LmaPA2G4, a Homolog of Human Ebp1, Is an Essential Gene and Inhibits Cell Proliferation in L. major

We have identified LmaPA2G4, a homolog of the human proliferation-associated 2G4 protein (also termed Ebp1), in aphosphoproteomic screening. Multiple sequence alignment and cluster analysis revealed that LmaPA2G4 is a non-peptidasemember of the M24 family of metallopeptidases. This pseudoenzyme is structurally related to methionine aminopeptidases. Anull mutant system based on negative selection allowed us to demonstrate that LmaPA2G4 is an essential gene inLeishmaniamajor. Over-expression of LmaPA2G4 did not alter cell morphology or the ability to differentiate into metacyclic and amastigotestages. Interestingly, the over-expression affected cell proliferation and virulence in mouse footpad analysis. LmaPA2G4 binds asynthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I:C)] as shown in an electrophoretic mobility shiftassay (EMSA). Quantitative proteomics revealed that the over-expression of LmaPA2G4 led to accumulation of factors involved intranslation initiation and elongation. Significantly, we found a strong reduction ofde novoprotein biosynthesis in transgenicparasites using a non-radioactive metabolic labeling assay. In conclusion, LmaPA2G4 is an essential gene and is potentiallyimplicated in fundamental biological mechanisms, such as translation, making it an attractive target for therapeutic intervention.Fil: Norris Mullins, Brianna. University Of Notre Dame-Indiana; Estados UnidosFil: VanderKolk, Kaitlin. University Of Notre Dame-Indiana; Estados UnidosFil: Vacchina, Paola. University Of Notre Dame-Indiana; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Joyce, Michelle V.. University Of Notre Dame-Indiana; Estados UnidosFil: Morales, Miguel A.. University Of Notre Dame-Indiana; Estados Unido

Phytochelatins in roots are earlier biochemical biomarkers than leaf fatty acids in tomato (Lycopersicon Esculentum) exposed to Cadmium

International audienc

Investigation of the response of wood-rotting fungi to copper stress by size-exclusion chromatography and capillary zone electrophoresis with ICP MS detection

International audienceA method based on the coupling of size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP MS) was developed for screening the changes in the bioligand composition of wood-rotting fungi as a function of their exposure to copper stress. Strains of four different species of wood-rotting fungi: Phanerochaete chrysosporium, Schizophyllum commune, Daedalea quercina and Pleurotus ostreatus were examined. Only one, namely Ph. chrysosporium, showed any significant difference in terms of the fingerprint of Cu-binding ligands between control and exposed cells which suggest trapping of Cu(II) by cell walls as the only resistance mechanisms. In the case of Ph. chrysosporium the bioinduction of a new Cu-binding ligand was demonstrated. The presence of a new compound in the SE chromatographic fraction of interest was confirmed by CZE-ICP MS. Attempts to identify the new compound by electrospray MS/MS failed because of insufficient sensitivity. © Springer-Verlag 2001

Identification of phytochelatin-related peptides in maize seedlings exposed to cadmium and obtained enzymatically in vitro

International audienceAn analytical strategy based on the sensitivity of electrospray tandem mass spectrometry following a simplified and reproducible sample preparation procedure was evaluated for the determination of Cd-induced phytochelatins (PC) and related peptides in four maize varieties. In addition to the three known families of PC (PC, desGly-PC and iso-PC(Glu)) that were observed, novel PC and desGly-PC homologues lacking the N-terminal ?-linked Glu were isolated from maize root extracts for the first time. Additionally the complete sequence of iso-PC3(Glu) was determined by tandem mass spectrometry. Peptides obtained in vivo and in vitro as the result of the reaction of glutathione with the enzyme phytochelatin synthase were compared. Minor forms detected from in vitro reactions include compounds with intramolecular or intermolecular disulfide bonds resulting from the oxidation of SH groups, phytochelatin homologues lacking the N-terminal ?-linked Glu, and new PC-related peptides with a Cys-Cys motif. Since peptides lacking a ?Glu residue could be generated as artifacts in electrospray mass spectrometry, the application of capillary electrophoresis with online electrospray mass spectrometry allowed the separation and detection of such peptides as endogenous molecules present in planta and as products of in vitro reactions. © 2001 Elsevier Science Ltd

Zinc fate in animal husbandry systems

International audienceZinc (Zn) is considered in animal production systems as both an essential nutrient and a possible pollutant. While it is generally supplemented at low levels in animal diets, with less than 200 mg kg(-1) in complete feeds, it is under scrutiny due to potential accumulation in the environment. This explains why international regulations limit maximum supplementation levels in animal feeds in a stricter way. This article gives an overview of the current knowledge on the fate of zinc in animal production systems, from animal diets to animal wastes. Some analytical methods can be used for the quantification and qualification of Zn chemical forms: X-ray crystallography, electrospray tandem mass spectrometry, separation techniques, hyphenated techniques... Analysis of chelated forms issued from complex matrices, like hydrolysed proteins, remains difficult, and the speciation of Zn in diluted carriers (premix and feed) is a challenge. Our understanding of Zn absorption has made progress with recent research on ZnT/Zip families and metallothioneins. However, fine-tuned approaches towards the nutritional and metabolic interactions for Zn supplementation in farm conditions still require further studies. The speciation of zinc in pig manure and poultry litter has been a priority as monogastric animals are usually raised under intensive conditions and fed with high quantities of trace minerals, leading to high animal density and elevated quantities of zinc from animal wastes

Relevancy of speciation analysis for geographical origin discrimination of red wines

International audienceWine traceability has become of primary importance for consumers as well as for producers. Indeed, because of its high market price, it's a product highly subject to fraud. Traceability analysis are, among others, performed by trace element analysis. Indeed, their content may change depending on the soils on which the vine has grown and also as a function of the agricultural practices. But in addition, these different origins may also impact these trace elements speciation. Therefore, the objective of our work was to check if the speciation analysis could be more discriminant than the single total element content for geographical discrimination purpose. In a first step, the development and the validation of the speciation methods of different elements (As, Se) in red wine will be described. Then, the application of these methods to the analysis of red wines from different geographical origin will be presented. Finally, the statistical treatment of these data will allow to the conclusion about the relevancy of using speciation analysis to discriminate the geographical origin of red wines

Determination of phytochelatins by capillary zone electrophoresis with electrospray tandem mass spectrometry detection (CZE-ES MS/MS)

International audienceThe coupling of capillary zone electrophoresis with electrospray mass spectrometry was optimized for the direct determination of phytochelatins (PCs) in extracts obtained from cells and plants that had been exposed to metal stress. Gluthathione and phytochelatins belonging to the different families (?Glu-Cys)nGly (n-PC), (?Glu-Cys)nSer, (?Glu-Cys)n?Ala and (?Glu-Cys)n were separated in an uncoated capillary at pH 4 using a 5 mM ammonium acetate buffer, and detected by electrospray (ES) MS in the full scan mode (300-1100 u). The use of on-line tandem MS detection in the product ion scan mode of putative protonated molecules of PCs allowed the unambiguous confirmation of the identity of the compounds detected by ES MS. The operational conditions were optimized and the figures of merit were evaluated using n-PC2, n-PC3 and n-PC4 standards purified from a mixture obtained after the reaction of glutathione in the presence of Cd2+ and the enzyme PC-synthase. The method was applied to the characterization of bioinduced ligands in cell cultures of soybeans (Glycine max) and in rice (Oryza sativa) roots without the need for a preliminary sample cleanup by size-exclusion and/or reversed phase chromatography

Rapid ion-exchange matrix removal for a decrease of detection limits in the analysis of salt-rich reservoir waters for fluorobenzoic acids by liquid chromatography coupled with tandem mass spectrometry

International audienceA matrix removal procedure with ion-exchange resin prior to analysis for 18 fluorinated benzoic acids (FBAs) tracers in saline (\textgreater25% salt) reservoir water was optimized. The elimination of \textgreater98% of salt and the simultaneous matrix sample cleanup allowed the direct analysis using the supernatant by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This resulted in a gain in detection limits for most of the tracers in comparison with the reference method (direct analysis after minimum required dilution). The limits of detection (LODs) were in the range of 0.01–0.15 ng/ml and compared to other studies the developed method provided comparable limits of detection and advantage of simplified and shorter sample preparation. The presented method offers a considerable gain in simplicity and analysis time. Recoveries for all the tracers reached 80–100%, except for 2-FBA and 2,6-dFBA for which they were ca. 60%. The low recoveries were corrected by the use of five isotopically labeled internal standards. The method was validated by the analysis of spiked samples and by an independent comparison of the results with those obtained by solid-phase extraction LC-MS/MS method