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Functional Divergence in Shrimp Anti-Lipopolysaccharide Factors (ALFs): From Recognition of Cell Wall Components to Antimicrobial Activity ArchiMer
Rosa, Rafael Diego; Vergnes, Agnes; De Lorgeril, Julien; Goncalves, Priscila; Perazzolo, Luciane Maria; Saune, Laure; Romestand, Bernard; Fievet, Julie; Gueguen, Yannick; Bachere, Evelyne; Destoumieux-garzon, Delphine.
Antilipopolysaccharide factors (ALFs) have been described as highly cationic polypeptides with a broad spectrum of potent antimicrobial activities. In addition, ALFs have been shown to recognize LPS, a major component of the Gram-negative bacteria cell wall, through conserved amino acid residues exposed in the four-stranded beta-sheet of their three dimensional structure. In penaeid shrimp, ALFs form a diverse family of antimicrobial peptides composed by three main variants, classified as ALF Groups A to C. Here, we identified a novel group of ALFs in shrimp (Group D ALFs), which corresponds to anionic polypeptides in which many residues of the LPS binding site are lacking. Both Group B (cationic) and Group D (anionic) shrimp ALFs were produced in a...
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Ano: 2013 URL: http://archimer.ifremer.fr/doc/00160/27077/25236.pdf
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Generation and analysis of a 29,745 unique Expressed Sequence Tags from the Pacific oyster (Crassostrea gigas) assembled into a publicly accessible database: the GigasDatabase ArchiMer
Fleury, Elodie; Huvet, Arnaud; Lelong, Christophe; De Lorgeril, Julien; Boulo, Viviane; Gueguen, Yannick; Bachere, Evelyne; Tanguy, Arnaud; Moraga, Dario; Fabioux, Caroline; Lindeque, Penelope; Shaw, Jenny; Reinhardt, Richard; Prunet, Patrick; Davey, Grace; Lapegue, Sylvie; Sauvage, Christopher; Corporeau, Charlotte; Moal, Jeanne; Gavory, Frederick; Wincker, Patrick; Moreews, Francois; Klopp, Christophe; Mathieu, Michel; Boudry, Pierre; Favrel, Pascal.
Background: Although bivalves are among the most-studied marine organisms because of their ecological role and economic importance, very little information is available on the genome sequences of oyster species. This report documents three large-scale cDNA sequencing projects for the Pacific oyster Crassostrea gigas initiated to provide a large number of expressed sequence tags that were subsequently compiled in a publicly accessible database. This resource allowed for the identification of a large number of transcripts and provides valuable information for ongoing investigations of tissue-specific and stimulus-dependant gene expression patterns. These data are crucial for constructing comprehensive DNA microarrays, identifying single nucleotide...
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Ano: 2009 URL: http://archimer.ifremer.fr/doc/2009/publication-6770.pdf
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High polymorphism in big defensin gene expression reveals presence-absence gene variability (PAV) in the oyster Crassostrea gigas ArchiMer
Rosa, Rafael D.; Alonso, Pascal; Santini, Adrien; Vergnes, Agnes; Bachere, Evelyne.
We report here the first evidence in an invertebrate, the oyster Crassostrea gigas, of a phenomenon of Presence-Absence Variation (PAV) affecting immune-related genes. We previously evidenced an extraordinary interindividual variability in the basal mRNA abundances of oyster immune genes including those coding for a family of antimicrobial peptides, the big defensins (Cg-BigDef). Cg-BigDef is a diverse family composed of three members: Cg-BigDef1 to -3. Here, we show that besides a high polymorphism in Cg-BigDef mRNA expression, not all individual oysters express simultaneously the three Cg-BigDefs. Moreover, in numerous individuals, no expression of Cg-BigDefs could be detected. Further investigation at the genomic level revealed that in individuals in...
Tipo: Text Palavras-chave: Mollusk bivalve; Antimicrobial peptide; Polymorphism; Gene copy number variation; High-throughput microfluidic RT-qPCR; Vibrio infection.
Ano: 2015 URL: https://archimer.ifremer.fr/doc/00239/35009/33545.pdf
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In vitro culture of mollusc hemocytes. Functional study of burst respiratory activity and analysis of interactions with protozoan and procaryotic pathogens ArchiMer
Boulo, Viviane; Hervio, Dominique; Morvan, Annie; Bachere, Evelyne; Mialhe, Eric.
The chief importance of hemocytes as immune effectors in molluscs has led to develop researches for in vitro culture. The chemiluminescence technique has been used to estimate the phagocytosis capacity of Crassostrea gigas hemocytes according to different culture media and times. Sea water was used as a basic medium. Additives, such as antibiotics or glucose and buffers, such as Tris or Hepes, were tested as weIl as artificial sea water. Hemocyte chemiluminescence activities were recorded at t=o in relation to the medium composition. The highest values were observed when hemocytes were kept in sea water without any modification. Whichever medium, chemiluminescence activity was substantially decreased when hemocytes were previously kept in vitro for a few...
Tipo: Text Palavras-chave: Crassotrea gigas; Oyster; Bonamia ostreae; Pathogens; Molluscs; Hemocytes.
Ano: 1991 URL: http://archimer.ifremer.fr/doc/1991/acte-4115.PDF
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Inefficient immune response is associated with microbial permissiveness in juvenile oysters affected by mass mortalities on field ArchiMer
De Lorgeril, Julien; Escoubas, Jean Michel; Loubiere, Vincent; Pernet, Fabrice; Le Gall, Patrik; Vergnes, Agnes; Aujoulat, Fabien; Jeannot, Jean-luc; Jumas-bilak, Estelle; Got, Patrice; Gueguen, Yannick; Destoumieux-garzon, Delphine; Bachere, Evelyne.
Since 2008, juvenile Crassostrea gigas oysters have suffered from massive mortalities in European farming areas. This disease of complex etiology is still incompletely understood. Triggered by an elevated seawater temperature, it has been associated to infections by a herpes virus named OsHV-1 as well as pathogenic vibrios of the Splendidus clade. Ruling out the complexity of the disease, most of our current knowledge has been acquired in controlled experiments. Among the many unsolved questions, it is still ignored what role immunity plays in the capacity oysters have to survive an infectious episode. Here we show that juvenile oysters susceptible to the disease mount an inefficient immune response associated with microbial permissiveness and death. We...
Tipo: Text Palavras-chave: Host pathogen interaction; Innate immunity; Invertebrate; Mollusk; In situ mortality; Total bacteria; Crassostrea gigas.
Ano: 2018 URL: https://archimer.ifremer.fr/doc/00433/54470/55845.pdf
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Insight into Invertebrate Defensin Mechanism of Action OYSTER DEFENSINS INHIBIT PEPTIDOGLYCAN BIOSYNTHESIS BY BINDING TO LIPID II ArchiMer
Schmitt, Paulina; Wilmes, Miriam; Pugniere, Martine; Aumelas, Andre; Bachere, Evelyne; Sahl, Hans-georg; Schneider, Tanja; Destoumieux-garzon, Delphine.
Three oyster defensin variants (Cg-Defh1, Cg-Defh2, and Cg-Defm) were produced as recombinant peptides and characterized in terms of activities and mechanism of action. In agreement with their spectrum of activity almost specifically directed against Gram-positive bacteria, oyster defensins were shown here to be specific inhibitors of a bacterial biosynthesis pathway rather than mere membrane-active agents. Indeed, at lethal concentrations, the three defensins did not compromise Staphylococcus aureus membrane integrity but inhibited the cell wall biosynthesis as indicated by the accumulation of the UDP-N-acetylmuramyl-pentapeptide cell wall precursor. In addition, a combination of antagonization assays, thin layer chromatography, and surface plasmon...
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Ano: 2010 URL: http://archimer.ifremer.fr/doc/00014/12503/9369.pdf
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Isolement et purification de Marteilia (Protozoa : Ascetospora) parasites de bivalves marins ArchiMer
Mialhe, Eric; Bachere, Evelyne; Le Bec, Claude; Grizel, Henri.
Marteilia (Protozoa: Ascetospora) parasites of Ostrea edulis and Mytilus edulis, have been isolated and purified by an original method. The electron microscopie study of the purified pansporoblasts and spores confirms the integrity of the parasite and shows some new characteristics.
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Ano: 1985 URL: http://archimer.ifremer.fr/doc/1985/publication-3223.pdf
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Les surmortalités des naissains d’huîtres creuses, Crassostrea gigas : acquis des recherches en 2010 ArchiMer
Cochennec-laureau, Nathalie; Baud, Jean-pierre; Pepin, Jean-francois; Benabdelmouna, Abdellah; Soletchnik, Patrick; Lupo, Coralie; Garcia, Celine; Arzul, Isabelle; Boudry, Pierre; Huvet, Arnaud; Pernet, Fabrice; Bachere, Evelyne; Bedier, Edouard; Petton, Bruno; Gaussem, Florian; Stanisiere, Jean-yves; Degremont, Lionel.
Depuis 2008, la filière ostréicole française doit faire face à des surmortalités exceptionnelles des naissains d’huîtres creuses, Crassostrea gigas, (huîtres de moins d’un an) comprises entre 60 et 90% dans tous les sites d’élevage. Des cas de mortalité similaires ont été décrits en Irlande, dans les îles anglo-normandes, au Portugal en 2009 et au Royaume-Uni en 2010. Récemment deux épisodes de mortalité, associés à la présence d’un virus OsHV1 μvar (ou un variant très proche) ont été également décrits en Nouvelle Zélande (décembre 2010) et en Australie (janvier 2011) soulignant la vulnérabilité de la filière ostréicole dans le monde. L’objectif de ce document est de faire un point sur les recherches menées depuis l’apparition de ce phénomène et de...
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Ano: 2011 URL: http://archimer.ifremer.fr/doc/00033/14423/11719.pdf
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Luminol-dependent chemiluminescence by hemocytes of two marine bivalves, Ostrea edulis and Crassostrea gigas ArchiMer
Bachere, Evelyne; Hervio, Dominique; Mialhe, Eric.
Microbicidal phagocytic function and oxidative metabolism of the hemocytes of 2 oyster species, Ostrea edulis and Crassostrea gigas, were investigated using a luminol-enhanced chemiluminescence (technique. First, experimental parameters adapted to marine bivalve hemocytes were established on hemolymph pools of C. gigas in order to obtain qualitatively and quantitatively homogeneous samples and so to perform statistically comparable assays. The use of Modified Alsever Solution (allowed hemocytes to be kept non-aggregated and in a non-stimulated state until starting the assays. A number of aliquots of 2 X 10(5) hemocytes with an MAS final concentration of 3.5 % showed high chemiluminescent response after stimulation by zymosan particles A particle:hemocyte...
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Ano: 1991 URL: http://archimer.ifremer.fr/doc/1991/publication-2761.pdf
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Mise au point de techniques d'isolement de parasites - Compte rendu d'activité ArchiMer
Grizel, Henri; Bachere, Evelyne; Gagneraud, Sylvie; Audic, Guylaine.
Les recherches faisant l'objet de ce contrat ont porté sur la mise au point de techniques adaptées aux bivalves marins et à l'étude du parasite Bonamia ostreae responsable de la maladie hémocytaire de l'huître plate, Ostrea edulis.
Tipo: Text Palavras-chave: Pathologie; Huître plate; Ostrea edulis; Bonamia ostreae; Techniques laboratoire.
Ano: 1982 URL: http://archimer.ifremer.fr/doc/00015/12626/9514.pdf
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Mise en évidence d'Haplosporidium sp. (Haplosporida - Haplosporidiidae) parasite de l'huître plate Ostrea edulis L. ArchiMer
Bachere, Evelyne; Grizel, Henri.
A new Haplosporidie has been observed in the flat oyster Ostrea edulis from the Mediterranean Sea. Different development stages have been described in light and electron microscopy. The characteristics of the spores and especially the presence of at least four sporal filaments enable us to distinguish this Haplosporidie from those already described in the Atlantic flat oysters. Different haplosporidies have been described in the oyster. Some are known as being pathogenic, Minchinia costalis (Andrews et al., 1962 and 1978; Couch 1967) and Minchinia nelsoni (Haskin et al., 1966; Farley 1968 and 1975), others, identified only recently, are considered as occasional parasites (Van Banning, 1977). During the research conducted on Bonamia ostreae (Pichot et al.,...
Tipo: Text Palavras-chave: Pathologie; Haplosporidium; Parasite; Ostrea edulis; Huître plate.
Ano: 1982 URL: http://archimer.ifremer.fr/doc/1982/publication-6576.PDF
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Molecular characterization of penaeidins from two Atlantic Brazilian shrimp species, Farfantepenaeus paulensis and Litopenaeus schmitti ArchiMer
Barracco, Margherita; De Lorgeril, Julien; Gueguen, Yannick; Bachere, Evelyne.
We report here the molecular cloning of new members of the penaeidin family from two Atlantic penaeids from Brazil, Litopenaeus schmitti and Farfantepenaeus paulensis. The presence of penaeidins in the granular hemocytes of both shrimps was first evidenced by immunofluorescence, using polyclonal antibodies raised against L. vannamei penaeidin Litvan PEN3-1. cDNAs from the hemocytes of both Brazilian species were obtained by reverse transcription and the sequences encoding penaeidins were amplified by PCR, using primers based on penaeidin consensus sequences. Five penaeidin clones were obtained. According to the international penaeidin classification (PenBase, http://www.penbase.immunaqua.com), the deduced amino acid sequences of two clones from L. schmitti...
Tipo: Text Palavras-chave: Immuno detection; Molecular cloning; Litopenaeus schmitti; Farfantepenaeus paulensis; Brazilian penaeid shrimps; Hemocytes; Antimicrobial peptides; Penaeidins.
Ano: 2005 URL: http://archimer.ifremer.fr/doc/2005/publication-3582.pdf
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Molecular characterization of two isoforms of defensin from hemocytes of the oyster Crassostrea gigas ArchiMer
Gonzalez, Marcelo; Gueguen, Yannick; Desserre, Geraldine; De Lorgeril, Julien; Romestand, Bernard; Bachere, Evelyne.
Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. We previously characterized the first AMP from an oyster, a defensin, that was shown to be continuously expressed in the mantle of Crassostrea gigas. In this study, we report the cDNA cloning of two new isoforms of the defensin AMP family (Cg-defh1 and Cg-defh2) from the hemocytes of the oyster. The deduced amino acid sequences reveal two peptides of 73 amino acid residues with a mature portion consisting of 43 amino acid residues. Cy-Defh1 and CyDefh2 share 86% amino acid identity and belong to the "arthropod-molluscs defensin family". qRT-PCR analyses indicate that Cg-defh2 is continuously expressed in the hemocytes of C. gigas. In...
Tipo: Text Palavras-chave: Defensin; Antimicrobial peptide; Hemocytes; Pacific oyster; Marine invertebrate; Mollusc bivalve.
Ano: 2007 URL: http://archimer.ifremer.fr/doc/2007/publication-2331.pdf
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Molecular diversity of antimicrobial effectors in the oyster Crassostrea gigas ArchiMer
Schmitt, Paulina; Gueguen, Yannick; Desmarais, Erick; Bachere, Evelyne; De Lorgeril, Julien.
Background: To gain insight into the molecular diversity of antimicrobial peptides and proteins in the oyster Crassostrea gigas, we characterized and compared the sequence polymorphism of the antimicrobial peptides (AMPs), Cg-Defensins (Cg-Defs) and Cg-Proline Rich peptide (Cg-Prp), and of the bactericidal permeability increasing protein, Cg-BPI. For that, we analyzed genomic and transcript sequences obtained by specific PCR amplification and in silico searches. Results: High diversification among the three antimicrobial effectors was evidenced by this polymorphism survey. On the basis of sequence phylogenies, each AMP aggregates into clearly defined groups of variants and is the product of a multigenic family displaying a variety of gene structures. In...
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Ano: 2010 URL: http://archimer.ifremer.fr/doc/00001/11182/6691.pdf
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Monoclonal antibodies against Bonamia ostreae (Protozoa: Ascetospera), an intrahaemocytic parasite of flat oyster Ostrea edulis (Mollusca : Bivlavia) ArchiMer
Rogier, Hervé; Hervio, Dominique; Boulo, Viviane; Clavies, Christine; Hervaud, Eliane; Bachere, Evelyne; Mialhe, Eric; Grizel, Henri; Pau, Bernard; Paolucci, Francis.
The protozoan Bonamia ostreae (Ascetospora), a paasite of the flat oyster Ostrea edulis, was purified by differential and isopycnic centrifugations. Mice of the strain Balb/c were immunized with purified parasites and hybridomas were prepared by fusion of immunized mouse splenocytes with the mouse myeloma cell line P3-X63-Ag8-653. From the fusion, 12 clones were isolated and saved. The resulting antibodies were characterized with a solid phase radioimmunoassay (RIA). Antibodies from 7 clones reacted only with B. ostreae and not with normal host tissue. Although some of the antibiodies identified. One of these antibodies (20B2-1B12) has an apparent association constant of ca 3 X 10(8) M-1 and bound to a number of different sites on the parasite. These...
Tipo: Text Palavras-chave: Monoclonal antibodies; Oyster; Ostrea edulis; Bonamia ostreae.
Ano: 1991 URL: http://archimer.ifremer.fr/doc/1991/publication-2756.pdf
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Mortalité du naissain d’Huître creuse Crassostrea gigas dans l’étang de Thau en 2009 ArchiMer
Pernet, Fabrice; Barret, Jean; Le Gall, Patrik; Malet, Nathalie; Pastoureaud, Annie; Munaron, Dominique; De Lorgeril, Julien; Bachere, Evelyne; Vaquer, Andre; Huvet, Arnaud; Corporeau, Charlotte; Normand, Julien; Boudry, Pierre; Moal, Jeanne; Quere, Claudie; Quilien, Virgile; Daniel, Jean-yves; Pepin, Jean-francois; Saulnier, Denis; Gonzalez, Jean-louis.
This study was conducted in the Mediterranean area during a mass mortality event of Pacific oyster Crassostrea gigas. The objectives of this work were to increase our understanding of the mass mortality phenomena in Pacific oysters, to identify risky cultivation practises and to propose mitigation strategies. In the first part of this work, we followed environmental parameters of the Thau lagoon in relation with mortality events. Then, we compared mortality and growth parameters of oysters as a function of their origins, their development stages and cultivation sites in relation with energetic, reproduction and pathogens. Finally, we conducted transfer experiments where oysters maintained free of mortality in open sea were transferred in the Thau lagoon...
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Ano: 2010 URL: http://archimer.ifremer.fr/doc/00002/11354/7927.pdf
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NMR Structure of rALF-Pm3, an Anti-Lipopolysaccharide Factor from Shrimp: Model of the Possible Lipid A-Binding Site ArchiMer
Yang, Yinshan; Boze, Helene; Chemardin, Patrick; Padilla, Andre; Moulin, Guy; Tassanakajon, Anchalee; Pugniere, Martine; Roquet, Francoise; Destoumieux Garzon, Delphine; Gueguen, Yannick; Bachere, Evelyne; Aumelas, Andre.
The anti-lipopolysaccharide factor ALF-Pm3 is a 98-residue protein identified in hemocytes from the black tiger shrimp Penaeus monodon. It was expressed in Pichia pastoris from the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter as a folded and N-15 uniformly labeled rALF-Pm3 protein. Its 3D structure was established by NMR and consists of three alpha-helices packed against a four-stranded beta-sheet. The C-34-C-55 disulfide bond was shown to be essential for the structure stability. By using surface plasmon resonance, we demonstrated that rALF-Pm3 binds to LPS, lipid A and to OM(R)-174, a soluble analogue of lipid A. Biophysical studies of rALF-Pm3/LPS and rALF-Pm3/OM(R)-174 complexes indicated rather high molecular sized aggregates, which...
Tipo: Text Palavras-chave: Ultracentrifugation; Surface plasmon resonance; Septic shock; Structure; NMR; Lipid A; Lipopolysaccharide; Anti lipopolysaccharide factor.
Ano: 2009 URL: http://archimer.ifremer.fr/doc/2009/publication-6320.pdf
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Oyster hemocytes express a proline-rich peptide displaying synergistic antimicrobial activity with a defensin ArchiMer
Gueguen, Yannick; Romestand, Bernard; Fievet, Julie; Schmitt, Paulina; Destoumieux Garzon, Delphine; Franck, Vandenbulcke; Philippe, Bulet; Bachere, Evelyne.
A cDNA sequence that encodes a 61-amino acid polypeptide precursor with homologies to proline-rich antimicrobial peptides (AMPs) was identified in the oyster Crassostrea gigas. After release of a hydrophobic signal peptide. the resulting 37-amino acid peptide, Cg-Prp, is composed of an acidic region and a cationic proline-rich region. To evaluate the biological properties of Cg-Prp, multiple proline-rich peptides corresponding to putative processing of the full-length Cg-Prp were synthesized. A limited antimicrobial activity was observed for two of them, which also showed strong synergistic antimicrobial activity with Cg-Def, a defensin from C gigas. To our knowledge, this is the first evidence of synergy between a defensin and another AMP in an...
Tipo: Text Palavras-chave: Antimicrobial peptide; Mollusk; Synergy; Invertebrate; Innate immunity; Pacific oyster.
Ano: 2009 URL: http://archimer.ifremer.fr/doc/2009/publication-6232.pdf
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Penaeidins, a new family of antimicrobial peptides isolated from the shrimp Penaeus vannamei (Decapoda) ArchiMer
Destoumieux, Delphine; Bulet, Philippe; Loew, Damarys; Van Dorsselaer, Alain; Rodriguez, Jenny; Bachere, Evelyne.
We report here the isolation of three members of a new family of antimicrobial peptides from the hemolymph of shrimps Penaeus vannamei in which immune response has not been experimentally induced. The three molecules display antimicrobial activity against fungi and bacteria with a predominant activity against Gram-positive bacteria. The complete sequences of these peptides were determined by a combination of enzymatic cleavages, Edman degradation, mass spectrometry, and cDNA cloning using a hemocyte cDNA library. The mature molecules (50 and 62 residues) are characterized by an NH2-terminal domain rich in proline residues and a COOH-terminal domain containing three intramolecular disulfide bridges. One of these molecules is post-translationally modified by...
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Ano: 1997 URL: http://archimer.ifremer.fr/doc/00335/44649/57344.pdf
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Penaeidins, antimicrobial peptides with chitin-binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge ArchiMer
Destoumieux, Delphine; Munoz, Marcello; Cosseau, Céline; Rodriguez, Jenny; Bulet, Philippe; Comps, Marie-annick; Bachere, Evelyne.
Penaeidins are members of a new family of antimicrobial peptides isolated from a crustacean, which present both Gram-positive antibacterial and antifungal activities. We have studied the localization of synthesis and storage of penaeidins in the shrimp Penaeus vannamei. The distribution of penaeidin transcripts and peptides in various tissues reveals that penaeidins are constitutively synthesized and stored in the shrimp haemocytes. It was shown by immunocytochemistry, at both optical and ultrastructural levels, that the peptides are localized in granulocyte cytoplasmic granules, The expression and localization of penaeidins were further analysed in shrimp subjected to microbial challenge. We found that (1) penaeidin mRNA levels decrease in circulating...
Tipo: Text Palavras-chave: Crustacean immunity; Penaeid shrimp; Haemocyte; Chitin binding; Antimicrobial peptide.
Ano: 2000 URL: http://archimer.ifremer.fr/doc/2000/publication-412.pdf
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