| Miki Nakao | Last modified date：2024.04.18 |
Professor /
Division of Molecular Bioscience /
Department of Bioscience and Biotechnology /
Faculty of Agriculture
Papers
| 1. | Masaki Sukeda, Harsha Prakash, Takahiro Nagasawa, Miki Nakao, Tomonori Somamoto, Non-specific cytotoxic cell receptor protein-1 (NCCRP-1) is involved in anti-parasite innate CD8+ T cell-mediated cytotoxicity in ginbuna crucian carp, Fish Shellfish Immunol, DOI: 10.1016/j.fsi.2023.108904, Article No. 108904, 2023.06, CD8+ cytotoxic T cells (CTLs) are a main cellular component of adaptive immunity. Our previous research has shown that CD8+ cells demonstrate spontaneous cytotoxic activity against the parasite Ichthyophthirius multifiliis in ginbuna crucian carp, suggesting that CD8+ cells play an important role in innate immunity. Herein, we investigated the molecules and cellular signal pathways involved in the cytotoxic response of ginbuna crucian carp. We considered non-specific cytotoxic receptor protein-1 (NCCRP-1) as candidate molecule for parasite recognition. We detected NCCRP-1 protein in CD8+ cells and the thymus as well as in other cells and tissues. CD8+ cells expressed mRNA for NCCRP-1, Jak2, and T cell-related molecules. In addition, treatment with a peptide containing the presumed antigen recognition site of ginbuna NCCRP-1 significantly inhibited the cytotoxic activity of CD8+ cells against the parasites. The cytotoxic activity of CD8+ cells was significantly inhibited by treatment with the JAK1/2 inhibitor baricitinib. These results suggest that teleost CTLs recognize I. multifiliis through NCCRP-1 and are activated by JAK/STAT signaling.. |
| 2. | Nehlah R, Yamamoto A, Nagasawa T, Somamoto T, Nakao M, Functional analysis of two divergent C4 isotypes in the classical and lectin pathway of complement activation in the common carp (Cyprinus carpio), J. Marine Sci. Eng., 10.3390/jmse11040707, １１, 4, 707, 2023.03. |
| 3. | Meidong R, Muatong A, Nakao M, Sakai K, Tongpim S, Mixed culture of Bacillus aerius B81e and Lactiplantibacillus paraplantarum L34b-2 derived from in vivo screening using hybrid catfish exhibits high probiotic effects on Pangasius bocourti, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2021.06.006, 132, 4, 423-428, 2021.10, A mixed culture of probiotics, one from the genus Bacillus and one lactic acid bacterium (LAB), was developed to be used as a feed additive for enhancing growth, innate immunity and disease resistance in Pangasius bocourti. From our earlier work, three probiotic Bacillus species, Bacillus siamensis B44v, Bacillus sp. B51f and Bacillus aerius B81e, and three probiotic LABs, Streptococcus lutetiensis L7c, Lactiplantibacillus paraplantarum (synonym. Lactobacillus paraplantarum) L34b-2 and Lactiplantibacillus plantarum (synonym. Lactobacillus plantarum) L42g, were selected for comparison. These bacteria, which express probiotic properties including bacteriocin-like activity against Aeromonas hydrophila, were subjected to in vivo screening in hybrid catfish (Clarias macrocephalus × Clarias gariepinus). A 30-day feed-trial followed by a challenge test in screening experiments resulted in the prominent B. aerius B81e and L. paraplantarum L34b-2 being selected. A mixture of these bacteria was added to a diet for P. bocourti. After 60-day feeding, the fish fed with mixed probiotics had weight gain, specific growth rate and feed conversion ratio improved significantly (p |
| 4. | Prakash H, Sato M, Kojima K, Sato A, Maruyama S, Nagasawa T, Nakao M, Somamoto T, Development of a filter device for the prevention of aquatic bacterial disease using a single-chain variable fragment (scFv)-conjugated affinity silk, Scientific Reports, 10.1038/s41598-022-13408-6, 12, 9475, 2022.06, Infectious disease is one of the most serious problems in the aquaculture industry for ornamental or edible fsh. This study attempted to develop a new device for preventing an aquatic bacterial disease, ulcer disease, caused by Aeromonas salmonicida (As), using “afnity silk”. Afnity silk is a silk proteincontaining fbroin L-chain (FibL) fused to the single-chain variable fragment (scFv). It can be easily processed into diferent formats such as fbers, gels, sponges, or flms. A transgenic silkworm that could express a cDNA construct containing FibL fused to an scFv derived from a monoclonal antibody (MAb) against As was successfully generated. An enzyme-linked immunosorbent assay was used to detect As by employing 96-well plates coated with scFv-conjugated afnity silk. As could be captured efciently by glass wool coated with afnity silk in the column. Furthermore, the air-lift water flter equipped with the afnity silk-coated wool could considerably reduce the concentration of As in water and was estimated to have sufcient ability to trap a lethal dose of As. These fndings show that the “afnity silk flter” is a potential device for the prophylaxis of aquatic animal diseases.. |
| 5. | ＃Shiota K, Sukeda M, Prakash H, Kondo M, Nakanishi T, Nagasawa T, Nakao M, Somamoto T , Local immune responses to two stages of Ichthyophthirius multifiliis in ginbuna crucian carp, Fish and Shellfish Immunology, 10.1016/j.fsi.2021.08.013, 118, 19-24, 2021.11, Ichthyophthirius multifiliis is a ciliated protozoan parasite and is known to infect many freshwater teleosts. Characterizing the immune system in epithelial tissues, where the parasites penetrate and settle, is key to understanding host–parasite interactions. This study examined local immune responses in vivo to the infective stage (theront and trophont) of the parasites using intra-fin administration, which has been developed to analyze in vivo immune responses using fish fin. CD8α+ and CD4+ T-cell compositions were increased significantly in the fin cavity injected with theront or trophont antigens. The expression of GATA-3 and T-bet mRNA, which regulate differentiation of helper T-cells, was upregulated significantly in leukocytes from the trophont antigen–injected site. In contrast, the percentages of macrophages and neutrophils, which are innate immunity components, were decreased significantly in the injection sites. These results suggest that I. multifiliis antigens inhibit the migration of macrophages and neutrophils, and T-cells are the first responders to I. multifiliis. Thus, to better understand the interaction of host immunity and I. multifiliis, further studies should focus on exploring the inhibitory factors from I. multifiliis or examining innate functions of teleost T-cells.. |
| 6. | Prakash H, Motobe S, Nagasawa T, Somamoto T, Nakao M, Homeostatic Functions of Tecrem, a CD46-Like Regulatory Protein of Complement Activation, on Epithelial Cells in Carp Fish, Journal of Marine Science and Engineering, https://doi.org/10.3390/jmse9070687, 97, 7, 687, 2021.06, Fish mucosal surface is a significant interface for pathogens to infect from an aqueous environment. In addition to mucosal innate and adaptive immune factors, epithelial cells are considered as a significant physical barrier against microbial invasion. Previously, we identified a mammalian CD46-like complement regulatory protein (Tecrem) in teleost and detected its expression on epithelial cells derived from fin, suggesting its physiological role on the fish surface. This study examines the homeostatic roles of Tecrem in maintaining the fish epithelium, by analyzing the expression behavior of Tecrem on the fin-derived epithelial cell lines (KF-1 from the common carp and CFS from ginbuna crucian carp) using monoclonal and polyclonal anti-Tecrem antibodies. Expression of KF-1 protein was associated with the adhesion of KF-1, and the adhesion was enhanced by anti-Tecrem treatments of the cells. Stimulation of the epithelial cells with anti-Tecrem enhanced wound healing, protein expression of tight-junction proteins, and cell density of the KF-1 and CFS monolayer culture. These results suggest that Tecrem on epithelial cells play a homeostatic role in maintaining intactness of the surface epithelial barrier, implying that modification of Tecrem expression may develop a novel tool to improve the first-line defense against pathogens in aquaculture.. |
| 7. | Sukeda M, Shiota K, Kondo M, Nagasawa T, Nakao M, Somamoto T, Innate cell-mediated cytotoxicity of CD8+ T cells against the protozoan parasite Ichthyophthirius multifiliis in the ginbuna crucian carp, Carassius auratus langsdorfii, Developmental and Comparative Immunology, doi.org/10.1016/j.dci.2020.103886, 115, Article No. 103885, 2021.02, Cytotoxic T cells are known to have the ability to kill microbe-infected host cells, which makes them essential in the adaptive immunity processes of various vertebrates. In this study, we demonstrated innate cell-mediated cytotoxicity of CD8+ T cells against protozoan parasites found in the ginbuna crucian carp. When isolated effector cells such as CD8+, CD4+ (CD4-1+), or CD8− CD4− (double-negative, DN), from naïve ginbuna crucian carp were co-incubated with target parasites (Ichthyophthirius multifiliis), CD8+ cells from the kidney and gill showed the highest cytotoxic activity. On the other hand, DN cells, which include macrophages and CD4− CD8− lymphocytes, showed the lowest cytotoxic activity against I. multifiliis. Additionally, the cytotoxic activity of CD8+ cells was found to significantly decrease in the presence of a membrane separating the effector cells from I. multifiliis. Furthermore, the serine protease inhibitor 3,4-dichloroisocoumarin and perforin inhibitor concanamycin A significantly inhibited the cytotoxic activity of CD8+ cells. These results demonstrate that CD8+ T cells of ginbuna crucian carp can kill extracellular parasites in a contact-dependent manner via serine proteases and perforin. Therefore, we conclude that CD8+ T cells play an essential role in anti-parasite innate immunity of teleost fish..  |
| 8. | Meidong R, Nakao M, Sakai K, Tongpim S, Lactobacillus paraplantarum L34b-2 derived from fermented food improves the growth, disease resistance and innate immunity in Pangasius bocourti, Aquaculture, doi.org/10.1016/j.aquaculture.2020.735878, 531, Article No. 735878, 2021.01, Probiotics have increasingly gained interest as alternatives to antibiotics in controlling infectious diseases in aquaculture. This research aimed to isolate and evaluate lactic acid bacteria (LAB) for potential use as probiotics for Pangasius bocourti. A total of 656 LAB isolates were obtained from fish gut and fermented beef samples, of which 367 isolates displayed antagonistic activity against Aeromonas hydrophila FW52 and/or Streptococcus agalactiae F3S which were employed as indicator fish pathogens. Among these antagonistic LAB, only 18 isolates produced bacteriocin-like activity. These were further evaluated in vitro for other probiotic properties. After in vitro screening, the isolate L34b-2 obtained from Thai indigenous fermented beef was chosen as a suitable probiotic LAB for in vivo studies based on its remarkable probiotic characteristics. These included broad-spectrum bacteriocin-like activity, tolerance to simulated gastrointestinal tract conditions, mucin adhesion ability, co-aggregation ability with fish pathogens tested, non-blood hemolysis, antibiotic susceptibility and protease enzyme production. Identification of the isolate L34b-2 using conventional methods together with 16S rDNA analysis revealed that it belonged to Lactobacillus paraplantarum. Then, the effects of 60-day-dietary administration of L. paraplantarum L34b-2 on fish growth, innate immune responses and disease resistance were investigated in Pangasius bocourti. In feed-trial studies, after feeding for both 30 and 60 days it was found that the Pangasius fish fed with basal diet containing 107 CFU g−1 L. paraplantarum L34b-2 had significantly (p 0.05) in phagocytic and respiratory burst activities. After 60-day feeding, fish were subjected to challenge tests by intraperitoneal injection with 106 CFU of a virulent Aeromonas hydrophila FW52 and rearing was continued for two more weeks. As expected, fish that received L34b-2 had a significantly higher (p |
| 9. | Seisuke Tajimi, Masakazu Kondo, Teruyuki Nakanishi, Takahiro Nagasawa, Miki Nakao, Tomonori Somamoto, Generation of virus-specific CD8 + T cells by vaccination with inactivated virus in the intestine of ginbuna crucian carp, Developmental and Comparative Immunology, 10.1016/j.dci.2018.12.009, 93, 37-44, 2019.04, Although a previous study using ginbuna crucian carp suggested that cell-mediated immunity can be induced by the oral administration of inactivated viruses, which are exogenous antigens, there is no direct evidence that CD8 + cytotoxic T cells (CTLs) in teleost fish are generated by vaccination with exogenous antigens. In the present study, we investigated whether antigen-specific CD8 + CTLs in ginbuna crucian carp can be elicited by intestinal immunization with an exogenous antigen without any adjuvant. The IFNγ-1 and T-bet mRNA expressions were up-regulated in intestinal leukocytes following the administration of formalin-inactivated crucian hematopoietic necrosis virus (FI-CHNV), whereas the down-regulation of these genes was observed in kidney leukocytes. Furthermore, an increase in the percentage of proliferating CD8 + cells was detected in the posterior portion of the hindgut, suggesting that the virus-specific CTLs are locally generated in this site. In addition, cell-mediated cytotoxicity against CHNV-infected syngeneic cells and the in vivo inhibition of viral replication were induced by immunization with FI-CHNV. Unexpectedly, intraperitoneal immunization with FI-CHNV induced a type I helper T cell (Th1)-response in the intestine, but not in the kidney; however, its effect was slightly lower than that reported after intestinal immunization. These findings suggest that the posterior portion of the intestine is an important site for generating virus-specific CTLs by vaccination with the inactivated vaccine.. |
| 10. | Somamoto, T., Maruyama, S., Nagasawa, T., Nakao, M., Sato, A., Hatta, H., Sato, M., Murakami-Yamaguchi, Y., Kizu-Mori, K., Hirakawa, Y., Narita, H., Development of anti-atypical Aeromonas salmonicida monoclonal antibodies for diagnosis of “new ulcer disease" in koi carp, Fish Pathology, 10.3147/jsfp.53.36, 53, 1, 36-39, 2018.06. |
| 11. | Ratchanu Meidong, Kulwadee Khotchanalekha, Sompong Doolgindachbaporn, Takahiro Nagasawa, Miki Nakao, Kenji Sakai, Saowanit Tongpim, Evaluation of probiotic Bacillus aerius B81e isolated from healthy hybrid catfish on growth, disease resistance and innate immunity of Pla-mong Pangasius bocourti, Fish and Shellfish Immunology, 10.1016/j.fsi.2017.11.032, 73, 1-10, 2018.02, Infectious diseases have been found to be a major cause of mortality in fish hatcheries. Probiotics have been introduced to replace antibiotics commonly used for treatment of bacterial infection in aquaculture. This study was conducted to isolate, screen, and evaluate the probiotic Bacillus spp. for potential use as a feed supplement to enhance fish growth, disease resistance and innate immunity of Pla-mong Pangasius bocourti. Bacillus aerius strain B81e was selectively isolated from the intestine of healthy catfish and chosen based on its probiotic properties both in vitro and in vivo. This bacterium produced a bacteriocin-like substance and exhibited a broad-spectrum antibacterial activity inhibiting both Gram-positive and Gram-negative bacteria especially the fish pathogens Aeromonas hydrophila and Streptococcus agalactiae. The susceptibility to all 8 antibiotics tested implies that it is unlikely to be an antibiotic-resistant bacterium. B. aerius strain B81e possessed interesting adhesion properties as shown by its high percentages of hydrophobicity, auto-aggregation, co-aggregation with fish pathogens A. hydrophila FW52 and S. agalactiae F3S and mucin binding. The strain B81e survived simulated gastrointestinal conditions, producing protease and lipase but not β-haemolysin. The study also evaluated the effects of dietary supplementation with strain B81e on growth performance, innate immunity, and the disease resistance of P. bocourti against A. hydrophila infection. Fish with a mean body weight of 69 g were fed strain B81e at 0 (control) and 107 CFU g−1 feed (test) for 60 days. Various growth and immune parameters were examined at 30 and 60 days post-feeding. Fish were challenged with A. hydrophila 60 days post-feeding and mortalities were recorded over 14 days post-infection. Results showed that the administration of strain B81e for 60 days had significant effects (p |
| 12. | Yumie Tokunaga, Masamichi Shirouzu, Ryota Sugahara, Yasutoshi Yoshiura, Ikunari Kiryu, Mitsuru Ototake, Takahiro Nagasawa, Tomonori Somamoto, Miki Nakao, Comprehensive validation of T- and B-cell deficiency in rag1-null zebrafish Implication for the robust innate defense mechanisms of teleosts, Scientific Reports, 10.1038/s41598-017-08000-2, 7, 1, 2017.12, rag1 -/- zebrafish have been employed in immunological research as a useful immunodeficient vertebrate model, but with only fragmentary evidence for the lack of functional adaptive immunity. rag1-null zebrafish exhibit differences from their human and murine counterparts in that they can be maintained without any specific pathogen-free conditions. To define the immunodeficient status of rag1 -/- zebrafish, we obtained further functional evidence on T- and B-cell deficiency in the fish at the protein, cellular, and organism levels. Our developed microscale assays provided evidence that rag1 -/- fish do not possess serum IgM protein, that they do not achieve specific protection even after vaccination, and that they cannot induce antigen-specific CTL activity. The mortality rate in non-vaccinated fish suggests that rag1 -/- fish possess innate protection equivalent to that of rag1 -/- fish. Furthermore, poly(I:C)-induced immune responses revealed that the organ that controls anti-viral immunity is shifted from the spleen to the hepatopancreas due to the absence of T- and B-cell function, implying that immune homeostasis may change to an underside mode in rag-null fish. These findings suggest that the teleost relies heavily on innate immunity. Thus, this model could better highlight innate immunity in animals that lack adaptive immunity than mouse models.. |
| 13. | Kolder IC, van der Plas-Duivesteijn SJ, Tan G, Wiegertjes GF, Forlenza M, Guler AT, Travin DY, Miki Nakao, Moritomo T, Irnazarow I, den Dunnen JT, Anvar SY, Jansen HJ, Dirks RP, Palmblad M, Lenhard B, Henkel CV, Spaink HP, A full-body transcriptome and proteome resource for the European common carp., BMC Genomics, 10.1186/s12864-016-3038-y, 17, 701-1-701-12, 2016.09, [URL], BACKGROUND: The common carp (Cyprinus carpio) is the oldest, most domesticated and one of the most cultured fish species for food consumption. Besides its economic importance, the common carp is also highly suitable for comparative physiological and disease studies in combination with the animal model zebrafish (Danio rerio). They are genetically closely related but offer complementary benefits for fundamental research, with the large body mass of common carp presenting possibilities for obtaining sufficient cell material for advanced transcriptome and proteome studies. RESULTS: Here we have used 19 different tissues from an F1 hybrid strain of the common carp to perform transcriptome analyses using RNA-Seq. For a subset of the tissues we also have performed deep proteomic studies. As a reference, we updated the European common carp genome assembly using low coverage Pacific Biosciences sequencing to permit high-quality gene annotation. These annotated gene lists were linked to zebrafish homologs, enabling direct comparisons with published datasets. Using clustering, we have identified sets of genes that are potential selective markers for various types of tissues. In addition, we provide a script for a schematic anatomical viewer for visualizing organ-specific expression data. CONCLUSIONS: The identified transcriptome and proteome data for carp tissues represent a useful resource for further translational studies of tissue-specific markers for this economically important fish species that can lead to new markers for organ development. The similarity to zebrafish expression patterns confirms the value of common carp as a resource for studying tissue-specific expression in cyprinid fish. The availability of the annotated gene set of common carp will enable further research with both applied and fundamental purposes.. |
| 14. | Nur I, Abdelkhalek NK, Motobe S, Nakamura R, Tsujikura M, Tomonori Somamoto, Miki Nakao, Functional analysis of membrane-bound complement regulatory protein on T-cell immune response in ginbuna crucian carp, Molecular Immunology, 10.1016/j.molimm.2015.11.010, 70, 1-7, 2016.02, [URL], Complements have long been considered to be a pivotal component in innate immunity. Recent researches, however, highlight novel roles of complements in T-cell-mediated adaptive immunity. Membrane-bound complement regulatory protein CD46, a costimulatory protein for T cells, is a key molecule for T-cell immunomodulation. Teleost CD46-like molecule, termed Tecrem, has been newly identified in common carp and shown to function as a complement regulator. However, it remains unclear whether Tecrem is involved in T-cell immune response. We investigated Tecrem function related to T-cell responses in ginbuna crucian carp. Ginbuna Tecrem (gTecrem) proteins were detected by immunoprecipitation using anti-common carp Tecrem monoclonal antibody (mAb) and were ubiquitously expressed on blood cells including CD8α(+) and CD4(+) lymphocytes. gTecrem expression on leucocyte surface was enhanced after stimulation with the T-cell mitogen, phytohaemagglutinin (PHA). Coculture with the anti-Tecrem mAb significantly inhibited the proliferative activity of PHA-stimulated peripheral blood lymphocytes, suggesting that cross-linking of Tecrems on T-cells interferes with a signal transduction pathway for T-cell activation. These findings indicate that Tecrem may act as a T-cell moderator and imply that the complement system in teleost, as well as mammals, plays an important role for linking adaptive and innate immunity.. |
| 15. | Takahiro Nagasawa, Tomonori Somamoto, Miki Nakao, Carp thrombocyte phagocytosis requires activation factors secreted from other leukocytes, Developmental and Comparative Immunology, 10.1016/j.dci.2015.05.002, 52, 2, 107-111, 2015.07, Thrombocytes are nucleated blood cells in non-mammalian vertebrates, which were recently focused on not only as hemostatic cells but also as immune cells with potent phagocytic activities. We have analyzed the phagocytic activation mechanisms in common carp (Cyprinus carpio) thrombocytes. MACS-sorted mAb+ thrombocytes showed no phagocytic activity even in the presence of several stimulants. However, remixing these thrombocytes with other anti-thrombocyte mAb− leukocyte populations restored their phagocytic activities, indicating that carp thrombocyte phagocytosis requires an appropriate exogenous stimulation. Culture supernatant from anti-thrombocyte mAb− leukocytes harvested after PMA or LPS stimulation, but not culture supernatant from unstimulated leukocytes, could activate thrombocyte phagocytosis. This proposed mechanism of thrombocyte phagocytosis activation involving soluble factors produced by activated leukocytes suggests that thrombocyte activation is restricted to areas proximal to injured tissues, ensuring suppression of excessive thrombocyte activation and a balance between inflammation and tissue repair.. |
| 16. | Tomonori Somamoto, Yuhei Miura, Teruyuki Nakanishi, Miki Nakao, Local and systemic adaptive immune responses toward viral infection via gills in ginbuna crucian carp, Developmental and Comparative Immunology, 10.1016/j.dci.2015.04.016, 52, 1, 81-87, 2015.07, Recent studies on fish immunity highlighted the significance of gills as mucosal immune tissues. To understand potential of gills as vaccination sites for inducing adaptive systemic immunity, we investigated virus-specific cell-mediated and humoral immune responses following a “per-gill infection method”, which directly exposes virus only to gills. The viral load in crucian carp hematopoietic necrosis virus (CHNV)-infected gills decreased after peaking at a particular time point. Furthermore, the viral titers in the gills following the secondary infection were lower than that after the primary infection, indicating that local adaptive immunity helped the elimination of virus. Gene expression analysis demonstrated that IFN-γ in gills and perforin in kidney were increased after the gill infection. CD8+ cells in kidney leukocytes increased after the secondary infection, whereas IgM+ cells decreased. These results suggest that IFN-γ and CTL contribute in controlling CHNV-replication in gills and kidney. Gill infection could induce specific cell-mediated cytotoxicity of peripheral blood leukocytes (PBL) and secretion of CHNV-specific IgM in serum, indicating that local priming of the gill site can generate adaptive systemic immunity. Thus, the gills could be prospective antigen-sensitization sites for mucosal vaccination.. |
| 17. | Gan H, He H, Sato A, Hatta H, Nakao M, Somamoto T, Ulcer disease prophylaxis in koi carp by bath immersion with chicken egg yolk containing anti-Aeromonas salmonicida IgY, RESEARCH IN VETERINARY SCIENCE, 10.1016/j.rvsc.2015.01.016, 99, 82-86, 2015.04. |
| 18. | Masakazu Tsujikura, Takahiro Nagasawa, Satoko Ichiki, Ryota Nakamura, Tomonori Somamoto, Miki Nakao, A CD46-like Molecule Functional in Teleost Fish Represents an Ancestral Form of Membrane-Bound Regulators of Complement Activation, JOURNAL OF IMMUNOLOGY, 10.4049/jimmunol.1303179, 194, 1, 262-272, 2015.01, In the complement system, the regulators of complement activation (RCA) play crucial roles in controlling excessive complement activation and in protecting host cell from misdirected attack of complement. Several members of RCA family have been cloned from cyclostome and bony fish species and classified into soluble and membrane-bound type as in mammalian RCA factors. Complement-regulatory functions have been described only for soluble RCA of lamprey and barred sand bass; however, little is known on the biological function of the membrane-bound RCA proteins in the lower vertebrates. In this study, a membrane-bound RCA protein, designated teleost complement-regulatory membrane protein (Tecrem), was cloned and characterized for its complement-regulatory roles. Carp Tecrem, an ortholog of a zebrafish type 2 RCA, ZCR1, consists of four short consensus repeat modules, a serine/threonine/proline-rich domain, a transmembrane region, and a cytoplasmic domain, from the N terminus, as does mammalian CD46. Tecrem showed a ubiquitous mRNA expression in carp tissues, agreeing well with the putative regulatory role in complement activation. A recombinant Chinese hamster ovary cell line bearing carp Tecrem showed a significantly higher tolerance against lytic activity of carp complement and less deposition of C3-S, the major C3 isotypes acting on the target cell, than control Chinese hamster ovary (mock transfectant). Anti-Tecrem mAb enhanced the depositions of carp C3 and two C4 isotypes on autologous erythrocytes. Thus, the present findings provide the evidence of complement regulation by a membrane-bound group 2 RCA in bony fish, implying the host-cell protection is an evolutionarily conserved mechanism in regulation of the complement system.. |
| 19. | Takahiro Nagasawa, Chihaya Nakayasu, Aja M. Rieger, Daniel R. Barreda, Tomonori Somamoto, Miki Nakao, Phagocytosis by thrombocytes is a conserved innate immune mechanism in lower vertebrates. , Frontiers in Immunology, doi: 10.3389/fimmu.2014.00445, 5, 445, Article 445, 2014.09, Thrombocytes, nucleated hemostatic blood cells of non-mammalian vertebrates, are regarded as the functional equivalent of anucleated mammalian platelets. Additional immune functions, including phagocytosis, have also been suggested for thrombocytes, but no conclusive molecular or cellular experimental evidence for their potential ingestion and clearance of infiltrating microbes has been provided till date. In the present study, we demonstrate the active phagocytic ability of thrombocytes in lower vertebrates using teleost fishes and amphibian models. Ex vivo, common carp thrombocytes were able to ingest live bacteria as well as latex beads (0.5-3 μm in diameter) and kill the bacteria. In vivo, we found that thrombocytes represented nearly half of the phagocyte population in the common carp total peripheral blood leukocyte pool. Phagocytosis efficiency was further enhanced by serum opsonization. Particle internalization led to phagolysosome fusion and killing of internalized bacteria, pointing to a robust ability for microbe elimination. We find that this potent phagocytic activity is shared across teleost (Paralichthys olivaceus) and amphibian (Xenopus laevis) models examined, implying its conservation throughout the lower vertebrate lineage. Our results provide novel insights into the dual nature of thrombocytes in the immune and homeostatic response and further provide a deeper understanding of the potential immune function of mammalian platelets based on the conserved and vestigial functions.. |
| 20. | Tomonori Somamoto, Masakazu Kondo, Teruyuki Nakanishi, Miki Nakao, Helper function of CD4⁺ lymphocytes in antiviral immunity in ginbuna crucian carp, Carassius auratus langsdorfii, Developmental and Comparative Immunology, 10.1016/j.dci.2013.12.008., 44, 1, 111-115, 2014.05. |
| 21. | Tomokazu Yamaguchi, Kazufumi Takamune, Masakazu Kondo, Yukinori Takahashi, Yoko Kato-Unoki, Miki Nakao, Tamotsu Fujii, Hagfish C1q: Its Unique Binding Property, Developmental and Comparative Immunology, https://doi.org/10.1016/j.dci.2013.10.009, 43, 1, 47-53, 2014.03, Hagfish C1q (HaC1q) was identified and characterized as a pattern-recognition molecule (PRM) in the hagfish complement system. The serum from hagfish, Eptatretus burgeri, was applied to a GlcNAc-agarose column and eluted sequentially with GlcNAc and EDTA. Four (31, 27, 26, and 19 kDa) and one (26 kDa) proteins were detected as bound molecules in the GlcNAc- and the EDTA-eluates, respectively. Among these, the 26 kDa protein from the EDTA eluate was found to be a homologue of mammalian C1q through cDNA analysis. HaC1q had an ability to bind to various microbes in a Ca2+-dependent manner and its target ligands on the microbes were lipopolysaccharide, lipoteichoic acid, and peptidoglycan. The binding of HaC1q to GlcNAc-agarose was not inhibited by an excess amount of monosaccharide such as GlcNAc. While HaC1q bound to Sepharose 6B with a matrix of GlcNAc-agarose (polymer of agarobiose), it did not bind to Sepharose 4B that contained lower concentration of agarobiose than Sepharose 6B. Therefore, the target of HaC1q on GlcNAc-agarose was concluded to be agarobiose and high density of the target moiety seemed to be required for the stable binding. This finding was in accordance with the known behavior of other lectins involved in the complement system. We have concluded that HaC1q recognizes agarobiose-like structures present on the surface of microbes and acts as a pattern-recognition molecule in the process for elimination of invading microbes.. |
| 22. | Abdel-Salam, Soha G. R., Masakazu Kondo, Masakazu Tsujikura, Tomonori Somamoto, Miki Nakao, Purification and functional characterization of complement C3 and a novel zymosan-binding protein in tilapia serum, Fisheries Science, 10.1007/s12562-014-0700-7, 80, 2, 301-310, 2014.03, Zymosan, a yeast cell wall preparation that binds activated forms of complement C3, is a useful model target to activate the complement system. In our trial to analyze C3 diversity in Nile tilapia at the protein level using zymosan, we found that a novel 240-kDa serum protein (ZBP-240) also bound to zymosan in addition to C3-derived fragments. In the present study, we aimed to characterize tilapia C3 and ZBP-240, focusing on their immune-related functions. Four distinct C3 isoforms were purified from tilapia serum and shown to possess an intrachain thioester bond. ZBP-240 was also isolated from tilapia serum and examined for its binding properties to various microbial targets. As a result, ZBP-240 showed a wide spectrum of binding to Gram-positive and Gram-negative bacteria and yeasts. Amino acid sequence analysis of CNBr fragments of ZBP-240 suggested that this is a novel protein with no homologous sequence in protein databases. It was also suggested that the binding of ZBP-240 to microbes largely depends on hydrophobic interactions in a divalent-cation-independent manner, and that there may be a divalent-cation-dependent factor that enhances the binding of ZBP-240 in tilapia serum. Interestingly, ZBP-240 showed opsonic activity for tilapia kidney phagocytes at a level comparable to that of C3, implying that ZBP-240 is a novel teleost opsonic serum protein.. |
| 23. | Indriyani Nur, Hikari Harada, Masakazu Tsujikura, Tomonori Somamoto, Miki Nakao, Molecular characterization and expression analysis of three membrane-bound complement regulatory protein isoforms in the ginbuna crucian carp Carassius auratus langsdorfii., Fish Shellfish Immunol., 10.1016/j.fsi.2013.08.002, 35, 4, 1333-1337, 2013.10, Regulators of complement activation (RCA) play a role in protecting cells from excessive complement activation in humans. cDNA corresponding to three isoforms of teleost membrane-bound RCA protein (gTecrem) have been identified in the ginbuna crucian carp. gTecrem-1 consists of seven short consensus repeats (SCRs), whereas gTecrem-2 and gTecrem-3 have four SCRs. While gTecrem-1 possesses a tyrosine phosphorylation site in its cytoplasmic region, gTecrem-2 and gTecrem-3 lack the site. Tissue distribution analysis showed that gTecrem-1 and gTecrem-2 mRNAs were expressed in almost all tissues examined, whereas gTecrem-2 expression was not significantly detected in gill, liver, or intestine. Furthermore, analysis showed that gTecrem-1 was expressed in both peripheral blood leukocytes (PBLs) and erythrocytes and was also expressed in T cell subsets such as CD4+, CD8+ T cells, and IgM+ B cells. gTecrem-2 expression was not detected in either PBLs or erythrocytes, whereas gTecrem-3 was expressed only in erythrocytes. These results suggested that gTecrem isoforms may serve different functional roles; gTecrem-1, which is expressed in T cells and possesses a tyrosine phosphorylation site, may act as a complement regulator and a cellular receptor in adaptive immunity.. |
| 24. | Tomonori Somamoto, Masakazu Kondo, Teruyuki Nakanishi, Miki Nakao, Identification of anti-viral cytotoxic effector cells in the ginbuna crucian carp, Carassius auratus langsdorfii, Developmental and Comparative Immunology, 10.1016/j.dci.2012.11.001, 39, 4, 111-115, 2013.04, Previous studies have suggested that anti-viral cytotoxic effector cells induced by infection with a sublethal dose of crucian carp hematopoietic necrosis virus (CHNV) correspond to mammalian cytotoxic T-lymphocytes (CTLs), because the mRNA expression patterns of the effector cells are similar to those of mammalian CTLs. To further characterize the effector population in cell-mediated cytotoxic (CMC) activity, we isolated the effector cells using an anti-CD8α monoclonal antibody, a density gradient and plastic adherence. As expected, the purified CD8α-positive cells killed CHNV-infected cells, indicating that the fish CTLs are one of anti-viral effector cells similar those to in mammals. However, it appeared that cytotoxic cells other than CTLs were the dominant effectors, because CTL-depleted peripheral blood leukocytes (PBL) exhibited significant cytotoxic activity against CHNV-infected cells. In addition, the adoptive transfer of CTL-depleted PBL provided as efficient protection against CHNV-infection as the transfer of PBL containing CTLs. Further analyses showed that sIg/CD8α-negative cells and monocyte-enriched effectors possessed activities that were comparable to or were higher than that of CD8α-positive cells, suggesting that natural killer (NK)-like cells and monocytes are among the dominant effector cells. CMC inhibition assays with concanamycin A suggested that CTLs and CD8α-negative lymphocytes lysed virus-infected cells by a perforin-based cytotoxic pathway. These results indicate that CMC induced by viral-infection is executed by not only CTLs but monocytes and CD8/IgM-negative lymphocytes.. |
| 25. | Yamauchi T, Takenaka K, Urata S, Shima T, Kikushige Y, Tokuyama T, Iwamoto C, Nishihara M, Iwasaki H, Miyamoto T, Honma N, Nakao M, Matozaki T, Akashi K., Polymorphic Sirpa is the genetic determinant for NOD-based mouse lines to achieve efficient human cell engraftment., Blood, 10.1182/blood-2012-06-440354, 121, 8, 1316-1325, 2013.01, Current mouse lines efficient for human cell xenotransplantation are backcrossed into NOD mice to introduce its multiple immunodeficient phenotypes. Our positional genetic study has located the NOD-specific polymorphic Sirpa as a molecule responsible for its high xenograft efficiency: it recognizes human CD47 and the resultant signaling may cause NOD macrophages not to engulf human grafts. In the present study, we established C57BL/6.Rag2nullIl2rgnull mice harboring NOD-Sirpa (BRGS). BRGS mice engrafted human hematopoiesis with an efficiency that was equal to or even better than that of the NOD.Rag1nullIl2rgnull strain, one of the best xenograft models. Consequently, BRGS mice are free from other NOD-related abnormalities; for example, they have normalized C5 function that enables the evaluation of complement-dependent cytotoxicity of antibodies against human grafts in the humanized mouse model. Our data show that efficient human cell engraftment found in NOD-based models is mounted solely by their polymorphic Sirpa. The simplified BRGS line should be very useful in future studies of human stem cell biology.. |
| 26. | Ichiki S, Kato-Unoki Y, Somamoto T, Nakao M., The binding spectra of carp C3 isotypes against natural targets independent of the binding specificity of their thioester., Developmental and Comparative Immunology, 10.1016/j.dci.2012.03.004., 38, 1, 10-16, 2012.08, The central component of complement, C3, plays a versatile role in innate immune defense of vertebrates and some invertebrates. A notable molecular characteristic of this component is an intra-chain thioester site that enables C3 to bind covalently to its target. It has been reported that the binding preference of the thioester to hydroxyl or amino groups is primarily defined by presence or absence of the catalytic histidine residue at position 1126 in human C3. In teleosts, a unique C3 (non-His type) has been found, in addition to the common His type C3. These distinct C3 isoforms may provide diversity in the target-binding attributable to the different binding specificities of their thioesters. In the present study, we examine the hypothesized correlation of the catalytic histidine with the binding spectra of two major C3 isotypes of carp towards various model and natural targets. The results reveal that non-His type C3, rather than His type C3, has a wider range of binding spectrum, despite the binding specificity of its thioester being limited to amino groups. It is therefore hypothesized that the binding spectra of C3 isotypes are not defined by the binding specificity of the thioester but is more affected by differences in microbe-associated molecular patterns that activate complement. Overall, the present data imply that non-His type C3 plays a significant role against bacterial infections in the fish defense system.. |
| 27. | Sameshima S, Nakao M, Somamoto T., Diversity of CD2 subfamily receptors in cyprinid fishes., Result Immunol, 10.1016/j.rinim.2012.01.003, 2, 25-34, 2012.02, CD2 family receptor (CD2f) is evolutionarily conserved and is widely expressed by various types of leukocytes. To elucidate the phylogenetic diversity of the CD2f, we characterized CD2f in teleosts using ginbuna crucian carp and zebrafish. The identified CD2f isoforms of the ginbuna carp (caauCD2f) exhibited high sequence similarity to the mammalian CD2 subsets CD48, CD244, and CD319, but it was difficult to classify them into their respective mammalian CD2f based on sequence similarity, the presence of an immunoreceptor tyrosine-based switch motif (ITSM), and phylogenetic tree analysis. Although the four caauCD2f isoforms share an extracellular domain with quite high identity (83-94% identity at the nucleic acid level), they differ in the number of ITSM motifs in their cytoplasmic tail. RT-PCR and in situ hybridization analyses showed that the caauCD2f isoforms are expressed by different cell populations, suggesting that they, like mammalian CD2f, have diverse roles. Interestingly, immunoglobulin (Ig) domain-like sequences with high identity to caauCD2fs are clustered close together within 0.6. Mbp on zebrafish chromosomes 1 and 2 (at least 8 and 35 sequences, respectively), and many pairs of the Ig domains share more than 90% identity at the amino acid level. Therefore, the teleost CD2fs with considerably high identity have been probably generated from a common ancestral Ig-domain gene by a very recent gene duplication event. These findings suggest that the identified CD2f acquired functional diversification through successive duplications together with the acquisition of ITSM.. |
| 28. | Urabe S, Somamoto T, Sameshima S, Unoki-Kato Y, Nakanishi T, Nakao M., Molecular characterization of MHC class I and beta-2 microglobulin in a clonal strain of ginbuna crucian carp, Carassius auratus langsdorfii., Fish Shellfish Immunol., 10.1016/j.fsi.2011.06.004, 31, 3, 469-474, 2011.09, Clonal ginbuna crucian carp is, a naturally gynogenetic fish, and is a useful model animal for studying T-cell-mediated immunity. To gain molecular information on MHC class I molecules from this species, we have identified four types of MHC class I (caauUA-S3n, caauUF-S3n, caauZE-S3n, and caauZB-S3n) and five beta 2-microglobulin (β2m) (caauβ2m-1a, caauβ2m-1b, caauβ2m-2, caauβ2m-3a and caauβ2m-3b) by an expressed sequence tag (EST) analysis and using homology cloning with degenerated primers. Like UA class I genes in other cyprinid fish, the caauUA-S3n shows features of classical MHC class I, such as conservation of all key amino acids interacting with antigenic peptides, and ubiquitous tissue expression. A phylogenetic analysis shows that the β2m-1 and β2m-2 isoforms are clustered with those of other cyprinid fishes, while β2m-3 isoforms make a cluster that is separated from a common ancestor of salmonid and cyprinid fishes. This finding suggests that the β2m isoforms of ginbuna cruician carp comprise two lineages and may possess different functions. The MHC class I and β2m sequences from one clonal strain will facilitate our understanding of the interaction of MHC class I with β2m in teleosts.. |
| 29. | Oba Y, Yamauchi A, Hashiguchi Y, Satone H, Miki S, Nassef M, Shimasaki Y, Kitano T, Nakao M, Kawabata S, Honjo T, Oshima Y, Purification and characterization of tributyltin-binding protein of tiger puffer, Takifugu rubripes, Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, http://dx.doi.org/10.1016/j.cbpc.2010.07.009, 153, 1, 17-23, 2011.01, We successfully purified Trub.TBT-bpα, a tributyltin (TBT) binding protein (bp) of the tiger puffer, Takifugu rubripes. Tiger puffer was injected intraperitoneally with TBT (1.0 mg/kg body weight) and Trub.TBT-bpα was purified from serum by ammonium sulfate fractionation, gel filtration chromatography and polyacrylamide gel electrophoresis. Gel electrophoresis revealed that the Trub.TBT-bpα has a molecular mass of approximately 48.5 kDa and contains at least 40% N-glycan. The deduced 212 amino acid sequence of the protein showed the highest identity (41%, 212 amino acid overlap and E-value: 9e−42) with TBT-binding protein type 1 (TBT-bp1) of Paralichthys olivaceus (Japanese flounder). Analysis of the gene structure of Trub.TBT-bpα suggests that this protein belongs to the lipocalin superfamily, which may be important in the accumulation and elimination of TBT. Phylogenetic analysis suggests that functionalization of TBT-bps has occurred during evolution, and that the functions of this group of proteins might be important for fish survival. . |
| 30. | VO Kha Tam, Masakazu TSUJIKURA, Tomonori SOMAMOTO, and Miki NAKAO, Identification of cDNA sequences encoding the complement components of zebrafish (Danio rerio), Journal of Faculty of Agriculture, Kyushu University, 54, 2, 373-387, 2009.10. |
| 31. | VO Kha Tam, Masakazu TSUJIKURA, Tomonori SOMAMOTO, and Miki NAKAO, Expression responses of the complement components in zebrafish organs after stimulation with poly I:C, mimicry of viral infection, Journal of Faculty of Agriculture, Kyushu University, 54, 2, 389-395, 2009.10. |
| 32. | Forlenza M, Nakao M, Wibowo I, Joerink M, Arts JA, Savelkoul HF, Wiegertjes GF, Nitric oxide hinders antibody clearance from the surface of Trypanoplasma borreli and increases susceptibility to complement-mediated lysis., Molecular Immunology, 10.1016/j.molimm.2009.08.011, 46, 16, 3188-3197, 2009.10, Trypanoplasma borreli is an extracellular blood parasite of carp belonging to the same Order (Kinetoplastida) as African trypanosomes. These mammalian parasites have developed different strategies to evade the host immune system including antigenic variation, immunosuppression and clearance of surface-bound antibodies. The latter mechanism allows trypanosomes to use their swimming movement to cause surface-bound antibodies to 'sail' and accumulate at the posterior end of the parasite, to be internalized via the flagellar pocket and be degraded. There is no evidence that T. borreli shows antigenic variation, but during the late phases of infection NO-mediated immunosuppression is observed. High levels of nitric oxide (NO) lead to extensive tissue nitration whereas the parasite itself is not affected. Therefore, the induction of NO has thus far been considered a parasite-driven response with immunosuppressive effects. In the present study, we show that the induction of NO, particularly during the early phase of T. borreli infections, should be re-considered an effective part of the host immune response. We show that T. borreli rapidly removes surface-bound IgM. In addition, moderate concentrations of NO, by hindering surface antibody clearance, maintain high the concentrations of membrane-bound IgM, thereby favoring antibody-dependent complement-mediated parasite lysis. We performed a comprehensive quantitative gene expression analysis of in total seven different complement factors involved in all three activation pathways, differentiating between 1 and 4 isoforms for each complement gene. Our gene expression analysis supports an important role for antibody-dependent complement-mediated lysis of T. borreli in vivo. To our knowledge, NO-dependent inhibition of antibody clearance from the surface of kinetoplastid parasites has not been investigated. Our data support a role for NO as an important player in host-parasite interactions, not only as immune suppressor (late response) but also as immune effector (early response) in infections with bloodstream parasites such as T. borreli.. |
| 33. | Katakura F, Takizawa F, Yoshida M, Yamaguchi T, Araki K, Tomana M, Nakao M, Moritomo T, Nakanishi T, Co-culture of carp (Cyprinus carpio) kidney haematopoietic cells with feeder cells resulting in long-term proliferation of T-cell lineages., Vet Immunol Immunopathol., 10.1016/j.vetimm.2009.03.007, 131, 1-2, 127-136, 2009.09, To characterise fish haematopoietic stem/progenitor cells, it is necessary to develop a culture system that supports proliferation and differentiation of these cells. In the present study, we established cell lines from various tissues of carp (Cyprinus carpio) and ginbuna (Carassius auratus langsdorfii). By using these cell lines, we developed a culture system in which carp haematopoietic cells proliferated and were successively passaged. Cell lines from carp thymus (KoT), carp fin (KoF1) and ginbuna thymus (GTS6 and GTS9) were newly established. In addition to these cell lines, ginbuna fin (CFS) cell lines were also used as feeder layers. Kidney haematopoietic cells co-cultured with these feeder layers proliferated rapidly and were passaged over 20 times for more than 60 days. To characterise the proliferating cells, expression of marker genes for blood cell development were analysed. In the primary culture, marker genes for myeloid/erythroid progenitors (gata1), haematopoietic stem cells (gata2), neutrophils (mpx/mpo), B-cells (IgH) and T-cells (lck, TCRβ and gata3) were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Expression of most of the genes disappeared after the third passage, only T-cell marker genes were highly expressed after passages. These results indicate that multiple blood cells developed in the primary culture and then T-cell lineages dominantly proliferated after several passages.. |
| 34. | Nevien K. Abdelkhalek, Asuka Komiya, Yoko Kato-Unoki, Tomonori Somamoto, Miki Nakao, Molecular evidence for the existence of two distinct IL-8 lineages of teleost CXC-chemokines, Fish & Shellfish Immunology, 10.1016/j.fsi.2009.08.004, 27, 6, 763-767, 2009.09, Interleukin-8 (IL-8) is a CXC-type chemokine with a chemotactic activity mainly on neutrophils and plays a key role in promoting inflammation. In teleosts, several CXC-chemokines have been cloned and characterized as being IL-8-like. Phylogenetic data however indicate that the reported teleost IL-8-like chemokines are substantially remote from mammalian IL-8, forming a fish-specific clade of IL-8-like chemokines distinct from that of tetrapod IL-8. In the present study, a novel IL-8-like chemokine, designated CaIL-8, has been found in the expressed sequence tags of carp gills and identified as an orthologue of mammalian IL-8. The CaIL-8 transcript encodes 99 amino acids containing a typical CXC motif but lacks an ELR motif, as in most teleost IL-8-like chemokines. Phylogenetic tree constructed by the maximum likelihood method suggests a closer relationship of CaIL-8 with mammalian IL-8 than with other teleost CXC-chemokines reported to be IL-8-like. In a normal unstimulated carp, CaIL-8 mRNA was detected by RT-PCR only in gills, kidney, spleen, heart and peripheral blood leukocytes, in contrast to a previously reported carp IL-8-like chemokine CXCa, which shows ubiquitous basal expression. The results, taken together, are strongly indicative of the presence of two major IL-8-like lineages of CXC-chemokines in teleost.. |
| 35. | Tomonori Somamoto, Nobuaki Okamoto, Teruyuki Nakanishi, Mitsuru Ototake, and Miki Nakao, In vitro generation of viral-antigen dependent cytotoxic T-cells from ginbuna crucian carp, Carassius auratus langsdorfii, Virology, 10.1016/j.virol.2009.04.008, 389, 1-2, 26-33, 2009.06, Little is known about antigen-specific T-cell responses to viruses in teleosts due to a lack of a suitable experimental system using inbred or clonal animals. In the present study we have successfully induced an in vitro generation of virus-specific cytotoxic T-cells (CTLs) from isogeneic ginbuna crucian carp. Responder cells (primarily lymphocytes) from crucian carp haematopoietic necrosis virus (CHNV)-infected fish were capable of proliferating after stimulation in vitro with CHNV-infected syngeneic stimulator cells (primarily lymphocytes and macrophages). The effector cells collected 8 and 12 days after the in vitro stimulation efficiently lysed CHNV-infected syngeneic cells, but not CHNV-infected allogeneic cells or different virus (EVA)-infected syngeneic cells. Furthermore, in situ hybridization analysis showed that some effector cells binding to a CHNV-infected target were TCRβ or CD8α positive. These results provide evidence that the teleost effector cells generated in vitro correspond to virus-specific CTL and they recognize virus-infected target cells in a similar manner of mammalian counterparts.. |
| 36. | Dong-Ho Shin, Barbara M. Webb, Miki Nakao, Sylvia L. Smith, Characterization of shark complement factor I gene(s): Genomic analysis of a novel shark-specific sequence, Molecular Immunology, 10.1016/j.molimm.2009.04.002, 46, 11-12, 2299-2308, 2009.05, Complement factor I is a crucial regulator of mammalian complement activity. Very little is known of complement regulators in non-mammalian species. We isolated and sequenced four highly similar complement factor I cDNAs from the liver of the nurse shark (Ginglymostoma cirratum), designated as GcIf-1, GcIf-2, GcIf-3 and GcIf-4 (previously referred to as nsFI-a, -b, -c and -d) which encode 689, 673, 673 and 657 amino acid residues, respectively. They share 95% (≤) amino acid identities with each other, 35.4-39.6% and 62.8-65.9% with factor I of mammals and banded houndshark (Triakis scyllium), respectively. The modular structure of the GcIf is similar to that of mammals with one notable exception, the presence of a novel shark-specific sequence between the leader peptide (LP) and the factor I membrane attack complex (FIMAC) domain. The cDNA sequences differ only in the size and composition of the shark-specific region (SSR). Sequence analysis of each SSR has identified within the region two novel short sequences (SS1 and SS2) and three repeat sequences (RS1-3). Genomic analysis has revealed the existence of three introns between the leader peptide and the FIMAC domain, tentatively designated intron 1, intron 2, and intron 3 which span 4067, 2293 and 2082 bp, respectively. Southern blot analysis suggests the presence of a single gene copy for each cDNA type. Phylogenetic analysis suggests that complement factor I of cartilaginous fish diverged prior to the emergence of mammals. All four GcIf cDNA species are expressed in four different tissues and the liver is the main tissue in which expression level of all four is high. This suggests that the expression of GcIf isotypes is tissue-dependent.. |
| 37. | Yasuhiro Yamasaki, Tomoyuki Shikata, Atsushi Nukata, Satoko Ichiki, Sou Nagasoe,
Tadashi Matsubara, Yohei Shimasaki, Miki Nakao, Kenichi Yamaguchi, Yuji Oshima,
Tatsuya Oda, Makoto Ito, Ian R Jenkinson, Makio Asakawa and Tsuneo Honjo, Extracellular polysaccharide-protein complexes of a harmful alga mediate the allelopathic control it exerts within the phytoplankton community, The ISME Journal, 10.1038/ismej.2009.24, 3, 7, 808-817, 2009.03, The goal of this study was to examine the significance of allelopathy by the raphidophyte Heterosigma akashiwo in a multispecies phytoplankton community in the field. Towards this aim, we sought allelochemicals of H. akashiwo, which had allelopathic effect both in laboratory experiments and in the field. As an initial step, we showed that the allelopathic effects of H. akashiwo filtrate were both species-specific and dependent upon the cell density of the target species. Secondly, we found for the first time that extracellular, high-molecular weight allelochemicals that is, polysaccharide-protein complexes (APPCs) were produced by a marine phytoplankton species, H. akashiwo. Thirdly, we indicated that the purified APPCs selectively inhibited the growth of the diatom Skeletonema costatum that is a major competitor of H. akashiwo, and thereby tended to promote the formation of monospecific H. akashiwo blooms. Furthermore, we demonstrated that the inhibitory effect of APPCs on the growth of the diatoms was determined by binding to the cell surface of the target species. Finally, we succeeded in the detection of APPCs in the field samples at concentrations exceeding their experimentally determined action threshold during the H. akashiwo bloom. Strategies for ecosystem control, including mitigation of harmful algal blooms (HABs), should take into account that red-tide organisms like H. akashiwo are already part of complex webs involving inter-specific allelopathic inhibition and ecosystem control during their dense blooms.. |
| 38. | Nomiyama H, Hieshima K, Osada N, Kato-Unoki Y, Otsuka-Ono K, Takegawa S, Izawa T, Yoshizawa A, Kikuchi Y, Tanase S, Miura R, Kusada J, Nakao M, Yoshie O, Extensive Expansion and Diversification of the Chemokine Gene Family in Zebrafish: Identification of a Novel Chemokine Subfamily CX, BMC Genomics, 10.1186/1471-2164-9-222, 9, Article No. 222, 2008.05, Background: The chemokine family plays important roles in cell migration and activation. In humans, at least 44 members are known. Based on the arrangement of the four conserved cysteine residues, chemokines are now classified into four subfamilies, CXC, CC, XC and CX3C. Given that zebrafish is an important experimental model and teleost fishes constitute an evolutionarily diverse group that forms half the vertebrate species, it would be useful to compare the zebrafish chemokine system with those of mammals. Prior to this study, however, only incomplete lists of the zebrafish chemokine genes were reported. Results: We systematically searched chemokine genes in the zebrafish genome and EST databases, and identified more than 100 chemokine genes. These genes were CXC, CC and XC subfamily members, while no CX3C gene was identified. We also searched chemokine genes in pufferfish fugu and Tetraodon, and found only 18 chemokine genes in each species. The majority of the identified chemokine genes are unique to zebrafish or teleost fishes. However, several groups of chemokines are moderately similar to human chemokines, and some chemokines are orthologous to human homeostatic chemokines CXCL12 and CXCL14. Zebrafish also possesses a novel species-specific subfamily consisting of five members, which we term the CX subfamily. The CX chemokines lack one of the two N-terminus conserved cysteine residues but retain the third and the fourth ones. (Note that the XC subfamily only retains the second and fourth of the signature cysteines residues.) Phylogenetic analysis and genome organization of the chemokine genes showed that successive tandem duplication events generated the CX genes from the CC subfamily. Recombinant CXL-chr24a, one of the CX subfamily members on chromosome 24, showed marked chemotactic activity for carp leukocytes. The mRNA was expressed mainly during a certain period of the embryogenesis, suggesting its role in the zebrafish development. Conclusion: The phylogenic and genomic organization analyses suggest that a substantial number of chemokine genes in zebrafish were generated by zebrafish-specific tandem duplication events. During such duplications, a novel chemokine subfamily termed CX was generated in zebrafish. Only two human chemokines CXCL12 and CXCL14 have the orthologous chemokines in zebrafish. The diversification observed in the numbers and sequences of chemokines in the fish may reflect the adaptation of the individual species to their respective biological environment.. |
| 39. | Nonaka S, Somamoto T, Kato-Unoki Y, Ototake M, Nakanishi T, Nakao M, Molecular cloning of CD4 from ginbuna crucian carp Carassius auratus langsdorfii, Fisheries Science, 10.1111/j.1444-2906.2008.01530.x, 74, 2, 341-346, 2008.04, The clonal triploid ginbuna crucian carp Carassius auratus langsdorfii, a naturally occurring gynogenetic fish, is a useful model for studying T-cell-mediated immunity. CD4, a T-cell receptor (TCR) coreceptor, is a membrane-bound glycoprotein found on helper T-cells, and assists in the binding of major histocompatibility complexes. In the present study, full-length cDNAs encoding the CD4 molecule from the S3n strain of ginbuna crucian carp were cloned and characterized. 5′-rapid amplification of cDNA ends (RACE) and 3′-RACE yielded two distinct cDNA clones of CD4 homolog from the ginbuna, and these sequences share 95% identity at the amino acid level. These ginbuna CD4 molecules consisted of a signal peptide, immunoglobulin superfamily (IgSf) like domains, a transmembrane domain, and a cytoplasmic domain similar to other known CD4. A tyrosine protein kinase p56lck binding motif is conserved in the cytoplasmic tail of ginbuna CD4. Phylogenetic tree analysis indicated that ginbuna CD4 sequences are closely related to CD4L-1 from other fish species. Expression of ginbuna CD4 mRNA was detected in the gill, thymus, head kidney, trunk kidney and peripheral blood leukocytes, indicating that its expression pattern is similar to that of ginbuna TCRβ mRNA. The results suggest that ginbuna CD4 sequences are useful as molecular probes for helper T-cells.. |
| 40. | Shin DH, Webb B, Nakao M, Smith SL, Molecular cloning, structural analysis and expression of complement component Bf/C2 genes in the nurse shark, Ginglymostoma cirratum, Dev Comp Immunol, 10.1016/j.dci.2007.03.001, 31, 11, 1168-1182, 2007.04, Factor B and C2 are serine proteases that provide the catalytic subunits of C3 and C5 convertases of the alternative (AP) and classical (CP) complement pathways. Two Bf/C2 cDNAs, GcBf/C2-1 and -2 (previously referred to as nsBf/C2-A and nsBf/C2-B), were isolated from the nurse shark, Ginglymostoma cirratum. GcBf/C2-1 and -2 are 3364 and 3082 bp in length and encode a leader peptide, three CCPs, one VWFA, the serine protease domain and have a putative factor D/C1s/MASP cleavage site. Southern blots show that there might be up to two Bf/C2-like genes for each of the two GcBf/C2 isoforms. GcBf/C2-1 and -2 are constitutively expressed, albeit at different levels, in all nine tissues examined. Expression in erythrocytes is a novel finding. Structural analysis has revealed that the localization of glycosylation sites in the SP domain of both putative proteins indicates that the molecular organization of the shark molecules is more like C2 than factor B. Phylogenetic analysis indicates that GcBf/C2-1 and -2 and TrscBf of Triakis scyllia (another shark species) originated from a common ancestor and share a remote ancestor with Bf and C2 of mammals and bony fish.. |
| 41. | Nakao M, Kajiya T, Sato Y, Somamoto T, Kato-Unoki Y, Matsushita M, Nakata M, Fujita T, Yano T, Lectin pathway of bony fish complement: Identification of two homologues of the mannose-binding lectin associated with MASP2 in the common carp (Cyprinus carpio), Journal of Immunology, 10.4049/jimmunol.177.8.5471, 177, 8, 5471-5479, 2006.10, The lectin pathway of complement is considered to be the most ancient complement pathway as inferred from identification of ancient homologs of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) in some invertebrates. MBL homologs with galactose selectivity and an MASP3-like sequence also occur in bony fish, linking the evolution of the lectin complement pathway from invertebrates to higher vertebrates. However, these cannot be considered authentic complement components until confirmatory functional evidence is obtained. Here, we report the isolation and characterization of two MBL homologs from a cyprinid teleost, the common carp, Cyprinus carpio. One, designated GalBL, corresponds to the MBL-like molecule with the galactose specificity. The other is an authentic MBL with mannose specificity. Both were found to associate with a serine protease that cleaves native human C4 into C4b but not C4i with a hydrolyzed thioester. Molecular cloning and phylogenetic analysis revealed this C4-activating protease to be carp MASP2, indicating that MASP2 arose before the emergence of bony fish. Database mining of MBL-like genes reveals that MBL and GalBL genes are arranged in tandem in the zebrafish genome and that both lectins are conserved in the distantly related puffer fish. These results imply that bony fish have developed a diverged set of MBL homologs that function in the lectin complement pathway.. |
| 42. | Somamoto T, Yoshiura Y, Sato A, Nakao M, Nakanishi T, Okamoto N, Ototake M., Expression profiles of TCRbeta and CD8alpha mRNA correlate with virus-specific cell-mediated cytotoxic activity in ginbuna crucian carp., Virology, 10.1016/j.virol.2006.01.019, 348, 2, 370-377, 2006.05, Our previous studies have demonstrated that virus-specific cell-mediated cytotoxicity of sensitized leukocytes can be induced using clonal ginbuna crucian carp and their syngeneic cell lines. In the present study, we attempt to determine if virus-specific cytotoxic cell populations of fish express CD8α and TCRβ genes. Leukocytes from ginbuna crucian carp were separated into four fractions by immunomagnetic separation and density gradient centrifugation: Fraction A, leukocytes with a density of 1.08 g/ml (primarily lymphocytes); Fraction B, sIg-negative leukocytes with density of 1.08 g/ml; Fraction C, sIg-positive cells (primarily B-lymphocytes); Fraction D, leukocytes with a density of 1.08-1.09 g/ml (primarily neutrophils). Leukocytes in all fractions from uninfected fish do not exhibit cytotoxic activity against virus-infected syngeneic cells and weakly express CD8α and TCRβ mRNAs. In contrast, leukocytes in fractions A and B from virus-infected fish exhibit a high level of cytotoxic activity and strongly express CD8α and TCRβ mRNAs. In addition, mRNA expressions of CD8α and TCRβ in effector cells are upregulated by cocultivation with virus-infected target cells but not uninfected ones. The present study suggests that fish possess virus-specific cytotoxic cells with phenotype and gene expression pattern similar to those of CTLs in mammals.. |
| 43. | Mutsuro J, Tanaka N, Kato Y, Dodds AW, Yano T, Nakao M, Two divergent isotypes of the fourth complement component from a bony fish, the common carp (Cyprinus carpio)., Journal of Immunology, 175, 7, 4508-4517, 175(7): 4508-4517, 2005.10, Duplication and diversification of several complement components is a striking feature of bony fish complement systems. It gives an interesting insight into an evolutionary strategy for the possible enhancement of the repertoire of innate immunity. The present study is aimed at examining diversity in bony fish C4, a member of the thioester-containing complement components. Two diverged cDNA sequences sharing only ∼32% identity at the amino acid level were isolated from the common carp and designated C4-1 and C4-2. C4-1 and C4-2 share a number of C4-like structural signatures, such as the thioester site and a disulfide-linked three-chain structure. Interestingly, they differ at the residue corresponding to the thioester-catalytic histidine, as seen in the human C4A and C4B isotypes, suggesting their distinct substrate specificities in the binding reaction of the thioester. Phylogenetic analysis indicates that the divergence of C4-1 and C4-2 predated the separation of the cartilaginous and bony fish lineages. Genomic Southern hybridization suggests the presence of single copy genes each encoding C4-1 and C4-2 in the carp genome. An activation fragment, C4a, was shown to be released from each isotype in carp serum activated via the classical aad/or lectin pathways. Synthetic peptides representing a putative C2 binding site on C4-1 and C4-2 inhibited the classical pathway-mediated hemolytic activity of carp seram in a dose-dependent manner. The results suggest that C4-1 and C4-2 represent two major lineages of C4 that are present in carp serum, have distinct binding specificities, and are functional in the classical/lectin pathways of complement activation.. |
| 44. | Savan R, Aman A, Nakao M, Watanuki H, Sakai M, Discovery of a novel immunoglobulin heavy chain gene chimera from common carp (Cyprinus carpio L.), Immunogenetics, 10.1007/s00251-005-0015-z, 57, 6, 458-463, 57(6): 458-463, 2005.07, In fish, two types of immunoglobulin heavy chain (IGH) genes, namely, IgM and IgD, have been cloned and characterized. Recently, a new IGH isotype specific to teleosts had been identified from zebra fish, rainbow trout, and fugu. In zebra fish, the domains of this new gene are present upstream of the μ region along the IGH locus. During this study, a novel IGH chimera (IgM-IgZ) has been discovered from common carp. The cloned cDNA encodes a typical leader peptide, a variable region, two constant regions, and a secretory tail. The first constant region is made up of the CH1 domain of carp IgM, while the second constant region shares a high similarity to the C H4 domain of the IgZ from zebrafish. Southern hybridization studies of the μ and ζ domains, conducted separately, revealed the presence of at least three copies of the respective genes, and μ and ζ domains might be present on the same loci, although far apart. Expression studies of the IGH genes suggest that there is an increase in chimeric immunoglobulin gene transcription when stimulated with lipopolysaccharide.. |
| 45. | Hammond JA, Nakao M, Smith VJ, Cloning of a glycosylphosphatidylinositol-anchored alpha-2-macroglobulin cDNA from the ascidian, Cionaintestinalis, and its possible role in immunity, Molecular Immunology, 10.1016/j.molimm.2004.09.017, 42, 6, 683-694, 42(6): 683-694, 2005.04, Molecular approaches were used to study thiolester-containing genes in the ascidian, Ciona intestinalis. RT-PCR, RACE and genome mining revealed that this animal expresses not only conventional alpha-2-macroglobulin (α2m) and two forms of C3 but also a gene encoding a glycosylphosphatidylinositol (GPI)-anchored α2m. Previously, GPI-anchored α2ms have been reported only for humans and mice. We propose that GPI-anchored α2ms constitute a third subgroup of the α2m superfamily and may represent an important evolutionary stage in the phylogeny of the thiolester containing proteins. Its occurrence in an ascidian shows its origin pre-dates the evolution of the vertebrates. In C. intestinalis this GPI-anchored α2m, designated Ciona α2m-GPI, is expressed in the hepatopancreas, circulating coelomic blood cells and the gut of adults. It is also expressed in 3-5 days old larvae. Its tissue distribution coupled with its sequence characteristics and unusual domain structure indicate that the encoded protein probably assists in host defence by entrapping and inhibiting proteases from micro-organisms.. |
| 46. | Tsujita T, Tsukada H, Nakao M, Oshiumi H, Matsumoto M, Seya T, Sensing bacterial flagellin by membrane and soluble orthologs of Toll-like receptor 5 in rainbow trout (Onchorhynchus mikiss), Journal of Biological Chemistry, 10.1074/jbc.M407634200, 279, 47, 48588-48597, 279 (47) 48588-48597, 2004.11, Rainbow trout (Onchorhynchus mikiss) possess two genes encoding putative leucine-rich repeat (LRR)-containing proteins similar to human TLR5. Molecular cloning of these two LRR proteins suggested the presence of a TLR5-like membrane form (rtTLR5M) and a soluble form (rtTLR5S). Here we elucidated the primary structures and the unique combinational functions of these fish versions of TLR5. The LRR regions of rtTLR5S and rtTLR5M exhibited 81% homology and relatively high (35.6 and 33.7%) homology to the extracellular domains of human TLR5 (huTLR5). Thus, two distinct genes encode the TLR5 orthologs in fish, one of which has a consensus intracellular domain (TIR). In order to test their functions, we constructed fusion proteins with the LRR region of rtTLR5S (S-chimera) or thai of rtTLR5M and the TIR of huTLR5 (M-chimera). The S- and M-chimeras expressed in HeLa or CHO cells signaled the presence of Vibrio anguillarum flagellin, resulting in NF-κB activation. rtTLR5M was ubiquitously expressed, whereas rtTLR5S was predominantly expressed in the liver. In the hepatoma cell lines of the rainbow trout RTH-149, stimulation of rtTLR5M with V. anguillarum or its flagellin allowed the up-regulation of rtTLR5S. Flagellin-mediated NF-κB activation was more significant in the presence of or simultaneous expression of rtTLR5S. Therefore, a two-step flagellin response occurred for host defense against bacterial infection in fish: (a) flagellin first induced basal activation of NF-κB via membrane TLR5, facilitating the production of soluble TLR5 and minimal acute phase proteins, and (b) the inducible soluble TLR5 amplifies membrane TLR5-mediated cellular responses in a positive feedback fashion.. |
| 47. | Ishikawa J, Imai E, Moritomo T, Nakao M, Yano T, Tomana M, Characterisation of a fourth immunoglobulin light chain isotype in the common carp., Fish & Shellfish Immunology, 10.1016/j.fsi.2003.06.002, 16, 3, 369-379, 16 (3), 369-379, 2004.03, Three isotypes of immunoglobulin (Ig) light (L) chain, designated L1A, L1B, and L3, have been characterised in the common carp (Cyprinus carpio L.) to date. In this paper the molecular cloning of a fourth IgL isotype in carp, designated L2, is described. A reverse transcription-polymerase chain reaction (RT-PCR) method including 5′- and 3′-RACE was used to isolate carp L2 cDNA clones. The VL sequences could be divided into two distinct VL families, designated VL2-1 and VL2-2, most similar to rainbow trout (68% similarity) and zebrafish (78%) VL2 amino acid sequences, respectively. The CL amino acid sequences showed the highest similarity to zebrafish L2 (80%), and contained the characteristic cysteines necessary for intradomain or interchain disulphide bridges as did the VL sequences. Neither the VL nor CL sequences demonstrated such a high similarity to the other carp IgL isotypes, L1A, L1B, and L3. For JL segments, sequence variations appeared to be confined to a few positions. In the course of 5′- and 3′-RACE, cDNA clones containing recombination signal sequence (RSS), representatives of IgL sterile transcripts, were obtained. Southern blot analyses suggested that the locus of carp L2 has a cluster-like organisation. Phylogenetic analyses showed that both carp VL2 and CL2 amino acid sequences highly clustered with other teleost L2 sequences.. |
| 48. | Nakao M, Miura C, Itoh S, Nakahara M, Okumura K, Mutsuro J, Yano T, A complement C3 fragment equivalent to mammalian C3d from the common carp (Cyprinus carpio): Generation in serum after activation of the alternative pathway and detection of its receptor on the lymphocyte surface., Fish & Shellfish Immunology, 10.1016/S1050-4648(03)00057-3, 16, 2, 139-149, 16 (2), 129-149, 2004.02, A terminal degradation product (C3d) of mammalian complement component C3 plays an important role in modulation of the adaptive immune response through the interaction with complement receptor type 2 (CR2) on B cells. The present study is aimed at determining whether this is a functional bridge between the innate and adaptive immune systems in bony fish. The fragmentation of the complement component C3 in carp (Cyprinus carpio) serum, activated with zymosan, was analysed to ascertain if carp C3 also generates a mammalian C3d-like fragment under physiological conditions. A 35 kDa peptide reactive to the anti-carp C3 α-chain was detected on the zymosan particles and in the activated serum. Its N-terminal amino acid sequence identified it as carp C3d derived from the C3-H1 isoform. Another C3 isoform, C3-S, of carp was found to yield a C3d fragment at lower efficiency than C3-H1. Recombinant C3d fragments derived from C3-H1 and C3-S were produced in Escherichia coli as fusion proteins with glutathione-S-transferase (GST), and used for ligands to examine the presence of a possible CR2-like C3d receptor on carp lymphocytes. An enzyme-linked immunoadsorbent assay system, using the recombinant C3d proteins and anti-GST on a microplate to which was attached carp peripheral lymphocytes, detected a significant binding of carp C3d to the lymphocyte. The degree of binding of C3-H1-derived C3d was higher than that of C3-S-derived C3d. In addition, the binding of both ligands was inhibited by anti-C3 α-chain, but not by EDTA or EGTA, indicating that the putative C3d receptor does not require divalent cation. These properties agree well with those reported for mammalian CR2.. |
| 49. | Nakao M, Nakahara M, Yano T, Isolation of carp complement factor B and formation of the C3-convertase, Journal of Faculty of Agriculture, Kyushu University, 49, 1, 101-110, 49 (1), 101-110, 2004.01. |
| 50. | Kato Y, Nakao M, Shimizu M, Wariishi H, Yano T, Purification and functional assessment of C3a, C4a and C5a of the common carp (Cyprinus carpio) complement, Developmental and Comparative Immunology, 10.1016/j.dci.2004.01.006, 28, 9, 901-910, 28, 901-910, 2004.01, Promotion of inflammatory response is an important role of the complement system, but this kind of function is poorly documented for the lower vertebrates. Here we report chemotactic activity of purified anaphylactic fragments derived from the complement components C3, C4 and C5 of the common carp. The purified anaphylatoxins are two C5a-desArg peptides derived from the C5-I isotype, an intact form and a desArg form of C4a from C4-2 isotype, and an intact form and a desArg form of C3a from C3-H1 isoform. These were identified by N-terminal sequencing, mass spectrometry, and peptide mass fingerprinting. In the chemotaxis assay using carp kidney neutrophils, the two C5a-desArg fragments, which are probably allotypic variants, showed a potent chemotactic activity at 0.5-1nM, whereas C3a or C4a showed no significant activity. The results suggest that C3a, C4a and C5a of bony fish have functionally diverged to the state similar to their mammalian homologs.. |
| 51. | Nakao M, Uemura T, Yano T, Characterization of the soluble membrane attack complex (SMAC) of carp (Cyprinus carpio) complement., Journal of Faculty of Agriculture, Kyushu University, 48, 1-2, 127-134, 48: 127-134., 2003.01. |
| 52. | Nakao M, Fujiki K, Kondo M, Yano T, Detection of complement receptors on head kidney phagocytes of the common carp (Cyprinus carpio)., Fisheries Science, 10.1046/j.1444-2906.2003.00709.x, 69, 5, 929-935, 69: 927-933., 2003.01, Opsonization is a crucial function of the complement system in immune clearance. To identify receptors responsible for the complement-mediated opsonization in teleost, head kidney leukocytes from β-1,3-glucan-injected and non-injected common carp (Cyprinus carpio) were subjected to a rosette-forming assay with fixed sheep erythrocytes (Ef). Leukocyte fractions rich in granulocytes and macrophages from the glucan-injected carp had an ability to form the rosette with complement-coated Ef ut the corresponding fractions from non-injected fish did not, suggesting that β-1,3-glucan elicits expression of complement receptors on those cells. While sensitization of Ef with antibodies slightly elevated the rosette formation, complement-coated Ef showed a significant increase in rosette formation and this formation was inhibited with anti-carp C3. Rosette formation was also inhibited by EDTA but not by EGTA. These results suggest that complement-dependent opsonization in carp is mediated by CS-receptors analogous to mammalian complement receptor type 3 (CD11b/CD18 or integrin αM/β2), which recognizes the iC3b fragment in a divalent cation-dependent manner.. |
| 53. | Fujiki K, Nakao M, Dixon B, Molecular cloning and characterization of a carp (Cyprinus carpio) cytokine-like cDNA that shares sequence similarity with IL-6 subfamily cytokines CNTF, OSM and LIF., Developmental and Comparative Immunology, 10.1016/S0145-305X(02)00074-5, 27, 2, 127-136, 27: 127-136., 2003.01, In the course of suppression subtractive hybridisation between sodium alginate-induced peritoneal cells (SA-PC) and normal head kidney cDNAs in common carp (Cyprinus carpio), a cytokine-like cDNA clone was found. The clone, named M17, contains a 1600bp nucleotide sequence that encodes a 215 amino acid putative protein that would have a pI of 9.01 and would include a 33 amino acid signal peptide. The 3′ untranslated region has seven ATTTA mRNA destabilising motifs that are common in cytokines and oncogenes. In a BLASTP search, M17 was most similar to chicken ciliary neurotrophic factor (CNTF) with 25% amino acid identity, followed by mammalian CNTF, cardiotrophin-1 and leukemia inhibitory factor (LIF) all of which belong to the IL-6 subfamily. However, M17 has some differences with CNTF in that CNTF has no signal sequence, the gene organisation of M17 is three exons and two introns, whereas that of CNTF is two exons and one intron, M17 has seven cysteines while CNTF has one cysteine, and M17 mRNA is detected in peripheral blood leukocytes as well as brain, whereas CNTF is expressed only in the nervous system. Compared to other members in the IL-6 subfamily cytokines, M17's cysteine positions and gene organisation are similar to those of oncostatin M and LIF, although amino acid identities are only 15-17%. Southern hybridisation suggested that M17 is a single copy gene. SA-PC showed significantly higher M17 mRNA levels than normal head kidney cells, which are considered to be a source of the SA-PC, indicating that M17 is inducible by inflammatory stimulation.. |
| 54. | Nakao M, Hisamatsu S, Nakahara M, Kato Y, Smith SL, Yano T, Molecular cloning of the complement regulatory factor I isotypes from the common carp (Cyprinus carpio)., Immunogenetics, 10.1007/s00251-002-0518-9, 54, 11, 801-806, 54: 801-806., 2003.01, Factor I is a novel serine protease that regulates complement activation. Here we report the complete primary structure of two isotypic factor Is isolated from the common carp (Cyprinus carpio), a pseudotetraploid teleost. A carp hepatopancreas cDNA library was screened using two RT-PCR-amplified cDNA fragments encoding part of the carp factor I-like serine protease domain. Two distinct cDNA clones, designated FI-A and FI-B, were isolated. Their deduced amino acid sequences share 75.2% identity with each other. FI-A has a typical factor I-like domain organization composed of two disulfide-linked polypeptides (H-chain and L-chain). On the other hand, FI-B contains a novel sequence of 115 amino acids inserted at the N-terminus of the H-chain. Genomic Southern hybridization suggests that FI-A and FI-B are encoded by distinct genes in the carp genome. Expression analysis by RT-PCR revealed that the major site of FI-A expression is the ovary, whereas FI-B expression is detected mainly in the hepatopancreas at a level higher than that of FI-A. The present data, taken together, suggest that carp have duplicated genes coding for factor I, and FI-B with the novel insertion plays a dominant role in the complement system. In addition, homology search of the fugu genome database using the carp FI-A and FI-B sequences identified a putative fugu factor I gene, which has an exon/intron organization different from that of the human orthologue.. |
| 55. | Kato Y, Nakao M, Mutsuro J, Zarkadis IK, Yano T (2003), The complement component C5 of the common carp (Cyprinus carpio) : cDNA cloning of two distinct isotypes that differ in a functional site., Immunogenetics, 10.1007/s00251-002-0528-7, 54, 11, 807-815, 54: 807-815., 2003.01, The complement component C5 plays important roles in inflammatory responses and complement-mediated cytolysis. In bony fish, although C5 has been identified at the DNA or the protein level in trout, carp and gilthead seabream, only partial C5 sequences are available. The present study was designed to obtain the complete primary structure of C5 from the common carp (Cyprinus carpio) and to examine its possible structural diversity. Reverse-transcribed polymerase chain reaction amplification from carp hepatopancreatic RNA resulted in isolation of six distinct C5-like cDNA segments, which were grouped into two divergent types (type I and type II). Using two sequences representative of the two types as probes, two distinct full-length cDNA clones (C5-1 and C5-2) were isolated, in addition to a truncated isoform of C5-1 (C5-1'). The deduced amino acid sequences of C5-1 and C5-2 share 83% identity and predict a typical two-chain structure of the mature protein that lacks the thioester bond, as in C5 from other animals. Southern hybridization of genomic DNA suggested the presence of multiple genes encoding C5-type I and a single gene encoding C5-type II. Interestingly, carp C5-type I contains novel subtypes like C5-1 that have a histidine instead of the well-conserved arginine at the cleavage site for the C5 convertase, both in the complete and truncated forms. Northern blotting analysis suggested that C5-type I and C5-type II are mainly expressed in hepatopancreas, and the expression levels are significantly increased by stimulating carp with lipopolysaccharide or β-1,3-glucan. Possible functional divergence among the C5 isotypes in carp is discussed.. |
| 56. | Tomana M, Ishikawa J, Imai E, Moritomo T, Nakao M, Yano T, Characterization of immunoglobulin light chain isotypes in the common carp., Immunogenetics, 10.1007/s00251-002-0447-7, 54, 2, 120-129, 54: 120-129., 2002.01. |
| 57. | Nakao M, Matsumoto M, Nakazawa M, Fujiki K and Yano T, Diversity of complement factor B/C2 in the common carp (Cyprinus carpio): Three isotypes of B/C2-A expressed in different tissues., Developmental and Comparative Immunology, 10.1016/S0145-305X(01)00083-0, 26, 6, 533-541, 26: 533-541., 2002.01. |
| 58. | Shimasaki Y, Oshima Y, Yokota Y, Kitano T, Nakao M, Kawabata S, Imada N and Honjo T, Purification and identification of a tributyltin-binding protein from serum of Japanese flounder, Paralichthys olivaceus., Environmental Toxicology and Chemistry, 10.1897/1551-5028(2002)0212.0.CO;2, 21, 6, 1229-1235, 21: 1229-1235., 2002.01. |
| 59. | Kono T, Fujiki K, Nakao M, Yano T, Endo M, Sakai M, The immune responses of common carp, Cyprinus carpio L., injected with carp interleukin-1b gene., Journal of Interferon and Cytokine Research, 10.1089/10799900252952190, 22, 4, 413-419, 22: 413-419., 2002.01. |
| 60. | Bayne CJ, Gerwick L., Fujiki K., Nakao M, Yano T, Immune relevant genes identified in the liver of rainbow trout, Oncorhynchus mykiss, by means of suppression subtractive hybridization., Developmental and Comparative Immunology, 10.1016/S0145-305X(00)00057-4, 25, 3, 205-217, 24: 205-217., 2001.01. |
| 61. | Fujiki K, Bayne CJ, Shin DH, Nakao M, Yano T, Molecular cloning of carp (Cyprinus carpio) C-type lectin and pentraxin by use of suppression subtractive hybridisation., Fish & Shellfish Immunology, 10.1006/fsim.2000.0331, 11, 3, 275-279, 11:275-279., 2001.01. |
| 62. | Nakao M, Osaka K, Kato Y, Fujiki K, Yano T, Molecular cloning of the complement C1r/C1s/MASP2-like serine proteases from the common carp (Cyprinus carpio)., Immunogenetics, 52, 3-4, 255-263, 52: 255-263., 2001.01. |
| 63. | Shin DH, Fujiki K, Nakao M, Yano T, Organization of the NKEF gene and its expression in the common carp (Cyprinus carpio)., Developmental and Comparative Immunology, 10.1016/S0145-305X(01)00021-0, 25, 7, 597-606, 25: 597-606., 2001.01. |
| 64. | Nagai T, Mutsuro J, Kimura M, Kato Y, Fujiki K, Yano T, Nakao M, A novel truncated isoform of the mannose-binding lectin-associated serine protease (MASP) from the common carp (Cyprinus carpio)., Immunogenetics, 10.1007/s002510050031, 51, 3, 193-200, 51: 193-200., 2000.01. |
| 65. | Fujiki K, Shin DH, Nakao M, Yano T, Molecular cloning and expression analysis of carp (Cyprinus carpio) interleukin-1b, high affinity immunoglobulin E Fc receptor g subunit and serum amyloid A., Fish & Shellfish Immunology, 10.1006/fsim.1999.0253, 10, 3, 229-242, 10: 229-241., 2000.01. |
| 66. | Fujiki K, Shin DH, Nakao M, Yano T, Molecular cloning and expression analysis of the putative carp (Cyprinus carpio) pre-B cell enhancing factor., Fish & Shellfish Immunology, 10.1006/fsim.2000.0263, 10, 4, 383-385, 10: 383-385., 2000.01. |
| 67. | Nakao M, Mutsuro J, Obo R, Fujiki K, Nonaka M, Yano T, Molecular cloning and protein analysis of divergent forms of the complement component C3 from a bony fish, the common carp (Cyprinus carpio): presence of variants lacking the catalytic histidine., European Journal of Immunology, 10.1002/1521-4141(200003)30:33.3.CO;2-D, 30, 3, 858-866, 30: 858-866., 2000.01. |
| 68. | Fujiki K, Shin DH, Nakao M, Yano T, Molecular cloning of carp (Cyprinus carpio) leukocyte cell-derived chemotaxin 2, glia maturation factor b, CD45 and lysozyme C by use of suppression subtractive hybridization., Fish & Shellfish Immunology, 10.1006/fsim.2000.0294, 10, 7, 643-650, 10: 642-650., 2000.01. |
| 69. | Muturo J, Nakao M, Fujiki K, Yano T, Multiple forms of a2-macroglobulin from a bony fish, the common carp (Cyprinus carpio): Striking sequence diversity in functional sites., Immunogenetics, 10.1007/s002510000216, 51, 10, 847-855, 51: 847-855., 2000.01. |
| 70. | Tomana M, Nakao M, Moritomo T, Fujiki K, Yano T, Isolation of cDNA encoding immunoglobulin light chain from common carp (Cyprinus carpio)., Fish & Shellfish Immunology, 9: 71-80., 1999.01. |
| 71. | Fujiki K, Shin DH, Nakao M, Yano T, Molecular cloning of carp (Cyprinus carpio) CC chemokine, CXC chemokine receptors, allograft inflammatory factor-1, and natural killer cell enhancing factor by use of suppression subtractive hybridization., Immunogenetics, 10.1007/s002510050573, 49, 10, 909-914, 49: 909-914., 1999.01. |
| 72. | Oshima Y, Nirmala K, Yokota Y, Go J, Shimasaki Y, Nakao M, Lee RF, Imada N, Kobayashi K, Accumulation of tributhyltin (TBT) in the blood of flounder and dab intraperitoneally administered with TBT., Marine Environment Research, 10.1016/S0141-1136(97)00082-2, 46, 1-5, 587-590, 46: 587-590., 1998.01. |
| 73. | Nakao M, Moritomo T., Tomana M, Fujiki K, Yano T, Isolation of cDNA encoding the constant region of the immunoglobulin heavy-chain from common carp (Cyprinus carpio L.)., Fish & Shellfish Immunology, 10.1006/fsim.1998.0149, 8, 6, 425-434, 8: 425-434., 1998.01. |
| 74. | Nakao M, Fushitani Y, Fujiki K, Nonaka M, Yano T, Two diverged complement factor B/C2-like cDNA sequences from a teleost, the common carp (Cyprinus carpio)., Journal of Immunology, 161, 9, 4811-4818, 161: 4811-4818., 1998.01. |
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