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Dive into the research topics where Masanori Ochiai is active.

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Featured researches published by Masanori Ochiai.


Journal of Biological Chemistry | 1999

Prophenoloxidase-activating Enzyme of the Silkworm, Bombyx mori PURIFICATION, CHARACTERIZATION, AND cDNA CLONING

Dan Satoh; Akio Horii; Masanori Ochiai; Masaaki Ashida

Prophenoloxidase-activating enzyme (PPAE) was purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis from larval cuticles of the silkworm, Bombyx mori. The purified PPAE preparation was shown to be a mixture of the isozymes of PPAE (PPAE-I and PPAE-II), which were eluted at different retention times in reversed-phase high performance liquid chromatography. PPAE-I and PPAE-II seemed to be post translationally modified isozymes and/or allelic variants. Both PPAE isozymes were proteins composed of two polypeptides (heavy and light chains) that are linked by disulfide linkage(s) and glycosylated serine proteases. The results of cDNA cloning, peptide mapping, and amino acid sequencing of PPAE revealed that PPAE is synthesized as prepro-PPAE with 441 amino acid residues and is activated from pro-PPAE by cleavage of a peptide bond between Lys152 and Ile153. The homology search showed 36.9% identity of PPAE to easter, which is a serine protease involved in dorso-ventral pattern formation in theDrosophila embryo, and indicated the presence of two consecutive clip-like domains in the light chain. A single copy of the PPAE gene was suggested to be present in the silkworm genome. In the fifth instar larvae, PPAE transcripts were detected in the integument, hemocytes, and salivary glands but not in the fat body or mid gut. A polypeptide cross-reactive to mono-specific anti-PPAE/IgG was transiently detected in the extract of eggs between 1 and 3 h after they were laid.


Journal of Biological Chemistry | 1999

A Pattern Recognition Protein for Peptidoglycan CLONING THE cDNA AND THE GENE OF THE SILKWORM, BOMBYX MORI

Masanori Ochiai; Masaaki Ashida

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect. The PGRP is an essential component for peptidoglycan to trigger the prophenoloxidase cascade that is now recognized to be an important insect defense mechanism. We cloned cDNA encoding PGRP from the silkworm fat body cDNA library. Northern blot analysis showed that the PGRP gene is constitutively expressed in the fat body, epithelial cell, and hemocytes of naive silkworms. Furthermore, a bacterial challenge intensified the gene expression, with the maximal period being from 6 to 36 h after infection. The upstream sequence of the cloned PGRP gene was shown to contain putativecis-regulatory elements similar to the NF-κB-like element, interferon-response half-element, and GATA motif element, which have been found in the promoters of the acute phase protein genes of mammals and insects. A homology search revealed that the homologs of silkworm PGRP are present in mice, nematodes, and bacteriophages. This suggests that the recognition of peptidoglycan as foreign is effected in both vertebrates and invertebrates by PGRP homologs with an evolutionally common origin.


Biochemical and Biophysical Research Communications | 1986

β-1,3-glucan receptor and peptidoglycan receptor are present as separate entities within insect prophenoloxidase activating system

Hideya Yoshida; Masanori Ochiai; Masaaki Ashida

Silkworm plasma was passed over a peptidoglycan-Sepharose 4B column or a CPB column [CPB, curdlan type polysaccharide (beta-1,3-glucan) bead] in the absence of divalent cation and the effluents from the columns were named plasma-PG and plasma-CPB, respectively. Prophenoloxidase activating system in plasma-PG was triggered by beta-1,3-glucan but not by peptidoglycan and the system in plasma-CPB was triggered by peptidoglycan but not by beta-1,3-glucan, suggesting that the peptidoglycan-Sepharose 4B column and the CPB column remove peptidoglycan-receptor and beta-1,3-glucan-receptor, respectively, from plasma. This result indicates that both receptors exist as separate entities in silkworm plasma. It is suggested that plasma-PG and plasma-CPB may be used as specific reagents to detect minute amounts of beta-1,3-glucan and peptidoglycan.


Tissue & Cell | 1988

Immunolocalization of prophenoloxidase among hemocytes of the silkworm, Bombyx mori.

Masaaki Ashida; Masanori Ochiai; Teruo Niki

Silkworm (Bombyx mori) hemocytes were fixed immediately after collection. Thin sections of the hemocytes were stained by an indirect immunogold staining method using rabbit anti-prophenoloxidase/IgG as a primary antibody and colloidal gold coated with goat anti-rabbit/IgG as a secondary antibody. Electron micrographs of the sections revealed that only plasmatocytes and oenocytoids have prophenoloxidase both in cytoplasm and nucleus whereas granulocytes, spherulocytes and prohemocytes do not have appreciable amounts of the proenzyme. Cytoplasmic inclusions of oenocytoids also contain the proenzyme. A wide variety of concentrations of prophenoloxidase was observed among oenocytoids. Plasmatocytes appeared to have less prophenoloxidase than any oenocytoids. Once materials in the granules of granulocyte were discharged into the plasma and formed coagula, they cross-reacted with antiprophenoloxidase/IgG, suggesting that prophenoloxidase was trapped in the coagula by unknown mechanisms. This observation is discussed in relation to the dispute concerning the presence of prophenoloxidase or phenoloxidase in the granulocyte.


Scientific Reports | 2012

Drosophila growth-blocking peptide-like factor mediates acute immune reactions during infectious and non-infectious stress

Seiji Tsuzuki; Masanori Ochiai; Hitoshi Matsumoto; Shoichiro Kurata; Atsushi Ohnishi; Yoichi Hayakawa

Antimicrobial peptides (AMPs), major innate immune effectors, are induced to protect hosts against invading microorganisms. AMPs are also induced under non-infectious stress; however, the signaling pathways of non-infectious stress-induced AMP expression are yet unclear. We demonstrated that growth-blocking peptide (GBP) is a potent cytokine that regulates stressor-induced AMP expression in insects. GBP overexpression in Drosophila elevated expression of AMPs. GBP-induced AMP expression did not require Toll and immune deficiency (Imd) pathway-related genes, but imd and basket were essential, indicating that GBP signaling in Drosophila did not use the orthodox Toll or Imd pathway but used the JNK pathway after association with the adaptor protein Imd. The enhancement of AMP expression by non-infectious physical or environmental stressors was apparent in controls but not in GBP-knockdown larvae. These results indicate that the Drosophila GBP signaling pathway mediates acute innate immune reactions under various stresses, regardless of whether they are infectious or non-infectious.


Proceedings of the National Academy of Sciences of the United States of America | 2009

Solution structure of the silkworm βGRP/GNBP3 N-terminal domain reveals the mechanism for β-1,3-glucan-specific recognition

Kiyohiro Takahasi; Masanori Ochiai; Masataka Horiuchi; Hiroyuki Kumeta; Kenji Ogura; Masaaki Ashida; Fuyuhiko Inagaki

The β-1,3-glucan recognition protein (βGRP)/Gram-negative bacteria-binding protein 3 (GNBP3) is a crucial pattern-recognition receptor that specifically binds β-1,3-glucan, a component of fungal cell walls. It evokes innate immunity against fungi through activation of the prophenoloxidase (proPO) cascade and Toll pathway in invertebrates. The βGRP consists of an N-terminal β-1,3-glucan-recognition domain and a C-terminal glucanase-like domain, with the former reported to be responsible for the proPO cascade activation. This report shows the solution structure of the N-terminal β-1,3-glucan recognition domain of silkworm βGRP. Although the N-terminal domain of βGRP has a β-sandwich fold, often seen in carbohydrate-binding modules, both NMR titration experiments and mutational analysis showed that βGRP has a binding mechanism which is distinct from those observed in previously reported carbohydarate-binding domains. Our results suggest that βGRP is a β-1,3-glucan-recognition protein that specifically recognizes a triple-helical structure of β-1,3-glucan.


Cell and Tissue Research | 1992

Immunocytochemical localization of β-1,3-glucan recognition protein in the silkworm, Bombyx mori

Masanori Ochiai; Teruo Niki; Masaaki Ashida

SummaryA monospecific antibody against β-1,3-glucan recognition protein (a 62 kDa protein) of the larval silkworm prophenoloxidase activating system was used to study the localization of the protein. Among tissues from 5th instar larvae, only hemocytes and plasma were shown to contain a 62 kDa polypeptide immunoreactive with the antibody. Ultra-thin sections of the hemocytes were stained by an indirect immunogold staining method. Labelling occurred in the granules and cytoplasm of granulocytes and in the spherules and cytoplasm of spherulocytes. It was most conspicuous in granules of granulocytes and uniformly labelled spherules of spherulocyte, whereas no labelling was evident in prohemocytes, plasmatocytes and oenocytoids. The results are discussed in relation to the mode of recognition of fungi as non-self in insect hemocoel.


Proceedings of the National Academy of Sciences of the United States of America | 2010

Adaptor protein is essential for insect cytokine signaling in hemocytes

Yasunori Oda; Hitoshi Matsumoto; Maiko Kurakake; Masanori Ochiai; Atsushi Ohnishi; Yoichi Hayakawa

Growth-blocking peptide (GBP) is an insect cytokine that stimulates a class of immune cells called plasmatocytes to adhere to one another and to foreign surfaces. Although extensive structure-activity studies have been performed on the GBP and its mutants in Lepidoptera Pseudaletia separata, the signaling pathway of GBP-dependent activation of plasmatocytes remains unknown. We identified an adaptor protein (P77) with a molecular mass of 77 kDa containing SH2/SH3 domain binding motifs and an immunoreceptor tyrosine-based activation motif (ITAM)–like domain in the cytoplasmic region of the C terminus. Although P77 showed no capacity for direct binding with GBP, its cytoplasmic tyrosine residues were specifically phosphorylated within seconds after GBP was added to a plasmatocyte suspension. Tyrosine phosphorylation of P77 also was observed when hemocytes were incubated with Enterobactor cloacae or Micrococcus luteus, but this phosphorylation was found to be induced by GBP released from hemocytes stimulated by the pathogens. Tyrosine phosphorylation of the integrin β subunit also was detected in plasmatocytes stimulated by GBP. Double-stranded RNAs targeting P77 not only decreased GBP-dependent tyrosine phosphorylation of the integrin β subunit, but also abolished GBP-induced spreading of plasmatocytes on foreign surfaces. P77 RNAi larvae also showed significantly higher mortality than control larvae after infection with Serratia marcescens, indicating that P77 is essential for GBP to mediate a normal innate cellular immunity in insects. These results demonstrate that GBP signaling in plasmatocytes requires the adaptor protein P77, and that active P77-assisted tyrosine phosphorylation of integrins is critical for the activation of plasmatocytes.


Plant Physiology | 2015

Reexamination of Chlorophyllase Function Implies Its Involvement in Defense against Chewing Herbivores

Xueyun Hu; Satoru Makita; Silvia Schelbert; Shinsuke Sano; Masanori Ochiai; Tohru Tsuchiya; Shigeaki F. Hasegawa; Stefan Hörtensteiner; Ayumi Tanaka; Ryouichi Tanaka

A jasmonate-inducible chlorophyllase catabolizes chlorophyll upon tissue disruption to generate compounds that are toxic to insect herbivores. Chlorophyllase (CLH) is a common plant enzyme that catalyzes the hydrolysis of chlorophyll to form chlorophyllide, a more hydrophilic derivative. For more than a century, the biological role of CLH has been controversial, although this enzyme has been often considered to catalyze chlorophyll catabolism during stress-induced chlorophyll breakdown. In this study, we found that the absence of CLH does not affect chlorophyll breakdown in intact leaf tissue in the absence or the presence of methyl-jasmonate, which is known to enhance stress-induced chlorophyll breakdown. Fractionation of cellular membranes shows that Arabidopsis (Arabidopsis thaliana) CLH is located in the endoplasmic reticulum and the tonoplast of intact plant cells. These results indicate that CLH is not involved in endogenous chlorophyll catabolism. Instead, we found that CLH promotes chlorophyllide formation upon disruption of leaf cells, or when it is artificially mistargeted to the chloroplast. These results indicate that CLH is responsible for chlorophyllide formation after the collapse of cells, which led us to hypothesize that chlorophyllide formation might be a process of defense against chewing herbivores. We found that Arabidopsis leaves with genetically enhanced CLH activity exhibit toxicity when fed to Spodoptera litura larvae, an insect herbivore. In addition, purified chlorophyllide partially suppresses the growth of the larvae. Taken together, these results support the presence of a unique binary defense system against insect herbivores involving chlorophyll and CLH. Potential mechanisms of chlorophyllide action for defense are discussed.


Journal of Invertebrate Pathology | 2015

Prophenoloxidase genes and antimicrobial host defense of the model beetle, Tribolium castaneum.

Kakeru Yokoi; Yuuki Hayakawa; Daiki Kato; Chieka Minakuchi; Toshiharu Tanaka; Masanori Ochiai; Katsumi Kamiya; Ken Miura

In this study, we characterized prophenoloxidase (proPO, (PPO)) genes of Tribolium castaneum and examined their involvement in antimicrobial host defense. Amino acid sequence comparison with well-characterized PPO proteins from other insect species suggested that T. castaneum PPO genes encoded functional proenzymes, with crucial sequence motifs being conserved. Developmental kinetics of the mRNA of two PPO genes, PPO1 and PPO2 in the pupal stage were different to each other. The PPO1 mRNA levels consistently decreased during pupal development while that of PPO2 peaked at mid-pupal stage. The two mRNAs also exhibited distinct responses upon immune challenges with heat-killed model microbes. The PPO1 mRNA stayed nearly unchanged by 6h post challenge, and was somewhat elevated at 24h. In contrast, the PPO2 mRNA significantly decreased at 3, 6 and 24h post challenge. These trends exhibited by respective PPO genes were consistent irrespective of the microbial species used as elicitors. Finally, we investigated the involvement of T. castaneum PPO genes in antimicrobial host defense by utilizing RNA interference-mediated gene silencing. Survival assays demonstrated that double knockdown of PPO genes, which was accompanied by weakened hemolymph PO activities, significantly impaired the host defense against Bacillus subtilis. By contrast, the knockdown did not influence the induction of any of the T. castaneum antimicrobial peptide genes that were studied here, except for one belonging to the gene group that shows very weak or negligible microbial induction. PPO knockdown as well weakened host defense against Beauveria bassiana moderately but significantly depending on the combination of infection methods and targeted genes. Our results indicated that the PPO genes represented constituents of both antibacterial and antifungal host defense of T. castaneum.

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