Mikimoto Pharmaceutical Co.

Ise, Japan

Mikimoto Pharmaceutical Co.

Ise, Japan
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Lin H.,Tokyo University of Marine Science and Technology | Lin H.,Guangdong Ocean University | Ishizaki S.,Tokyo University of Marine Science and Technology | Nagashima Y.,Tokyo University of Marine Science and Technology | And 3 more authors.
Fisheries Science | Year: 2017

The aim of this research was to characterize immune-related antibacterial substances from pearl oyster Pinctada fucata induced by bacterial invasion. Bacteria inoculation was performed by injecting 0.1 ml of 1.0 × 1012 colony-forming units/ml Vibrio parahaemolyticus into adductor muscle. Acidic extracts were prepared with 0.1% trifluoroacetic acid from different tissues after 8 h of injection, and antibacterial activity against V. parahaemolyticus was determined via the microdilution broth method. The acidic extracts from gills of inoculated oysters (AEg) showed stronger antibacterial activity than those from non-inoculated ones. Based on this result, antibacterial proteins were purified from AEg via two-step gel filtration chromatography, followed by high-performance liquid chromatography using a TSkgel G3000 column. Protein components were analyzed by both sodium dodecyl sulfate and native polyacrylamide gel electrophoresis. As a result, two antibacterial proteins, APg-1 (with a molecular mass of approximately 210 kDa) and APg-2 (of approximately 30 kDa), were obtained from AEg. Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis and partial amino acid sequences revealed that these proteins might be novel antibacterial proteins. These results indicate that antibacterial proteins are potentially upregulated in the gill of pearl oysters or released therefrom for defense against bacterial invasion. © 2017, Japanese Society of Fisheries Science.

Higuchi K.,K. Mikimoto and Co. | Nagai K.,K. Mikimoto and Co. | Hattori F.,Mikimoto Pharmaceutical Co. | Maeyama K.,Mikimoto Pharmaceutical Co. | And 3 more authors.
Nippon Suisan Gakkaishi (Japanese Edition) | Year: 2016

Waste from activities associated with pearl culturing has become a cause of environmental deterioration on pearl farms. As part of our researches on reducing the environmental burden of Akoya pearl culturing, we investigated methods of composting pearl culture waste and effectively using this compost. We conducted composting tests using the soft body of Pinctada fucata after pearl harvesting as the main material, supplemented with debris resulting from sessile organisms removed during shell cleaning, and plant materials. We found that it was possible to convert Akoya oyster meat to mature compost in approximately 45 days without desalinization, and it was observed that including the debris of removed sessile organisms improved the aeration of the composting materials and increased the fertilizer components of the mature compost. Moreover, a seedling test showed that the mature oyster meat compost, when used at a rate of 10 g compost/1000 cm3 soil, improved the fresh weight of komatsuna. These results indicated that compost made from discarded oyster meat and the debris of removed sessile organisms can be effectively utilized in agriculture. The compost is also expected to reduce the environmental burden on pearl farms. © 2016 The Japanese Society of Fisheries Science.

Wang N.,University of Tokyo | Kinoshita S.,University of Tokyo | Nomura N.,University of Tokyo | Riho C.,University of Tokyo | And 3 more authors.
Marine Biotechnology | Year: 2012

Recent researches revealed the regional preference of biomineralization gene transcription in the pearl oyster Pinctada fucata: it transcribed mainly the genes responsible for nacre secretion in mantle pallial, whereas the ones regulating calcite shells expressed in mantle edge. This study took use of this character and constructed the forward and reverse suppression subtractive hybridization (SSH) cDNA libraries. A total of 669 cDNA clones were sequenced and 360 expressed sequence tags (ESTs) greater than 100 bp were generated. Functional annotation associated 95 ESTs with specific functions, and 79 among them were identified from P. fucata at the first time. In the forward SSH cDNA library, it recognized mass amount of nacre protein genes, biomineralization genes dominantly expressed in the mantle pallial, calcium-ion-binding genes, and other biomineralization-related genes important for pearl formation. Real-time PCR showed that all the examined genes were distributed in oyster mantle tissues with a consistence to the SSH design. The detection of their RNA transcripts in pearl sac confirmed that the identified genes were certainly involved in pearl formation. Therefore, the data from this work will initiate a new round of pearl formation gene study and shed new insights into molluscan biomineralization. © 2011 Springer Science+Business Media, LLC.

Funabara D.,Mie University | Ohmori F.,University of Tokyo | Kinoshita S.,University of Tokyo | Koyama H.,University of Tokyo | And 10 more authors.
PLoS ONE | Year: 2014

In our previous publication, we identified novel gene candidates involved in shell formation by EST analyses of the nacreous and prismatic layer-forming tissues in the pearl oyster Pinctada fucata. In the present study, 14 of those genes, including two known genes, were selected and further examined for their involvement in shell formation using the RNA interference. Molecular characterization based on the deduced amino acid sequences showed that seven of the novel genes encode secretory proteins. The tissue distribution of the transcripts of the genes, as analyzed by RT-PCR and in situ hybridization, was mostly consistent with those obtained by the EST analysis reported previously. Shells in the pearl oysters injected with dsRNAs targeting genes 000027, 000058, 000081, 000096, 000113 (nacrein), 000118, 000133 and 000411(MSI60), which showed expression specific to the nacreous layer forming tissues, showed abnormal surface appearance in this layer. Individuals injected with dsRNAs targeting genes 000027, 000113 and 000133 also exhibited abnormal prismatic layers. Individuals injected with dsRNAs targeting genes 000031, 000066, 000098, 000145, 000194 and 000200, which showed expression specific to prismatic layer forming tissues, displayed an abnormal surface appearance in both the nacreous and prismatic layers. Taken together, the results suggest that the genes involved in prismatic layer formation might also be involved in the formation of the nacreous layers. © 2014 Funabara et al.

Takeuchi T.,Okinawa Institute of Science and Technology | Kawashima T.,Okinawa Institute of Science and Technology | Koyanagi R.,Okinawa Institute of Science and Technology | Gyoja F.,Okinawa Institute of Science and Technology | And 22 more authors.
DNA Research | Year: 2012

The study of the pearl oyster Pinctada fucata is key to increasing our understanding of the molecular mechanisms involved in pearl biosynthesis and biology of bivalve molluscs. We sequenced ∼1150-Mb genome at ∼40-fold coverage using the Roche 454 GS-FLX and Illumina GAIIx sequencers. The sequences were assembled into contigs with N50 = 1.6 kb (total contig assembly reached to 1024 Mb) and scaffolds with N50 = 14.5 kb. The pearl oyster genome is AT-rich, with a GC content of 34%. DNA transposons, retrotransposons, and tandem repeat elements occupied 0.4, 1.5, and 7.9% of the genome, respectively (a total of 9.8%). Version 1.0 of the P. fucata draft genome contains 23 257 complete gene models, 70% of which are supported by the corresponding expressed sequence tags. The genes include those reported to have an association with bio-mineralization. Genes encoding transcription factors and signal transduction molecules are present in numbers comparable with genomes of other metazoans. Genome-wide molecular phylogeny suggests that the lophotrochozoan represents a distinct clade from ecdysozoans. Our draft genome of the pearl oyster thus provides a platform for the identification of selection markers and genes for calcification, knowledge of which will be important in the pearl industry. © The Author 2012. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Kinoshita S.,University of Tokyo | Wang N.,University of Tokyo | Inoue H.,University of Tokyo | Maeyama K.,Mikimoto Pharmaceutical CO. | And 6 more authors.
PLoS ONE | Year: 2011

Background: Despite its economic importance, we have a limited understanding of the molecular mechanisms underlying shell formation in pearl oysters, wherein the calcium carbonate crystals, nacre and prism, are formed in a highly controlled manner. We constructed comprehensive expressed gene profiles in the shell-forming tissues of the pearl oyster Pinctada fucata and identified novel shell formation-related genes candidates. Principal Findings: We employed the GS FLX 454 system and constructed transcriptome data sets from pallial mantle and pearl sac, which form the nacreous layer, and from the mantle edge, which forms the prismatic layer in P. fucata. We sequenced 260477 reads and obtained 29682 unique sequences. We also screened novel nacreous and prismatic gene candidates by a combined analysis of sequence and expression data sets, and identified various genes encoding lectin, protease, protease inhibitors, lysine-rich matrix protein, and secreting calcium-binding proteins. We also examined the expression of known nacreous and prismatic genes in our EST library and identified novel isoforms with tissue-specific expressions. Conclusions: We constructed EST data sets from the nacre- and prism-producing tissues in P. fucata and found 29682 unique sequences containing novel gene candidates for nacreous and prismatic layer formation. This is the first report of deep sequencing of ESTs in the shell-forming tissues of P. fucata and our data provide a powerful tool for a comprehensive understanding of the molecular mechanisms of molluscan biomineralization. © 2011 Kinoshita et al.

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