The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Laurent Bedouet - One of the best experts on this subject based on the ideXlab platform.
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coupling proteomics and transcriptomics for the identification of novel and variant forms of mollusk shell proteins a study with p margaritifera
ChemBioChem, 2011Co-Authors: Sophie Berland, Christian Milet, Denis Duplat, Arul Marie, Jean Yves Sire, Laurent BedouetAbstract:Shell matrix proteins from Pinctada margaritifera were characterized by combining proteomics analysis of shell organic extracts and transcript sequences, both obtained from the shell-forming cell by using the suppression subtractive hybridization method (SSH) and from an expressed sequence tag (EST) database available from Pinctada maxima mantle tissue. Some of the identified proteins were homologues to proteins reported in other mollusk shells, namely lysine-rich matrix proteins (KRMPs), shematrins and molluscan prismatic and nacreous layer 88 kDa (MPN88). Sequence comparison within and among Pinctada species pointed to intra- and interspecies variations relevant to polymorphism and to evolutionary distance, respectively. In addition, a novel shell matrix protein, linkine was identified. BLAST analysis of the peptide sequences obtained from the shell of P. margaritifera against the EST database revealed the presence of additional proteins: two proteins similar to the Pif97 protein that was identified in the shell of P. fucata, a chitinase-like protein previously identified in Crassostrea gigas, two chitin-binding proteins, and two incomplete sequences of proteins unknown so far in mollusk shells. Combining proteomics and transcriptomics analysis we demonstrate that all these proteins, including linkine, are addressed to the shell. Retrieval of motif-forming sequences, such as chitin-binding, with functional annotation from several peptides nested in the shell could indicate protein involvement in shell patterning.
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Identification of calconectin, a calcium-binding protein specifically expressed by the mantle of Pinctada margaritifera.
FEBS Letters, 2006Co-Authors: Denis Duplat, Laurent Bedouet, Christian Milet, M. Puisségur, Marthe Rousseau, Hélène Boulzaguet, Daniel Y. Sellos, A. Van Wormhoudt, Evelyne LopezAbstract:Abstract Nacre or mother-of-pearl in the shell of Pinctada margaritifera is composed of 95–99% calcium carbonate and 1–5% organic matrix. In this study, we developed an original technique to characterize the genes differentially expressed in nacre-forming cells (NFC) by combining suppression subtractive hybridization (SSH), to establish a cDNA subtractive library, with rapid amplification of cDNA ends (RACE)-PCR. Seventy-two specific cDNA sequences have been obtained so far. These include a protein containing two EF-hand Ca2+-binding domains which was completely sequenced after amplification by RACE-PCR. Its specific expression as well as the specificity of the SSH method was confirmed by semi-quantitative RT-PCR on NFC and mantle cells.
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soluble proteins of the nacre of the giant oyster Pinctada maxima and of the abalone haliotis tuberculata extraction and partial analysis of nacre proteins
Comparative Biochemistry and Physiology B, 2001Co-Authors: Laurent Bedouet, Maria Jose Schuller, Christian Milet, Evelyne Lopez, Frederic Marin, Michel GiraudAbstract:Several proteins from nacre of the oyster Pinctada maxima and the abalone Haliotis tuberculata were extracted and partly characterized. Proteins dispersed in aragonite were solubilized during demineralization with acetic acid whereas proteins adsorbed on conchiolin were extracted with sodium dodecyl sulfate and β-mercaptoethanol. The matrix of Pinctada maxima nacre is composed of one main protein with an apparent molecular weight of 20 kDa (p20). This protein was found in the acetic acid soluble fraction of nacre, as well as in the Laemmli-solubilized extract of conchiolin. In addition, the p20 solubilized with acetic acid can form oligomers made of 6 monomers linked together by disulfide bridges. The first N-terminal 21 amino acids of p20 were determined and no homology with known proteins was found. In Haliotis tuberculata nacre, 5 main proteins were solubilized during demineralization and 3 glycoproteins were detected. Stains-all and Alcian blue staining revealed polyanionic proteins in the extracts isolated from Pinctada maxima and Haliotis tuberculata nacre.
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characterization and quantification of chitosan extracted from nacre of the abalone haliotis tuberculata and the oyster Pinctada maxima
Marine Biotechnology, 2001Co-Authors: Frederic Zentz, Laurent Bedouet, Christian Milet, Maria Almeida, Evelyne Lopez, Michel GiraudAbstract:This study was performed to characterize and quantify chitosan by simple physicochemical methods (infrared spectroscopy and potentiometric measurements). These procedures were validated with well-characterized chitosan before being used to investigate chitosan in nacre of the abalone Haliotis tuberculata and of the giant oyster Pinctada maxima. Potentiometric study revealed a chitosan extract from the nacre of H. tuberculata with a degree of deacetylation of around 88% and an intrinsic pK of 6.5. According to infrared and potentiometric data, a low yield (η) of extraction was calculated (η= 0.064%). For experiments performed on the nacre of P. maxima, and in spite of more stringent deacetylation conditions, results suggested that a chitin-protein complex (η= 0.053%) was isolated rather than chitosan.
Christian Milet - One of the best experts on this subject based on the ideXlab platform.
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coupling proteomics and transcriptomics for the identification of novel and variant forms of mollusk shell proteins a study with p margaritifera
ChemBioChem, 2011Co-Authors: Sophie Berland, Christian Milet, Denis Duplat, Arul Marie, Jean Yves Sire, Laurent BedouetAbstract:Shell matrix proteins from Pinctada margaritifera were characterized by combining proteomics analysis of shell organic extracts and transcript sequences, both obtained from the shell-forming cell by using the suppression subtractive hybridization method (SSH) and from an expressed sequence tag (EST) database available from Pinctada maxima mantle tissue. Some of the identified proteins were homologues to proteins reported in other mollusk shells, namely lysine-rich matrix proteins (KRMPs), shematrins and molluscan prismatic and nacreous layer 88 kDa (MPN88). Sequence comparison within and among Pinctada species pointed to intra- and interspecies variations relevant to polymorphism and to evolutionary distance, respectively. In addition, a novel shell matrix protein, linkine was identified. BLAST analysis of the peptide sequences obtained from the shell of P. margaritifera against the EST database revealed the presence of additional proteins: two proteins similar to the Pif97 protein that was identified in the shell of P. fucata, a chitinase-like protein previously identified in Crassostrea gigas, two chitin-binding proteins, and two incomplete sequences of proteins unknown so far in mollusk shells. Combining proteomics and transcriptomics analysis we demonstrate that all these proteins, including linkine, are addressed to the shell. Retrieval of motif-forming sequences, such as chitin-binding, with functional annotation from several peptides nested in the shell could indicate protein involvement in shell patterning.
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Identification of calconectin, a calcium-binding protein specifically expressed by the mantle of Pinctada margaritifera.
FEBS Letters, 2006Co-Authors: Denis Duplat, Laurent Bedouet, Christian Milet, M. Puisségur, Marthe Rousseau, Hélène Boulzaguet, Daniel Y. Sellos, A. Van Wormhoudt, Evelyne LopezAbstract:Abstract Nacre or mother-of-pearl in the shell of Pinctada margaritifera is composed of 95–99% calcium carbonate and 1–5% organic matrix. In this study, we developed an original technique to characterize the genes differentially expressed in nacre-forming cells (NFC) by combining suppression subtractive hybridization (SSH), to establish a cDNA subtractive library, with rapid amplification of cDNA ends (RACE)-PCR. Seventy-two specific cDNA sequences have been obtained so far. These include a protein containing two EF-hand Ca2+-binding domains which was completely sequenced after amplification by RACE-PCR. Its specific expression as well as the specificity of the SSH method was confirmed by semi-quantitative RT-PCR on NFC and mantle cells.
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soluble proteins of the nacre of the giant oyster Pinctada maxima and of the abalone haliotis tuberculata extraction and partial analysis of nacre proteins
Comparative Biochemistry and Physiology B, 2001Co-Authors: Laurent Bedouet, Maria Jose Schuller, Christian Milet, Evelyne Lopez, Frederic Marin, Michel GiraudAbstract:Several proteins from nacre of the oyster Pinctada maxima and the abalone Haliotis tuberculata were extracted and partly characterized. Proteins dispersed in aragonite were solubilized during demineralization with acetic acid whereas proteins adsorbed on conchiolin were extracted with sodium dodecyl sulfate and β-mercaptoethanol. The matrix of Pinctada maxima nacre is composed of one main protein with an apparent molecular weight of 20 kDa (p20). This protein was found in the acetic acid soluble fraction of nacre, as well as in the Laemmli-solubilized extract of conchiolin. In addition, the p20 solubilized with acetic acid can form oligomers made of 6 monomers linked together by disulfide bridges. The first N-terminal 21 amino acids of p20 were determined and no homology with known proteins was found. In Haliotis tuberculata nacre, 5 main proteins were solubilized during demineralization and 3 glycoproteins were detected. Stains-all and Alcian blue staining revealed polyanionic proteins in the extracts isolated from Pinctada maxima and Haliotis tuberculata nacre.
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characterization and quantification of chitosan extracted from nacre of the abalone haliotis tuberculata and the oyster Pinctada maxima
Marine Biotechnology, 2001Co-Authors: Frederic Zentz, Laurent Bedouet, Christian Milet, Maria Almeida, Evelyne Lopez, Michel GiraudAbstract:This study was performed to characterize and quantify chitosan by simple physicochemical methods (infrared spectroscopy and potentiometric measurements). These procedures were validated with well-characterized chitosan before being used to investigate chitosan in nacre of the abalone Haliotis tuberculata and of the giant oyster Pinctada maxima. Potentiometric study revealed a chitosan extract from the nacre of H. tuberculata with a degree of deacetylation of around 88% and an intrinsic pK of 6.5. According to infrared and potentiometric data, a low yield (η) of extraction was calculated (η= 0.064%). For experiments performed on the nacre of P. maxima, and in spite of more stringent deacetylation conditions, results suggested that a chitin-protein complex (η= 0.053%) was isolated rather than chitosan.
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Effect of water soluble extract of nacre (Pinctada maxima) on alkaline phosphatase activity and Bcl-2 expression in primary cultured osteoblasts from neonatal rat calvaria.
Journal of Materials Science: Materials in Medicine, 2001Co-Authors: F. Moutahir-belqasmi, Christian Milet, N. Balmain, M. Lieberrher, S. Borzeix, S. Berland, M. Barthelemy, J. Peduzzi, E. LopezAbstract:The nacre (mother of pearl) layer of the oyster Pinctada maxima shell can initiate bone formation by human osteoblasts in vivo and in vitro and is a new biomaterial that induces osteogenesis. This activity of nacre could be due to its water-soluble matrix. We examined the action of a water-soluble extract of nacre on the osteoblast phenotype of cells isolated from rat neonatal calvaria by measuring alkaline phosphatase (ALP) activity and by localization of the anti-apoptotic protein Bcl-2 by immunocytochemistry. ALP activity was increased 7% by 100 µg proteins/ml extract and 20% by 50 µg proteins/ml extract, but a low concentration of extract decreased the ALP activity by 8%. Cells treated with a high aspartic acid content fraction of the extract had increased ALP activity (23%). Nacre extract and the fraction have no effect on the proliferation of mature osteoblasts. Immunoreactive Bcl-2 was overproduced in the cytoplasm and nuclei of osteoblasts at all stages of culture. Bcl-2 was found over the whole chromatin in quiescent and mitotic cells at the end of mitosis in the two nuclei in one cell, before cytodieresis. Bcl-2 was also found over chromosomes. Thus, nacre extract stimulates Bcl-2 production in osteoblasts, that is correlated with the cell cycle. Bcl-2 was also abundant in the nucleoli of extract-treated cells. Thus, the concentration and subcellular distribution of Bcl-2 in osteoblasts in primary cultures is influenced by nacre extract, and related to the cell cycle and the regulation of gene expression. Hence, knowledge of how water-soluble extracts of Pinctada maxima nacre act on osteoblasts in vitro may reveal the mechanisms involved in its action in vivo on bone cells and bone regeneration.
Michel Giraud - One of the best experts on this subject based on the ideXlab platform.
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soluble proteins of the nacre of the giant oyster Pinctada maxima and of the abalone haliotis tuberculata extraction and partial analysis of nacre proteins
Comparative Biochemistry and Physiology B, 2001Co-Authors: Laurent Bedouet, Maria Jose Schuller, Christian Milet, Evelyne Lopez, Frederic Marin, Michel GiraudAbstract:Several proteins from nacre of the oyster Pinctada maxima and the abalone Haliotis tuberculata were extracted and partly characterized. Proteins dispersed in aragonite were solubilized during demineralization with acetic acid whereas proteins adsorbed on conchiolin were extracted with sodium dodecyl sulfate and β-mercaptoethanol. The matrix of Pinctada maxima nacre is composed of one main protein with an apparent molecular weight of 20 kDa (p20). This protein was found in the acetic acid soluble fraction of nacre, as well as in the Laemmli-solubilized extract of conchiolin. In addition, the p20 solubilized with acetic acid can form oligomers made of 6 monomers linked together by disulfide bridges. The first N-terminal 21 amino acids of p20 were determined and no homology with known proteins was found. In Haliotis tuberculata nacre, 5 main proteins were solubilized during demineralization and 3 glycoproteins were detected. Stains-all and Alcian blue staining revealed polyanionic proteins in the extracts isolated from Pinctada maxima and Haliotis tuberculata nacre.
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characterization and quantification of chitosan extracted from nacre of the abalone haliotis tuberculata and the oyster Pinctada maxima
Marine Biotechnology, 2001Co-Authors: Frederic Zentz, Laurent Bedouet, Christian Milet, Maria Almeida, Evelyne Lopez, Michel GiraudAbstract:This study was performed to characterize and quantify chitosan by simple physicochemical methods (infrared spectroscopy and potentiometric measurements). These procedures were validated with well-characterized chitosan before being used to investigate chitosan in nacre of the abalone Haliotis tuberculata and of the giant oyster Pinctada maxima. Potentiometric study revealed a chitosan extract from the nacre of H. tuberculata with a degree of deacetylation of around 88% and an intrinsic pK of 6.5. According to infrared and potentiometric data, a low yield (η) of extraction was calculated (η= 0.064%). For experiments performed on the nacre of P. maxima, and in spite of more stringent deacetylation conditions, results suggested that a chitin-protein complex (η= 0.053%) was isolated rather than chitosan.
Hiroshi Miyamoto - One of the best experts on this subject based on the ideXlab platform.
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the diversity of shell matrix proteins genome wide investigation of the pearl oyster Pinctada fucata
Zoological Science, 2013Co-Authors: Hiroshi Miyamoto, Kurin Limura, Takumi Miki, Yukinobu Isowa, Hirotoshi Endo, Shigeharu Kinoshita, Tetsuji Masaoka, Tomohiro Kotaki, Naoki Hashimoto, Seiji NakayamaAbstract:In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, four Pifs, nine shematrins, two prismalin-14 proteins, and 21 tyrosinases. This diversity of shell matrix proteins may be implicated in the morphological diversity of mollusc shells. The annotated genes reported here can be searched in P. fucata gene models version 1.1 and genome assembly version 1.0 ( http://marinegenomics.oist.jp/Pinctada_fucata ). These genes should provide a useful resource for studies of the genetic basis of biomineralization and evaluation of the role of shell matrix proteins as an evolutionary toolkit among the molluscs.
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the diversity of shell matrix proteins genome wide investigation of the pearl oyster Pinctada fucata
Zoological Science, 2013Co-Authors: Hiroshi Miyamoto, Kurin Limura, Takumi Miki, Yukinobu Isowa, Hirotoshi Endo, Shigeharu Kinoshita, Tetsuji Masaoka, Tomohiro Kotaki, Naoki Hashimoto, Seiji NakayamaAbstract:In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, fo...
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a novel nacre protein n19 in the pearl oyster Pinctada fucata
Biochemical and Biophysical Research Communications, 2007Co-Authors: Masato Yano, Kouhei Nagai, Koichi Morimoto, Hiroshi MiyamotoAbstract:Abstract A novel 19 kDa protein, which was named N19, was isolated from the nacreous layer of the pearl oyster Pinctada fucata . N19 is one of predominant proteins found in the water-insoluble fraction of the nacreous layer. MALDI–TOF/TOF analysis indicated that the three trypsin-digested peptides (791.45, 824.42, and 1118.65 m / z ) corresponded to the amino acid sequences predicted from a cDNA isolated from a mantle cDNA library of P. fucata . Northern blot analysis revealed that the N19 mRNA was a little more abundant in the pallial region than the edge region, in the mantle. In CaCO 3 precipitation assay, the recombinant N19 protein inhibited the crystallization of CaCO 3 . These results indicate that N19 is localized in the nacre and plays a negative regulatory role in calcification in the pearl oyster.
Evelyne Lopez - One of the best experts on this subject based on the ideXlab platform.
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Identification of calconectin, a calcium-binding protein specifically expressed by the mantle of Pinctada margaritifera.
FEBS Letters, 2006Co-Authors: Denis Duplat, Laurent Bedouet, Christian Milet, M. Puisségur, Marthe Rousseau, Hélène Boulzaguet, Daniel Y. Sellos, A. Van Wormhoudt, Evelyne LopezAbstract:Abstract Nacre or mother-of-pearl in the shell of Pinctada margaritifera is composed of 95–99% calcium carbonate and 1–5% organic matrix. In this study, we developed an original technique to characterize the genes differentially expressed in nacre-forming cells (NFC) by combining suppression subtractive hybridization (SSH), to establish a cDNA subtractive library, with rapid amplification of cDNA ends (RACE)-PCR. Seventy-two specific cDNA sequences have been obtained so far. These include a protein containing two EF-hand Ca2+-binding domains which was completely sequenced after amplification by RACE-PCR. Its specific expression as well as the specificity of the SSH method was confirmed by semi-quantitative RT-PCR on NFC and mantle cells.
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soluble proteins of the nacre of the giant oyster Pinctada maxima and of the abalone haliotis tuberculata extraction and partial analysis of nacre proteins
Comparative Biochemistry and Physiology B, 2001Co-Authors: Laurent Bedouet, Maria Jose Schuller, Christian Milet, Evelyne Lopez, Frederic Marin, Michel GiraudAbstract:Several proteins from nacre of the oyster Pinctada maxima and the abalone Haliotis tuberculata were extracted and partly characterized. Proteins dispersed in aragonite were solubilized during demineralization with acetic acid whereas proteins adsorbed on conchiolin were extracted with sodium dodecyl sulfate and β-mercaptoethanol. The matrix of Pinctada maxima nacre is composed of one main protein with an apparent molecular weight of 20 kDa (p20). This protein was found in the acetic acid soluble fraction of nacre, as well as in the Laemmli-solubilized extract of conchiolin. In addition, the p20 solubilized with acetic acid can form oligomers made of 6 monomers linked together by disulfide bridges. The first N-terminal 21 amino acids of p20 were determined and no homology with known proteins was found. In Haliotis tuberculata nacre, 5 main proteins were solubilized during demineralization and 3 glycoproteins were detected. Stains-all and Alcian blue staining revealed polyanionic proteins in the extracts isolated from Pinctada maxima and Haliotis tuberculata nacre.
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characterization and quantification of chitosan extracted from nacre of the abalone haliotis tuberculata and the oyster Pinctada maxima
Marine Biotechnology, 2001Co-Authors: Frederic Zentz, Laurent Bedouet, Christian Milet, Maria Almeida, Evelyne Lopez, Michel GiraudAbstract:This study was performed to characterize and quantify chitosan by simple physicochemical methods (infrared spectroscopy and potentiometric measurements). These procedures were validated with well-characterized chitosan before being used to investigate chitosan in nacre of the abalone Haliotis tuberculata and of the giant oyster Pinctada maxima. Potentiometric study revealed a chitosan extract from the nacre of H. tuberculata with a degree of deacetylation of around 88% and an intrinsic pK of 6.5. According to infrared and potentiometric data, a low yield (η) of extraction was calculated (η= 0.064%). For experiments performed on the nacre of P. maxima, and in spite of more stringent deacetylation conditions, results suggested that a chitin-protein complex (η= 0.053%) was isolated rather than chitosan.
