Stress-derived reactive oxygen species enable hemocytes to release activator of growth blocking peptide (GBP) processing enzyme.　　　　　　　　　　　　　　　
Hitoshi Matsumoto, Masanori Ochiai, Erina Imai, Takashi Matsumura, Yoichi Hayakawa
Journal of Insect Physiology, 131, 104225, 2021年05月, ［査読有り］
英語, 研究論文（学術雑誌）

A gene-driven recovery mechanism: Drosophila larvae increase feeding activity for post-stress weight recovery
Masasuke Ryuda, Miku Tabuchi, Hitoshi Matsumoto, Takashi Matsumura, Masanori Ochiai, Yoichi Hayakawa
Archives of Insect Biochemistry and Physiology, 97, 3, e21440, John Wiley and Sons Ltd, 2018年03月01日, ［査読有り］
英語, 研究論文（学術雑誌）, Recovery from weight loss after stress is important for all organisms, although the recovery mechanisms are not fully understood. We are working to clarify these mechanisms. Here, we recorded enhanced feeding activity of Drosophila melanogaster larvae from 2 to 4 h after heat stress at 35°C for 1 h. During the post-stress period, expression levels of sweet taste gustatory receptor genes (Grs), Gr5a, Gr43a, Gr64a, and Gr64f, were elevated, whereas bitter taste Grs, Gr66a, and Gr33a, were decreased in expression and expression of a non-typical taste receptor Gr, Gr68a, was unchanged. Similar upregulation of Gr5a and downregulation of Gr66a was recorded after cold stress at 4°C. Expression levels of tropomyosin and ATP synthase ß subunit were significantly increased in larval mouth parts around 3 to 5 h after the heat stress. We infer that up-regulation of post-stress larval feeding activity, and weight recovery, is mediated by increasing capacity for mouth part muscular movements and changes in taste sensing physiology. We propose that Drosophila larvae, and likely insects generally, express an efficient mechanism to recover from weight loss during post-stress periods.

Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress
Eui Jae Sung, Masasuke Ryuda, Hitoshi Matsumoto, Outa Uryu, Masanori Ochiai, Molly E. Cook, Na Young Yi, Huanchen Wang, James W. Putney, Gary S. Bird, Stephen B. Shears, Yoichi Hayakawa
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 114, 52, 13786, 13791, NATL ACAD SCIENCES, 2017年12月, ［査読有り］
英語, 研究論文（学術雑誌）, A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed Drosophila, a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca2+ signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca2+ mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. We further report that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these various homeostatic attributes of GBP: We found that elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan. We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity.

Effects of urea and NaCl on the fibrinogen cryogelation
Yoshiharu Toyama, Masahiro Shimizu, Masanori Ochiai, Toshiaki Dobashi
Journal of Biorheology, 31, 1, 12, 15, Japanese Society of Biorheology, 2017年, ［査読有り］
英語, 研究論文（学術雑誌）, Fibrinogen is converted to fibrin monomer and forms a three-dimensional gel network in a step wise manner by the enzymatic action of thrombin (thrombin-induced fibrin gelation). On the other hand, when a fibrinogen solution is incubated at low temperatures (e.g. below 4°C), it is converted to a gel without thrombin. The fibrinogen gel induced by lowering temperature is called “cryogel” and is closely related to some diseases. However, the mechanism of fibrinogen cryogelation has not been understood yet. In this study, the effects of urea and NaCl on fibrinogen cryogelation and thrombin-induced fibrin gelation have been studied turbidimetrically. The addition of urea retarded both gelation, and the extent of retardation increased with increasing the concentration of urea. The effects of urea on fibrinogen cryogelation were much larger than those on thrombin-induced fibrin gelation, and fibrinogen cryogelation was completely inhibited by the addition of 150 mM urea. The addition of NaCl retarded fibrinogen cryogelation with increasing the concentration of NaCl. By contrast, the effects of NaCl on thrombin-induced fibrin gelation were only limited. Based on the experimental results, the contributions of hydrogen and ionic bonds to each gelation are discussed.

Group-housed females promote production of asexual ootheca in American cockroaches
Ko Katoh, Masazumi Iwasaki, Shouhei Hosono, Atsushi Yoritsune, Masanori Ochiai, Makoto Mizunami, Hiroshi Nishino
Zoological Letters, 3, 1, 3, BioMed Central Ltd., 2017年, ［査読有り］
英語, 研究論文（学術雑誌）, Background Facultative parthenogenesis, seen in many animal phyla, is a reproductive strategy in which females are able to generate offspring when mating partners are unavailable. In some subsocial and eusocial insects, parthenogenesis is often more prevalent than sexual reproduction. However, little is known about how social cooperation is linked to the promotion of parthenogenesis. The domiciliary cockroach Periplaneta americana is well-suited to addressing this issue as this species belongs to the superfamily Blattoidea, which diverged into eusocial termites and shows facultative parthenogenesis. Results We studied environmental factors that influence asexual production of ootheca using behavioral assays in P. americana. When more than three virgin females immediately after the imaginal molt were kept together in a small sealed container, they tended to produce egg cases (oothecae) via parthenogenesis earlier than did isolated females, resulting in apparent synchronization of ootheca production, even among females housed in different containers. In contrast, virgin females housed with genitalia-ablated males or group-housed females with antennae ablated did not significantly promote ootheca production compared to isolated females. Daily addition of the primary sex pheromone component to the container did not promote ootheca production in isolated females. Another line of study showed that grouped females make parthenogenesis more sustainable than previously known
a founder colony of 15 virgin females was sufficient to produce female progeny for a period of more than three years. Conclusions Group-housed females promote and stabilize asexual ootheca production compared to isolated females, and that this promotion is triggered by female-specific chemosensory signals (other than sex pheromone) primarily detected by antennae. Promotion of ootheca production between females is likely to be an early stage of social cooperation, reminiscent of the foundation and maintenance of a colony by female pairs in the eusocial termite Reticulitermes speratus.

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
JOURNAL OF INVERTEBRATE PATHOLOGY, 132, 190, 200, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2015年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 6 h post challenge, and was somewhat elevated at 24 h. In contrast, the PPO2 mRNA significantly decreased at 3, 6 and 24 h 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. (C) 2015 Elsevier Inc. All rights reserved.

Function of desiccate in gustatory sensilla of drosophila melanogaster
Takeshi Kawano, Masasuke Ryuda, Hitoshi Matsumoto, Masanori Ochiai, Yasunori Oda, Teiichi Tanimura, Gyorge Csikos, Megumi Moriya, Yoichi Hayakawa
SCIENTIFIC REPORTS, 5, 17195, NATURE PUBLISHING GROUP, 2015年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Desiccate (Desi), initially discovered as a gene expressing in the epidermis of Drosophila larvae for protection from desiccation stress, was recently found to be robustly expressed in the adult labellum; however, the function, as well as precise expression sites, was unknown. Here, we found that Desi is expressed in two different types of non-neuronal cells of the labellum, the epidermis and thecogen accessory cells. Labellar Desi expression was significantly elevated under arid conditions, accompanied by an increase in water ingestion by adults. Desi overexpression also promoted water ingestion. In contrast, a knockdown of Desi expression reduced feeding as well as water ingestion due to a drastic decrease in the gustatory sensillar sensitivity for all tested tastants. These results indicate that Desi helps protect insects from desiccation damage by not only preventing dehydration through the integument but also accelerating water ingestion via elevated taste sensitivities of the sensilla.

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 Hoertensteiner, Ayumi Tanaka, Ryouichi Tanaka
PLANT PHYSIOLOGY, 167, 3, 660, +, AMER SOC PLANT BIOLOGISTS, 2015年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 methyljasmonate, 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.

A NOVEL TYPE OF HEMOCYTES LOCALIZING MELANIZATION WITH HIGH-SPREADING BEHAVIOR IN MYTHIMNA SEPARATA
Yoshiaki Kato, Tatsuhiro Yoshida, Ken Miura, Toshiharu Tanaka, Yutaka Nakamatsu, Masanori Ochiai
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, 86, 4, 220, 239, WILEY-BLACKWELL, 2014年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Lepidopteran larvae show a cellular response to invading foreign substances that are larger than hemocytes, for example, parasitoid eggs or larvae. This response is called hemocyte encapsulation and is often accompanied by phenoloxidase (PO)-catalyzed melanization. In the present study, we artificially transplanted endoparasitoid larvae and small glass fragments into the hemocoel of the common armyworm, Mythimna separata. We observed that the host larva showed a cellular response and that, 2-4 h after transplantation, melanin formation was spatially confined to the surface of the encapsulated substances. We further noted that specific morphological hemocytes surrounded by melanin formation became attached to the surface of the foreign substances. We designated these hemocytes hyperspread cells (HSCs) on the basis of their specific characteristics and circumferential spread. We confirmed the occurrence of prophenoloxidase (PPO)/phenoloxidase (PO) on the periphery of the HSCs and in the substance secreted around the HSCs by using anti-PPO antibody. We were unable to detect PPO-mRNA in HSCs by using in situ hybridization, although we showed that oenocytoids contained PPO-mRNA and PPO protein. We used light microscopy and scanning electron microscopy to discriminate five main types of circulating M. separata hemocytes. We observed that HSCs differed from plasmatocytes, but spread out well. Further, during the encapsulation process, HSCs appeared to provide a localized melanization spot on the surface of foreign invaders. (C) 2014 Wiley Periodicals, Inc.

Analysis Of The Gene Products Related To Osseointegration In The Early Stage Of Titanium Implantation
Masataka Horiuchi, Rumi Horiuchi, Masanori Ochiai, Atsuro Yokoyama
PROTEIN SCIENCE, 23, 102, 102, WILEY-BLACKWELL, 2014年07月, ［査読有り］
英語

Immunoevasive protein (IEP)-containing surface layer covering polydnavirus particles is essential for viral infection
Shunsuke Furihata, Kohjiro Tanaka, Masasuke Ryuda, Masanori Ochiai, Hitoshi Matsumoto, Gyorge Csikos, Yoichi Hayakawa
JOURNAL OF INVERTEBRATE PATHOLOGY, 115, 26, 32, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2014年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Polydnaviruses (PDVs) are unique symbiotic viruses associated with parasitoid wasps: PDV particles are injected into lepidopteran hosts along with the wasp eggs and express genes that interfere with aspects of host physiology such as immune defenses and development. Recent comparative genomic studies of PDVs have significantly improved our understanding of their origin as well as the genome organization. However, the structural features of functional PDV particles remain ambiguous. To clear up the structure of Cotesia kariyai PDV (CkPDV) particles, we focused on immunoevasive protein (IEP), which is a mediator of immunoevasion by the wasp from the encapsulation reaction of the host insect's hemocytes, because it has been demonstrated to be present on the surface of the virus particle. We discovered that IEP tends to polymerize and constitutes a previously unidentified thin surface layer covering CkPDV particles. This outermost surface layer looked fragile and was easily removed from CkPVD particles by mechanical stressors such as shaking, which prevented CkPDV from expressing the encoded genes in the host target tissues such as fat body or hemocytes. Furthermore, we detected IEP homologue gene expression in the wasp's venom reservoirs, implying IEP has another unknown biological function in the wasp or parasitized hosts. Taken together, the present results demonstrated that female C kariyai wasps produce the fragile thin layer partly composed of IEP to cover the outer surfaces of CkPDV particles; otherwise, they cannot function as infectious agents in the wasp's host. The fact that IEP family proteins are expressed in both venom reservoirs and oviducts suggests an intimate relationship between both tissues in the development of the parasitism strategy of the wasp. (C) 2013 Elsevier Inc. All rights reserved.

A low-cost affinity purification system using ß-1,3-glucan recognition protein and curdlan beads
Horiuchi, M, Takahasi, K, Kobashigawa, Y, Ochiai, M, Inagaki, F
PEDS., 25, 8, 405, 413, OXFORD UNIV PRESS, 2012年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Silkworm -1,3-glucan recognition protein (GRP) tightly and specifically associates with -1,3-glucan. We report here an affinity purification system named the oGRP system', which uses the association between the -1,3-glucan recognition domain of GRP (GRP-tag), as an affinity tag, and curdlan beads. Curdlan is a water-insoluble -1,3-glucan reagent, the low cost of which (about 100 JPY/g) allows the economical preparation of beads. Curdlan beads can be readily prepared by solubilization in an alkaline solution, followed by neutralization, sonication and centrifugation. We applied the GRP system to preparation of several proteins and revealed that the expression levels of the GRP-tagged proteins in soluble fractions were two or three times higher than those of the glutathione S-transferase (GST)-tagged proteins. The purity of the GRP-tagged proteins on the curdlan beads was comparable to that of the GST-tagged proteins on glutathione beads. The chemical stability of the GRP system was more robust than conventional affinity systems under various conditions, including low pH (46). Biochemical and structural analyses revealed that proteins produced using the GRP system were structurally and functionally active. Thus, the GRP system is suitable for both the large- and small-scale preparation of recombinant proteins for functional and structural analyses.

Enhanced expression of stress-responsive cytokine-like gene retards insect larval growth
Koichiro Yamaguchi, Hitoshi Matsumoto, Masanori Ochiai, Seiji Tsuzuki, Yoichi Hayakawa
INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 42, 3, 183, 192, PERGAMON-ELSEVIER SCIENCE LTD, 2012年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Growth rates of immature animals are governed by their feeding activities. A reduction in feeding sometimes causes serious growth retardation in insects; a typical case is often seen in host insects parasitized by a solitary endoparasitoid wasp. However, understanding of the mechanisms underlying the physiological repression of parasitized insects is fragmentary. Here we analyzed brain gene expression of the host common cutworm, Spodoptera litura, parasitized by a solitary endoparasitoid, Microplitis manilae, and identified a novel gene whose expression was significantly enhanced by parasitization. The gene encoded a pre-pro-peptide of a cytokine-like molecule and its expression was observed mainly in nervous tissues, hemocytes, and integuments. The 25 amino acid cytokine-like peptide encoded by the C-terminus of this gene was demonstrated to exist in the hemolymph of S. litura larvae and to change hemocytes from non-adhesive to adhesive in vitro. Further, injection of the active peptide reduced feeding activities of test larvae and consequently delayed their growth. The enhanced gene expression was also observed in larvae under severe stress conditions: abdominal ligature, proleg cutting, mechanical vibration, low temperature, and heat shock at 45 C. Elevated gene expression was maintained only in seriously growth-retarded larvae but not in recovered larvae at 24 h or 48 h after heat treatment. Thus, it is reasonable to conclude that stress-induced elevation of the peptide gene expression highly correlates with reduced feeding activities and growth retardation of the host larvae parasitized by M. manilae. Based on the conclusion, we named this peptide stress-responsive peptide (SRP). (C) 2011 Elsevier Ltd. All rights reserved.

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
SCIENTIFIC REPORTS, 2, 210, NATURE PUBLISHING GROUP, 2012年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Additive effects of bestaines on the fibrinogen cryogelation induced by low temperature
Toyama,Y, Miyamoto, K, Kubota, K, Wakamatsu, K, Nameki, N, Saheki, T, Ochiai, M
Trans. MRS-J., 36, 3, 393, 396, The Materials Research Society of Japan, 2011年, ［査読有り］
英語, 研究論文（学術雑誌）, When fibrinogen solution is incubated at low temperature, it converts to gel state spontaneously without the action of thrombin. The fibrinogen gel induced by low temperature is called "cryogel" and is associated with various diseases such as thromboembolic disorders, Raynaud's disease and rheumatoid arthritis. In our previous paper, we have reported that cryogelation is inhibited by the addition of glucose and/or mannose. In this study, the additive effects of betaines on the cryogelation were examined. Betaines are known as the mild solubilization agents that prevent protein aggregation. Betaines used in this study are glycine betaine, non-detergent sulfobetaine (NDSB) -195 and Choline-O-Sulfate (COS) which is similar to NDSB-195. Bovine fibrinogen was dissolved in a phosphate buffer saline solution at the concentration of 3 mg/ml. Respective betaine was added to the fibrinogen solution at various concentrations, and turbidity measurements of those solutions were carried out over the wavelength range of 350 to 800 nm at 2°C. The characteristic quantities to the fiber structure composing the cryogel network (mass/length ratio, radius and density of the fibers) were analyzed according to the procedure employed by Carr et al. Fibrinogen cryogelation was inhibited by the addition of the betaines. Among the betaines used in this study, the order of inhibiting effect on the cryogelation was in the order of COS > NDSB-195 > glycine betaine. Fibrinogen in the presence of COS or NDSB-195 formed loose fibers with low density.

Effect of plasmin treatment on the fibrin gel formation
Yatagai,Y, Kubota, K, Toyama,Y, Nameki, N, Ochiai, M
Trans. MRS-J., 36, 3, 371, 374, The Materials Research Society of Japan, 2011年, ［査読有り］
英語, 研究論文（学術雑誌）, In order to clarify the effective regions that are substantially involved in the gelling process of plasma glycoprotein fibrinogen, we examined the aggregating properties of plasmin-treated fibrinogen, fragment-X. Two types of fragment-X were prepared by the digestion at 6°C and 37°C (fragment-XY and -XN, respectively). αC regions were cleaved thoroughly in both samples, but the amount of cleavage of BβN region differed between them (higher in the fragment-XN). Thrombin- and reptilase-catalyzed fibrin polymerizations were studied for both of the samples. Although the B-knob:b-hole interaction does neither work in the reptilase-catalyzed fibrin polymerization nor the αC-αC interaction is present, network formation proceeded in the fragment-XY with delayed polymerization. In the fragment-XN, protofibril formation occurred, but lateral aggregation did not. Mixing effect of intact fibrinogen showed that BβN region might play an important role in the lateral aggregation process cooperatively with αC-domain. .

Mixing effect of deglycosylated fibrinogen on the fibrin polymerization
Kubota, K, Yatagai,Y, Watanabe, N, Fukudal, T, Toyama,Y, Nameki, N, Ochiai, M
Trans. MRS-J., 36, 3, 375, 378, The Materials Research Society of Japan, 2011年, ［査読有り］
英語, 研究論文（学術雑誌）, The role of N-linked carbohydrate chains, bound to the polypeptide chains Bβ and γ of fibrinogen, in the process of fibrin gel formation has not been understood well yet, although it has been well known that the deglycosylation accelerates the fibrin polymerization. We investigated the time course of the fibrin polymerization employing the turbidity and light scattering measurements focusing on the role of carbohydrate chains. Deglycosylated bovine fibrinogen was prepared by using peptide N-glycosidase F, PNGF, and the effects of its mixing with the intact one were examined. Fibrinopeptide release and protofibril growth were not affected by the deglycosylation at all. However, only a slight mixing of deglycosylated fibrinogen by 5 and 10 % resulted in a markedly accelerated polymerization and the highly promoted switchover from the protofibril growth to the lateral aggregation of protofibrils. It was suggested that N-linked carbohydrate chains could interact with αC and BβN regions so as to suppress their releases from the central region of fibrinogen molecule, and play a regulating role of the switchover from the protofibril growth to the lateral aggregation.

Adaptor protein is essential for insect cytokine signaling in hemocytes
Yasunori Oda, Hitoshi Matsumoto, Maiko Kurakake, Masanori Ochiai, Atsushi Ohnishi, Yoichi Hayakawa
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 107, 36, 15862, 15867, NATL ACAD SCIENCES, 2010年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 beta subunit also was detected in plasmatocytes stimulated by GBP. Double-stranded RNAs targeting P77 not only decreased GBP-dependent tyrosine phosphorylation of the integrin beta 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.

Calmodulin Binding-Heat Shock Proteins Form a Ring Structure in the Rat Testis
Jon Nield, Megumi Moriya, Masanori Ochiai
MOLECULAR REPRODUCTION AND DEVELOPMENT, 77, 9, 738, 738, WILEY-LISS, 2010年09月, ［査読有り］
英語, 研究論文（学術雑誌）

Solution structure of the silkworm ß-GRP/GNBP3 N-terminal domain reveals the mechanism for ß-1,3-glucan specific recognition
Takahasi K, Ochiai M, Horiuchi M, Kumeta H, Ogura K, Ashida M, Inagaki F
Proc Natl Acad Sci USA, 106, 28, 11679, 11684, NATL ACAD SCIENCES, 2009年07月, ［査読有り］
英語, 研究論文（学術雑誌）, The beta-1,3-glucan recognition protein (beta GRP)/Gram-negative bacteria-binding protein 3 (GNBP3) is a crucial pattern-recognition receptor that specifically binds beta-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 beta GRP consists of an N-terminal beta-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 beta-1,3-glucan recognition domain of silkworm beta GRP. Although the N-terminal domain of beta GRP has a beta-sandwich fold, often seen in carbohydrate-binding modules, both NMR titration experiments and mutational analysis showed that beta GRP has a binding mechanism which is distinct from those observed in previously reported carbohydarate-binding domains. Our results suggest that beta GRP is a beta-1,3-glucan-recognition protein that specifically recognizes a triple-helical structure of beta-1,3-glucan.

Formation of Recombinant Fibrinogen Lacking alpha C Termini
Kenji Kuboa, Yuka Masuda, Yoshiharu Toyama, Nobukazu Nameki, Nobuo Okumura, Masanori Ochiai
GELS: STRUCTURES, PROPERTIES, AND FUNCTIONS, 136, 187, +, SPRINGER-VERLAG BERLIN, 2009年, ［査読有り］
英語, 研究論文（国際会議プロシーディングス）, In order to examine the role of alpha C domains, especially the terminal region of it, of fibrinogen A alpha chain in the fibrin gel formation, we prepared a recombinant fibrinogen, A alpha 570 fibrinogen. A alpha 570 fibrinogen is the fibrinogen that is truncated at A alpha 570 and lacks 40 amino acids at the terminus of the alpha C domain. We examined the thrombin-catalyzed polymerization by transmission spectroscopy and confocal laser scanning microscopy (CLSM). We found that A alpha 570 fibrinogen exhibited a significantly delayed aggregation showing the importance of the terminal region of the alpha C domain in the polymerization process. Contrary to the fact that the addition of glucose to the mixture of fibrinogen and thrombin results in a substantial delay of the lateral aggregation of protofibrils for the native fibrinogen, delaying effect due to the addition of glucose disappeared thoroughly in the case of A alpha 570 fibrinogen. Turbidity measurements dependent upon the wavelength in the time course of gelation showed that mass per unit fiber length of A alpha 570 fibrinogen decreased significantly compared to the native fibrinogen, and the lateral aggregation of protofibrils was hindered significantly. Those results are consistent with the CLSM measurements that the bundles of protofibrils of A alpha 570 fibrinogen are thinner and denser with more branching than those of the native one. It was confirmed that C-terminal region of the alpha C domain plays an important role in the lateral aggregation and glucose interferes the interacting process between the alpha C domains.

Heat Shock Proteins in Rat Testis as calmodulin-binding proteins　　　　　　　　　　　　　　　
Moriya, M, Ochiai, M
Trend in Developmental Biology, 3, 1, 10, 2008年, ［査読有り］
英語, 研究論文（学術雑誌）

Stereoisomeric effect of saccharides on the fibrin gel formation　　　　　　　　　　　　　　　
Yuka Masuda, Hiroyuki Kogure, Takeshi Ishii, Yoshiharu Toyama, Kaori Wakamatsu, Kenji Kubota, Masanori Ochiai
Polymer Preprints, Japan, 55, 1, 1048, 2006年
日本語, 研究論文（国際会議プロシーディングス）, The final stage of blood coagulation cascade is the fibrinogen-fibrin conversion induced by thrombin. We have reported the unique effect by the addition of various saccharides on this system. Glucose and oligosaccharides retarded the fibrin gelation, and the retarding effect becomes less significant with the increase in the number of glucopyranose units composing the saccharides. The enzymatic activity of thrombin examined by using a synthetic substrate showed no effect by saccharides, glucose and oligosaccharides. That is, these saccharides do not interact with thrombin, but do with fibrinogen and/or fibrin. In the present study, we examined the stereoisomeric effects of saccharides on the interaction with the fibrinogen. In order to investigate such an effect, light scattering and turbidity measurements were carried out.

Stereoisomer effect of disaccharide on the interaction with fibrinogen
Yuka Masuda, Hiroyuki Kogure, Takeshi Ishii, Yoshiharu Toyama, Kaori Wakamatsu, Kenji Kubota, Masanori Ochiai
Transactions of the Materials Research Society of Japan, Vol 31, No 3, 31, 3, 751, 754, ELSEVIER SCIENCE BV, 2006年, ［査読有り］
英語, 研究論文（国際会議プロシーディングス）, Stereoisomer effects of the addition of disaccharides to the fibrin gel. formation process induced by serine protease, thrombin, were investigated systematically by using light scattering combined with turbidity measurements. Three disaccharides, maltose, isomaltose, and cellobiose, were of glycoside-glucose type, and the temporal growth of fibrin gel formation in the presence of them were compared. with each other. Maltose and cellobiose retarded the fibrin gelation, and the resultant fibrin fibers were thin and loose ones. In contrast, the result in the presence,of isomaltose agreed with the control (Without dissacharides), and the fibrin gel formation and the characteristics of fibrin fibers were not affected. According to the HPLC measurements, the release of fibrinopeptides A and B were found not to be affected by the addition of those disaccharides so much. Disaccharides do not interact with thrombin, but do with fibrinogen and/or fibrin.

Effects of saccharides on fibrinogen gelation induced by low temperature
Y. Toyama, N. Kawashima, Y. Masuda, H. Kogure, K. Kubota, M. Ochiai
Transactions of the Materials Research Society of Japan, Vol 31, No 3, 31, 3, 747, 750, ELSEVIER SCIENCE BV, 2006年, ［査読有り］
英語, 研究論文（国際会議プロシーディングス）, When fibrinogen solution is incubated at a low temperature, it converts to gel state without the action of thrombin. The fibrinogen gel induced by low temperature is called "cryogel" and is associated with some diseases such as thromboembolic disorders, Raynaud's disease and rheumatoid arthritis. In order to clarify the details of fiber structure composing of cryogel network: mass/length ratio (mu), radius (r) and density (mu/r(2)) of the fibers, turbidity measurements were carried out over the wavelength range of 400 to 800 run at 2 degrees C. Furthermore, the additive effects of saccharides, glucose, mannose and dextrans with various molecular weights, on the cryogelation were examined. The addition of glucose and mannose retarded the gelation and produced very thin and loose fibers with small values of mu, r and mu/r(2). Stereo isomeric effects were observed between the two saccharides, and mannose showed the large effects than glucose. On the contrary, the gelation was enhanced by the addition of dextrans especially with high molecular weights more than 42000. These findings obtained for the cryogel were essentially consistent with those obtained for fibrin gel induced by the action of thrombin, suggesting that there may exist a common mechanism between these two different gel formations.

Effect of NDSB on the fibrinogen gelation by thrombin
Hiroyuki Kogure, Yuka Masuda, Takeshi Ishii, Kaori Wakamatsu, Kenji Kubota, Masanori Ochiai
TRANSACTIONS OF THE MATERIALS RESEARCH SOCIETY OF JAPAN, VOL 31, NO 3, 31, 3, 787, 790, ELSEVIER SCIENCE BV, 2006年, ［査読有り］
英語, 研究論文（国際会議プロシーディングス）, Fibrinogen is converted to fibrin gel by the action of thrombin. In our previous study, it has been found that the fibrin gelation was inhibited by the addition of P-cyclodextrin to the mixture of fibrinogen and thrombin. In the present work, the additive effect of non-detergent sulphobetaine (NDSB) was examined. NDSB is known to have an inhibiting effect on the aggregation of proteins. NDSB-195 was used in this work. The gel formation of fibrinogen initiated by the addition of thrombin was greatly affected by the presence of NDSB-195: in the presence of NDSB-195, transparent soft gel was formed different from the usual cloudy stiff gel. This phenomenon was characterized by dynamic light scattering (DLS), enzymatic activity, and HPLC measurements. In the light scattering measurement, scattered light intensity increased rapidly with gelation in a stepwise manner without NDSB. However, when NDSB-195 was added, no remarkable change in the scattered intensity was observed. From the enzymatic activity measurement, it was found that NDSB-195 inhibited about half of the thrombin activity. In the HPLC measurement the release of fibrinopeptides was inhibited by the addition NDSB-195. These facts suggest that NDSB-195 weakens the thrombin activity, and, in addition, inter-acts with the fibrinopeptide A and B of fibrinogen, too. As a result, transparent soft gel is formed, and the gelation is delayed.

A Spätzle-Processing Enzyme Required for Toll Signaling Activation in Drosophila Innate Immunity.
Jang IH, Chosa N, Kim SH, Nam HJ, Lemaitre B, Ochiai M, Kambris Z, Brun S, Hashimoto C, Ashida M, Brey PT, Lee WJ
Dev. Cell, 10, 1, 45, 55, CELL PRESS, 2006年01月, ［査読有り］
英語, 研究論文（学術雑誌）, The Toll receptor was originally identified as an indispensable molecule for Drosophila embryonic development and subsequently as an essential component of innate immunity from insects to humans. Although in Drosophila the Easter protease processes the pro-Spatzle protein to generate the Toll ligand during development, the identification of the protease responsible for pro-Spatzle processing during the immune response has remained elusive for a decade. Here, we report a protease, called Sp5tzle-processing enzyme (SPE), required for Toll-dependent antimicrobial response. Flies with reduced SPE expression show no noticeable pro-Spatzle processing and become highly susceptible to microbial infection. Furthermore, activated SPE can rescue ventral and lateral development in embryos lacking Easter, showing the functional homology between SPE and Easter. These results imply that a single ligand/receptor-mediated signaling event can be utilized for different biological processes, such as immunity and development, by recruiting similar ligand-processing proteases with distinct activation modes.

Effect of DNA structure on the formation of collagen–DNA complex.
Kaya, M, Toyama, Y, Kubota, K, Nodasaka, Y, Ochiai M, Nomizu, M, Nishi, N
Int. J. Biol. Macromol, 35, 1-2, 39, 46, ELSEVIER SCIENCE BV, 2005年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Using various types of DNAs prepared from plasmid DNA, complete double-stranded DNA (ds.DNA) with linear and cyclic forms and double-stranded DNA coexisting with single-stranded DNA (ss.DNA), the structure and fibrillogenesis of the collagen-DNA complex were investigated by means of turbidity, transmission I electron microscopy, and confocal laser-scanning microscopy. The rate of fibrillogenesis of the collagen-DNA complex significantly depends on the DNA structure. The structure of the fibrils formed in the complexes showed a marked difference between the ds.DNA and ss.DNA complexes with collagen. Spatial distribution of the DNA and collagen in the complexes suggests that the characteristic collagen-DNA interaction depends on the DNA forms. (c) 2004 Elsevier B.V. All rights reserved.

Effect of NDSB on protein-protein interaction　　　　　　　　　　　　　　　
Hiroyuki Kogure, Yuka Masuda, Takeshi Ishii, Mayuko Nakamura, Kaori Wakamatsu, Kenji Kubota, Masanori Ochiai
Polymer Preprints, Japan, 54, 1, 2069, 2005年
日本語, 研究論文（国際会議プロシーディングス）, NDSBs (non-detergent sulphobetaines) are a group of small zwitterionic molecules having a hydrophilic sulphobetaine group and a short hydrophobic group. Because of the small sizes of their hydrophobic groups, they do not form micelles even at high concentrations. NDSBs are known to increase the solubility and stability of proteins. We have found that NDSB's has the anti-aggregative effect on several proteins so far. In this study we examined, whether NDSB's influences the function of several enzymes. NDSB's hardly affected the activity of lysozyme and thrombin even at a concentration as high as 1.0 M. The effects of NDSB's on other enzymes will also be reported.

Stereoisomer effect of saccharides to the interaction with the fibrinogen　　　　　　　　　　　　　　　
Yuka Masuda, Hiroyuki Kogure, Takeshi Ishii, Yoshiharu Toyama, Kaori Wakamatsu, Kenji Kubota, Masanori Ochiai
Polymer Preprints, Japan, 54, 1, 1277, 2005年
日本語, 研究論文（国際会議プロシーディングス）, The final stage of blood coagulation reaction is the fibrinogen-fibrin conversion induced by thrombin. We have reported the unique effect by the addition of various saccharides on this system. Glucose and oligosaccharides retarded the fibrin gelation, and the retarding effect becomes less significant with the increase in the number of glucophyranose units composing the saccharides. In the present study, we examined the stereoisomer effect of saccharides to the interaction with the fibrinogen. In order to investigate such an effect, light scattering and turbidity measurements were carried out. Those results will be reported.

Genetic Analyses of Genus Cypripedium Found in Northern Japanese Islands and Related Species Endemic to Northeast China.
Jo, S, Ochiai M, Furuta, K, Yagi, K
J. Japan. Soc. Hort. Sci., 74, 3, 234, 241, 一般社団法人 園芸学会, 2005年, ［査読有り］
英語, 研究論文（学術雑誌）, 日本海を囲む日本列島の北部と中国の東北地方に生育しているCypripedium 属植物について, 遺伝子分析によって比較研究を行った. ホテイアツモリソウとレブンアツモリソウは日本列島で採取したもので, 中国の東北地方で採取したC . macranthos の変種または品種と考えられている. 葉緑体にあるribulose-1, 5-diphosphate decarboxylase/oxygenaseの大サブユニット (rbc L) のDNAを比較すると, 3種はまったく区別できなかった. ところが, 核DNA上にあるリボソームRNAのITS部分を比較すると, ホテイアツモリソウとレブンアツモリソウ・中国産C . macranthos との間で, 1塩基の置換が認められた. また以上3種のゲノムを鋳型にランダムプライマーOPA16を用いたRAPD電気泳動を行った結果, レブンアツモリソウにのみ430 bpのDNAフラグメントが認められた. 中国の東北地方で採取したC . calceolus のrbc LとITSの塩基配列はいずれも同じ地域で採集したC . macranthos と異なっていた. C . × ventricosum はrbc LとITSの遺伝子分析から, C . macranthos とC . calceolus の雑種であると確認された. これらのことよりrbc LとITS遺伝子の分析は, Cypripedium 属の種や変種の分類に有用であることが示された.

Identification of Ca2+-dependent calmodulin-binding proteins in rat spermatogenic cells as complexes of the heat-shock proteins
M Moriya, M Ochiai, HJ Yuasa, N Suzuki, M Yazawa
MOLECULAR REPRODUCTION AND DEVELOPMENT, 69, 3, 316, 324, WILEY-BLACKWELL, 2004年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Ca2+-calmodulin (CaM)-binding proteins in rat testes were characterized by assays for CaM-binding activity using the CaM-overlay method on transblots of electrophoresed gels and purification by gel-filtration, ion exchange, and adsorption chromatographies. A major CaM-binding protein complex (CaMBP) was identified and found to be comprised of three proteins with molecular masses 110, 100, and 70 kDa. Amino acid sequence analyses of lysylendo-peptidase digests from these proteins indicated that all of the constituents of CaMBP are very similar to the members of the heat-shock protein family, i.e., the 110-kDa protein is similar to the APG-2/94 kDa rat ischemia-responsive protein, the 100-kDa protein is similar to the rat counterpart of the mouse APG-1/94 kDa osmotic stress protein, and the 70-kDa protein is similar to the rat testis-specific major heat-shock protein (HSP70). Immunohistochemistry using anti-CaMBP and anti-CaM antibodies demonstrated that CaMBP was co-localized with CaM in the cytoplasm of pachytene spermatocytes and nuclei of round spermatids. In addition, CaMBP, but not CaM, was localized at a high level in the residual bodies of elongated spermatids. The possible relevance of CaMBP to regulation of cell cycle progression and spermatogenesis is discussed in this paper. (C) 2004 Wiley-Liss, Inc.

Effects of Mono-, Oligo- and Polysaccharides on Fibrin Gelation.　　　　　　　　　　　　　　　
Masuda, Y, Toyama, Y, Kogure, H, Kubota, K, Ochiai, M
Trans. MRS-J., 29, 3331, 3335, 2004年, ［査読有り］
英語, 研究論文（学術雑誌）

Effect of saccharides on the fibrinogen-Fibrin conversion by thrombin.　　　　　　　　　　　　　　　
Kogure, H, Kitazawa, M, Toyama, Y, Kubota, K, Ochiai, M
Trans. MRS-J., 28, 949, 952, 2003年, ［査読有り］
英語, 研究論文（学術雑誌）

Effect of DNA structure on collagen fibrillogenesis.　　　　　　　　　　　　　　　
Kaya, M, Toyama, Y, Kubota, K, Nodasaka, Y, Ochiai, M, Nomizu, M, Nishi, N
Peptide Sci., 379, 382, 2003年, ［査読有り］
英語, 研究論文（学術雑誌）

Existence of phenol oxidase in the argasid tick Ornithodoros moubata
K Kadota, E Satoh, M Ochiai, N Inoue, N Tsuji, Igarashi, I, H Nagasawa, T Mikami, FG Claveria, K Fujisaki
PARASITOLOGY RESEARCH, 88, 8, 781, 784, SPRINGER, 2002年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Phenol oxidase (PO, EC 1.10.3.1) activity was detected in the hemolymph of the fourth instar nymphs of the argasid tick, Ornithodoros moubata, with peak levels corresponding to the days before the majority of the nymphs had molted, suggestive of a protective role of PO during the ecdysial phase. Higher PO activity was detected in plasma relative to the hemolymph and was negligible in hemocytes. The concentration of the hemolymph and plasma assayed clearly influenced the level of PO activity, and was significantly reduced (P < 0.005) after treatment with 1-phenyl-2 thiourea, a specific PO inhibitor. This is the first report of the existence of PO in the hemolymph and plasma of a soft tick species. The regulation of PO activity and its precise role in soft tick immunity, particularly during the ecdysial phase, are interesting and need to be examined further.

Purification and characterization of biliverdin-binding vitellogenin from the hemolymph of the common cutworm, Spodoptera litura
K Maruta, T Yoshiga, C Katagiri, M Ochiai, S Tojo
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, 50, 2, 97, 106, WILEY-LISS, 2002年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Biliverdin-binding vitellogenin (Vg) was purified from adult female hemolymph of the common cutworm, Spodoptera litura, by using gel filtration and ion exchange chromatographies. The molecular mass of the protein was 490 kDa and it was composed of two 188-kDa subunits. Three internal amino acid sequences obtained by digestion of the protein with lysylendopeptidase showed high similarity to those of Bombyx mori Vg, supporting the purified blue protein to be vitellogenin. latroscan analyses demonstrated the presence of biliverdin in Vg that occupied 2.4% of total lipid components. Among the lipids of Vg (9.5 mug total lipids per 100 mug protein), diacylglycerol was the most predominant, followed by phospholipid, hydrocarbons, and then triacylglycerol, while in biliverdin-binding proteins (BPs) purified from larval hemolymph (3.1 mug total lipids per 100 mug protein), phospholipid was the most abundant lipid followed by diacylglycerol; hydrocarbons and triacylglycerol were minor components. Vg was first defected in the hemolymph of female pupae one day before eclosion, but injection of 5 mug of methoprene into a 3-day-old pupa induced Vg in the hemolymph 4 days earlier than in the control. Methoprene also induced a faster decline in BP-A and BP-B titers in the hemolymph with a corresponding increase of the Vg titer. These results suggest that juvenile hormone (JH) induces not only vitellogenesis but also the uptake of these proteins by stimulating the metamorphosis of fat body during the pupal stage. (C) 2002 Wiley-liss, Inc.

Purification and characterization of biliverdin-binding protein from larval hemolymph of the swallowtail butterfly, Papilio xuthus L.
A Yamanaka, T Ito, D Koga, T Sato, M Ochiai, K Endo
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 64, 9, 1978, 1981, TAYLOR & FRANCIS LTD, 2000年09月, ［査読有り］
英語, 研究論文（学術雑誌）, The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The N-amino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.

A pattern-recognition protein for ß-1,3-glucan – The binding domain and the cDNA cloning of ß-1,3-glucan recognition protein from the silkworm, Bombyx mori.
Ochiai, M, Ashida, A
J. Biol. Chem., 275, 7, 4995, 5002, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2000年02月, ［査読有り］
英語, 研究論文（学術雑誌）, The beta-1,S-glucan recognition protein (beta GRP) has strong specific affinity for beta-1,3-glucan, a component of the fungal cell wall. Its interaction with beta-1,3-glucan initiates the activation of the prophenoloxidase cascade, which is an important defense system in invertebrates of many species. We cloned the cDNA of the beta GRP of the silkworm Bombyx mori. The beta GRP mRNA transcript was constitutively expressed in the hemocytes, fat body, and epithelial cells of the naive silkworm. At the same time, a bacterial or yeast challenge was indicated to intensify the transcription, Comparison of the deduced amino acid sequence with known sequences revealed that the beta GRP contained a region (Thr(264) to Pro(386)) displaying significant similarity to the catalytic regions of bacterial beta-1,3-glucanases and much higher similarity to the glucanase-like regions of Gram-negative bacteria-binding proteins found in the silkworm B, mori and the mosquito Anopheles gambiae, The region (Thr(264) to Pro(386)) Of the beta GRP, however, was demonstrated not to have appreciable affinity for beta-1,3-glucan. A recombinant peptide corresponding to an N-terminal region (Tyr(1) to Ala(102)) of the beta GRP bound strongly to beta-1,3-glucan. These results indicate that the binding domain of the beta GRP for beta-1,3-glucan is located in the N-terminal region. Glucanases and the current pattern-recognition proteins that contain a glucanase-like region seem to have a common origin in their molecular evolution.

A pattern-recognition protein for peptidoglycan – Cloning the cDNA and the gene of the silkworm, Bombyx mori.
Ochiai, M, Ashida, A
J. Biol. Chem., 274, 17, 11854, 11858, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 1999年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 putative cis-regulatory elements similar to the NF-kappa B-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.

Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori – Purification, characterization, and cDNA cloning.
Satoh, D, Horii, A, Ochiai, M, Ashida, M
J. Biol. Chem., 274, 11, 7441, 7453, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 1999年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 Lys(152) and Ile(153), The homology search showed 36.9% identity of PPAE to easter, which is a serine protease involved in dorsoventral pattern formation in the Drosophila 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.

Identification of chondromodulin I as a novel endothelial cell growth inhibitor - Purification and its localization in the avascular zone of epiphysical cartilage
Y Hiraki, H Inoue, K Iyama, A Kamizono, M Ochiai, C Shukunami, S Iijima, F Suzuki, J Kondo
JOURNAL OF BIOLOGICAL CHEMISTRY, 272, 51, 32419, 32426, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 1997年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Cartilage is unique among tissues of mesenchymal origin in that it is resistant to vascular invasion due to an intrinsic angiogenic inhibitor, During endochondral bone formation, however, calcified cartilage formed in the center of the cartilaginous bone rudiment allows vascular invasion, which initiates the replacement of cartilage by bone, The transition of cartilage from the angioresistant to the angiogenic status thus plays a key role in bone formation, However, the molecular basis of this phenotypic transition of cartilage has been obscure, We report here purification of an endothelial cell growth inhibitor from a guanidine extract of bovine epiphyseal cartilage, The N-terminal amino acid sequence indicated that the inhibitor was identical to chondromodulin I (ChM-I), a cartilage-specific growth-modulating factor, Purified ChM-I inhibited DNA synthesis and proliferation of vascular endothelial cells as well as tube morphogenesis in vitro, Expression of ChM-I cDNA in COS7 cells indicated that mature ChM-I molecules were secreted from the cells after post-translational modifications and cleavage from the transmembrane precursor at the predicted processing signal, Recombinant ChM-I stimulated DNA synthesis and proteoglycan synthesis of cultured growth plate chondrocytes, but inhibited tube morphogenesis of endothelial cells, In situ hybridization and immunohistochemical studies indicated that ChM-I is specifically expressed in the avascular zone of cartilage in developing bone, but not present in calcifying cartilage, These results suggest a regulatory role of ChM-I in vascular invasion during endochondral bone formation.

MOLECULAR-CLONING OF INSECT PRO-PHENOL OXIDASE - A COPPER-CONTAINING PROTEIN HOMOLOGOUS TO ARTHROPOD HEMOCYANIN
T KAWABATA, Y YASUHARA, M OCHIAI, S MATSUURA, M ASHIDA
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 92, 17, 7774, 7778, NATL ACAD SCIENCES, 1995年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] is present in the hemolymph plasma of the silkworm Bombyx mori. Pro-PO is a heterodimeric protein synthesized by hemocytes, A specific serine proteinase activates both subunits through a limited proteolysis. The amino acid sequences of both subunits were deduced from their respective cDNAs; amino acid sequence homology between the subunits was 51%. The deduced amino acid sequences revealed domains highly homologous to the copper-binding site sequences (copper-bidding sites A and B) of arthropod hemocyanins. The overall sequence homology between silkworm pro-PO and arthropod hemocyanins ranged from 29 to 39%, Phenol oxidases from prokaryotes, fungi, and vertebrates have sequences homologous to only the copper-binding site B of arthropod hemocyanins, Thus, silkworm pro-PO DNA described here appears distinctive and more closely related to arthropod hemocyanins, The pro-PO-activating serine proteinase was shown to hydrolyze peptide bonds at the carboxyl side of arginine in the sequence -Asn-49-Arg-50-Phe-51-Gly-52- of both subunits. Amino groups of N termini of both submits were indicated to be N-acetylated. The cDNAs of both pro-PO submits lacked signal peptide sequences, This result supports our contention that mature pro-PO accumulates in the cytoplasm of hemocytes and is released by cell rupture, as for arthropod hemocyanins.

A SERINE-PROTEASE ZYMOGEN IN INSECT PLASMA - PURIFICATION AND ACTIVATION BY MICROBIAL CELL-WALL COMPONENTS
Y KATSUMI, H KIHARA, M OCHIAI, M ASHIDA
EUROPEAN JOURNAL OF BIOCHEMISTRY, 228, 3, 870, 877, SPRINGER VERLAG, 1995年03月, ［査読有り］
英語, 研究論文（学術雑誌）, A protease zymogen present in the plasma fraction of the hemolymph of silkworm, Bombyx mori, was purified to homogeneity as judged by SDS/PAGE and IEF/PAGE. An activating system for the zymogen was also isolated from the plasma fraction and was shown to be triggered by zymosan (yeast cell wall polysaccharide containing beta-1,3-glucan) or peptidoglycan. Using this system, the purified zymogen was activated and the active enzyme was purified to homogeneity. The physiological function of the zymogen or its active form is not yet known, but the active form was shown to have narrower substrate specificity than trypsin. Among 33 peptide derivatives examined, Boc-Gln-Arg-Arg-NH-Mec and Boc-Val-Pro-Arg-NH-Mec (Boc = tert-butoxycarbonyl, NH-Mec = 4-methylcoumaryl-7-amide) were the best and the second best substrates, respectively. The purified zymogen was determined to be a 39-kDa protein consisting of a single polypeptide. The active form of the zymogen was labeled with [H-3]diisopropylfluorophosphate and was completely inactivated by (p-amidinophenyl)methanesulfonyl fluoride. The molecular mass of the [H-3]-labeled enzyme was determined to be 38 kDa in SDS/PAGE under reducing conditions. These results indicate that the 39-kDa protein purified in the present study is a zymogen of a serine-type protease and that the activation of the zymogen occurs by limited proteolysis.

ACTIVATION OF THE ZYMOGEN OF HEPATOCYTE GROWTH-FACTOR ACTIVATOR BY THROMBIN
T SHIMOMURA, J KONDO, M OCHIAI, D NAKA, K MIYAZAWA, Y MORIMOTO, N KITAMURA
JOURNAL OF BIOLOGICAL CHEMISTRY, 268, 30, 22927, 22932, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 1993年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Hepatocyte growth factor activator (HGF activator) is a serine protease which converts single-chain HGF to the active two-chain form. HGF activator purified from human serum has a molecular mass of 34 kDa and consists of two chains held together by a disulfide bond. The nucleotide sequence of HGF activator cDNA shows that HGF activator is derived from the COOH-terminal region of a precursor of 655 amino acids by proteolytic cleavage of the bonds between Arg372 and Val373 and between Arg407 and Ile408 and that the precursor consists of multiple domains homologous to those observed in blood coagulation factor XII. In this study, we identified the precursor of HGF activator in human plasma using an enzyme-linked immunosorbent assay system. The precursor was purified from plasma by a five-step procedure. The purified precursor did not activate single-chain HGF. The precursor was efficiently cleaved in vitro by thrombin, at the bond between Arg407 and Ile408, in the presence of negatively charged substances. The cleaved precursor activated single-chain HGF. These findings led us to conclude that HGF activator is present in plasma as an inactive zymogen and that the zymogen is activated by the cleavage of the bond between Arg407 and Ile408 by thrombin. Characteristic structural domains in the NH2-terminal region of the zymogen may be involved in the binding of the zymogen to negatively charged substances, which stimulates the activation of the zymogen by thrombin.

Immunocytochemical localization of ß-1,3-glucan recognition protein in the silkworm, Bombyx mori.
Ochiai, M, Niki, T, Ashida, M
Cell Tissue Res., 268, 3, 431, 437, SPRINGER VERLAG, 1992年06月, ［査読有り］
英語, 研究論文（学術雑誌）, A monospecific antibody against beta-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.

A NOVEL PROTEASE OBTAINED FROM FBS-CONTAINING CULTURE SUPERNATANT, THAT PROCESSES SINGLE CHAIN FORM HEPATOCYTE GROWTH-FACTOR TO 2 CHAIN FORM IN SERUM-FREE CULTURE
T SHIMOMURA, M OCHIAI, J KONDO, Y MORIMOTO
CYTOTECHNOLOGY, 8, 3, 219, 229, KLUWER ACADEMIC PUBL, 1992年, ［査読有り］
英語, 研究論文（学術雑誌）, The recombinant human hepatocyte growth factor (r-hHGF) produced by Chinese hamster ovary cells transfected with hHGF cDNA (CHO BD-24 cells) was the two chain form in fetal bovine serum (FBS) containing culture. However, in serum-free culture the non-processed r-hHGF, single chain form, was detected with two chain form r-hHGF. We purified the protease that proteolytically processed single chain r-hHGF to two chain form r-hHGF. A protease was purified to give a single peak from the culture supernatant by use of several column chromatographies. When this protease was added to serum-free culture of CHO BD-24 cells, the proteolytic processing of single chain r-hHGF to two chain form r-hHGF was completely achieved. This protease was found to be composed of two peptide chains with molecular mass of 38 kDa under non-reducing condition by SDS-PAGE. The results of N-terminal amino acid sequence analysis and inhibitor selectivity suggested that this protease was a novel serine protease originating from fetal bovine serum.

Purification of a ß-1,3-glucan recognition protein in the prophenoloxidase activating system from hemolymph of the silkworm, Bombyx mori.
Ochiai, M, Ashida, M
J. Biol. Chem., 263, 24, 12056, 12062, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 1988年08月, ［査読有り］
英語, 研究論文（学術雑誌）

IMMUNOLOCALIZATION OF PROPHENOLOXIDASE AMONG HEMOCYTES OF THE SILKWORM, BOMBYX-MORI
M ASHIDA, M OCHIAI, T NIKI
TISSUE & CELL, 20, 4, 599, 610, CHURCHILL LIVINGSTONE, 1988年, ［査読有り］
英語, 研究論文（学術雑誌）

ß-1,3-Glucan receptor and peptidoglycan receptor are present as separate entities within insect prophenoloxidase activating system
Yoshida, H, Ochiai, M, Ashida, M
Biochem. Biophys. Res. Commun., 141, 3, 1177, 1184, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 1986年12月, ［査読有り］
英語, 研究論文（学術雑誌）

NADPH再生系を伴ったアゾレダクターゼによるアゾ染料分解反応のNMR解析
堀内正隆, 永田崇, 片平正人, 小橋川敬博, 鈴木定彦, 落合正則, 日本分子生物学会年会プログラム・要旨集(Web), 41st, 2018年

Development of the artificial multi-domain enzymes immobilized on the curdlan sheet at low cost.　　　　　　　　　　　　　　　
Masataka Horiuchi, Takashi Nagata, Masato Katahira, Yoshihiro Kobashigawa, Yasuhiko Suzuki, Masanori Ochiai, The 8th International Symposium of Advanced Energy Science, Kyoto Univ., Uji, 2017.9.5-6, 2017年09月
英語, 研究発表ペーパー・要旨（国際会議）

Low-cost degradation of azo dyes using the GRP-tagged azoreductase combined with NADPH regeneration enzymes immobilized on the curdlan sheet　　　　　　　　　　　　　　　
Masataka Horiuchi, Yasuhiko Suzuki, Masanori Ochiai, Takashi Nagata, Masato Katahira, The 7th International Symposium of Advanced Energy Science.京都大学（宇治市）,2016.9.5-9.7, 2016年09月
英語, 研究発表ペーパー・要旨（国際会議）

GRP-tag化酵素を利用したアゾ染料分解システムの構築
堀内正隆, 鈴木定彦, 落合正則, 日本生化学会大会(Web), 88th, 2015年

カイコの微生物感染によるメラニン形成と異物認識　　　　　　　　　　　　　　　
落合 正則, 蚕糸・昆虫バイオテック, 84, 3, 195, 204, 2015年, ［査読有り］, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

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堀内留美, 堀内正隆, 平田恵理, 落合正則, 横山敦郎, チタンと歯科臨床, 10, 1, 2012年

β-1,3-glucan recognition proteinの分泌機構を利用した昆虫細胞培地からの組換え分泌タンパク質精製システム
HORIUCHI Masataka, TAKAHASI Kiyohiro, OCHIAI Masanori, INAGAKI Fuyuhiko, 日本分子生物学会年会プログラム・要旨集(Web), 34th, 2011年

オッセオインテグレーション獲得および非獲得ラットモデルにおける遺伝子の網羅的解析
堀内留美, 堀内正隆, 平田恵理, 落合正則, 横山敦郎, 日本バイオマテリアル学会大会予稿集, 33rd, 2011年

カイコβGRP/GNBP3のN末端ドメインの構造解析
高橋清大, 落合正則, 芦田正明, 稲垣冬彦, 日本蛋白質科学会年会プログラム・要旨集, 9th, 39, 2009年04月24日
日本語

Cold adaptation of New Zealand weta and their lipids
Masazumi Iwasaki, Yukako Gotoh, Masanori Ochiai, Chihiro Katagiri, COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, 151, 4, 452, 453, 2008年12月
ELSEVIER SCIENCE INC, 英語, 研究発表ペーパー・要旨（国際会議）

カイコbeta‐1,3‐glucan recognition proteinの構造解析
高橋清大, 落合正則, 久米田博之, 堀内正隆, 小椋賢治, 稲垣冬彦, 生化学, 4T24-2, 2008年
日本語

Purification and characterization of silkworm paralytic peptide processing enzyme from hemolymph
Hitoshi Matsumoto, Masanori Ochiai, Manabu Kamimura, Yoichi Hayakawa, ZOOLOGICAL SCIENCE, 22, 12, 1497, 1497, 2005年12月
ZOOLOGICAL SOC JAPAN, 英語, 研究発表ペーパー・要旨（国際会議）

GRAM-NEGATIVE BACTERIA-BINDING PROTEIN IS AN ESSENSIAL COMPONENT OF THE PEPTIDOGLYCAN-MEDIATED PATHWAY IN THE PROPHENOLOXIDASE CASCADE(Biochemistry)Proceedings of the Seventy-First Annual Meeting of the Zoological Society of Japan :
Ochiai M., Ashida M., Zoological science, 17, 49, 49, 2000年
Zoological Society of Japan, 英語

LIMITED PROTEOLYSIS OF DROSOPHILA SPATZLE PROTEIN BY A SERINE PROTEASE(BAEEase) IN PLASMA OF THE SILKWORM, B.MORI(Biochemistry)Proceedings of the Seventy-First Annual Meeting of the Zoological Society of Japan :
Chosa N., Yamao M., Ochiai M., Ashida M., Zoological science, 17, 50, 50, 2000年
Zoological Society of Japan, 英語

CHARACTERIZATION OF A SERINE PROTEASE ZYMOGEN (proBAEEase) IN PLASMA OF THE SILKWORM, BOMBYX MORI(Biochemistry)(Proceedings of the Seventieth Annual Meeting of the Zoological Society of Japan) :
Chosa N., Ochiai M., Ashida M., Zoological science, 16, 44, 44, 1999年
Zoological Society of Japan, 英語

PROPHENOLOXIDASE ACTIVATING ENZYME IN EGGS OF THE SILKWORM, BOMBYX MORI(Biochemistry)(Proceedings of the Seventieth Annual Meeting of the Zoological Society of Japan) :
Horii A., Ochiai M., Ashida M., Zoological science, 16, 44, 44, 1999年
Zoological Society of Japan, 英語

β-1,3-GLUCAN RECOGNITION PROTEIN II FROM THE SILKWORM, Bombyx mori(Biochemistry)(Proceedings of the Seventieth Annual Meeting of the Zoological Society of Japan) :
Ochiai M., Ashida M., Zoological science, 16, 44, 44, 1999年
Zoological Society of Japan, 英語

cDNA CLONING OF A SERIN PROTEASE ZYMOGEN IN PLASMA OF THE SILKWORM, BOMBYX MORI(Biochemistry)(Proceedings of the Sixty-Ninth Annual Meeting of the Zoological Society of Japan) :
Chosa N., Ochiai M., Ashida M., Zoological science, 15, 43, 43, 1998年
Zoological Society of Japan, 英語

PEPTIDOGLYCAN RECOGNITION PROTEIN OF SILKWORM, Bombyx mori IS INDUCED UPON BACTERIAL CHALLENGE(Biochemistry)(Proceedings of the Sixty-Ninth Annual Meeting of the Zoological Society of Japan) :
Ochiai M., Ashida M., Zoological science, 15, 43, 43, 1998年
Zoological Society of Japan, 英語

LOCALIZATION OF [BETA-], 3-GLUCAN RECEPTOR IN HEMOLYMPH OF SILKWORM, BOMBYX-MORI
M OCHIAI, M ASHIDA, ZOOLOGICAL SCIENCE, 4, 6, 1026, 1026, 1987年12月
ZOOLOGICAL SOC JAPAN, 英語, 研究発表ペーパー・要旨（国際会議）

PURIFICATION AND CHARACTERIZATION OF BETA-1, 3-GLUCAN RECEPTOR IN HEMOLYMPH OF SILKWORM, BOMBYX-MORI
M OCHIAI, M ASHIDA, ZOOLOGICAL SCIENCE, 3, 6, 1015, 1015, 1986年12月
ZOOLOGICAL SOC JAPAN, 英語, 研究発表ペーパー・要旨（国際会議）

動物の事典
末光, 隆志, 第11章 動物の生息環境 11.9.1 寒冷耐性
朝倉書店, 2020年11月, 9784254171662, xxiii, 746p, 日本語, ［分担執筆］

低温環境の科学事典　　　　　　　　　　　　　　　
落合 正則, 第7章 8. 昆虫の耐寒性
朝倉書店, 2016年, ［分担執筆］

低温科学便覧　　　　　　　　　　　　　　　
落合 正則, 第14章 昆虫の環境ストレス応答
丸善出版, 2015年, ［分担執筆］

カイコ幼虫外皮の創傷時に放出されるサイトカイン　　　　　　　　　　　　　　　
落合正則
令和２年度蚕糸・昆虫機能利用学術講演会, 2020年03月, 口頭発表（一般）

NADPH再生系を内包するハイブリッド型アゾレダクターゼの開発　　　　　　　　　　　　　　　
堀内 正隆, 永田 崇, 片平 正人, 小橋川 敬博, 鈴木 定彦, 落合 正則
第42回 日本分子生物学会年会, 2019年12月04日, ポスター発表

ニカメイガ越冬幼虫における氷結晶成長抑制物質の探索　　　　　　　　　　　　　　　
泉洋平, 佐﨑元, 落合正則
日本昆虫学会第79回大会, 2019年09月15日

Continuous degradation of azo dyes using a hybrid azoreductase including NADPH regeneration system　　　　　　　　　　　　　　　
Horiuchi M, Nagata T, Katahira M, Kobashigawa Y, Suzuki Y, Ochiai M
The 10th International Symposium of Advanced Energy Science., 2019年09月05日

昆虫の自然免疫とメラニン形成系　　　　　　　　　　　　　　　
落合 正則
公益財団法人 科学技術交流財団 第6回メラニン機能科学研究会, 2019年03月11日, 日本語, 口頭発表（招待・特別）
［国内会議］

Role of αC-domain in fibrinogen cryogelation　　　　　　　　　　　　　　　
Ito Y, Dobashi T, Ochiai M, Toyama Y
5th International Symposium of Gunma University Medical Innovation and 9th International Conference on Advanced Micro-Device Engineering, 2018年12月06日, 英語, シンポジウム・ワークショップパネル（公募）
［国際会議］

モンシロチョウ成虫における山口および札幌個体群間の毛状鱗粉　　　　　　　　　　　　　　　
山中 明, 勇村悠介, 北沢千里, 落合正則
日本動物学会第89回, 2018年09月13日, 日本語, 口頭発表（一般）
［国内会議］

フィブリノゲンクライオゲル形成に与えるカルシウムイオンの影響　　　　　　　　　　　　　　　
伊藤 優吾, 外山 吉治, 土橋 敏明, 落合 正則
第65回レオロジー討論会, 2017年10月18日, 日本語, 口頭発表（一般）
［国内会議］

カイコ幼虫における傷害関連分子パターン(DAMPs)の探索　　　　　　　　　　　　　　　
石川 晴菜, 落合 正則
日本動物学会第88回大会, 2017年

ジャコウアゲハにおける短日および長日休眠蛹のホルモン応答性の比較　　　　　　　　　　　　　　　
酒井 勇輝, 河 秀平, 北沢 千里, 落合 正則, 山中 明
日本動物学会第88回大会, 2017年

Development of the artificial multi-domain enzymes immobilized on the curdlan sheet at low cost.　　　　　　　　　　　　　　　
Horiuchi M, Nagata A, Katahira M, Kobashigawa Y, Suzuki Y, Ochiai M
The 8th International Symposium of Advanced Energy Science, 2017年

モンシロチョウ成虫における毛状鱗粉形成の内分泌調節機構　　　　　　　　　　　　　　　
勇村 悠介, 前平 剛史, 落合 正則, 北沢 千里, 山中 明
第61回日本応用動物昆虫学会, 2017年

Seasonal plasticity of the small copper butterfly Lycaena phlaeas daimio Seitz.　　　　　　　　　　　　　　　
Yamanaka A, Masumoto Y, Abe N, Ochiai M, Kitazawa C
XXV international Congress of Entomology, 2016年

ルリタテハ成虫の季節型の違いによる翅の紋様と形状の解析　　　　　　　　　　　　　　　
平木 佳奈, 山本 響, 北沢 千里, 落合 正則, 山中 明
日本農芸化学会中四国支部第44回講演会, 2016年

ヒメアカタテハの蛹体色の白色化に関する内分泌学的研究　　　　　　　　　　　　　　　
長谷川 尋士, 林 絵里, 河崎 秀平, 森岡 律, 渕上 惣一郎, 北沢 千里, 落合 正則, 山中 明
日本農芸化学会中四国支部第44回講演会, 2016年

GRP-tagを用いた低コストなアゾ染料分解システムの構築　　　　　　　　　　　　　　　
堀内 正隆, 鈴木 定彦, 落合 正則
第88回日本生化学会大会, 2015年

数種のチョウ成虫の翅に生じる毛状鱗粉と季節型との関連性　　　　　　　　　　　　　　　
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第59回日本応用動物昆虫学会, 2015年

Analysis of the gene products related to osseointegration in the early stage of titanium implantation　　　　　　　　　　　　　　　
Horiuchi M, Horiuchi R, Ochiai M, Yokoyama A
The 28th Annual Symposium of the Protein Society, 2014年07月27日, 英語
［国際会議］

宿主アワヨトウ幼虫 からのカリヤコマユバチ脱出誘導因子の発見　　　　　　　　　　　　　　　
砂山 貴志, 内川 雄貴, 落合 正則, 早川 洋一
第58回日本応用動物昆虫学会, 2014年

ベニシジミ成虫の毛状鱗粉形成に及ぼす日長と温度の影響　　　　　　　　　　　　　　　
山中明, 益本祐希, 落合正則, 北沢千里
第58回日本応用動物昆虫学会, 2014年

シアル酸切除は高イオン強度下でのフィブリン凝集を加速する　　　　　　　　　　　　　　　
第23回日本MRS年次大会, 2013年

高イオン強度下でのフィブリン凝集に対するシアル酸添加効果　　　　　　　　　　　　　　　
第23回日本MRS年次大会, 2013年

N結合型糖鎖とフィブリノーゲン分解産物との相互作用解析　　　　　　　　　　　　　　　
第23回日本MRS年次大会, 2013年

フィブリン重合におけるBβ鎖N末端領域の抑制効果　　　　　　　　　　　　　　　
第23回日本MRS年次大会, 2013年

ベニシジミ成虫の表現型可塑性について　　　　　　　　　　　　　　　
平成25年度日本応用動物昆虫学会・日本昆虫学会中国支部号例会, 2013年

カイコの微生物に対する生体防御機構　　　　　　　　　　　　　　　
第65回日本衛生動物学会大会・病害動物の生理分子生物談話会, 2013年

Possible involvement of chlorophyllase in a plant defense system　　　　　　　　　　　　　　　
第54回日本植物生理学会, 2013年

エビガラスズメ真皮細胞に存在する色素結合タンパク質凝集成分Xの性状について　　　　　　　　　　　　　　　
日本蚕糸学会支部合同大会, 2012年

ジャコウアゲハの帯糸の黒色化機構の解析　　　　　　　　　　　　　　　
日本農芸化学会中四国支部大会, 2012年

微生物非感染時に自然免疫を活性化するサイトカイン　　　　　　　　　　　　　　　
日本動物学会第83回大会, 2012年

チタンインプラントにおけるオッセオインテグレーション獲得に関する遺伝子の網羅的解析　　　　　　　　　　　　　　　
第25回歯科チタン学会学術講演会, 2012年

Low-cost recombinant protein immobilization and purification system using β-1,3-glucan recognition protein from insect cells　　　　　　　　　　　　　　　
第34回日本分子生物学会, 2011年

オッセオインテグレーション獲得および非獲得ラットモデルにおける遺伝子の網羅的解析　　　　　　　　　　　　　　　
第33回日本バイオマテリアル学会, 2011年

Activation of the Prophenoloxidase Cascade in Hemocytes of the Locust, Locusta migratoria.　　　　　　　　　　　　　　　
Sixth International Symposium on Molecular Insect Science, 2011年

ジャコウアゲハの帯糸の黒色化に関わる因子　　　　　　　　　　　　　　　
日本動物学会第82回大会, 2011年

カイコのアポリポホリンIIIによるセクロピン抗菌活性の増強作用　　　　　　　　　　　　　　　
日本動物学会第82回大会, 2011年

フィブリン凝集に対するN-結合糖鎖の部分的切除効果　　　　　　　　　　　　　　　
第60回高分子年次大会, 2011年

フィブリン重合におけるフィブリノペプチドリリースの解析　　　　　　　　　　　　　　　
第60回高分子年次大会, 2011年

フィブリノゲンクライオゲル形成へのベタインの添加効果　　　　　　　　　　　　　　　
第20回日本MRS学術シンポジウム, 2010年

フィブリンゲル形成に対するプラスミン処理効果　　　　　　　　　　　　　　　
第20回日本MRS学術シンポジウム, 2010年

フィブリンゲル形成に対する糖鎖除去フィブリノゲンの混合効果　　　　　　　　　　　　　　　
第20回日本MRS学術シンポジウム, 2010年

GRP-tag融合蛋白質のカードランによる低コストなアフィニティー精製法の開発　　　　　　　　　　　　　　　
第33回日本分子生物学会年会、第83回日本生化学会大会 合同大会, 2010年

Deglycosylation Effect on the Fibrin Gel Formation　　　　　　　　　　　　　　　
International Union of Materials Research Societies 11th International Conference, 2010年

フィブリノーケンFragment-Xの凝集特性　　　　　　　　　　　　　　　
第59回高分子討論会, 2010年

水晶振動子マイクロバランスを用いたフィブリノゲン及びフィブリン集合過程の測定　　　　　　　　　　　　　　　
第33回日本バイオレオロジー学会年会, 2010年

昆虫の液性生体防御系と異物認識　　　　　　　　　　　　　　　
第19回WSフォーラム タンパク質・ペプチド研究の現状と展望, 2009年

カイコß-1,3-グルカン認識蛋白質の認識ドメインの立体構造　　　　　　　　　　　　　　　
日本動物学会第80回大会, 2009年

カイコのアポリポホリン-IIIの自然免疫における役割　　　　　　　　　　　　　　　
日本動物学会第80回大会, 2009年

フィブリノゲンクライオゲル形成に与える付加糖鎖の影響　　　　　　　　　　　　　　　
第32回日本バイオレオロジー学会年会, 2009年

フィブリノゲンクライオゲル形成に与えるフラグメントX及びD添加の影響　　　　　　　　　　　　　　　
第32回日本バイオレオロジー学会年会, 2009年

カイコ ßGRP/GNBP3のN末端ドメインの構造解析　　　　　　　　　　　　　　　
第9回日本蛋白質科学会年会, 2009年

カイコ ß-1,3-glucan recognition proteinの構造解析　　　　　　　　　　　　　　　
第31回日本分子生物学会年会、第81回日本生化学会大会 合同大会, 2008年

トノサマバッタ血球細胞のフェノ−ル酸化酵素前駆体活性化系
日本動物学会第79回大会, 2008年

ニュージーランド固有昆虫種ウェタのlipophorin　　　　　　　　　　　　　　　
日本動物学会第79回大会, 2008年

精子形成細胞のカルシュウム・カルモジュリン結合性タンパク質；電子顕微鏡による観察　　　　　　　　　　　　　　　
日本動物学会第79回大会, 2008年

フィブリノーゲンクライオゲル形成に与える糖添加の効果　　　　　　　　　　　　　　　
第30回バイオレオロジー学会年会, 2007年, ポスター発表

Biochemical analysis of lipophorin in tree weta, Hemideina femorata.　　　　　　　　　　　　　　　
2nd International Symposium on the Environmental Physiology of Ectotherms and Plants, 2007年, ポスター発表

カイコのフェノ−ル酸化酵素前駆体活性化系におけるFactor H の役割　　　　　　　　　　　　　　　
日本動物学会第78回大会, 2007年

フィブリンゲル形成に及ぼす糖の異性体効果　　　　　　　　　　　　　　　
第55回高分子学会年次大会, 2006年

カイコフェノ−ル酸化酵素前駆体活性化系のモザイク構造をもつ新規プロテアーゼ前駆体　　　　　　　　　　　　　　　
日本動物学会第77回大会, 2006年

Functional role of prophenoloxidase cascade of the silkworm, Bombyx mori　　　　　　　　　　　　　　　
Symposium on Pathogen Recognition and Signal Transduction in Innate Immunity, 2005年

フィブリノーゲンとの相互作用における糖の異性体効果　　　　　　　　　　　　　　　
第54回高分子学会年次大会, 2005年, ポスター発表

NDSBsが蛋白質—蛋白質相互作用に及ぼす効果　　　　　　　　　　　　　　　
第54回高分子学会年次大会, 2005年

フェノ−ル酸化酵素前駆体活性化系のFactor Sの役割　　　　　　　　　　　　　　　
文部科学省科学研究費特定領域研究(B)平成17年度公開シンポジウム「自然免疫による異物認識の分子基盤」, 2005年

メラニン合成に関わるドーパクロム変換酵素の精製と一次構造　　　　　　　　　　　　　　　
日本動物学会第76回大会, 2005年, ポスター発表

カイコの新たなペプチドグリカン認識蛋白質　　　　　　　　　　　　　　　
日本動物学会第76回大会, 2005年, ポスター発表

カイコ幼虫体液のlipophorinと細菌リポ多糖の複合体形成　　　　　　　　　　　　　　　
日本動物学会第76回大会, 2005年, ポスター発表

フィブリノーゲンとの相互作用における二糖の異性体効果　　　　　　　　　　　　　　　
第16回日本MRS学術シンポジウム, 2005年, ポスター発表

蛋白質の凝集に対するNDSBsの効果　　　　　　　　　　　　　　　
第16回日本MRS学術シンポジウム, 2005年, ポスター発表

フェノ−ル酸化酵素前駆体カスケ−ドにおけるグラム陰性菌結合蛋白質の役割　　　　　　　　　　　　　　　
日本動物学会第75回大会, 2004年, ポスター発表

フィブリンゲル形成過程に及ぼす糖類の影響 –フィブリノペプチド切断との関係　　　　　　　　　　　　　　　
第53回高分子討論会, 2004年, ポスター発表

DNA-コラーゲン複合体の構造制御について　　　　　　　　　　　　　　　
第53回高分子討論会, 2004年

フィブリンゲル形成におけるトロンビン酵素活性への糖類の影響　　　　　　　　　　　　　　　
第53回高分子学会年次大会, 2004年, ポスター発表

DNA-コラーゲン複合体ゲルの構造制御　　　　　　　　　　　　　　　
第52回レオロジー討論会, 2004年

Peptidoglycan-mediated activation of the proBAEEase pathway of the prophenoloxidase cascade　　　　　　　　　　　　　　　
文部科学省科学研究費特定領域研究(B)平成16年度公開シンポジウム「自然免疫による異物認識の分子基盤」, 2004年

DNA存在下におけるコラーゲン線維　　　　　　　　　　　　　　　
第50回マトリックス研究会記念大会, 2003年

カイコ幼虫斑紋形成に関与する顆粒フェノール酸化酵素(gPO)の精製とその性質　　　　　　　　　　　　　　　
日本蚕糸学会第73回大会, 2003年

コラーゲン-DNA複合体形成における添加DNAの形状の影響　　　　　　　　　　　　　　　
第52回高分子学会年次大会, 2003年

トロンビンによるフィブリノーゲン-フィブリン転換における添加物の効果　　　　　　　　　　　　　　　
第52回高分子学会年次大会, 2003年, ポスター発表

An overview of the prophenoloxidase cascade of insects　　　　　　　　　　　　　　　
文部科学省科学研究費特定領域研究(B)平成15年度公開シンポジウム「自然免疫による異物認識の分子基盤」, 2003年

A new serine protease zymogen of the silkworm prophenoloxidase cascade　　　　　　　　　　　　　　　
文部科学省科学研究費特定領域研究(B)平成15年度公開シンポジウム「自然免疫による異物認識の分子基盤」, 2003年

カイコのフェノ−ル酸化酵素前駆体活性化系の未同定因子の検出とproBAEEase活性化経路　　　　　　　　　　　　　　　
日本動物学会第74回大会, 2003年

フェノール酸化酵素前駆体(proPO)カスケードから得られた新しい活性型プロテアーゼ　　　　　　　　　　　　　　　
日本動物学会第74回大会, 2003年

The peptidoglycan-mediated pathway of the silkworm prophenoloxidase cascade　　　　　　　　　　　　　　　
The 16th Naito Conference on Innate Immunity in Medicine and Biology [I], 2003年, ポスター発表

Structure and Formation Process of Collagen–DNA Complex　　　　　　　　　　　　　　　
The 8th Pacific Polymer Conference, 2003年, ポスター発表

カイコのペプチドグリカン認識蛋白質ファミリー　　　　　　　　　　　　　　　
日本動物学会第73回大会, 2002年, ポスター発表

フェノール酸化酵素前駆体活性化系の新たに単利された構成因子(プロテアーゼ前駆体)　　　　　　　　　　　　　　　
日本動物学会第73回大会, 2002年, ポスター発表

精子形成細胞のカルモジュリンと関連するヒートショックプロテイン複合体について　　　　　　　　　　　　　　　
日本動物学会第73回大会, 2002年, ポスター発表

コラーゲン-DNAコンポジットの形成について　　　　　　　　　　　　　　　
第51回高分子討論会, 2002年

Effect of DNA structure on collagen fibrillogenesis　　　　　　　　　　　　　　　
第39回ペプチド討論会, 2002年

フェノール酸化酵素前駆体カスケードのペプチドグリカン活性化経路　　　　　　　　　　　　　　　
文部科学省科学研究費特定領域研究(B)平成14年度公開シンポジウム「自然免疫による異物認識の分子基盤」, 2002年

トロンビンによるフィブリノーゲン-フィブリン転換における糖類の影響　　　　　　　　　　　　　　　
第14回日本MRS学術シンポジウム, 2002年, ポスター発表

カイコのβ-1,3-グルカン認識蛋白質II　　　　　　　　　　　　　　　
日本比較免疫学会第13回学術集会, 2001年, ポスター発表

カイコ体液中のフェノール酸化酵素前駆体カスケードの液性生体防御における役割　　　　　　　　　　　　　　　
文部科学省科学研究費特定領域研究(B)平成13年度公開シンポジウム「自然免疫による異物認識の分子基盤」, 2001年

A new component of the prophenoloxidase cascade, ß-1,3-glucan recognition protein II from the silkworm, Bombyx mori　　　　　　　　　　　　　　　
XXI international Congress of Entomology, 2000年, ポスター発表

Prophenoloxidase cascade of the silkworm, Bombyx mori　　　　　　　　　　　　　　　
XXI international Congress of Entomology, 2000年

グラム陰性菌結合蛋白質はフェノ−ル酸化酵素前駆体カスケ−ドの構成因子である　　　　　　　　　　　　　　　
日本動物学会第71回大会, 2000年, ポスター発表

家蚕血漿中のセリンプロテアーゼ(BAEEase)によるspatzleの限定分解　　　　　　　　　　　　　　　
日本動物学会第71回大会, 2000年, ポスター発表

カイコのβ-1,3-グルカン認識蛋白質II　　　　　　　　　　　　　　　
日本動物学会第70回大会, 1999年

家蚕血漿中に存在するセリンプロテアーゼ前駆体(proBAEEase)の性状解析　　　　　　　　　　　　　　　
日本動物学会第70回大会, 1999年

カイコ卵のフェノール酸化酵素前駆体活性化酵素に関する研究　　　　　　　　　　　　　　　
日本動物学会第70回大会, 1999年

昆虫フェノール酸化酵素の接着性に関与するアミノ酸配列について　　　　　　　　　　　　　　　
日本動物学会第70回大会, 1999年

Pro-phenol oxidase cascade in insect hemolymph and cuticle　　　　　　　　　　　　　　　
Third International Symposium on Molecular Insect Science, Utah, USA, 1998年

カイコの細菌感染によるペプチドグリカン認識蛋白質の誘導合成　　　　　　　　　　　　　　　
日本動物学会第69回大会, 1998年

家蚕血液から外皮へ移行したフェノール酸化酵素前駆体が受けている修飾について　　　　　　　　　　　　　　　
日本動物学会第69回大会, 1998年

家蚕血漿中に存在するセリンプロテアーゼ前駆体のcDNAクローニング　　　　　　　　　　　　　　　
日本動物学会第69回大会, 1998年

カイコの初期発生におけるフェノール酸化酵素前駆体の局在　　　　　　　　　　　　　　　
日本動物学会第69回大会, 1998年

2020年01月
日本蚕糸学会　　　　　　　　　　　　　　　

ホルミシス誘導と持続の分子機構の解明
科学研究費助成事業 基盤研究(B)
2021年04月 - 2025年03月
早川 洋一, 落合 正則, 龍田 勝輔
予め弱いストレスを経験した生物個体が、次に来る強いストレスに対する耐性を獲得する能力がホルミシス（あるいは順応性）と総称される生理的潜在能力である。幅広い生物学分野においてその重要性は認識されているものの、分子レベルでのホルミシス誘導機構・持続機構については未解明な生理現象と言える。本研究では、特に後者の持続時間の決定分子機構に焦点を当てて解析を推進している。今年度は主にコナチャタテとキイロショウジョウバエの２種類の研究材料を対象に研究を進めた。
先ず、コナチャタテ類のヒラタチャタテは特に乾燥に弱い小型昆虫であることから、予めの乾燥ストレスによる乾燥に対する耐性（順応性）誘導現象の有無について検証を行った。その結果、湿度20%の前乾燥ストレスを２日以上与えたヒラタチャタテ個体においては、有意に乾燥耐性が増強される事が確認できた。従って、昆虫類のホルミシス現象は、従来より報告のあったチョウ目やハエ目という進化的に新しい完全変態昆虫のみならず、比較的原始的な不完全変態昆虫においても保持されている能力である事が明らかになった。
次に、キイロショウジョウバエを用いた解析では、３齢幼虫を用いて形成されるホルミシス持続時間の決定機構に関する基礎実験を行った。即ち、予めの熱ストレス（37度／30分間）供与の回数や間隔を変化させる事によって、その後形成されるストレス耐性の持続時間への影響を評価した。その結果、１回の前ストレス供与よりは、30分間のインターバルを設けて２回、３回と複数回前ストレスを与えた方が長いストレス耐性持続時間を維持できる事が分かった。次に、インターバルを30分から12時間へと延長したところ、同じ2回のストレス回数でも、12時間のインターバルの方がより長いストレス耐性持続時間となる事が明らかになった。今後、更に、前ストレス供与条件の検討が必要となる。
日本学術振興会, 基盤研究(B), 佐賀大学, 研究分担者, 21H02207

ßGRPとカードランによる簡便で低コストな組換えタンパク質固定化法の研究　　　　　　　　　　　　　　　
科学研究費補助金
2012年 - 2013年
堀内 正隆
日本学術振興会, 競争的資金

細胞性免疫に関与する昆虫サイトカインの微生物感染による活性化機構の解析　　　　　　　　　　　　　　　
科学研究費補助金
2011年 - 2013年
落合 正則
日本学術振興会, 研究代表者, 競争的資金

自然免疫による異物認識の分子基盤の研究成果とりまとめ
科学研究費助成事業
2001年 - 2006年
川畑 俊一郎, 藤田 禎三, 倉田 祥一郎, 落合 正則, 芦田 正明
本特定領域研究で対象とする非自己認識蛋白質や受容体、さらに生体防御レクチンは、すべて微生物表層成分を直接認識する真のパターン認識分子であり、それらの構造と機能解析を進めることにより、パターン認識という魅惑的な概念に潜む本質の解明が可能と考えている。これまで、具体的には、配列決定された蛋白質全体あるいは、機能ドメインを用いてX線結晶解析とNMRにより立体構造を決定し、パターン認識を支える分子基盤を明らかにしてきた。また、生体防御レクチンや関連蛋白質のノックアウトマウスの解析を行って、補体系におけるレクチン経路の役割を解明した。一方では、ショウジョウバエで確立したゲノムワイド同定系を用いて、これまで未知であったパターン認識受容体としてペプチドグリカン認識タンパク質(PGRP)-LEを同定した。そして、PGRP-LEが感染細菌の有するペプチドグリカンの構造上の違いを識別して、その感染に適した免疫応答を誘導することを示すと共に、PGRP-LEが抗菌ペプチド産生経路(imd経路)を活性化するだけでなく、異物の隔離と創傷治癒に関わるフェノール酸化酵素系(proPO系)をも活性化することを明らかにしてきた。
18年度は、計画研究代表者、共同研究者、海外研究協力者、評価委員が一同に会する機会をもうけるために、平成18年8月10日、11日の両日にかけて、福岡リーセントホテルにおいて「成果とりまとめ公開シンポジウム」を開催するとともに、計画班会議を行って、研究成果のとりまとめを行った。さらに、未発表データについても今年度中にも原著論文を投稿できるように討論した。
日本学術振興会, 特定領域研究, 九州大学, 13143101

自然免疫による異物認識の分子基盤の総括と評価　　　　　　　　　　　　　　　
科学研究費補助金
2004年 - 2005年
落合 正則
日本学術振興会, 研究代表者, 競争的資金

昆虫のフェノール酸化酵素前駆体活性化系構成因子の同定　　　　　　　　　　　　　　　
科学研究費補助金
2003年 - 2005年
落合 正則
日本学術振興会, 研究代表者, 競争的資金

自然免疫を制御するシグナル伝達カスケードとネットワーク形成　　　　　　　　　　　　　　　
科学研究費補助金
2005年
倉田 祥一郎
日本学術振興会, 競争的資金

昆虫のフェノール酸化酵素前駆体活性化系の新規構成因子の同定　　　　　　　　　　　　　　　
特定研究助成金
2004年
落合 正則
内藤記念科学振興財団, 研究代表者, 競争的資金

昆虫の液性及び細胞性生体防御におけるプロテアーゼカスケードの役割　　　　　　　　　　　　　　　
科学研究費補助金
2001年 - 2003年
芦田 正明
日本学術振興会, 競争的資金

昆虫のフェノール酸化酵素カスケード活性化の分子機構と生体防御における役割　　　　　　　　　　　　　　　
科学研究費補助金
1997年 - 2001年
芦田 正明
日本学術振興会, 競争的資金

フェノ-ル酸化酵素前駆体活性化系の時間的・空間的制御機構　　　　　　　　　　　　　　　
科学研究費補助金
1996年 - 1997年
芦田 正明
日本学術振興会, 競争的資金

細菌細胞壁構成成分の微量検出・定量試薬の開発　　　　　　　　　　　　　　　
科学研究費補助金
1994年 - 1996年
芦田 正明
日本学術振興会, 競争的資金

マラリア媒介蚊の非感受精機構に係わる生体防御因子の分子生物学的解析　　　　　　　　　　　　　　　
科学研究費補助金
1996年
芦田 正明
日本学術振興会, 競争的資金

昆虫体液の異物認識蛋白質に関する研究-異物認識の分子機構と生体防御機構-　　　　　　　　　　　　　　　
科学研究費補助金
1995年
落合 正則
日本学術振興会, 研究代表者, 競争的資金

カイコ体液中のフェノール酸化酵素前駆体活性化カスケードについて
科学研究費助成事業
1993年 - 1993年
落合 正則
フェノール酸化酵素前駆体活性化系(proPOカスケード)は昆虫の生体防御の要であり、無脊椎動物の体液などに存在する。このカスケードはカビや細菌の細胞壁成分により活性化され、これら異物成分各々に対する認識蛋白質と複数のプロテアーゼ前駆体によって構成されていることは判明しているが、詳細は明かではない。本研究はこのカスケード系の生化学的解析を目的としている。以前、研究代表者が発見したカビを認識する蛋白質の機能の解明、及びこの蛋白質が関与するカスケード系内のカビにより活性化する径路を明かにするためにカスケードの再構成系の確立に努めた。
カビを認識する蛋白質(beta-1,3-glucan recognition protein;betaGRP)の機能:カイコのbetaGRPとカビの細胞壁成分であるbeta-1,3-glucanの結合様式を探るため、betaGRP中の結合部位を調べた。betaGRPをキモトリプシンで分解すると42kDと20kDのペプチドに分かれ、このうち20kDペプチドがbeta-1,3-glucan結合能を有していることを見いだした。20kDペプチドとbetaGRPのN末端配列が一致することから、20kDペプチドはbetaGRPのN末端側にあり、beta-1,3-glucan結合部位はbetaGRPのN末端付近にあることがわかった。今後、この結果部位の一次構造を決定する予定である。
カスケードの再構成実験:カイコの血漿成分を原理の異なる複数のカラムを連結したものに供与することにより、カスケードの構成成分を不活性なまま三画分に分けることに成功した。この方法で得た画分を用いた再構成系は上記の異物成分で活性化され、分画前の血漿成分に含まれるカスケードと同じ性質を有していることが明らかになった。また、これらの画分のうち二つの画分にこれまで検出できなかった活性を見いだし、現在、これらの活性を示す構成因子を精製中である。
日本学術振興会, 奨励研究(A), 北海道大学, 05740502

昆虫の生体防御機構とフェノール酸化酵素前駆体活性化カスケードの解析　　　　　　　　　　　　　　　
科学研究費補助金
1993年
落合 正則
日本学術振興会, 研究代表者, 競争的資金

Insect immunity and prophenoloxidase cascade　　　　　　　　　　　　　　　
1993年
競争的資金