An electron microscope study of the muscle attachment in the mollusc Pinctada radiata.
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An electron microscope study of ingestion of thorotrast by amoebocytes of Pinctada radiata.
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Animals↗
In situ chemical speciation of sulfur in calcitic biominerals and the simple prism concept.
The microstructure and composition of two mollusc shells were investigated using a combination of light microscopy, SEM, EPMA, and XANES. The shells of Pinna and Pinctada are composed of calcite prisms separated by organic walls. The prismatic units of Pinna are monocrystalline, and those of Pinctada are polycrystalline with internal organic radial membranes. High-spatial-resolution XANES maps for the different S species across adjacent prisms show that sulfate is the principal component in both the intraprismatic organic matrices and the outer membranes. Additionally, these maps confirm that the inner structures of the prismatic units are different for both genera. In many ways, the prisms of Pinna and Pinctada are different and invalidate the "simple prism" concept.
Animals↗
A survey of some parasites and diseases of several species of bivalve mollusc in northern Western Australia.
Pteriid oysters (Pinctada maxima, Pinctada margaritifera, Pinctada albina, Pteria penguin), rock oysters (Saccostrea glomerata, Saccostrea cuccullata, Saccostrea echinata) and representatives of other taxa (Malleidae, Isognomonidae, Pinnidae, Mytilidae, Spondylidae, Arcidae) from the wild, and 4670 hatchery-reared P. maxima, from northern and Western Australia, were examined for parasites and diseases. Rickettsiales-like inclusions and metacestodes of Tylocephalum occurred in most species. Intranuclear virus-like inclusions occurred in 1/415 wild P. maxima, 1/1254 S. cuccullata, 3/58 Isognomon isognomum, 1/80 Pinna bicolor and 1/45 Pinna deltodes. Perkinsus was histologically observed in 1/4670 P. maxima spat, 2/469 P. albina, 1/933 S. glomerata, 16/20 Malleus meridianus, 12/58 I. isognomum, 1/45 P. deltodes, 5/12 Spondylus sp., 1/16 Septifer bilocularis and 3/6 Barbatia helblingii. One of 1254 S. cuccullata was heavily systematically infected with Perkinsus merozoites, meronts and schizonts, and was patently diseased. Other potentially serious pathogens included Haplosporidium sp. in 6/4670 P. maxima spat, Marteilia sydneyi from 1/933 S. glomerata, and Marteilia sp. (probably M. lengehi) (1/1254) and Haplosporidium sp. (125/1254) from S. cuccullata. The latter were associated with epizootics on offshore islands, with heaviest prevalence (45%) in oysters with empty gonad follicles. Marteilioides sp. infected the oocytes of 9/10 female S. echinata from Darwin Harbour. Details of geographical distribution and pathology are given, and the health of the bivalves examined is discussed.
Animals↗
[Raman spectra of nacre from shells of main pearl-culturing mollusk in China].
The Raman spectra of nacre and cultured pearl from main pearl-culturing mollusks in China have been studied systematically in this paper. It shows that: 1) the Raman bands caused by organic matrix have not been observed in nacre from shell of pinctada maxima; 2) in nacre from shell of pinctada martensii and hyriopsis cumingii, besides the bands caused by aragonite mineral, two bands resulted from all-trans carotenoid are obviously observed, and the number of C=C double bonds in molecular of this carotenoid is predicted to be 10 based on the position of its Raman bands; 3) a number of complicated bands are observed in nacre from shell of peteria penguin, and we predicted that this bands perhaps are caused by metalloporphyrin compounds; 4) the relative concentrations of carotenoid and metalloporphyrin in nacre are closely associated with the color of nacre. The Raman spectra research on organic matrix in nacre and cultured pearl provides insight into the applications of them in cosmetics and food, as well as in application of pearl bleaching.
Animals↗
Pearl formation: persistence of the graft during the entire process of biomineralization.
Most bivalves species of the genus Pinctada are well known throughout the world for production of white or black pearls of high commercial value. For cultured pearl production, a mantle allograft from a donor is implanted into the gonad of a recipient oyster, together with a small inorganic bead. Because of the dedifferentiation of cells during the first steps of the host oyster's immunological reaction, so far the fate of the graft and its exact role in the process of pearl formation could not be determined via classical histological methods. Here we report the first molecular evidence of the resilience of the graft in the recipient organism by showing that cells containing genome from the donor are still present at the end of pearl formation. These results suggest the existence of a unique biological cooperation leading to the successful biomineralization process of nacreous secretion in pearl formation.
Animals↗
Identification of low molecular weight molecules as new components of the nacre organic matrix.
Nacre of Pinctada margaritifera displays a number of interesting biological activities on bone, mainly concentrated in a water-soluble organic matrix representing 0.24% of the nacre weight. Dialysis of that matrix through 8 kDa and 1 kDa cut-off membranes showed that 60% of it is made of small molecules of molecular masses below 1 kDa. Reversed-phase high-performance liquid chromatography of the small molecule fractions and subsequent electrospray ionization mass spectrometric analysis of 19 fractions thereof indicated the presence of at least 110 different molecules, in the range 100 Da-700 Da. Evidence for aggregate-forming properties of the small molecules was given. Amino acid analysis revealed that most of the small molecules were not peptides and tandem mass spectrometric gas-phase fragmentations clearly indicated a structural relationship between several molecules. Intriguingly, differences of a single Dalton between mono-charged ions peaks were observed. Further, approximately 40 analytes could be arranged in a ladder-like manner with mass spaces of 57 Da. Some of the water-soluble peptide sequences obtained after MS/MS fragmentation revealed that the 57 Da shift corresponds to the repetition of glycine residues. Furthermore, the exchange of glycine against alanine explains the 14 Da shift observed between some peptides. These data show for the first time that small molecules, especially peptides, are prevalent components of nacre. The molecular species described in this report might have a functional role in nacre.
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Restoration of stratum corneum with nacre lipids.
To discover potential new products for the atopic dermatitis treatment, lipids extracted from nacre from the oyster Pinctada margaritifera were tested on artificially dehydrated skin explants. Expression of filaggrin and transglutaminase 1 was investigated after treatment of dehydrated skin with P. margaritifera lipid extracts according to light microscopy after labelling with specific monoclonal antibodies. The lipids were extracted from the nacre with methanol/chloroform mixture at room temperature and the extract composition was determined according to TLC and densitometry measures. Relative to the dry nacre material, a yield of extraction in lipids of 0.54% (w/w) was determined. Fatty acids, triglycerides, cholesterol and ceramides were in low abundance. Then, application of lipid formulations on skin explants previously dehydrated gave after 3 h an overexpression of filaggrin and a decrease of transglutaminase expression as shown by light microscopy. Using immunofluorescence labelling, we showed that lipids extracted from the mother of pearl of P. margaritifera induced a reconstitution of the intercellular cement of the stratum corneum. The signaling properties of the nacre lipids could be used for a development of new active product treatment against the symptoms of the dermatitis.
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Tyrosinase localization in mollusc shells.
In molluscan shellfish, pigmentation is frequently observed in the calcified shell, but the molecular basis of this process is not understood. Here, we report two tyrosinase proteins (Pfty1 and Pfty2) found in the prismatic shell layer of the pearl oyster Pinctada fucata; this layer is recognized as the pigmented region in P. fucata. The protein sequences were deduced from the corresponding cDNAs and confirmed by MALDI-TOF/TOF analysis. The sequences suggest that both tyrosinases have two copper-binding sites in similar N-terminal domains that are homologous to tyrosinases of cephalopods and hemocyanins of gastropods. In turn, this suggests that bivalve tyrosinases are evolved from a common ancestral copper-binding protein in the mollusc. Pfty1 and Pfty2 were specifically expressed in the mantle, and their expression in the mantle is different from each other, suggesting that these tyrosinases have distinctive roles in melanogenesis in shells.
Amino Acid Sequence↗
The Akoya pearl oyster shell as an archival monitor of lead exposure.
The Akoya pearl oyster (Pinctada imbricata) was experimentally exposed to (a) constant levels of lead (Pb) at 180 microg L(-1) for nine weeks, or (b) two short term (pulse) exposures of Pb at 180 microg L(-1) (three weeks each) with an intervening depuration period (three weeks), to assess its utility as an (i) accumulative monitor of Pb contamination and an (ii) archival monitor for discriminating constant versus pulsed Pb exposure events. P. imbricata showed similar reductions in growth (based on shell morphology and wet weight) and Pb accumulation patterns for whole tissue and shell in response to both Pb exposure regimes. Thus the whole oyster was deemed an inappropriate accumulative monitor for assessing short-term temporal variation of Pb exposure and effect. However, using secondary ion mass spectrometry, Pb was shown to accumulate in the successively deposited nacreous layers of the shell of P. imbricata, documenting the exposure history of constant versus pulsed Pb events. Patterns of Pb deposition not only reflected the frequency of Pb exposure events but also their relative durations. Thus, the shell of P. imbricata may be employed as a suitable biological archive of Pb exposure.
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CGRP stimulates gill carbonic anhydrase activity in molluscs via a common CT/CGRP receptor.
The physiological significance of calcitonin gene-related peptide (CGRP) during biomineralization was investigated by assessing the effect of human CGRP on the carbonic anhydrase activity in gill membranes of the pearl oyster, Pinctada margaritifera. Salmon CT and human CGRP were able to induce a 150% increase of the basal activity. No additive effect was observed suggesting that both activities are mediated by the same receptor. The CGRP-stimulated effect was specific as demonstrated by the inhibition produced by the CGRP antagonist, hCGRP8-37. So, CGRP by its specific action on gill carbonic anhydrase controls the calcification process, an ancient role both in invertebrates and non-mammalian vertebrates.
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Conformational study of silklike peptides modified by the addition of the calcium-binding sequence from the shell nacreous matrix protein MSI60 using 13C CP/MAS NMR spectroscopy.
The calcium-binding site of the pearl oyster (Pinctada fucata) nacreous layer matrix protein MSI60 was introduced between different Ala-Gly repeating regions derived from the primary sequences of several silk fibroins. Several different organic solvents whose effect on the repetitive domains of silk peptides is well-understood were used to modify the secondary structure of the flanking Ala-Gly repeating regions. The local conformations of the flanking Ala-Gly repeating regions as well as the calcium-binding motif, MSI60, were determined by 13C CP/MAS NMR spectroscopy. The secondary structures of the polyalanine, poly(Ala), domains were modified by the solvent treatments in a predictable fashion, suggesting that only the solvent treatment and not the conformation of the MSI60 domain affected the conformation of poly(Ala) regions. Ala-Gly domains behaved differently, taking random coil conformation regardless of the choice of solvent, indicating that their secondary structure is affected by the central MSI60 domain. The conformation of the MSI60 domain is not altered by the solvent treatments, suggesting that it may retain its ability to bind calcium ions. This was confirmed using a calcium-binding assay. The assay further showed that the calcium-binding capability of MSI60 in the synthetic peptides was most effective when the flanking domain was in the beta-sheet structure.
Amino Acid Sequence↗
Comparative effects of nacre water-soluble matrix and dexamethasone on the alkaline phosphatase activity of MRC-5 fibroblasts.
In this study we demonstrate, for the first time, that dexamethasone and BMP-2 stimulated alkaline phosphatase (ALP) activity in MRC-5 fibroblasts, a cell line derived from human fetal lung. Previously we reported that the water-soluble matrix (WSM) of nacre obtained from the inner shell layer of the oyster Pinctada maxima, promoted an increase in ALP activity that was dose-dependent. In this work, we show that the effect of WSM is also time-dependent. As a comparison, the effect of WSM was also tested in bone marrow stromal cells because marrow and other bone surface-derived osteoblast stem cells have the inherent direct potential for osteogenesis. WSM promotes cell proliferation and ALP activity when tested with bone marrow cells in concentrations between 135 and 540 microg protein/mL. The effect of WSM on ALP activity of bone marrow stromal cells is similar to that obtained by dexamethasone. These results imply that MRC-5 fibroblasts respond to differentiating factors that promote osteoblastic phenotype in bone-derived cell cultures.
Alkaline Phosphatase↗
Nacre initiates biomineralization by human osteoblasts maintained in vitro.
When nacreous shell produced by the marine oyster Pinctada maxima, used as a biomaterial in oral surgery, is implanted in human bone, new bone formation occurs, resulting in a tight welding of the bone to the nacre [16]. These findings are consistent with the possibility that nacre adjacent to bone can locally stimulate osteogenic activity. To test this hypothesis, we have evaluated the effect of the simultaneous presence of bone and nacre on human osteoblasts in vitro. Nacre chips (1 mm3) were placed at approximately 1 mm distance from a similarly sized bone chip on a layer of first passage human osteoblasts. None of the chemical inducers generally required to obtain bone mineralization in vitro (in particular, beta-glycerophosphate) was added to the cultures. Mineralized sections of the cultures were evaluated by light and electron microscopy, contact microradiography, and Laser Raman Spectroscopy. The results demonstrated that nacre has strong osteogenic effects on human osteoblasts when placed in proximity to bone in vitro. New bone formation occurred by both appositional growth on the existing bone and by the formation of mineralized nodules within the matrix adjacent to the bone explant. Electron microscopic evaluation of these sites demonstrated findings typical of those described in the course of bone formation in vivo, and no evidence of toxicity was observed. In addition, under the conditions of culture used, nacre can also promote the formation by osteoblasts of a structure with characteristics similar to nacre (e.g., lamellar organic matrix mineralized with aragonite, as demonstrated by Laser Raman Spectroscopy).(ABSTRACT TRUNCATED AT 250 WORDS)
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Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes.
In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster. Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20 degrees C in 5% CO2:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2'-deoxyuridine was added to the culture at 30 microns. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2'-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.
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Structure and expression of an unusually acidic matrix protein of pearl oyster shells.
We report identification and characterization of the unusually acidic molluscan shell matrix protein Aspein, which may have important roles in calcium carbonate biomineralization. The Aspein gene (aspein) encodes a sequence of 413 amino acids, including a high proportion of Asp (60.4%), Gly (16.0%), and Ser (13.2%), and the predicted isoelectric point is 1.45; this is the most acidic of all the molluscan shell matrix proteins sequenced so far, or probably even of all known proteins on earth. The main body of Aspein is occupied by (Asp)(2-10) sequences punctuated with Ser-Gly dipeptides. RT-PCR demonstrated that the transcript of aspein is expressed at the outer edge of the mantle, corresponding to the calcitic prismatic layer, but not at the inner part of the mantle, corresponding to the aragonitic nacreous layer. Our findings and previous in vitro experiments taken together suggest that Aspein is responsible for directed formation of calcite in the shell of the pearl oyster Pinctada fucata.
Amino Acid Sequence↗
Multiscale structure of sheet nacre.
This work was conducted on Pinctada maxima nacre (mother of pearl) in order to understand its multiscale ordering and the role of the organic matrix in its structure. Intermittent-contact atomic force microscopy with phase detection imaging reveals a nanostructure within the tablet. A continuous organic framework divides each tablet into nanograins. Their shape is supposed to be flat with a mean extension of 45nm. TEM performed in the darkfield mode evidences that at least part of the intracrystalline matrix is crystallized and responds like a 'single crystal'. The tablet is a 'hybrid composite'. The organic matrix is continuous. The mineral phase is thus finely divided still behaving as a single crystal. It is proposed that each tablet results from the coherent aggregation of nanograins keeping strictly the same crystallographic orientation thanks to a hetero-epitaxy mechanism. Finally, high-resolution TEM performed on bridges from one tablet to the next, in the overlying row, did not permit to evidence a mineral lattice but crystallized organic bridges. The same organic bridges were evidenced by SEM in the interlaminar sequence.
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Significance of the extra C-terminal tail of CaLP, a novel calmodulin-like protein involved in oyster calcium metabolism.
Oyster (Pinctada fucata) calmodulin-like protein (CaLP), containing a C-terminally extra hydrophilic tail (150D-161K), is a novel protein involved in the regulation of oyster calcium metabolism. To investigate the importance of the extra fragment to the Ca(2+)/Mg(2+)-dependent conformational changes in the intact CaLP molecule and the interactions between CaLP and its target proteins, a truncated CaLP mutant (M-CaLP) devoid of the extended C-terminus was constructed and overexpressed in Escherichia coli. The conformational characteristics of M-CaLP were studied by CD and fluorescence spectroscopy and compared with those of the oyster CaM and CaLP. The far-UV CD results reveal that the extra tail has a strong effect on the Ca(2+)-induced, but a relatively weak effect on the Mg(2+)-induced conformational changes in CaLP. However, upon Ca2+ or Mg2+ binding, only slight changes for intrinsic phenylalanine and tyrosine fluorescence spectra between M-CaLP and CaLP are observed. Our results also indicate that the extra tail can significantly decrease the exposure of the hydrophobic patches in CaLP. Additionally, affinity chromatography demonstrates that the target binding of CaLP is greatly influenced by its additional tail. All our results implicate that the extra tail may play some important roles in the interactions between CaLP and its targets in vivo.
Amino Acid Sequence↗