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PubMed2006

Identification of calconectin, a calcium-binding protein specifically expressed by the mantle of Pinctada margaritifera.

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.

Amino Acid Sequence↗

PubMed2007

Identification and characterization of a biomineralization related gene PFMG1 highly expressed in the mantle of Pinctada fucata.

To elucidate the mechanism of nacre biomineralization, the mantle of Pinctada fucata (P. fucata) from the South China Sea was used. Using the mantle cDNA library and the ESTs we have cloned through suppression subtractive hybridization (SSH), ten novel genes including PFMG1 were obtained through nested PCR. Bioinformative results showed that PFMG1 had a high homology (40%) with Onchocerca volvulus calcium-binding protein CBP-1 and had two EF-hand calcium-binding domains from the 81st to the 93rd amino acid and from the 98th to the 133rd amino acid in the deduced amino acid sequence. The results of multitissue RT-PCR and in situ hybridization demonstrated the high expression of PFMG1 in the mantle of P. fucata and confirmed the SSH method. The results of GST-PFMG1 on CaCO3 crystallization showed significant effects on nucleation and precipitation of CaCO3. PFMG1 was cloned into the pcDNA.3.1/myc-HisA vector and was subsequently transfected into MC3T3-E1 cells. RT-PCR revealed upregulation of the marker genes related to cell growth, differentiation, and mineralization, and BMP-2, osterix, and osteopontin were upregulated as a result. This research work suggests that PFMG1 plays an important role in the nacre biomineralization, and the SSH method can pave the way for the bulk cloning and characterization of new genes involved in biomineralization in P. fucata and may accelerate research on the mechanism of pearl formation.

Amino Acid Sequence↗

PubMed2025

The chromosome-level genome assembly and annotation of the silver-lipped pearl oyster, Pinctada maxima.

The silver-lipped pearl oyster (Pinctada maxima) is a valuable tropical aquaculture species, playing a crucial economic role in the global pearl industry. However, the lack of genomic reference limits our in-depth understanding of this species in genome-based breeding, conservation, evolution and adaptation. Here, annotated chromosome-level reference genome for P. maxima was generated by integrating PacBio long-read sequencing, Illumina short-read sequencing, and Hi-C sequencing data. The total genome size is 1,264.93 Mb, with contig N50 and scaffold N50 of 649 kb and 89.19 Mb, respectively. The majority (97.94%) of the assembled genome was anchored to the 14 chromosomes by Hi-C analysis. The relatively high genome completeness was observed, with 97.38% (metazoa_odb10 database) and 95.26% (mollusca_odb10 database) in BUSCO analysis. Genome annotation revealed approximately 65.46% of the repeat sequences and 26,315 protein-coding genes. Comparative genome analysis revealed 28 expanded and 48 contracted families (p < 0.05) in P. maxima, with 3.2% of genes (894) being species-specific. This chromosome-level genome serves as an essential resource for research in evolutionary genomics, phylogenetics, and biomineralization.

Animals↗

PubMed1999

Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction analyses of mineral and organic matrix during heating of mother of pearl (nacre) from the shell of the mollusc Pinctada maxima.

Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction patterns were used to analyze the mineral structure and organic matrix composition and thermal behavior of the internal nacreous layer (mother of pearl or nacre) of the shell of the giant oyster Pinctada maxima. Nacre is a natural biomaterial with osteogenic properties. The mineral of nacre is calcium carbonate crystallized as aragonite and it is highly crystallized. The FT-IR spectra showed amide, amine, and carboxylic acid groups in the organic matrix of the whole (organic and mineral) nacreous layer, with the HCO(-)(3) groups possibly at the organic-mineral interface. The insoluble organic matrix remaining after decalcification contained amide, amine, and carboxylic groups. The heated aragonite mineral structure of nacre underwent two transformations (X-ray diffraction), aragonite to calcite at 300-400 degrees C, and calcite to calcium oxide (CaO) at 500-600 degrees C. The organic matrix of nacre was destroyed around 550-600 degrees C, the same temperature as the calcite to CaO transformation, revealing the great thermal stability of the organic matrix and the organic-mineral bonding. This could be an useful feature for the in vivo use of this natural biomaterial as an implant.

Animals↗

PubMed2000

Effect of water-soluble matrix fraction extracted from the nacre of Pinctada maxima on the alkaline phosphatase activity of cultured fibroblasts.

A new approach to the isolation of the water-soluble factors from nacre without any demineralization is described and examined their effect on fibroblast cells in culture. The soluble matrix in pure water from the nacre of Pinctada maxima was analysed by size-exclusion HPLC. Four fractions (SE1-SE4) of the water-soluble matrix (WSM) were further analysed by anion-exchange HPLC. The amino acid composition of the WSM showed that it is mainly composed of glycine and alanine. SE1 and SE4 had different amino acid compositions from the whole WSM. The WSM and SE4 tested on a culture of human foetus lung tissue fibroblasts increased the alkaline phosphatase (ALP) activity. SE1 caused a decrease in ALP activity. Our results support the hypothesis that WSM promotes the differentiation of cells in vitro.

Alkaline Phosphatase↗

PubMed2000

Molecular mechanism of the nacreous layer formation in Pinctada maxima.

We have cloned the cDNAs that encode two kinds of molluscan shell matrix proteins, namely N66 and N14, in the nacreous layer of Pinctada maxima. N66 is composed of carbonic anhydrase-like and repeat domains, as described for nacrein (1) in the pearls of P. fucata. N14 is homologous to N16, recently found in the nacreous layer of P. fucata (2) and is characterized by high proportions of Gly, Tyr, and Asn together with NG repeat sequences. The molecular weights of these proteins were estimated as 59,814 and 13,734 Da, respectively. Structural differences were clearly indicated in the alignment and length of the repeat sequences of the sets of the homogeneous proteins (N66/nacrein and N14/N16). The longer repeat sequences of N66 and N14 may be responsible for P. maxima's excellent property of calcification. The in vitro crystallization experiments revealed that the mixture of N66 and N14 could induce platy aragonite layers highly similar to the nacreous layer, once adsorbed onto the membrane of the water-insoluble matrix.

Animals↗

PubMed1998

Haplosporidium sp. (Haplosporidia) in Hatchery-Reared Pearl Oysters, Pinctada maxima (Jameson, 1901), in north Western Australia

A species of Haplosporidium is described from 6 of 105 pearl oyster (Pinctada maxima) spat from a hatchery at Carnarvon in north Western Australia. Presporulation and sporulation stages occurred in the connective tissue surrounding the digestive gland and were common in mantle connective tissue, but only light to moderate infections were observed in the heart, gills, foot, and adductor muscle. No stages were observed in epithelia, and the gut and diverticulae remained intact. Ultrastructurally, sporoblasts were binucleate with the two nuclei either closely apposed to form a diplokaryon or separated, with the cytoplasm containing round to pyriform haplosporosomes. In spores the sporoplasm contained a basal, or sometimes equatorial, nucleus, dense ovoid vesicles that appeared to develop to ovoid haplosporosomes with an internal axehead-shaped membrane, and spherical haplosporosomes. Apparently senescent spores with a dense content were also present and they, and normal spores, were ornamented with filaments placing the parasite in the genus Haplosporidium. Copyright 1998 Academic Press. Copyright 1998 Academic Press

Journal Article↗

PubMed1999

Studies on rickettsia-like organism disease of the tropical marine pearl oyster I: the fine structure and morphogenesis of pinctada maxima pathogen rickettsia-like organism

The present paper reports for the first time the discovery of a rickettsia-like organism (RLO) in the cultured tropical marine pearl oyster Pinctada maxima with mass mortality in the Hainan Province of China. This organism parasitizes the cytoplasm of host cells and forms intracytoplasmic eosinophilic inclusions. These organisms are extremely pleiomorphic in shape and average 967 x 551 nm in size, as measured in cross sections of transmission electron micrographs. The organisms exhibit clearly recognizable ultrastructural characteristics of prokaryotic bacteria-like cells, including two trilaminar membranes, an increasing electron-dense periplasmic ribosome zone, and a thread-like DNA nucleoidal structure. In addition to the above prokaryotic characteristics, the following unique biological characteristics were confirmed by TEM: (i) These organisms are usually located in host cells in two ways, namely, free in the cell cytoplasm and involved within membrane-limited phagolysosomes; (ii) The organisms exist in two morphological cell types, namely a small cell variant (SCV) and a large cell variant (LCV). The most important morphological difference between two cell types is that the SCV is obviously ribosome-rich in the periphery of the body, which makes SCV more electron-dense in the cytoplasm and narrower in the central nucleoid area than the LCV; (iii) Two propagative modes of the organisms, transverse binary fission and budding, are observed in cytoplasm and phagolysosomes of host cells under TEM, in which the budding is more often seen in phagolysosomes. These characteristics indicate that the organism is a separate species in the family Rickettsiaceae and should be classified into the genus Rickettsia. On the basis of the existence of the two propagative modes and two cell types, and intracellular location, we propose a developmental cycle for this organism which includes a vegetative differentiation stage to develop LCV by transverse binary fisson and a budding differentiation stage to develop resistant SCV. Copyright 1999 Academic Press.

Journal Article↗

PubMed2000

Comparison of the soluble organic matrices of healthy and diseased shells of Pinctada margaritifera (L.) and Pecten maximus L. (Mollusca, Bivalvia).

Pinctada margaritifera and Pecten maximus are among the mollusks of commercial value. Both are known to show abnormal calcification processes that strongly increase the mortality rate. Several parameters of the soluble organic matrices extracted from the shells of P. margaritifera and P. maximus are analyzed: bulk composition, molecular weights, and acidity. The composition of the matrices of healthy and diseased shells were compared, showing that the protein and the sugar contents are variously modified. Differences between healthy and diseased shells are dissimilar in the two species. This is in accordance with the previously described macroscopic and microscopic alterations (red color, numerous brown membranes). This study does not allow identification the origin of the disease, but provides new insights on the role of sugars in biomineralization processes.

Acids↗

PubMed2000

Seasonal change of infective state of marine birnavirus in Japanese pearl oyster Pinctada fucata.

This study examines the seasonal occurrence and infective state of marine birnavirus (MABV) in cultured Japanese pearl oyster (Pinctada fucata). Planted oysters were sampled monthly in 1997 and 1998. To detect MABV in the oysters, PCR and virus isolation were carried out. Also, the indirect fluorescent antibody technique (IFAT) was performed to know the organs expressing viral antigens. The detection rate of the MABV genome by PCR was low during July to October, but increased after November. This virus was isolated only after October, with a 10-40% isolation rate. Results of the IFAT showed that the specific fluorescence was observed in hemocytes in September. Fluorescence in hemocytes decreased in January, but increased in liver parenchymal cells. These results suggest that MABV persistently infected hemocytes in summer with a small amount of genome and protein, and then the virus spread in winter into the parenchymal cells.

Animals↗

PubMed2003

Molecular cloning and expression of a pearl oyster (Pinctada fucata) homologue of mammalian putative tumor suppressor QM.

The QM gene was originally identified as a putative tumor suppressor gene from a Wilms' tumor cell line by subtractive hybridization assay. Later studies showed that the QM protein is multifunctional, involved in cell growth and differentiation, energy metabolism, respiration, and cytoskeletal function. In this report a full-length complementary DNA encoding a QM counterpart in pearl oyster (Pinctada fucata) was isolated. Phylogenetic analysis shows that oyster QM is more closely related to its insect homologues than to the mammalian homologues. Analysis of the tissue expression pattern of the oyster QM gene showed that oyster QM messenger RNA is expressed in all tissues tested, with highest levels in the digestive gland and mantle. Furthermore, we expressed the QM protein in Escherichia coli; Western blotting showed that the antibody of human QM is immunoreactive to the expressed oyster QM protein. Incubation of the oyster QM with Zn2+ resulted in the reduction of intrinsic emission fluorescence and a red-shift in the lambda(max) emission, indicating the occurrence of Zn(2+)-induced conformational changes. This evidence presents a possible mechanism for the critical function of zinc ion in the interaction of QM with Jun.

Amino Acid Sequence↗

PubMed2001

Characterization and quantification of chitosan extracted from nacre of the abalone Haliotis tuberculata and the oyster Pinctada maxima.

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 (eta) of extraction was calculated (eta = 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 (eta = 0.053%) was isolated rather than chitosan.

Journal Article↗

PubMed2004

Lipid and FA composition of the pearl oyster Pinctada fucata martensii: influence of season and maturation.

The lipid and FA composition of the total lipids of the pearl oyster Pinctada fucata martensii, in different seasons and in different areas, were analyzed to clarify its lipid physiology and to estimate the possible influence of its prey phytoplankton. During the spawning season (June and July), the lipid contents were slightly higher than in the growing season (November and March). TAG and sterols were the major components in the neutral lipids in all conditions, whereas high levels of phospholipids (PE and PC) were found in the polar lipids. In addition, significant levels of ceramide aminoethyl phosphonate but low levels of sphingolipids were found in the polar lipids. The major FA in the TAG in all samples were 14:0, 16:0, and 18:0 as saturated FA (saturates); 16:1 n-7, 18:1 n-9, and 18:1 n-7 as monoenoic FA (monoenes); and 20:4n-6 (arachidonic acid: AA), 20:5n-3 (EPA), and 22:6n-3 (DHA) as PUFA. The major components found in the polar lipids were 16:0 and 18:0 as saturates; 22:2n-9,15 and 22:2n-7,15 as non-methylene-interrupted dienes (NMID), and AA, 22:3n-6,9,15, EPA, and DHA as PUFA. Similar to the high levels of total PUFA in the phospholipids, comparatively high PUFA levels were found in TAG in both the growing and the spawning season. This may be a characteristic of the species as a typical bivalve, because the lipids were similar to those of other bivalves. Although it is a marine animal, uncharacteristically high levels of AA were found in both the TAG and phospholipids. This result suggests that lipids of P. fucata may be influenced by those of its phytoplanktonic prey. The increase in levels of NMID from TAG to PE with a decrease in those of monoenes suggests that the tissues of this species are able to biosynthesize only the less unsaturated PUFA, such as NMID. In particular, NMID derivatives are considered to be biosynthesized in the PE; thus, they might play a particular role in the membrane, because NMID were characteristically localized only in the PE.

Animals↗

PubMed1989

A galactose-specific lectin from the hemolymph of the pearl oyster, Pinctada fucata martensii.

1. A lectin in the serum of Pinctada fucata martensii was purified by a combination of affinity chromatography on Sepharose 4B coupled with bovine submaxillary gland mucine, anion exchange chromatography on Mono Q and gel filtration on Superose 6. 2. The purified lectin was indicated to be homogeneous by polyacrylamide electrophoresis and rechromatography on Mono Q. 3. The purified lectin was approximately 440,000 in molecular weight and was composed of identical subunits with a molecular weight of approximately 20,000. 4. D-galactose and N-acetylgalactosamine gave a 50% inhibition of agglutination of horse erythrocytes by the lectin at 0.3 and 1.2 mM, respectively. 5. The antibody obtained from rabbit immunized with the purified lectin was monospecific to the lectin judged from the hemagglutination blocking test, immunoelectrophoresis and immunoblotting.

Animals↗

PubMed2005

Chemical modification studies on alkaline phosphatase from pearl oyster (Pinctada fucata): a substrate reaction course analysis and involvement of essential arginine and lysine residues at the active site.

Alkaline phosphatases (ALP, EC 3.1.3.1) are ubiquitous enzymes found in most species. ALP from a pearl oyster, Pinctada fucata (PALP), is presumably involved in nacreous biomineralization processes. Here, chemical modification was used to investigate the involvement of basic residues in the catalytic activity of PALP. The Tsou's plot analysis indicated that the inactivation of PALP by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and phenylglyoxal (PG) is dependent upon modification of one essential lysine and one essential arginine residue, respectively. Substrate reaction course analysis showed that the TNBS and PG inactivation of PALP followed pseudo-first-order kinetics and the second-order inactivation constants for the enzyme with or without substrate binding were determined. It was found that binding substrate slowed the PG inactivation whereas had little effect on TNBS inactivation. Protection experiments showed that substrates and competitive inhibitors provided significant protection against PG inactivation, and the modified enzyme lost its ability to bind the specific affinity column. However, the TNBS-induced inactivation could not be prevented in presence of substrates or competitive inhibitors, and the modified enzyme retained the ability to bind the affinity column. In a conclusion, an arginine residue involved in substrate binding and a lysine residue involved in catalysis were present at the active site of PALP. This study will facilitate to illustrate the role ALP plays in pearl formation and the mechanism involved.

Alkaline Phosphatase↗

PubMed2004

Cloning and expression of a pivotal calcium metabolism regulator: calmodulin involved in shell formation from pearl oyster (Pinctada fucata).

The shells of bivalves are mainly composed of calcium carbonate, a product of calcium metabolism. In the process of shell formation, the uptake, transport and recruitment of calcium ion are highly regulated and involved in many factors. Among these regulatory factors, calmodulin (CaM), a pivotal multifunction regulator of calcium metabolism in nearly all organisms, is thought to play an important role in the calcium metabolism involved in shell formation. In this study, a full-length CaM cDNA was isolated from the pearl oyster (Pinctada fucata). The oyster calmodulin encodes a 16.8 kDa protein which shares high similarity with vertebrate calmodulin. The oyster CaM mRNA shows the highest level of expression in the gill, a key organ involved in calcium uptake in oyster calcium metabolism. In situ hybridization results revealed that oyster CaM mRNA is expressed at the folds and the outer epithelial cells of the dorsal region of the mantle, suggesting that CaM is involved in regulation of calcium transport and secretion. Oyster CaM also showed a typical Ca2+ dependent electrophoretic shift characterization and calcium binding activity. Taken together, we have identified and characterized a pivotal calcium metabolism regulator of the oyster that may play an important role in regulation of calcium uptake, transport and secretion in the process of shell formation.

Amino Acid Sequence↗

PubMed2004

Cloning and characterization of an mRNA encoding a novel G protein alpha-subunit abundant in mantle and gill of pearl oyster Pinctada fucata.

Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel G(s)alpha (pfG(s)alpha) from the pearl oyster was isolated. The G(s)alpha cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) G(s)alpha. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfG(s)alpha has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster G(s)alpha mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfG(s)alpha in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfG(s)alpha that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the G(s)alpha in MC3T3-E1 cells. These results indicated that G(s)alpha may be involved in regulation of physiological function, particularly in biological biomineralization.

Alkaline Phosphatase↗

PubMed2003

The influence of cooling rate, developmental stage, and the addition of sugar on the cryopreservation of larvae of the pearl oyster Pinctada fucata martensii.

This paper examines the effects of cooling rate, developmental stage, and the addition of sugar on the cryopreservation of the larvae of the pearl oyster, Pinctada fucata martensii. The survival rates of frozen-thawed trochophores was 43.1% at a cooling rate of 1.0 degrees C/min. The survival rate of frozen-thawed larvae increased with developmental stage, except for umbo stage larvae, and the late D-shaped larvae showed a survival rate as high as 91%. The addition of sugar (0.2M glucose or sucrose) improved the survival rate of larvae. These results indicate that the preferred cooling rate, developmental stage, and sugar for the cryopreservation of pearl oyster larvae are 1 degrees C/min, late D-shaped larvae and 0.2M glucose or sucrose.

Animals↗