Characterization of fish-specific IFNγ-related binding with a unique receptor complex and signaling through a novel pathway.

Unlike mammals, fish express two type II interferons, IFNγ and fish-specific IFNγ (IFNγ-related or IFNγrel). We previously reported the presence of two IFNγrel genes, IFNγrel 1 and IFNγrel 2, which exhibit potent antiviral activity in the Ginbuna crucian carp, Carassius auratus langsdorfii. We also found that IFNγrel 1 increased allograft rejection; however, the IFNγrel 1 receptor(s) and signaling pathways underlying this process have not yet been elucidated.

Investigation of geriatric syndromes associated with medication in Japan using insurance claims data.

Aim: Multiple risk factors are involved in geriatric syndrome (GS) occurring in older adults. Although drug therapy often contributes to GS, the specific causes among older adults in Japan remain unclear. In this study, we examined the possible prescribing cascade rate among older outpatients eligible for Late-stage Elderly Health Insurance and elucidated the differences between GS and GS associated with medication (GSAM) trends.

Alteration of hemoglobin ß gene expression in mucosal tissues of Japanese flounder, , in response to heat stress, infection, and immunostimulants administration.

Hemoglobin beta (Hbß) is a heme-binding protein capable of oxygen delivery. The oligopeptides derived from Hbβ in fish mucus are active against a variety of gram-negative bacteria and protozoa. To gain information on the physiological and immunological roles of Hbβ in the mucosal tissues of fish, we analyzed changes in Hbß gene expression levels in the epidermis, gills, and intestine of Japanese flounder, , in response to heat stress, infection, and trial feeding of immunostimulants, high-concentration ascorbic acid (AsA) or lactoferrin (LF).

Peculiar Expression of CD3-Epsilon in Kidney of Ginbuna Crucian Carp.

TCR/CD3 complex is composed of the disulfide-linked TCR-αβ heterodimer that recognizes the antigen as a peptide presented by the MHC, and non-covalently paired CD3γε- and δε-chains together with disulfide-linked ζ-chain homodimers. The CD3 chains play key roles in T cell development and T cell activation. In the present study, we found nor or extremely lower expression of CD3ε in head- and trunk-kidney lymphocytes by flow cytometric analysis, while CD3ε was expressed at the normal level in lymphocytes from thymus, spleen, intestine, gill, and peripheral blood.

Effects of IFNγ administration on allograft rejection in ginbuna crucian carp.

In vertebrates, the rejection of allografts is primarily accomplished by cell-mediated immunity. We recently identified four IFNγ isoforms with antiviral activity in ginbuna crucian carp, Carassius auratus langsdorfii. However, involvement of the IFNγ isoforms in cell-mediated immunity, especially in T cell function remains unknown.

Sequential steps of macroautophagy and chaperone-mediated autophagy are involved in the irreversible process of posterior silk gland histolysis during metamorphosis of Bombyx mori.

To elucidate the degradation process of the posterior silk gland during metamorphosis of the silkworm ITALIC! Bombyx mori, tissues collected on the 6th day after entering the 5th instar (V6), prior to spinning (PS), during spinning (SP) and after cocoon formation (CO) were used to analyze macroautophagy, chaperone-mediated autophagy (CMA) and the adenosine triphosphate (ATP)-dependent ubiquitin proteasome. Immediately after entering metamorphosis stage PS, the levels of ATP and phosphorylated p70S6 kinase protein decreased spontaneously and continued to decline at SP, followed by a notable restoration at CO. In contrast, phosphorylated AMP-activated protein kinase α (AMPKα) showed increases at SP and CO.

Purification and characterization of a fish granzymeA involved in cell-mediated immunity.

Granzymes are serine proteases involved in the induction of cell death against non-self cells. The enzymes differ in their primary substrate specificity and have one of four hydrolysis activities: tryptase, Asp-ase, Met-ase and chymase. Although granzyme genes have been isolated from several fishes, evidence for their involvement in cytotoxicity has not yet been reported.

Recombinant carp IL-4/13B stimulates in vitro proliferation of carp IgM(+) B cells.

Teleost IL-4/13B is a cytokine related to mammalian IL-4 and IL-13, of which hitherto the function had not been studied at the protein level. We identified an IL-4/13B gene in common carp (Cyprinus carpio) and expressed the recombinant protein (rcIL-4/13B). RcIL-4/13B was shown to stimulate proliferation of IgM(+) B cells, because after four days of stimulation the IgM(+) fraction of carp kidney and spleen leukocytes had formed many cell colonies, whereas such colonies were not found in the absence of rcIL-4/13B stimulation.

Exploring erythropoiesis of common carp (Cyprinus carpio) using an in vitro colony assay in the presence of recombinant carp kit ligand A and erythropoietin.

The use of in vitro colony assays in mammals has contributed to identification of erythroid progenitor cells such as burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors, and serves to examine functions of erythropoietic growth factors like Erythropoietin (Epo) and Kit ligand. Here, we established an in vitro colony-forming assay capable of investigating erythropoiesis in carp (Cyprinus carpio), cloned and functionally characterized recombinant homologous molecules Epo and Kit ligand A (Kitla), and identified three distinct erythroid progenitor cells in carp. Recombinant carp Epo induced the formation of CFU-E-like and BFU-E-like erythroid colonies, expressing erythroid marker genes, β-globin, epor and gata1.

Kinetics of lymphocyte subpopulations in allogeneic grafted scales of ginbuna crucian carp.

In mammals the rejection of allografts is primarily accomplished by cell-mediated immunity including T cells. Recently, considerable studies reveal the existence of helper and cytotoxic T cell subsets in fish. Here we investigate the kinetics of CD4(+) and CD8α(+) T cells along with sIgM(+) cells and phagocytic cells in an allogeneic scale graft model using ginbuna crucian carp for understanding the mechanisms of cell-mediated immune response.

Identification of a novel fish granzyme involved in cell-mediated immunity.

Granzymes (Gzms) are serine proteases released from cytoplasmic granules within cytotoxic T lymphocytes and natural killer (NK) cells. Gzms induce apoptosis within virus-infected and transformed cells. In fish as well as mammals, Gzms appear to play a major role in inducing target cell death.

Peculiar monomeric interferon gammas, IFNγrel 1 and IFNγrel 2, in ginbuna crucian carp.

The existence of fish-specific isoforms of interferon (IFN)γ, known as IFNγ-related (IFNγrel), has been reported in several fish species. However, comparisons with deduced amino acid sequences of known IFNγrels among several fish species have indicated significant differences at the C-terminus basic amino acid continuous sequences, which indicate the existence of multiple IFNγrel isoforms. Two distinct cDNAs, encoding two IFNγrels, ifngrel 1 and ifngrel 2, were cloned from ginbuna crucian carp (Carassius auratus langsdorfii).

Selenoneine, a novel selenium-containing compound, mediates detoxification mechanisms against methylmercury accumulation and toxicity in zebrafish embryo.

The selenium (Se)-containing antioxidant selenoneine (2-selenyl-N α,N α,N α-trimethyl-L-histidine) has recently been discovered to be the predominant form of organic Se in tuna blood. Although dietary intake of fish Se has been suggested to reduce methylmercury (MeHg) toxicity, the molecular mechanism of MeHg detoxification by Se has not yet been determined. Here, we report evidence that selenoneine accelerates the excretion and demethylation of MeHg, mediated by a selenoneine-specific transporter, organic cations/carnitine transporter-1 (OCTN1).

Molecular cloning of two gonadotropin receptors in mummichog Fundulus heteroclitus and their gene expression during follicular development and maturation.

Two cDNAs encoding gonadotropin receptors, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) were cloned from mummichog (Fundulus heteroclitus) ovary. Deduced amino acid sequences of the mummichog FSHR (fhFSHR) and LHR (fhLHR) showed high homologies to teleost FSHRs (77-53%) and teleost LHRs (76-62%), respectively. Both the fhFSHR and fhLHR are composed of a typical structural architecture of glycoprotein hormone receptors consisting of the large N-terminal extracellular domain, the transmembrane domain containing seven cell surface membrane-spanning regions, and the intracellular domain.

Protective role of cell division cycle 48 (CDC48) protein against neurodegeneration via ubiquitin-proteasome system dysfunction during zebrafish development.

Cell division cycle 48 (CDC48), a ubiquitin-dependent molecular chaperone, is thought to mediate a variety of degradative and regulatory processes and maintain cellular homoeostasis. To investigate the protective function of CDC48 against accumulated ubiquitinated proteins during neurodevelopment, we developed an in vivo bioassay technique that detects expression and accumulation of fluorescent proteins with a polyubiquitination signal at the N terminus. When we introduced CDC48 antisense morpholino oligonucleotides into zebrafish embryos, the morphant embryos were lethal and showed defects in neuronal outgrowth and neurodegeneration, and polyubiquitinated fluorescent proteins accumulated in the inner plexiform and ganglion cell layers, as well as the diencephalon and mesencephalon, indicating that the degradation of polyubiquitinated proteins by the ubiquitin-proteasome system was blocked.

Induction of autophagy by amino acid starvation in fish cells.

Autophagy is well established as a starvation-induced process in yeast and mammalian cells and tissues. To elucidate the cellular mechanisms induced by starvation in fish, we characterized the induction of autophagy in cultured zebrafish cells under starvation conditions. As an autophagic marker protein, the microtubule-associated protein 1-light chain 3B protein (MAP1-LC3B) was cloned from the fish cells, and its expression and localization were characterized.

Antiviral protection mechanisms mediated by ginbuna crucian carp interferon gamma isoforms 1 and 2 through two distinct interferon gamma-receptors.

Fish genomes possess three type II interferon (IFN) genes, ifnγ1, ifnγ2 and ifnγ-related (ifnγrel). The IFNγ-dependent STAT signalling pathway found in humans and mice had not been characterized in fish previously. To identify the antiviral functions and signalling pathways of the type II IFN system in fish, we purified the ifnγ1, ifnγ2 and ifnγrel proteins of ginbuna crucian carp expressed in bacteria and found them to elicit high antiviral activities against crucian carp hematopoietic necrosis virus.

Discovery of the strong antioxidant selenoneine in tuna and selenium redox metabolism.

A novel selenium-containing compound, selenoneine, has been isolated as the major form of organic selenium in the blood and tissues of tuna. Selenoneine harbors a selenium atom in the imidazole ring, 2-selenyl-N(α), N(α), N(α)-trimethyl-L-histidine, and is a selenium analog of ergothioneine. This selenium compound has strong antioxidant capacity and binds to heme proteins, such as hemoglobin and myoglobin, to protect them from iron auto-oxidation, and it reacts with radicals and methylmercury (MeHg).

Evaluating antigen-specific cytotoxicity of CD8+ T cells in fish by granzyme B-like activity.

Granzyme B plays an important role in granule-mediated apoptosis by CTL. It is a well characterized component of the cytolytic machinery in mammals and a candidate for the evaluation of cytotoxic activity of CTL as an alternative to conventional cytotoxicity assay. In this study, we examined the effects of granzyme inhibitors to assess the characteristics of fish granzymes in terms of substrate specificity and the involvement of granzyme B-like in the cytotoxic response.

Conservation of characteristics and functions of CD4 positive lymphocytes in a teleost fish.

The presence of helper and cytotoxic T cells in fish has been suggested, although T cell subsets have yet to be identified at the cellular level. In order to investigate the functions of CD4 and CD8α positive T cells we attempted to produce and characterize monoclonal antibodies (mAbs) against teleost CD4 and CD8α. Here we report the successful production of mAbs against CD4 and CD8α in clonal ginbuna crucian carp Carassius auratus langsdorfii and the function of CD4 positive T cells.

Differential gene expression of HSC70/HSP70 in yellowtail cells in response to chaperone-mediated autophagy.

A cell line derived from the tailfin of the marine teleost yellowtail fish Seriola quinqueradiata was established to examine cellular temperature regulation in an ectothermic animal. Three cytosolic members of the HSP70 family, heat-shock cognate proteins HSC70-1, HSC70-2 and heat-shock protein HSP70, were isolated from cultured yellowtail cells as stress-responsive biomarkers. Expression of hsp70 was heat-inducible, in contrast to the hsc70-1 gene product, which was expressed constitutively.

Clonal growth of carp (Cyprinus carpio) T cells in vitro.

Carp kidney leukocytes co-cultured with a supporting cell layer resulted in the rapid proliferation of various types of leukocytes including immature leukocytes. Expressions of marker genes for multiple blood cell lineages were observed in the primary culture. However, after several passages, the proliferating cells expressed only T cell and macrophage marker genes.

A novel mitochondrial sphingomyelinase in zebrafish cells.

Sphingolipids are important signaling molecules in many biological processes, but little is known regarding their physiological roles in the mitochondrion. We focused on the biochemical characters of a novel sphingomyelinase (SMase) and its function in mitochondrial ceramide generation in zebrafish embryonic cells. The cloned SMase cDNA encoded a polypeptide of 545 amino acid residues (putative molecular weight, 61,300) containing a mitochondrial localization signal (MLS) and a predicted transmembrane domain.