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At least 19 recordsLinked to original sources

PubMed2001

Soluble proteins of the nacre of the giant oyster Pinctada maxima and of the abalone Haliotis tuberculata: extraction and partial analysis of nacre proteins.

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 beta-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.

Amino Acid Sequence↗

PubMed2003

Soluble organic matrices of the calcitic prismatic shell layers of two Pteriomorphid bivalves. Pinna nobilis and Pinctada margaritifera.

The calcitic prisms of the shells of two bivalves, Pinna and Pinctada, are considered simple prisms according to some morphological and mineralogical characteristics. Scanning electron microscopic and atomic force microscopic studies show that the microstructures and nanostructures of these two shells are different. Pinna prisms are monocrystalline, whereas Pinctada prisms are not. Moreover, intraprismatic membranes are present only in the Pinctada prisms. The soluble organic matrices extracted from these prisms are acidic, but their bulk compositions differ. Ultraviolet and infrared spectrometries, fluorescence, high pressure liquid chromatography, and electrophoresis show that the sugar-protein ratios and the molecular weights are different. Sulfur is mainly associated with acidic sulfated sugars, not with amino acids, and the role of acidic sulfated sugars is still underestimated. Thus, the simple prism concept is not a relevant model for the biomineralization processes in the calcitic prismatic layer of mollusk shells.

Acids↗

PubMed2003

[Pinctada margaritifera nacre (mother-of-pearl): physico-chemical and biomechanical properties, and in vitro cytocompatibility].

PURPOSE OF THE STUDY: Pinctada maxima nacre (mother-of-pearl) has been recently proposed as a bone substitute. The purpose of this work was to assess the in vitro cytocompatibility of Pinctada margaritifera nacre and its physico-chemical and biomechanical properties. MATERIAL AND METHODS: The cytocompatiblity was assessed in contact with human osteoprogenetic cells. Attachment was measured at one hour and three hours by determining N-acetyl-beta-D-hexosaminidase activity. Proliferation was monitored by measuring metabolic activity with the MTT test. Cell morphology was studied under scanning electron microscopy and cell differentiation was assessed by immunocytochemistry monitoring of the synthesis of type I collagen and osteocalcin. Diffraction x-ray and scanning electron microscopy was used to study the physico-chemical structure. Two samples taken from the inner part of the shell and two other samples from the outer part of the shell were tested for resistance under compression and to calculate the Young module. RESULTS: The results showed that osteoprogenetic cells attached to the nacre (2/3 of the plastic control), proliferated according to a standard pattern (increased metabolic activity followed by a plateau then decreased activity), synthetized type I collagen and osteocalcin, and presented a morphology analogous to control cells cultured on the plastic culture wells. The diffraction spectrum of the crystalline structure corresponded to crystallized calcium carbonate in the form of calcite (CaCO(3)) for the outer part and in the form of aragonite for the inner part. The Young module was 46.1 Gpa and resistance to rupture was 185 Mpa. CONCLUSION: Pinctada margaritifera nacre is cytocompatible in vitro with mechanical properties very similar to cortical bone.

Animals↗

PubMed1990

Hemolymph lectin of the pearl oyster, Pinctada fucata martensii: a possible non-self recognition system.

To elucidate a mutual correlation between the hemolymph lectin and hemocytes of the pearl oyster, Pinctada fucata martensii, we searched for common epitopes and ligands. Neither the hemocyte plasma membrane nor cytoplasm was immunoreactive to anti-hemolymph lectin antibody. The hemolymph lectin strongly bound to D-galactose and N-acetyl-D-galactosamine, and the plasma membrane of both granulocytes and agranulocytes had affinity only for D-mannose and N-acetyl-D-glucosamine-binding plant lectins. In the gonad, the hemolymph lectin selectively adsorbed injected horse red blood cells (HRBC), and its hemagglutinating activity probably prevented them from dispersing. Pinctada sp. may possess system of recognition of non-self by the hemolymph lectin.

Animals↗

PubMed2001

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.

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% (p<0.001) by 100 microg proteins/ml extract and 20% (p<0.001) by 50 microg 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%, p<0.0001). 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.

Journal Article↗

PubMed2006

Biphasic and dually coordinated expression of the genes encoding major shell matrix proteins in the pearl oyster Pinctada fucata.

Regional expression patterns of shell matrix protein genes of Pinctada fucata were investigated using real-time quantitative polymerase chain reaction (PCR) and in situ hybridization. Six shell matrix proteins examined in this study indicated a distinct biphasic pattern of expression, falling into one of the following three groups: (1) expressed only in the more dorsal region of the mantle (MSI60 and N16); (2) expressed only in the more ventral region (MSI31, Prismalin-14, and Aspein); and (3) expressed in both regions (nacrein). The ubiquity of the last protein probably reflects its general role as a carbonate-producing enzyme, while the other groups are interpreted as corresponding to the distinction between the two varieties of shell layers, the aragonitic nacreous layer and the calcitic prismatic layer. In addition, the constituent genes of each of these two groups indicated similar levels of relative expression among different sites even among different individuals, suggesting that the genes of each group share a single upstream regulatory factor, respectively, and that these genes are expressed in a dually coordinated fashion.

Animal Structures↗

PubMed2006

A novel matrix protein p10 from the nacre of pearl oyster (Pinctada fucata) and its effects on both CaCO3 crystal formation and mineralogenic cells.

A novel matrix protein, designated as p10 because of its apparent molecular mass of 10 kDa, was isolated from the nacreous layer of pearl oyster (Pinctada fucata) by reverse-phase high-performance liquid chromatography. In vitro crystallization experiments showed that p10 could accelerate the nucleation of calcium carbonate crystals and induce aragonite formation, suggesting that it might play a key role in nacre biomineralization. As nacre is known to contain osteogenic factors, two mineralogenic cell lines, MRC-5 fibroblasts and MC3T3-E1 preosteoblasts, were used to investigate the biological activity of p10. The results showed that p10 could increase alkaline phosphatase activity, an early marker of osteoblast differentiation, while the viability of MRC-5 and MC3T3-E1 remained unchanged after treatment of p10. Taken together, the findings led to identification of a novel matrix protein from the nacre of P. fucata that plays a role in both the mineral phase and in the differentiation of the cells involved in biomineralization.

Animals↗

PubMed2026

Quantitative Proteomic Profiling of Pinctada fucata Shell Nacre Defines a Solubility-Based Type Classification of Shell Matrix Proteins.

Shell matrix proteins (SMPs) are key organic components of molluscan biominerals, yet previous nacre proteomics have remained largely qualitative, limiting evaluation of the abundance and fraction association of individual SMPs. Here, we established a quantitative proteomic approach for the nacreous layer of the pearl oyster Pinctada fucata by integrating optimized shell preservation, stepwise fractionation, and data-independent acquisition (DIA) proteomics. SMPs were separated into an ethylenediaminetetraacetic acid (EDTA)-soluble matrix (ESM), an EDTA-insoluble but sodium dodecyl sulfate/dithiothreitol (SDS/DTT)-soluble matrix (SSM), and an SDS/DTT-insoluble matrix (ISM). DIA outperformed data-dependent acquisition in proteome coverage and enabled quantification of 327 SMPs across a broad dynamic range. Fraction-resolved abundance profiling showed that each fraction was characterized by distinct SMP compositions. To summarize these distributions, we introduced a solubility-based type classification that grouped SMPs into four types according to their quantitative partitioning among fractions. Well-known SMPs, including nacrein, Pif 80, and MSI60, were assigned to intuitively consistent types, whereas proteases, protease inhibitors, and tyrosinases also showed biased type distributions. These results support a three-compartment model of the nacreous layer consisting of (i) an insoluble interlamellar membrane core, (ii) a relatively extractable interfacial layer, and (iii) a soluble matrix fraction enriched in proteins potentially involved in ionic regulation and protein maturation. This study provides a quantitative framework for understanding coordinated SMP functions during nacre formation and for comparative analyses of molluscan shell proteomes.

Animals↗

PubMed2006

Pf-ALMP, a novel astacin-like metalloproteinase with cysteine arrays, is abundant in hemocytes of pearl oyster Pinctada fucata.

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.

Amino Acid Sequence↗

PubMed2006

A novel putative tyrosinase involved in periostracum formation from the pearl oyster (Pinctada fucata).

Tyrosinase (monophenol, L-DOPA: oxygen oxidoreductase, EC 1.14.18.1), a kind of copper-containing phenoloxidase, arouses great interests of scientists for its important role in periostracum formation. A cDNA clone encoding a putative tyrosinase, termed OT47 because of its estimated molecular mass of 47kDa, was isolated from the pearl oyster, Pinctada fucata. This novel tyrosinase shares similarity with the cephalopod tyrosinases and other type 3 copper proteins within two conserved copper-binding sites. RT-PCR analysis showed that OT47 mRNA was expressed only in the mantle edge. Further in situ hybridization analysis and tyrosinase activity staining revealed that OT47 was expressed at the outer epithelial cells of the middle fold, different from early histological results in Mercenaria mercenaria, suggesting a different model of periostracum secretion in P. fucata. Taken together, these results suggest that OT47 is most likely involved in periostracum formation. The identification and characterization of oyster tyrosinase also help to further understand the structural and functional properties of molluscan tyrosinase.

Amino Acid Sequence↗

PubMed2006

A novel matrix protein family participating in the prismatic layer framework formation of pearl oyster, Pinctada fucata.

Understanding the molecular composition and the formation mechanism of shell matrix framework is of great interest for biomineralization in mollusk shell. The cDNAs encoding a novel matrix protein family (KRMP) were cloned from the mantle of pearl oyster, Pinctada fucata. Analysis of the deduced amino acid sequences revealed that KRMP have a high proportion of lysine, glycine, and tyrosine, and their predict isoelectric points are higher than any other identified shell matrix protein to our knowledge. The deduced amino acid sequences of KRMP can be divided into three regions, including an N-terminal signal peptide, a lysine-rich basic region interacting with acidic proteins or CO(3)(2-), and a Gly/Tyr-rich region involved in the protein cross-link via quinone-tanning process. RT-PCR and in situ hybridization demonstrated that KRMP mRNA was specifically expressed in the mantle edge, involved in the prismatic layer formation. Taken together, it seems that KRMP is a matrix protein family participating in the framework formation of prismatic layer.

Amino Acid Sequence↗

PubMed2006

Wound healing after excision of mantle tissue from the Akoya pearl oyster, Pinctada fucata.

Pearl oysters are usually sacrificed to donate mantle tissue for pearl production. However, if oysters are anaesthetized, they are able to survive mantle excision and regenerate this tissue. Mantle excision causes a large wound and severs the pallial artery that necessitates rapid wound repair to avoid death by bleeding. This study was undertaken to assess the wound healing process in the mantle of the Akoya pearl oyster, Pinctada fucata, following mantle excision. Forty-seven P. fucata were relaxed with 2.5 mL L(-1) propylene phenoxetol before mantle tissue was excised. Oysters were relaxed and sacrificed 1, 3, 6, 12, 25, 36, 48, 66, 80 and 105 h after excision to assess mantle healing using histological techniques. Muscular contraction that effectively reduced the size of the wound was observed within 1 h after mantle excision. Accumulation of haemocytes and connective tissue occurred 3-6 h after excision and wound plugging was achieved within 6 h of excision. Proliferation of epithelial cells to cover the wound site was observed within the first 25 h after mantle excision and growth of connective tissue and formation of the pallial artery were observed within 105 h after mantle excision.

Animals↗

PubMed2006

A novel carbonic anhydrase from the mantle of the pearl oyster (Pinctada fucata).

A novel carbonic anhydrase (CA) has been purified from the mantle of the pearl oyster, Pinctada fucata, by ammonium sulfate precipitation and affinity chromatography. Its molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be approximately 38 kDa. Native-PAGE shows that the novel CA can bind a fluorescent probe, 5-dimethylamino-1-naphthalenesulfonamide (DNSA), known to specifically bind carbonic anhydrase. Compared to carbonic anhydrase I (CAI) from human erythrocytes, the novel CA migrates faster indicating that it is more acidic. The effect of an inhibitor on the enzyme activity was also examined. The CA from the mantle showed a weak resistance to acetazolamide (AZ), a specific inhibitor of CA. When DNSA was bound to CA, it caused the wavelength of emission maximum intensity to blue shift to 454 nm upon excitation at 326 nm. Histochemical data indicates that the enzyme is distributed widely throughout the mantle tissue, being concentrated at the edge of the mantle. The evidence presented indicates a function for CA in the process of pearl formation and biomineralization.

Acetazolamide↗

PubMed2005

Purification and partial characterization of two acid phosphatase forms from pearl oyster (Pinctada fucata).

The present study describes the details about the acid phosphatase forms in the pearl oyster, Pinctada fucata. Two isoenzymes (AcPase I and II) of acid phosphatase were separated and purified from viscera of pearl oyster, P. fucata to homogeneity by chromatography on DEAE-Sepharose Fast Flow, Sephadex G-200 superfine and ConA Sepharose 4B, and partial biochemical properties of AcPase I and II were studied. AcPase I and AcPase II had molecular weights of 208.8 and 64.3 kDa, respectively. AcPase I was a single polypeptide chain, while AcPase II was a dimeric enzyme composed of two equivalent subunits. AcPase I and II showed optimal pHs at 4.6 and 3.2 with p-nitrophenylphosphate as substrate. The optimal catalytic reaction temperature was 47 degrees C for AcPase I and 57 degrees C for AcPase II. Both enzyme forms were stable when incubated at 50 degrees C for 40 min. Tartrate and fluoride were the most effective inhibitors of the enzymes. Fe(3+), Zn(2+), Cu(2+) and Pb(2+) inhibited the activity of AcPase I and II to differing extents. AcPase I and II were apparently nonspecific and hydrolyzed various phosphoric esters. The different properties of AcPase I and II suggested that the two enzymes may play different roles in the pearl oyster.

Acid Phosphatase↗

PubMed2006

Shematrin: a family of glycine-rich structural proteins in the shell of the pearl oyster Pinctada fucata.

Random sequencing of molecules from a cDNA library constructed from mantle mRNA of the pearl oyster Pinctada fucata was used to obtain information on organic matrix proteins in the shell. In the determined sequences, we identified 7 distinct cDNAs encoding similar glycine-rich domains. Complete sequence analysis of these cDNAs showed that the predicted sequences of the proteins, which we named shematrins, possessed similar domains comprising repeat sequences of two or more glycines, followed by a hydrophobic amino acid. In addition, in shematrin-1, -2 and -3, a repeat domain designated as XGnX (where X is a hydrophobic amino acid) was conserved. It is of further note that all the shematrin proteins have RKKKY, RRKKY or RRRKY as their C-terminal sequence. According to northern blot analysis, all shematrins are exclusively expressed in the mantle, and particularly in the edge region of the mantle; furthermore, peptide fragments similar to shematrin-1 and -2 were detected in the prismatic layer of shells by MALDI-TOF/TOF MS analysis. These findings suggest that many of shematrins are synthesized in the mantle edge and secreted into the prismatic layer of the shell, where the protein family is thought to provide a framework for calcification.

Amino Acid Sequence↗

PubMed2005

Cloning and characterization of a novel G protein beta-subunit of pearl oyster (Pinctada fucata), and its interaction sites with calmodulin.

A cDNA clone encoding a novel G protein beta subunit of beta1 subclass, pfGbeta1 was isolated from the pearl oyster (Pinctada fucata). The deduced amino acid sequence of pfGbeta1 (341 amino acids) shares high homology to northern European squid (Loligo pealei) and great pond snail (Lymnaea stagnalis) pfGbeta1, while it has diverged from bovine (Bos taurus) and human. The well-conserved amino acid domains in G protein beta subunit, seven WD repeats, were founded in the deduced amino acid sequence. Alignment analysis showed that the beginning amino acid residues in variable fragment of the seventh WD motif are different from any other Gbeta. The prediction of 3D structure of pfGbeta showed that pfGbeta belongs to beta-propeller family proteins whose members contain 4-8 antiparallel beta-sheets resembling the blades of a propeller. In situ hybridization and Northern blotting analysis revealed that the pfGbeta mRNA hybridization signals were widely expressed in various tissues except muscle, with abundantly in epithelia of gill, gonad and outer fold of mantle. We also investigated the interactions between Gbetagamma and calmodulin (CaM), and specific amino acid residues that may be critical for the binding of Gbetagamma to CaM were also identified. Furthermore, the functional studies of the interaction showed that the binding of CaM and Gbetagamma increases the alkaline phosphatase (ALP) activity, an indicator for mineralization in MC3T3-E1 cells. The ALP activity of the mutants of pfGbetagamma that impaired the interactions of Gbetagamma with CaM is higher than the Control group; however, it is lower than the WTC group. Together, these results suggest that the Gbetagamma might interact with CaM and point to the important physiological function in modulating cellular functions.

Alkaline Phosphatase↗

PubMed2006

Metal accumulation and enzyme activities in gills and digestive gland of pearl oyster (Pinctada fucata) exposed to copper.

The effects of exposure to copper under laboratory-controlled conditions were investigated in the pearl oyster, Pinctada fucata. Metal accumulation and the activity of five enzymes were measured: two immune defense involved enzymes [acid phosphatase (AcPase) and phenoloxidase (PO)], two antioxidant enzymes [superoxide dismutase (SOD) and Se-dependent glutathione peroxidase (Se-GPx)] and one metal-sensitive enzyme [alkaline phosphatase (ALP)]. Analyses were carried out in gills and digestive gland of oysters exposed to 0.05 microM and 0.5 microM copper, respectively, at 12, 24, 48 and 72 h of exposure. The digestive gland of P. fucata was the main copper accumulation organ when oysters were exposed to low concentrations, whereas gills became the target organ in oysters exposed to high concentrations. The adaptation and recovery of the oysters were observed in our study. Levels of the copper accumulation and the sensitivity to copper were the main, if not, part of the reasons for the various responses of the selected enzymes. Se-GPx may potentially be used as biomarkers in biotesting of marine heavy metal pollutions. The enzymatic responses were compared with those of other studies and the possible reasons were discussed.

Acid Phosphatase↗

PubMed2006

Effects of cryoprotectant agents and freezing protocol on motility of black-lip pearl oyster (Pinctada margaritifera L.) spermatozoa.

Gamete cryopreservation techniques have been applied to several bivalve mollusc species. However, research activity in this area has primarily focused on cryopreserving gametes from edible oysters (Ostreiidae). Few studies have examined the effect of cryoprotectants and freezing protocols in the preservation of spermatozoa from cultured pearl oysters (Pteriidae). Pearl oyster producers are increasingly looking towards the development of improved family lines and, as a consequence, the ability to cryopreserve gametes would bring about significant benefits to the cultured pearl industry. In response to this need, we evaluated the effect of three cryoprotectant additives (CPA) on motility of spermatozoa from the black-lip pearl oyster, Pinctada margaritifera. These additives have previously been used to cryopreserve gametes of other bivalve species. The following CPA mixtures were evaluated: (1) 0.45M trehalose and 0, 0.64, 1.02 and 1.53 M dimethyl sulfoxide (Me(2)SO); (2) 0.2M glucose and 2M Me(2)SO and (3) 1.31 M propylene glycol (PG). The effects of four different freezing protocols on motility of P. margaritifera spermatozoa were also evaluated (slow, medium, medium-rapid and rapid cooling). This study showed that total motility was best retained when spermatozoa were cryopreserved in 0.45 M trehalose and 0, 0.64, 1.02 or 1.53 M Me(2)SO and frozen using slow to medium-rapid cooling rates (2.1-5.2 degrees Cmin(-1)). Rapid freezing through direct plunging of spermatozoa into liquid nitrogen resulted in the lowest overall retention of motility regardless of the CPA additive; however, CPA mixture also influenced retention of motility, with 0.2M glucose in 2M Me(2)SO and 1.31 M PG retaining the lowest levels of motility for the CPAs evaluated.

Animals↗