological treatment inlet) and a zero-cost liquid residue from a chemical industry containing polyethylene glycol and sorbitol were employed as carbon source for denitrification. In this last case, a total organic carbon (TOC) requirement of 4.5 g TOC g⁻¹ NO2⁻-N was calculated. The denitrification rate was 0.26 g NO2⁻-N g VSS⁻¹ d⁻¹ (VSS: volatile suspended solids). These results show that a carbon-rich waste can serve as a no-cost feed for denitrifying bioreactors. An in-depth economic analysis considering the main investment and operating costs of the process was developed, showing that it can suppose yearly savings above 50% with respect to the most widely used alternative of returning anaerobic digester centrate untreated to the head of the facility.

Cloning and functional analysis of the ecdysteroid receptor complex in the opossum shrimp Neomysis integer (Leach, 1814).

In this paper, the non-target effects of tebufenozide were evaluated on the estuarine crustacean, the opposum shrimp Neomysis integer (Leach, 1814). Tebufenozide is a synthetic non-steroidal ecdysone agonist insecticide and regarded as potential endocrine-disrupting chemical (EDC). N. integer is the most used crustacean in ecotoxicological research in parallel to Daphnia sp. and has been proposed for the regulatory testing of potential EDCs in the US, Europe and Japan. Major results were: (i) cDNAs encoding the ecdysteroid receptor (EcR) and the retinoid-X-receptor (RXR), were cloned and sequenced, and subsequent molecular phylogenetic analysis (maximum likelihood and neighbor-joining) revealed that the amino acid sequence of the ligand binding domain (LBD) of N. integer EcR (NiEcR) clusters as an outgroup of the Crustacea, while NiRXR-LBD clusters in the Malacostracan clade (bootstrap percentage=75%). (ii) 3D-modeling of ligand binding to NiEcR-LBD demonstrated an incompatibility of the insecticide tebufenozide to fit into the NiEcR-ligand binding pocket. This was in great contrast to ponasterone A (PonA) that is the natural molting hormone in Crustacea and for which efficient docking was demonstrated. In addition, the heterodimerization of NiEcR-LBD with the common shrimp Crangon crangon (Linnaeus, 1758) RXR-LBD (CrcRXR-LBD) was also modeled in silico. (iii) With use of insect Hi5 cells, chimeric constructs of NiEcR-LBD and CrcRXR-LBD fused to either the yeast Gal4-DNA binding domain (DBD) or Gal4-activation domain (AD) were cloned into expression plasmids and co-transfected with a Gal4 reporter to quantify the protein-protein interactions of NiEcR-LBD with CrcRXR-LBD. Investigation of the ligand effect of PonA and tebufenozide revealed that only the presence of PonA could induce dimerization of this heterologous receptor complex. (iv) Finally, in an in vivo toxicity assay, N. integer juveniles were exposed to tebufenozide at a concentration of 100 µg/L, and no effects against the molting process and nymphal development were scored. In conclusion, the in vitro cell reporter assay, based on NiEcR-LBD/CrcRXR-LBD heterodimerization in Hi5 cells and validated with the natural ecdysteroid hormone PonA, represents a useful tool for the screening of putative EDCs. As a test example for non-steroidal ecdysone agonist insecticides, tebufenozide had no negative effects on NiEcR/RXR receptor dimerization in vitro, nor on the molting process and nymphal development of N. integer at the tested concentration (100 µg/L) in vivo.

A new sensitive organic/inorganic hybrid material based on titanium oxide for the potentiometric detection of iron(III).

The formation of a new hybrid material based on titanium dioxide as inorganic support and containing an iron organochelator (ICL670) is described. An organophosphorous coupling agent was used to graft the organic molecule on the oxide surface. The attachment of the organic substrate was well-confirmed by FTIR (DRIFT), solid-state (31)P and (13)C CPMAS NMR, thermal analysis and the integrity of the structural and morphological parameters were verified using XRD and TEM analyses. The interaction between the material and dissolved iron(III) was also investigated through potentiometric measurements and demonstrated the interest of this new non-siliceous based hybrid material. The obtained linear evolution of the open circuit potential from 10(-2) to 10(-6) mol L(-1) can be used for the analytical detection of iron(III).

Structural changes under low evolutionary constraint may decrease the affinity of dibenzoylhydrazine insecticides for the ecdysone receptor in non-lepidopteran insects.

Understanding how variations in genetic sequences are conveyed into structural and biochemical properties is of increasing interest in the field of molecular evolution. In order to gain insight into this process, we studied the ecdysone receptor (EcR), a transcription factor that controls moulting and metamorphosis in arthropods. Using an in silico homology model, we identified a region in the lepidopteran EcR that has no direct interaction with the natural hormone but is under strong evolutionary constraint. This region causes a small indentation in the three-dimensional structure of the protein which facilitates the binding of tebufenozide. Non-Mecopterida are considered much older, evolutionarily, than Lepidoptera and they do not have this extended cavity. This location shows differences in evolutionary constraint between Lepidoptera and other insects, where a much lower constraint is observed compared with the Lepidoptera. It is possible that the higher flexibility seen in the EcR of Lepidoptera is an entirely new trait and the higher constraint could then be an indication that this region does have another important function. Finally, we suggest that Try123, which is evolutionarily constrained and is up to now exclusively present in Lepidoptera EcRs, could play a critical role in discriminating between steroidal and non-steroidal ligands.

Halloween genes and nuclear receptors in ecdysteroid biosynthesis and signalling in the pea aphid.

The pea aphid (Acyrthosiphon pisum) is the first whole genome sequenced insect with a hemimetabolic development and an emerging model organism for studies in ecology, evolution and development. The insect steroid moulting hormone 20-hydroxyecdysone (20E) controls and coordinates development in insects, especially the moulting/metamorphosis process. We, therefore present here a comprehensive characterization of the Halloween genes phantom, disembodied, shadow, shade, spook and spookiest, coding for the P450 enzymes that control the biosynthesis of 20E. Regarding the presence of nuclear receptors in the pea aphid genome, we found 19 genes, representing all of the seven known subfamilies. The annotation and phylogenetic analysis revealed a strong conservation in the class of Insecta. But compared with other sequenced insect genomes, three orthologues are missing in the Acyrthosiphon genome, namely HR96, PNR-like and Knirps. We also cloned the EcR, Usp, E75 and HR3. Finally, 3D-modelling of the ligand-binding domain of Ap-EcR exhibited the typical canonical structural scaffold with 12 alpha-helices associated with a short hairpin of two antiparallel beta-strands. Upon docking, 20E was located in the hormone-binding groove, supporting the hypothesis that EcR has a role in 20E signalling.

[Towards a new standard in cytoreductive surgery for advanced ovarian cancer]. / Vers une évolution du dogme de la chirurgie << optimale >> dans les cancers de l'ovaire.

The dismal outcome of ovarian, fallopian tube, and primary peritoneal carcinomas calls for an increase in surgical aggressiveness. After a long era during which incomplete cytoreduction was considered acceptable, it has been established that the outcome is directly related to the amount of diseased tissue left in place. Probably as a result of technical imitations of surgeons and anesthesiologists, the majority of teams have fixed a cut-off value of 2 cm to define what was called "optimal" cytoreduction. Although it is now established that reaching the 2 cm cut-off value is the minimal required target, the target has moved towards complete removal of visible implants. However, the methods of assessment of residual disease and the very concept of complete cytoreduction suffer from limitations.

Continuous thermal hydrolysis and energy integration in sludge anaerobic digestion plants.

A thermal hydrolysis pilot plant with direct steam injection heating was designed and constructed. In a first period the equipment was operated in batch to verify the effect of sludge type, pressure and temperature, residence time and solids concentration. Optimal operation conditions were reached for secondary sludge at 170 degrees C, 7 bar and 30 minutes residence time, obtaining a disintegration factor higher than 10, methane production increase by 50% and easy centrifugation In a second period the pilot plant was operated working with continuous feed, testing the efficiency by using two continuous anaerobic digester operating in the mesophilic and thermophilic range. Working at 12 days residence time, biogas production increases by 40-50%. Integrating the energy transfer it is possible to design a self-sufficient system that takes advantage of this methane increase to produce 40% more electric energy.

[Analysis of incidents, monitoring and safety devices of cardiopulmonary bypass for cardiac surgery for the year 2005 in France]. / Analyse des accidents, des moyens de monitorage et de sécurité de la circulation extracorporelle pour chirurgie cardiaque en France en 2005.

INTRODUCTION: Several surveys demonstrated that cardiopulmonary bypass (CPB) is associated with incidents that negatively affect the outcome of cardiac surgery. OBJECTIVES: In 2004, the French "Haute Autorité de santé" (an independent public scientific authority) and the French "College of Perfusion" issued recommendations concerning safety and monitoring devices for CPB. The aims of the present study were to: 1) investigate the difference between the recommendations and the clinical practice of CPB shortly after publication of the recommendations; 1) investigate the type and rate of perfusion incidents and their outcome. STUDY DESIGN: Retrospective survey. METHODS: A 62 items questionnaire was sent to all 66 centres performing cardiac surgery in France. RESULTS: Fifty-seven centres totalling 34,496 CPB procedures (response rate 86%) returned the questionnaire. There was a wide difference between the recommendations and the reported use of safety and monitoring devices. An incident was reported for every 198 CPB procedures with death occurring 1:4,864 and permanent sequelae 1:11,349 procedures respectively (permanent injury or death 1:3,220 procedures). The most frequent perfusion incidents were adverse effects to protamine (1:1,702), dissection at the arterial canulation site (1:1,792) and coagulation of the circuit (1: 4,864). CONCLUSION: This survey demonstrates that an important effort must be made in order to fill the gap between the recommendations and clinical use of monitoring and safety devices. The analysis of CPB-related incidents suggests that, with the exception of protamine adverse effects, the majority of deaths and severe permanent injuries could probably be avoided by improved use of the monitoring and safety devices.

The lipid transfer proteins (LTP) essentially concentrate in the skin of Rosaceae fruits as cell surface exposed allergens.

The localization and distribution of non-specific lipid transfer proteins (nsLTP) allergens in the skin and pulp of Rosaceae fruits (apple, peach, apricot, plum) has been investigated. nsLTP essentially concentrate in the pericarp of the fruits whereas the pulp contains lower amounts of allergens. Immunolocalization showed they are primarily located in the cytosol but are subsequently excreted and finally accumulate at the plasmalemma-cell wall interface and in the cell wall. However, high discrepancies were observed in the content of allergens among, e.g. different cultivars of apple. As a consequence, the consumption of peeled-off fruits is recommended to reduce the risk of severe allergic reactions (anaphylactic shock) in individuals sensitized to Rosaceae fruits.

Desalination of mixed tannery effluent with membrane bioreactor and reverse osmosis treatment.

A limiting factor for the reuse and recycling of treated tannery wastewater for irrigation and other uses is the high salt content, which persists even after conventional treatment. Reverse osmosis (RO) membrane treatment has been shown to significantly reduce the salt contents of tannery effluents. However, the high organic content of tannery effluent leads to rapid scaling and biofouling of RO membranes with a consequent reduction in flux rates and performance. Membrane bioreactors (MBR) have been shown to be highly effective in the removal of organic pollutants and suspended solids from tannery effluent. This research investigated the use of a combined MBR and RO treatment process to treat tannery effluents to an acceptable level for irrigation purposes. The MBR was operated at 17-20 h retention time, at a F/M ratio of 0.52 kg COD x kg SS(-1) x day(-1) and a volumetric loading rate of 3.28 kg COD x m(-3) x day(-1). This treatment reduced the COD, BOD, and ammonia concentrations of the effluent by 90-100%. The MBR was shown to be an excellent pretreatment prior to RO technology, due to the high removal efficiency of organic compounds and suspended solids, with average concentrations of 344 mg x L(-1) COD and 20 mg x L(-1) BOD achieved in the permeate. RO treatment reduced the salt content of the MBR permeate by up to 97.1%. The results of the research demonstrated that the MBR system developed was appropriate for the treatment of tannery effluents and, in combination with the RO treatment, reduced the salt content to acceptable levels for irrigation. The MBR pretreatment reduced bio-fouling and scaling of subsequent RO treatment and improved the overall performance of the RO unit. It is believed that this is the first investigation of a combined MBR and RO treatment for tannery effluents. This research provided data for an outline design of a full-scale MBR and RO plant with a treatment capacity of 5000 m3 per day for mixed tannery effluents.

Structure of an RNase-related protein from Calystegia sepium.

The structure of a catalytically inactive RNase-related protein from Calystegia sepium (CalsepRRP) has been resolved by protein crystallography at a resolution of 2.05 A and an R factor of 20.74%. Although the protein is completely devoid of ribonuclease activity, it adopts the typical alpha + beta structure of non-base-specific RNases. Analysis of the structure revealed that two amino-acid substitutions in the 'active' P1 site, in combination with the less hydrophobic/aromatic character of the B1 base-recognition site and a completely disrupted B2 base-recognition site, might account for this complete lack of activity.

Purification, characterization, immunolocalization and structural analysis of the abundant cytoplasmic beta-amylase from Calystegia sepium (hedge bindweed) rhizomes.

An abundant catalytically active beta-amylase (EC 3.2.1.2) was isolated from resting rhizomes of hedge bindweed (Calystegia sepium). Biochemical analysis of the purified protein, molecular modeling, and cloning of the corresponding gene indicated that this enzyme resembles previously characterized plant beta-amylases with regard to its amino-acid sequence, molecular structure and catalytic activities. Immunolocalization demonstrated that the beta-amylase is exclusively located in the cytoplasm. It is suggested that the hedge bindweed rhizome beta-amylase is a cytoplasmic vegetative storage protein.

Mannose-binding plant lectins: different structural scaffolds for a common sugar-recognition process.

Mannose-specific lectins are widely distributed in higher plants and are believed to play a role in recognition of high-mannose type glycans of foreign micro-organisms or plant predators. Structural studies have demonstrated that the mannose-binding specificity of lectins is mediated by distinct structural scaffolds. The mannose/glucose-specific legume (e.g., Con A, pea lectin) exhibit the canonical twelve-stranded beta-sandwich structure. In contrast to legume lectins that interact with both mannose and glucose, the monocot mannose-binding lectins (e.g., the Galanthus nivalis agglutinin or GNA from bulbs) react exclusively with mannose and mannose-containing N-glycans. These lectins possess a beta-prism structure. More recently, an increasing number of mannose-specific lectins structurally related to jacalin (e.g., the lectins from the Jerusalem artichoke, banana or rice), which also exhibit a beta-prism organization, were characterized. Jacalin itself was re-defined as a polyspecific lectin which, in addition to galactose, also interacts with mannose and mannose-containing glycans. Finally the B-chain of the type II RIP of iris, which has the same beta-prism structure as all other members of the ricin-B family, interacts specifically with mannose and galactose. This structural diversity associated with the specific recognition of high-mannose type glycans highlights the importance of mannose-specific lectins as recognition molecules in higher plants.

Structure of a legume lectin from the bark of Robinia pseudoacacia and its complex with N-acetylgalactosamine.

The structure of the bark lectin RPbAI (isoform A4) from Robinia pseudoacacia has been determined by protein crystallography both in the free form and complexed with N-acetylgalactosamine. The free form is refined at 1.80 A resolution to an R-factor of 18.9% whereas the complexed structure has an R-factor of 19.7% at 2.05 A resolution. Both structures are compared to each other and to other available legume lectin structures. The polypeptide chains of the two structures exhibit the characteristic legume lectin tertiary fold. The quaternary structure resembles that of the Phaseolus vulgaris lectin, the soybean agglutinin, and the Dolichos biflorus lectin, but displays some unique features leading to the extreme stability of this lectin.