Miki Nakao

Immune-relevant (including acute phase) genes identified in the livers of rainbow trout, Oncorhynchus mykiss, by means of suppression subtractive hybridization

To develop tools for analysis of the acute phase response, we used suppression subtractive hybridization of cDNAs from the livers of trout in an unchallenged state and in the course of a response to injection with a Vibrio bacterin emulsified in Freunds Incomplete Adjuvant. The resulting cDNA library contains 300-600bp long fragments of 25 or more immune-relevant genes. Fifteen were previously unreported for salmonids, and 12 were not known from any fish species. Known acute phase proteins include serum amyloid A, transferrin and precerebellin-like protein; trout C-polysaccharide-binding protein 1 is probably also an acute phase protein. Components of both the complement system (n=5) and the clotting system (n=3), as well as lectins, various binding proteins, a putative antibacterial peptide, a chemotaxin, an anti-oxidant enzyme, as well as some likely cell-surface receptors and metabolic and lysosomal enzymes are represented in the library. One clone closely resembles a group of Toll-like receptors, including the human IL-1 receptor. Three cDNAs appear to represent complete open reading frames.

Extensive expansion and diversification of the chemokine gene family in zebrafish: Identification of a novel chemokine subfamily CX

BackgroundThe 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.ResultsWe 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.ConclusionThe 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.

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

The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases and have been assigned the accession numbers C88358 through C88445, AB010468, AB010469, AB010713, AB010959, AB012309 and AB012310. The accession numbers of sequences retrieved in this study are as follows: chicken MIP1b, Q90826; human (hu) MCP1, P13500; huMCP2, P80075; huMCP3, P80098; dog MCP1, P52203; pig MCP1, P42831; huEotaxin, P51671; mouse (mo) eotaxin, P48298; huMIP-1a, P10147; moMIP-1a, P10855; huMIP-1b, P13236; moMIP-1b, P14097; huRANTES, P13501; moRANTES, P30882; rainbow trout CXCR4, AJ001039; huCXCR1, P25024; huCXCR2, P25025; huCXCR3, P49682; huCXCR4, P30991; rabbit (ra) CXCR1, P21109; raCXCR2, P35344; rat CXCR2, P35407; moCXCR2, P35343; moCXCR4, P70658; rat CXCR4, O08565; cattle CXCR4, P25930; huCCR1, P32246; huCCR2, P41597; huCCR3, P51677; huCCR4, P51679; huCCR5, P51681; huAIF-1, P55008; rat AIF-1, P70491; pig AIF-1, P81076; rainbow trout NKEF, Q91191; newt TPx, Q90384; huNKEF-A, P35703; huNKEF-B, P32119

Isolation and characterization of collagen from rhizostomous jellyfish (Rhopilema asamushi)

Abstract As a part of the study into the potential development of unused and under-used resources, collagen was isolated from the mesogloea of the rhizostomous jellyfish, Rhopilema asamushi, by limited pepsin digestion and characterized. The yield of this collagen was high (35.2% on a dry weight basis). The primary structure was very similar to that of pepsin-solubilized collagen from edible jellyfish mesogloea, but it was different from those of the collagen from edible jellyfish exumbrella and the acid-soluble collagen from its mesogloea. The denaturation temperature (Td) of 28.8°C. This collagen contained a large amount of a fourth subunit that was provisionally designated α4. This collagen may have the chain composition of an α1α2α3α4 heterotetramer.

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

Unlike mammals, some bony fish species have been reported to possess multiple forms of the complement component C3. To explore the structural and functional diversity of bony fish C3, we have isolated eight distinct cDNA clones encoding C3 from a single carp (Cyprinus carpio). The eight sequences were grouped into five C3 types, designated C3‐H1, C3‐H2, C3‐S, C3‐Q1 and C3‐Q2, each sharing 80 – 86 % amino acid sequence identity with the others. A striking amino acid substitution was noted at the position corresponding to the catalytic histidine, which is conserved in C3 from all the animals analyzed to date and provides the thioester with the ability to bind covalently to hydroxy groups on the target cells or to be hydrolyzed quickly; C3‐S, C3‐Q1 and C3‐Q2 have serine, glutamine and glutamine residues, respectively, in place of the histidine which is conserved in C3‐H1 and C3‐H2. On the other hand, five distinct C3 forms, named C3‐1 to C3‐5, were purified from the serum of a single carp. N‐terminal sequencing and covalent binding to [3H]glycine identified C3‐1 as the translated product of C3‐S, while C3‐2 was that of C3‐H1, and C3‐5 that of C3‐H2. C3‐1 showed a hemolytic activity threefold higher than that of C3‐2, whereas C3‐5 was inactive, suggesting that the thioester catalytic mechanism is not a necessary determinant for C3 activity and that C3 lacking the catalytic histidine plays a significant role in the complement system of carp and probably other bony fish.

Collagen of edible jellyfish exumbrella

The edible jellyfish exumbrella collagen was prepared by limited pepsin digestion. The yield of collagen was very high; 46.4% on the basis of lyophilised dry weight. This collagen was comprised of α1α2α3-heterotrimers, moreover it was relatively stable at 26.0 °C for 60 min. Thus, the edible jellyfish exumbrella will have potential as an important collagen source for use in various industries and it is expected that the development thus so far unutilised resource will advance in the future.

Discovery of a novel immunoglobulin heavy chain gene chimera from common carp (Cyprinus carpio L.)

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 CH4 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.

The complement system in teleost fish: Progress of post-homolog-hunting researches

Studies on the complement system of bony fish are now finishing a stage of homologue-hunting identification of the components, unveiling existence of almost all the orthologues of mammalian complement components in teleost. Genomic and transcriptomic data for several teleost species have contributed much for the homologue-hunting research progress. Only an exception is identification of orthologues of mammalian complement regulatory proteins and complement receptors. It is of particular interest that teleost complement components often exist as multiple isoforms with possible functional divergence. This review summarizes research progress of teleost complement system following the molecular identification and sequence analysis of the components. The findings of extensive expression analyses of the complement components with special emphasis of their prominent extrahepatic expression, acute-phase response to immunostimulation and various microbial infections, and ontogenic development including maternal transfer are discussed to infer teleost-specific functions of the complement system. Importance of the protein level characterization of the complement components is also emphasized, especially for understanding of the isotypic diversity of the components, a unique feature of teleost complement system.

Polymorphic Sirpa is the genetic determinant for NOD-based mouse lines to achieve efficient human cell engraftment

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.Rag2(nullIl2rgnull) 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.Rag1(nullIl2rgnull) 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.

Expansion of genes encoding complement components in bony fish: biological implications of the complement diversity.

The complement system is a major humoral component of vertebrate defenses for tagging and killing target microorganisms. Recent molecular analyses have uncovered a striking feature of bony fish complement, namely that several complement components are encoded by multiple genes. In this review, the structural diversity of C3, C4, C5, factor B, C2, C1r/s and MASP are discussed with special reference to their functional differentiation, mainly focusing on the common carp (Cyprinus carpio), a tetraploidized teleost. In carp, all the members (C3, C4, C5 and a non-complement protein alpha2-macroglobulin) of the thioester-containing protein family are present in multiple isotypes, differing in the primary structures of various functional sites. Three factor B/C2-like isotypes identified in carp showed distinct expression pattern (sites and inducibility), with one behaving as an acute-phase reactant. Two C1r/C1s/MASP2-like isotypes also contain an amino acid substitution that likely affects their substrate specificity. Overall, the present data suggest that the expanded genes of the carp complement system produce more diversified functional components than are known for mammals. The biological significance of this diversity is discussed.