In vitro production of peroxynitrite by haemocytes from marine bivalves: C-ELISA determination of 3-nitrotyrosine level in plasma proteins from Mytilus galloprovincialis and Crassostrea gigas

BACKGROUND: Peroxynitrite is increasingly proposed as a contributor to defence system in marine bivalve. It can be formed by combinaison of superoxide and nitric oxide, and can react with tyrosine residues of proteins giving rise to 3-nitrotyrosine. RESULTS: The present article describes a competitive ELISA for the measurement of 3-nitrotyrosine contents of plasma proteins from marine bivalves by means of a monoclonal anti 3-nitrotyrosine antibody mouse IgG. CONCLUSIONS: This assay is sensitive enough to determine the amounts of 3-nitrotyrosine in plasma proteins from one animal only. Using the C-ELISA, we have shown that the phagocytosis of zymosan particles increased the 3-nitrotyrosine levels of plasma proteins from mussel M. galloprovincialis and oyster C. gigas 5.8 and 7.5 times respectively

Production of monoclonal antibodies against the Protozoa, Perkinsus marinus: estimation of parasite multiplication in vitro

Among the different stages in the protozoan Perkinsus marinus life cycle, the trophozoite stage is known to be the most infective stage in marine molluscs. To develop a direct method for in vitro studies of P. marinus proliferation under various environmental conditions, monoclonal antibodies (MAbs) specific for this pathogen were produced. Inbred strains of mice BALB/c were immunised with a trophozoite prepared from cloned isolate Perkinsus 1 cultured on JLODRP1 medium. The mouse polyclonal antiserum showing the highest antibody titre for pathogen trophozoites was chosen for lymphocyte hybridisation. The screening of positive hybridoma by indirect enzyme-linked immunosorbent assay (ELISA) revealed two probes (17B2D5 and 19G3H6) detecting P. marinus trophozoites and their protein lysates but also trophozoites from P. atlanticus. These MAbs belonged to the immunoglobulin IgG1 subclass. Their binding specificity was investigated by ELISA and fluorescein (FITC) methods. Both immunoreacted with trophozoite stage as well as hypnospore and zoospore stages of P. marinus, but neither with hemolymph and tissues of oysters, Crassostrea gigas and C. virginica, nor with parasites, Bonamia ostreae and Marteilia refringens. A competitive ELISA method was developed, using 17B2D5 MAb to evaluate parasite multiplication in culture media and to estimate the parasite burdens from infected oysters. This method is sensitive enough to detect 103 trophozoites in 50 μL assay sample

Nitrite released in haemocytes from

In order to demonstrate the involvement of nitric oxide in the defence systems of marine bivalves, we investigated the production of superoxide and nitrite, following in vitro phorbol myristate acetate stimulation of Mytilus galloprovincialis, Crassostrea gigas and Ruditapes decussatus haemocytes. Whereas M. galloprovincialis and C. gigas haemocytes were found to produce superoxide and nitrite, R. decussatus haemocytes were found to be unable to generate either of these mediators. Nitrite is a stable end product of nitric oxide and peroxynitrite as well; it appeared therefore that some marine bivalves, to kill microbial pathogens, use NADPH-oxidase and nitric oxide-synthase pathways. This was confirmed at an experimental level where inhibitors of both enzymatic pathways blocked the production of nitrite. Moreover, this notion was strengthened by the inability of the haemocytes from R. decussatus, which cannot produce superoxide, to release nitrite when stimulated

Recherche et caractérisation d'immunoglobulines dans les oeufs du loup (Dicentrarchus labrax)

The occurrence of maternal immunity transfer in Sea bass (Dicentrarchus labrax) was investigated using ELISA and Western blot technics. In order to determine the possible presence of immunoglobulin (Ig), egg proteins obtained from fertilized eggs were analyzed. After several extraction protocol assays (precipitation, chromatography), monoclonal and polyclonal antibody probes specific to Sea bass Ig allowed to confirm the presence of an immunoglobulin-like protein in eggs with very low level. Biochemical characterization was carried out using SDS-PAGE. Results indicated that egg Ig had a molecular weight of 206 kDa. They were smaller than seric IgM (800 kDa). The heavy (H) and light (L) chains of the egg Ig and of the seric IgM had similar molecular weight, 75 kDa and 28 kDa, respectively. These data suggest that egg Ig of Sea bass have monomeric structure. We hypothesize they could be sub-unit from the tetrameric IgMChez le loup (Dicentrarchus labrax L.), l'existence d'un transfert d'immunité maternelle a été étudiée par ELISA et immmunotransfert. Les protéines extraites d'oeufs fécondés ont été analysées afin de déterminer la présence éventuelle d'immunoglobulines (Ig). Après essais de plusieurs protocoles d'extraction (précipitations, chromatographies), l'utilisation de sondes anticorps monoclonales et polyclonales spécifiques des Ig sériques du loup a permis de confirmer la présence d'une protéine de type Ig en trés faible quantité dans les oeufs. Leur caractérisation biochimique a été réalisée par électrophorèse en gel dénaturant SDS. Les résultats indiquent que ces Ig ont un poids moléculaire de 206 kDa. Leur poids moléculaire est inférieur à celui de l'IgM sérique qui est d'environ 800 kDa. La chaîne lourde (H) et la chaîne légère (L) des Ig de l'oeuf ou des IgM sériques ont des poids moléculaires similaires de 75 kDa et de 28 kDa, respectivement. Ces données suggèrent que la structure de l'lg d'oeuf de loup est monomérique. Cette Ig pourrait dériver de sous-unités de l'immunoglobuline sérique qui est tétramérique

Synthesis and investigation of inhibition effect of fluorinated sulfonamide derivatives on carbonic anhydrase

International audienceSeries of perfluoroalkanesulfonamides 1, sodium salt of perfluoroalkanesulfonamides 2 and polyfluoroalkanesulfonamides 3 derivatives were synthesized and characterized by 1H NMR, 13C NMR, 19F NMR, IR and HRMS. Inhibition effects of these compounds on bovine carbonic anhydrase (bCA) and human carbonic anhydrase isoenzyme II (hCA) have been investigated. Comparing IC50 values of the synthesized molecules 1, 2 and 3, it has been found that compound 2b is a more potent inhibitor than acetazolamide on hCA. Moreover 2b does not present cellular toxicity on sheep red globules

Simultaneous Electrokinetic and Hydrodynamic Injection for High Sensitivity Bacteria Analysis in Capillary Electrophoresis

International audienceA repeatable preconcentration electrophoretic methodology for the analysis of bacteria was developed. This method is based on an isotachophoretic mode coupled with a simultaneous hydrodynamic-electrokinetic injection in conditions of field-amplified sample injection. This electrophoretic method allows the quantification of Enterobacter cloacae (studied as a model of Gram negative bacteria) with a limit of detection of 2 × 104 cells/mL. With the optimized conditions, a preconcentration factor of about 500-fold was obtained as compared to a standard hydrodynamic injection. The RSD (n = 5) on the migration time and on the peak area were 3% and 5%, respectively. This capillary electrophoretic methodology has been applied for the quantification of microbes in natural water (river and natural spring waters). Filtration of the sample prior to injection was required to remove ions present in the water and to keep the field-amplified sample injection condition at the injection. Filtrated bacteria were then recovered in terminating electrolyte diluted 10 times with water. Good agreements were obtained between cellular ATP measurements and the proposed CE methodology for the quantification of bacteria in waters

Study of Antibacterial Activity by Capillary Electrophoresis Using Multiple UV Detection Points

International audienceA new methodology for an antibacterial assay based on capillary electrophoresis with multiple UV detection points has been proposed. The possible antibacterial activity of cationic molecules on bacteria (Gram-positive and Gramnegative) is studied by detecting the bacteria before, during, and after their meeting with the cationic antibacterial compound. For that, a UV area imaging detector having two loops and three detection windows was used with a 95 cm ×100 μm i.d. capillary. In the antibacterial assay, the bacteria (negatively charged) and the cationic molecules were injected separately from each end of the capillary. The bacteria were mobilized by anionic ITP mode while cationic molecules migrate in the opposite direction under conditions close to CZE. The cationic molecules were injected into the capillary as a broad band (injected volume about 16% of the volume of the capillary) to prevent dilution of the sample during the electrophoretic process. Bacteriolytic activity, as well as strong interactions between the small antibacterial molecules and the bacteria, can be investigated within a few minutes. The assay was used to study the antibacterial activity of dendrigraft poly-L-lysines on Micrococcus luteus and Erwinia carotovora. Because dendrigraft poly-L-lysines are nonimmunogenic and have low toxicity, this new class of dendritic biomacromolecules is very promising for antibacterial applications

Dendrigraft Poly-l-lysine: A Non-Immunogenic Synthetic Carrier for Antibody Production

International audienceAn easily synthesized DendriGraft poly-lysine DGL-G3 (third generation) was shown to act as an efficient carrier for raising antibodies directed against small molecules. The immunological properties of three different forms of DGL-G3 were investigated: the native form (molecular weight 22 kDa bearing a mean number of 123 surface amino groups as TFA salts), a form modified at the C-terminus by fluorescein (fluorescein-DGL-G3), and last a surface-modified form bearing histamine (DGL-G3-Histamine). Our studies demonstrate the native DGL-G3 to be inefficient in eliciting antibody production in rabbits. Immunizations of rabbits using the core-modified fluorescein-DGL-G3 or the surface-modified DGL-G3-histamine conjugate failed in eliciting antibody production. Conversely, following a primary immunization using a BSA-histamine conjugate, a second immunization with DGL-G3-histamine conjugate improved the production of specific hapten-directed antibodies, which demonstrates the utility of DGL-G3 as a carrier for the production of highly specific antibody against haptens

Composition and antimicrobial activity of essential oils of Cinnamosma fragrans

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