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ABSTRACT: The organic matrices in the exoskeleton of the crayfish Procambarus clarkii are classified into three groups depending on solubility; acid soluble, acid insoluble-SDS/dithiothreito (DTT) soluble, and acid insoluble-SDS/DTT insoluble fractions. In our previous studies, Casp-1 and -2 were identified in the acid soluble fraction, and CAP-1 and -2 were identified in the acid insoluble-SDS/DTT soluble fraction. In this study, acid insoluble-SDS/DTT insoluble materials were digested with proteases and the resulting peptides were purified and sequenced. Based on the sequences, a cDNA encoding this protein was cloned. The whole primary sequence of the matrix protein named strong chitin-binding protein-1 (SCBP-1), was deduced. SCBP-1 consisted of 155 amino acid residues and had a Rebers-Riddiford consensus sequence for chitin binding. A recombinant protein of SCBP-N corresponding to the N-terminal part of SCBP-1 showed no chitin-binding ability, while SCBP-C corresponding to the C-terminal part of SCBP-1, showed weak affinity to chitin. These results suggest that the primary sequence of SCBP-1 does not have strong chitin-binding ability. Therefore, SCBP-1 probably binds covalently to chitin through a particular residue contained in the peptide part that was not obtained by protease digestion.
Bioscience Biotechnology and Biochemistry 02/2013; · 1.28 Impact Factor
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ABSTRACT: Although the formation mechanism of calcite crystals in the prismatic layer has been studied well in many previous works, the initial state of calcite formation has not been observed in detail using electron microscopes. In this study, we report that the soft prismatic layer with transparent color (the thin prismatic layer) in the tip of the fresh shell of Pinctada fucata was picked up to observe the early calcification phase. A scanning electron microscope (SEM) image showed that the growth tip of the thin prismatic layer was covered by the periostracum, which was also where the initial formation of calcite crystals began. A cross-section containing the thin calcite crystals in the thin prismatic layer with the periostracum was made using a focused ion beam (FIB) system. In a transmission electron microscope (TEM) observation, the thin calcite crystal (thickness is about 1μm) on the periostracum was found to be a single crystal with the c-axis oriented perpendicular to the shell surface. On the other hand, many aggregated small particles consisting of bassanite crystals were observed in the periostracum suggesting the possibility that not only organic sulfate but also inorganic sulfates exist in the prismatic layer. These discoveries in the early calcification phase of the thin prismatic layer may help to clarify the mechanism of regulating the nucleation and orientation of the calcite crystal in the shell.
Micron 10/2012; · 1.53 Impact Factor
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ABSTRACT: {110} Twin densities in biotic aragonite have been estimated quantitatively from the peak widths of specific reflections in powder X-ray diffraction (XRD) patterns, as well as direct confirmation of the twins using transmission electron microscopy (TEM). Influence of the twin density on the peak widths in the XRD pattern was simulated using DIFFaX program, regarding (110) twin as interstratification of two types of aragonite unit layers with mirrored relationship. The simulation suggested that the twin density can be estimated from the difference of the peak widths between 111 and 021, or between 221 and 211 reflections. Biotic aragonite in the crossed-lamellar microstructure (three species) and nacreous microstructure (four species) of molluscan shells, fish otoliths (two species), and a coral were investigated. The XRD analyses indicated that aragonite crystals in the crossed-lamellar microstructure of the three species contain high density of the twins, which is consistent with the TEM examination. On the other hand, aragonite in the nacre of the four species showed almost no difference of the peak widths between the paired reflections, indicating low twin densities. The results for the fish otoliths were varied between the species. Such variation of the twin density in biotic aragonites may reflect different schemes of crystal growth in biomineralization.
Journal of Structural Biology 09/2012; · 3.41 Impact Factor
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ABSTRACT: Various novel proteins have been identified from many kinds of mollusk shells. Although such matrix proteins are believed to play important roles in the calcium carbonate crystal formation of shells, no common proteins that interact with calcium carbonate or that are involved in the molecular mechanisms behind shell formation have been identified. Pif consists of two proteins, Pif 80 and Pif 97, which are encoded by a single mRNA. Pif 80 was identified as a key acidic protein that regulates the formation of the nacreous layer in Pinctada fucata, while Pif 97 has von Willebrand factor type A (VWA) and chitin-binding domains. In this study, we identified Pif homologues from Pinctada margaritifera, Pinctada maxima, Pteria penguin, Mytilus galloprovincialis, and in the genome database of Lottia gigantea in order to compare their primary protein sequences. The VWA and chitin-binding domains are conserved in all Pif 97 homologues, whereas the amino acid sequences of the Pif 80 regions differ markedly among the species. Sequence alignment revealed the presence of a novel significantly conserved sequence between the chitin-binding domain and the C-terminus of Pif 97. Further examination of the Pif 80 regions suggested that they share a sequence that is similar to the laminin G domain. These results indicate that all Pif molecules in bivalves and gastropods may be derived from a common ancestral gene. These comparisons may shed light on the correlation between molecular evolution and morphology in mollusk shell microstructure.
Marine Biotechnology 07/2012; · 3.43 Impact Factor
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ABSTRACT: Crustacean hyperglycemic hormone (CHH) is released from the X-organ/sinus gland complex located in the eyestalks. In this
study, the most abundant CHH in the sinus gland of the greasyback shrimp Metapenaeus ensis was purified by reversed-phase HPLC and identified by N-terminal amino acid sequencing. Although two CHH molecules (Mee-CHH-A
and Mee-CHH-B) have already been identified from M. ensis by cDNA cloning, this study revealed the presence of an additional CHH peptide based on differences in the N-terminal amino
acid sequences of the CHH-A and CHH-B. Therefore, this novel CHH was designated as Mee-CHH-C. A cDNA encoding the Mee-CHH-C
precursor was cloned by RT-PCR coupled with 5′- and 3′-RACE, and it was found that the mature Mee-CHH-C consisted of 72 amino
acid residues containing 6 conserved cysteine residues and possessed an amidated C terminus. Mee-CHH-C had 62 and 68% identities
with Mee-CHH-A and Mee-CHH-B, respectively, and was highly homologous to CHHs characterized from other penaeid shrimp species.
The hyperglycemic activity of Mee-CHH-C was examined by an in vivo bioassay using the kuruma prawn Marsupenaeus japonicus. Injection of Mee-CHH-C increased hemolymph glucose levels significantly and dose-dependently. These results indicate that
Mee-CHH-C is possibly one of the major molecules in M. ensis that regulate glucose levels in the hemolymph.
KeywordsCrustacea-Crustacean hyperglycemic hormone-Greasyback shrimp-
Metapenaeus ensis
-Sinus gland
Fisheries Science 04/2012; 76(4):605-611. · 0.94 Impact Factor
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ABSTRACT: The nacreous layer of molluscan shells consists of a highly organised, layered structure comprising calcium carbonate aragonite crystals, each surrounded by an organic matrix. In the Japanese pearl oyster Pinctada fucata, the Pif protein from the nacreous layer functions in aragonite binding, and plays a key role in nacre formation. Here, we investigated whether the blue mussel Mytilus galloprovincialis also has a protein with similar functions in the nacreous layer. By using a calcium carbonate-binding assay, we identified the novel protein blue mussel shell protein (BMSP) 100 that can bind calcium carbonate crystals of both aragonite and calcite. When the entire sequence of a cDNA encoding BMSP 100 was determined, it was found that BMSP is a preproprotein consisting of a signal peptide and two proteins, BMSP 120 and BMSP 100. BMSP 120 contains four von Willebrand factor A (VWA) domains and one chitin-binding domain, thus suggesting that it has a role in maintaining structure within the matrix. Immunohistochemical analysis revealed that BMSP 100 is present throughout the nacreous layer with dense localisation in the myostracum. Posttranslational modification analysis indicated that BMSP 100 is phosphorylated and glycosylated. These results suggest that there is a common molecular mechanism between P. fucata and M. galloprovincialis that underlies the nacreous layer formation.
ChemBioChem 09/2011; 12(16):2478-87. · 3.94 Impact Factor
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ABSTRACT: The fine structure of the calcite prism in the outer layer of a pearl oyster, Pinctada fucata, has been investigated using various electron beam techniques, in order to understand its characteristics and growth mechanism including the role of intracrystalline organic substances. As the calcite prismatic layer grows thicker, sinuous boundaries develop to divide the prism into a number of domains. The crystal misorientation between the adjacent domains is several to more than ten degrees. The component of the misorientation is mainly the rotation about the c-axis. There is no continuous organic membrane at the boundaries. Furthermore, the crystal orientation inside the domains changes gradually, as indicated by the electron back-scattered diffraction (EBSD) in a scanning electron microscope (SEM). Transmission electron microscopy (TEM) examination revealed that the domain consists of sub-grains of a few hundred nanometers divided by small-angle grain boundaries, which are probably the origin of the gradual change of the crystal orientation inside the domains. Spherular Fresnel contrasts were often observed at the small-angle grain boundaries, in defocused TEM images. Electron energy-loss spectroscopy (EELS) indicated the spherules are organic macromolecules, suggesting that incorporation of organic macromolecules during the crystal growth forms the sub-grain structure of the calcite prism.
Micron 10/2010; 41(7):821-6. · 1.53 Impact Factor
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ABSTRACT: The microstructure and its crystallographic aspect of the shell of a limpet, Lottiakogamogai, have been investigated, as the first step to clarify the mechanism of shell formation in limpet. The shell consists of five distinct layers stacked along the shell thickness direction. Transmission electron microscopy (TEM) with the focused ion beam (FIB) sample preparation technique was primarily adopted, as well as scanning electron microscopy (SEM) with electron back-scattered diffraction (EBSD). The five layers were termed as M+3, M+2, M+1, M, M-1 from the outside to the inside in previous works, where M means myostracum. The outmost M+3 layer consists of calcite with a "mosaic" structure; granular submicron sub-grains with small-angle grain boundaries often accompanying dislocation arrays. M+2 layer consists of flat prismatic aragonite crystals with a leaf-like cross section, stacked obliquely to the shell surface. It looks that the prismatic crystals are surrounded by organic sheets, forming a compartment structure. M+1 and M-1 layers adopt a crossed lamellar structure consisting of aragonite flat prisms with rectangular cross section. M layer has a prismatic structure of aragonite perpendicular to the shell surface and with irregular shaped cross sections. Distinct organic sheets were not observed between the crystals in M+1, M and M-1 layers. The {110} twins are common in all aragonite M+2, M+1, M and M-1 layers, with the twin boundaries parallel to the prisms. These results for the microstructure of each layer should be considered in the discussion of the formation mechanism of the limpet shell structure.
Journal of Structural Biology 08/2010; 171(2):223-30. · 3.41 Impact Factor
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ABSTRACT: Capnocytophagacanimorsus and Capnocytophagacynodegmi, fastidious gram-negative rods, are commensal microbes thriving in the oral cavities of dogs and cats. C. canimorsus can sometimes cause fatal systemic infections in humans. In the present study, we established a specific PCR which could identify and distinguish C. canimorsus from C. cynodegmi. The prevalence of Capnocytophaga spp. in dogs and cats was determined using this method. C. canimorsus was detected in 74% of dogs and 57% of cats. C. cynodegmi was detected in 86% of dogs and 84% of cats. The prevalence of Capnocytophaga spp. obtained in this study is somewhat higher than those reported previously where bacterial isolation method was used for identification. This is probably due to the fact that the PCR detection is more sensitive compared to bacterial isolation. Our findings suggest the importance of informing people who belong to high-risk groups as well as health care workers on C. canimorsus infection and its potential risk to people particularly to those who are immunocompromised.
Veterinary Microbiology 01/2010; 144(1-2):172-6. · 3.33 Impact Factor
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ABSTRACT: Capnocytophaga canimorsus, a commensal bacterium from the carine mouth, causes septicemia in human beings through bites or scratches. We report a case of a 60-year-old man contracting septicemia due to C. canimorsus infection after a dog bite who died less than 12 hours after admission. We observed neutrophils with intracytoplasmic bacilli in the peripheral blood smear. We discuss clinical presentation and autopsy findings.
Kansenshogaku zasshi. The Journal of the Japanese Association for Infectious Diseases 11/2009; 83(6):661-4.
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ABSTRACT: The mollusk shell is a hard tissue consisting of calcium carbonate crystals and an organic matrix. The nacre of the shell is characterized by a stacked compartment structure with a uniformly oriented c axis of aragonite crystals in each compartment. Using a calcium carbonate-binding assay, we identified an acidic matrix protein, Pif, in the pearl oyster Pinctada fucata that specifically binds to aragonite crystals. The Pif complementary DNA (cDNA) encoded a precursor protein, which was posttranslationally cleaved to produce Pif 97 and Pif 80. The results from immunolocalization, a knockdown experiment that used RNA interference, and in vitro calcium carbonate crystallization studies strongly indicate that Pif regulates nacre formation.
Science 09/2009; 325(5946):1388-90. · 31.20 Impact Factor
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ABSTRACT: A novel matrix protein named calcification-associated soluble protein-2 (Casp-2) was isolated from the acetic acid-soluble fraction of the exoskeleton of the crayfish Procambarus clarkii, and its primary structure was determined by a combination of peptide sequencing, mass spectral analysis, and cDNA cloning. Casp-2 consists of 117 amino acid residues and has a chitin-binding consensus sequence, the so-called Rebers-Riddiford (R-R) consensus sequence. Casp-2 exhibited an inhibitory activity on calcium carbonate precipitation from its supersaturated solution in vitro, suggesting association with calcification of the exoskeleton. Reverse transcription PCR and Northern blot analyses indicated that the Casp-2 gene was expressed only at the epidermis throughout the molting stages, and most strongly at the late pre-molt stage. Recombinant Casp-2 showed weak affinity to chitin in spite of having the R-R consensus sequence. These results indicate that Casp-2 interacts loosely with chitin fibrils and regulates calcification in the cuticle.
Bioscience Biotechnology and Biochemistry 11/2008; 72(10):2697-707. · 1.28 Impact Factor
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ABSTRACT: A microplate agglutination test (MAT) was compared with the tube agglutinin test (TAT), a standard test for the diagnosis of Brucella canis, in terms of the sensitivity and specificity. The results showed that MAT was more sensitive, simpler to perform and easier to read the results than TAT. On top of that the MAT allows us to handle a larger number of samples at once. Using this method we conducted sero-surveillance of the prevalence of B. canis in dogs kept in an Animal Shelter located in Kanagawa Prefecture. Twelve of 485 (2.5%) showed seropositive against B. canis. These results indicate that B. canis infection in dogs is still occurring in Japan.
Journal of Veterinary Medical Science 08/2008; 70(7):707-9. · 0.85 Impact Factor
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ABSTRACT: Streptobacillus moniliformis is an etiological agent of rat-bite fever and Haverhill fever in human infection. As the currently available methods for identifying the causative bacteria are not satisfactory, we attempted to establish them by PCR using newly designed primers for the 16S rRNA gene of S. moniliformis. We then determined the prevalence of Streptobacillus spp. in two species of feral rats that inhabit an urban region in Japan, because information on the prevalence of the bacteria in feral rats is obscure. The use of PCR with newly designed primers showed that an extremely high proportion of R. norvegicus harbored the bacteria (61/66, 92%), whereas the prevalence was only 58% in R. rattus (30/52). The nucleotide sequence analysis of the 16S rRNA gene of Streptobacillus spp. isolated from oral swabs of feral rats showed at least two different types of bacteria among isolates from R. norvegicus and R. rattus.
Microbiology and Immunology 02/2008; 52(1):9-15. · 1.30 Impact Factor
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ABSTRACT: The mollusk shell is a hard tissue consisting of calcium carbonate and organic matrices. The organic matrices are considered to play important roles in shell formation. We have previously identified a prismatic layer-specific protein named Prismalin-14, which consists of 105 amino acid residues and includes four structurally characteristic regions; a repeated sequence of Pro-Ile-Tyr-Arg, a Gly/Tyr-rich region and N- and C-terminal Asp-rich regions. Prismalin-14 showed an inhibitory activity on calcium carbonate precipitation and a calcium-binding ability in vitro. In this study, we prepared some molecular species of recombinant proteins including Prismalin-14 and its truncated proteins in an Escherichia coli expression system to reveal a structure-function relationship of Prismalin-14. The results showed that the Gly/Tyr-rich region was responsible for chitin binding and was identified as a novel chitin-binding sequence. On the other hand, both N- and C-terminal Asp-rich regions are related to inhibitory activity on calcium carbonate precipitation in vitro. Immunohistological observation revealed that Prismalin-14 was localized at the acid-insoluble organic framework including chitin. All these results strongly suggest that Prismalin-14 is a framework protein that mediates chitin and calcium carbonate crystals by using its acidic and chitin-binding regions.
FEBS Journal 11/2007; 274(19):5158-66. · 3.79 Impact Factor
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ABSTRACT: 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.
Bioscience Biotechnology and Biochemistry 08/2007; 71(7):1735-44. · 1.28 Impact Factor
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ABSTRACT: We have developed a combinatorial polymerase chain reaction (PCR) procedure to identify four major species of the genus Brucella simultaneously. Four pairs of primers targeting the genes encoding a cell surface protein (BCSP31) and outer membrane proteins (omp2b, omp2a and omp31) were prepared. PCR using these primers gave rise to specific patterns of amplification for each Brucella spp. examined in this study. B. abortus could be identified when fragments of BCSP31 and omp2b/2a were amplified by B. abortus-specific primers. B. melitensis could be identified by the amplification of fragments of BCSP31, omp2b/2a and omp31 using pair of primers B4/B5, JRF/JPR-ab and omp31. Identification of B. canis could be achieved when the amplicons of omp2b/2a were detected by B. canis-specific primers, as could the identification of BCSP31 and omp31. If specific amplifications occurred using all pairs of primers, the strain was identified as B. suis. Combinatorial PCR reported here thus appeared to be an ideal method of identifying Brucella spp., the causative pathogen of human brucellosis.
Japanese journal of infectious diseases 06/2007; 60(2-3):137-9. · 1.49 Impact Factor
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ABSTRACT: Hollow spherical particles of calcite with a diameter of 2−10 μm were synthesized on a polyvinyliden difluoride (PVDF) membrane blotted with crude organic matrices prepared from the pearl oyster shell in a supersaturated solution of calcium carbonate. Examination of the produced particles as a function of the reaction time showed that the surface topography of the particles changed from a smooth surface to a ragged one with fine {104} facets of calcite crystals. Cross-sections and thin specimens of the particles were prepared by using the focused ion beam technique to observe the inside and determine the crystalline phases, respectively. Transmission electron microscopy (TEM) analysis showed that the particles with a short reaction time consist of vaterite and amorphous calcium carbonate (ACC). The ragged particles are polycrystalline calcite with a hollow structure (especially in relatively smaller particles) or a void space inside the particles. Large calcite crystals form a crust of the sphere, and minute calcite crystallites exist inside with the void space. Such a hollow structure can be explained by the solid-to-solid transformation from vaterite or ACC to calcite initiating from the surface and proceeding toward the inside of the sphere, with volume reduction by the phase transition.
08/2006;
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ABSTRACT: Recombinant peptides related to vitellogenesis-inhibiting hormone (VIH) of the American lobster Homarus americanus were expressed in bacterial cells, and then purified after being allowed to refold. Biological activities of the recombinant VIHs having an amidated C-terminus (rHoa-VIH-amide) and a free carboxyl-terminus (rHoa-VIH-OH) were examined using an ovarian fragment incubation system derived from the kuruma prawn, Marsupenaeus japonicus. The rHoa-VIH-amide significantly reduced vitellogenin mRNA levels in the ovary, while rHoa-VIH-OH had no effect. This is the first report that describes the production of a crustacean VIH having biological activity and the importance of the C-terminal amidation for its vitellogenesis-inhibiting activity.
Peptides 07/2006; 27(6):1251-8. · 2.43 Impact Factor
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ABSTRACT: The mollusc shell is a hard tissue consisting of calcium carbonate and organic matrices. The organic matrices are believed to play important roles in shell formation. In the present study, we extracted and purified a novel matrix protein, named Prismalin-14, from the acid-insoluble fraction of the prismatic layer of the shell of the Japanese pearl oyster (Pinctada fucata), and determined its whole amino acid sequence by a combination of amino acid sequence analysis and MS analysis of the intact protein and its enzymic digests. Prismalin-14 consisted of 105 amino acid residues, including PIYR repeats, a Gly/Tyr-rich region and N- and C-terminal Asp-rich regions. Prismalin-14 showed inhibitory activity on calcium carbonate precipitation and calcium-binding activity in vitro. The scanning electron microscopy images revealed that Prismalin-14 affected the crystallization of calcium carbonate in vitro. A cDNA encoding Prismalin-14 was cloned and its expression was analysed. The amino acid sequence deduced from the nucleotide sequence of Prismalin-14 cDNA was identical with that determined by peptide sequencing. Northern-blot analysis showed that a Prismalin-14 mRNA was expressed only at the mantle edge. In situ hybridization demonstrated that a Prismalin-14 mRNA was expressed strongly in the inner side of the outer fold of the mantle. These results suggest that Prismalin-14 is a framework protein that plays an important role in the regulation of calcification of the prismatic layer of the shell.
Biochemical Journal 09/2004; 382(Pt 1):205-13. · 4.90 Impact Factor
