Identification of calconectin, a calcium-binding protein specifically expressed by the mantle of Pinctada margaritifera.
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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. (+info)
Marinomonas ostreistagni sp. nov., isolated from a pearl-oyster culture pond in Sanya, Hainan Province, China.
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A Gram-negative, aerobic, halophilic, neutrophilic, rod-shaped, non-pigmented, polar-flagellated bacterium, UST010306-043(T), was isolated from a pearl-oyster culture pond in Sanya, Hainan Province, China in January 2001. This marine bacterium had an optimum temperature for growth of between 33 and 37 degrees C. On the basis of 16S rRNA gene sequence analysis, the strain was closely related to Marinomonas aquimarina and Marinomonas communis, with 97.5-97.7 and 97.1 % sequence similarity, respectively. Levels of DNA-DNA relatedness to the type strains of these species were well below 70 %. Analyses of phylogenetic, phenotypic and chemotaxomonic characteristics showed that strain UST010306-043(T) was distinct from currently established Marinomonas species. A novel species with the name Marinomonas ostreistagni sp. nov. is proposed to accommodate this bacterium, with strain UST010306-043(T) (=JCM 13672(T)=NRRL B-41433(T)) as the type strain. (+info)
Pf-ALMP, a novel astacin-like metalloproteinase with cysteine arrays, is abundant in hemocytes of pearl oyster Pinctada fucata.
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The astacin family metalloproteinase is a family of zinc-dependent endopeptidases which play crucial roles in embryonic development, bone growth and morphogenesis. A cDNA clone encoding a putative astacin-like metalloproteinase (pf-ALMP) was isolated from hemocytes of pearl oyster, Pinctada fucata. The novel metalloproteinase presents a molecular organization close to the astacins, but has a novel C-terminal domain with cysteine arrays. RT-PCR analysis revealed that pf-ALMP was expressed dramatically high in hemocytes, which was affected by lipopolysaccharides (LPS) challenge. High expression of pf-ALMP was also found in gill, gonad and digestion gland, and in situ hybridization demonstrated that pf-ALMP was expressed in the epithelia cells of these tissues. Substrate analysis studies indicated that the recombinant pf-ALMP catalytic domain could digest gelatin. Interestingly, the pf-ALMP also could be involved in cell proliferation processes and the cysteine arrays were necessary for the proliferative activity. Taken together, these studies also help to further understand the functions of astacins which may be related to the processes of molluscan inflammatory response, embryo development, proliferation and shell formation. (+info)
A novel extracellular EF-hand protein involved in the shell formation of pearl oyster.
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Mollusk shell formation is a complicated and highly controlled calcium metabolism process. Previous studies revealed that several EF-hand calcium-binding proteins actively participate in the regulation of shell mineralization. In this study, we cloned a full-length cDNA encoding a novel extracellular EF-hand calcium-binding protein (named EFCBP) from the pearl oyster, Pinctada fucata, according to the EF-hand motifs of calmodulin. Although it shares high similarity with the calmodulin family in its EF-hand signatures, EFCBP just has two EF-hand motifs and belongs to a new separate group from the other EF-hand proteins according to a phylogenetic analysis. EFCBP is specifically expressed in shell mineralization-related tissues, viz. the mantle, the gill, and the hemocytes. Moreover, its expression responds quickly only to the shell damage, but not to the damage of other tissues and the infection of the lipopolysaccharides from Escherichia coli. These results suggest that EFCBP might be an important regulator of shell formation. This finding may help better understand the functions of EF-hand proteins on the regulation of mollusk shell formation. (+info)
A novel extrapallial fluid protein controls the morphology of nacre lamellae in the pearl oyster, Pinctada fucata.
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Mollusk shell nacre is known for its superior mechanical properties and precisely controlled biomineralization process. However, the question of how mollusks control the morphology of nacre lamellae remains unresolved. Here, a novel 38-kDa extrapallial fluid (EPF) protein, named amorphous calcium carbonate-binding protein (ACCBP), may partially answer this question. Although sequence analysis indicated ACCBP is a member of the acetylcholine-binding protein family, it is actively involved in the shell mineralization process. In vitro, ACCBP can inhibit the growth of calcite and induce the formation of amorphous calcium carbonate. When ACCBP functions were restrained in vivo, the nacre lamellae grew in a screw-dislocation pattern, and low crystallinity CaCO(3) precipitated from the EPF. Crystal binding experiments further revealed that ACCBP could recognize different CaCO(3) crystal phases and crystal faces. With this capacity, ACCBP could modify the morphology of nacre lamellae by inhibiting the growth of undesired aragonite crystal faces and meanwhile maintain the stability of CaCO(3)-supersaturated body fluid by ceasing the nucleation and growth of calcite. Furthermore, the crystal growth inhibition capacity of ACCBP was proved to be directly related to its acetylcholine-binding site. Our results suggest that a "safeguard mechanism" of undesired crystal growth is necessary for shell microstructure formation. (+info)
Identification of chitin in the prismatic layer of the shell and a chitin synthase gene from the Japanese pearl oyster, Pinctada fucata.
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The shell of the Japanese pearl oyster, Pinctada fucata, consists of two layers, the prismatic layer on the outside and the nacreous layer on the inside, both of which comprise calcium carbonate and organic matrices. Previous studies indicate that the nacreous organic matrix of the central layer of the framework surrounding the aragonite tablet is beta-chitin, but it remains unknown whether organic matrices in the prismatic layer contain chitin or not. In the present study, we identified chitin in the prismatic layer of the Japanese pearl oyster, Pinctada fucata, with a combination of Calcofluor White staining with IR and NMR spectral analyses. Furthermore, we cloned a cDNA encoding chitin synthase (PfCHS1) that produces chitin, contributing to the formation of the framework for calcification in the shell. (+info)
Pf-Rel, a Rel/nuclear factor-kappaB homolog identified from the pearl oyster, Pinctada fucata.
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Transcription factor Rel/nuclear factor-kappa B (NF-kappaB) has been the focus of many studies since its discovery in 1986. Different homologs of Rel/NF-kappaB have been found in both vertebrate and invertebrate. A cDNA clone encoding a putative Rel/NF-kappaB homolog (designated Pf-Rel) was isolated from the pearl oyster, Pinctada fucata. The sequence of Pf-Rel consists of the Rel homology domain, IPT NF-kappaB domain and C-terminal transactivation domain. Sequence analysis of Pf-Rel shows that it shares high similarity with other Rel/NF-kappaB family proteins, especially within the conserved domains. Reverse transcription-polymerase chain reaction analysis revealed that Pf-Rel mRNA was expressed ubiquitously. Further in situ hybridization analysis showed that Pf-Rel mRNA was expressed mainly at the outer epithelial cells of the middle fold and the inner epithelial cells of the outer fold. The identification and characterization of pearl oyster Pf-Rel help to further investigate the involvement of Rel/NF-kappaB in oyster immunity and other biological processes. (+info)
Biomineralization: functions of calmodulin-like protein in the shell formation of pearl oyster.
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Calmodulin-like protein (CaLP) was believed to be involved in the shell formation of pearl oyster. However, no further study of this protein was ever performed. In this study, the in vitro crystallization experiment showed that CaLP can modify the morphology of calcite. In addition, aragonite crystals can be induced in the mixture of CaLP and a nacre protein (at 16 kDa), which was detected and purified from the EDTA-soluble matrix of nacre. These results agreed with that of immunohistological staining in which CaLP was detected not only in the organic layer sandwiched between nacre (aragonite) and the prismatic layer (calcite), but also around the prisms of the prismatic layer. Take together, we concluded that (1) CaLP, as a component of the organic layer, can induce the nucleation of aragonite through binding with the 16-kDa protein, and (2) CaLP may regulate the growth of calcite in the prismatic layer. (+info)