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The structures of fish serum immunoglobulin differ among different fish species. In this study, we accidently isolated a rabbit immunoglobulin (Ig) light chain bound to serum immunoglobulin from different marine fish species using phage display. Fish Ig was separated using a protein A column.The phage library was generated from variable regions of rabbit spleen B cells immunized with bluefin tuna Thunnus orientalis Ig. Fish Ig–specific phages were enriched using two rounds of bio-panning with yellowtail Seriola quinqueradiata serum Ig, followed by two rounds of bio-panning with red seabream Pagrus major serum Ig.The enriched phages demonstrated an increase in binding specificity to the tuna, yellowtail, and red seabream Igs compared to the phages listed in the unpanned library.A recombinant protein of a single clonal phage, which encodes the rabbit Ig light chain, was produced, and the binding specificities to fish Igs were analyzed using enzyme-linked immunosorbent assay (ELISA) and western blotting.The recombinant protein exhibited binding properties to fish Igs in the ELISA. However, the recombinant protein that bound to serum protein(s), but not IgM, was detected via western blotting.The recombinant protein may provide a novel information on the common structural feature in the fish immunoglobulins., 公開日: 2023-07-21

Red sea bream iridovirus (RSIV) is the pathogen that causes red sea bream iridoviral disease. It causes a huge loss to the Japanese aquaculture industry. In 2021, outbreaks of red sea bream iridovirus occurred in South Japan. This study analysed nine whole-genome sequences of RSIV isolated in Oita and Ehime Prefectures in 2021 using a short-read next-generation sequencer. Nine isolates had highly uniform sequences, and there was no variant depending on locations or host species. Phylogenetic analyses with other reported megalocytivirus isolates showed that RSIV isolated in 2021 was genetically different from RSIV previously isolated in Oita and Ehime Prefectures in 2017-2019. These results suggest that RSIV isolated in Oita and Ehime Prefectures in 2021 might spread from a common ancestor different from the recent one. Additionally, it was found that RSIV isolated in 2021 had sequence mutations on protein-coding sequences that may be involved in viral pathogenicity and infectivity.

The digestive organs of terrestrial isopods harbor bacteria of the recently proposedmollicute family Hepatoplasmataceae. The only complete genome available for Hepatoplasmataceae is that of “Candidatus Hepatoplasma crinochetorum”. The scarcity of genome sequences has hampered our understanding of the symbiotic relationship between isopods and mollicutes. Here, we present four complete metagenome-assembled genomes (MAGs) of uncultured Hepatoplasmataceae members identified from shotgun sequencing data of isopods. We propose three novel species “Candidatus Tyloplasma littoralis” identified from the semiterrestrial isopod Tylos granuliferus, “Candidatus Hepatoplasma vulgare” identified from the common pill bug Armadillidium vulgare, and “Candidatus Hepatoplasma scaber” identified from the common rough woodlouse Porcellio scaber. Phylogenetic analysis of 16S ribosomal RNA sequences showed that “Candidatus Tyloplasma littoralis” and other semiterrestrial isopod-associated mollicutes form a sister clade to terrestrial Hepatoplasma members, justifying their assignment to a novel genus. Phylogenomic analysis of 151 mollicutes confirmed that Hepatoplasmataceae is a sister clade of Metamycoplasmataceae in the order Mycoplasmoidales. Our analysis also revealed that that Hepatoplasmataceae lack major metabolic pathways but has a likely intact type IIA CRISPR-Cas9 machinery, indicating that these mollicutes have an ectosymbiotic lifestyle with high nutritional dependence on their host. We did not find any evidence that Hepatoplasmataceae encode digestive enzymes that could provide nutritional benefits to the host, which suggests that they may act as defensive symbionts.

Crustacean genomes harbor sequences originating from nimaviruses, a family of large double-stranded DNA viruses infecting crustaceans. In this study, we recovered metagenome-assembled genomes of 25 endogenous nimaviruses from crustacean genome data. Phylogenetic analysis revealed four major lineages withinNimaviridae, and for three of these lineages, we propose novel genera of endogenous nimaviruses: “Majanivirus” and “Pemonivirus” identified from penaeid shrimp genomes, and “Clopovirus” identified from terrestrial isopods. Majanivirus genomes contain multiple eukaryotic-like genes such as baculoviral inhibitor of apoptosis repeat-containing genes, innexins, and heat shock protein 70-like genes, some of which contain introns. An alignment of long reads revealed that that each endogenous nimavirus species specifically inserts into host microsatellites or within 28S rDNA. This insertion preference was associated with the type of virus-encoded DNA recombination enzymes, the integrases. Majaniviruses, pemoniviruses, some whispoviruses, and possibly clopoviruses specifically insert into the arthropod telomere repeat motif (TAACC/GGTTA)n and all possessed a specific tyrosine recombinase family. Pasiphaea japonica whipovirus and Portunus trituberculatus whispovirus, the closest relatives of white spot syndrome virus, integrate into the host 28S rDNA and are equipped with members of another family of tyrosine recombinases that are distantly related to telomere-specific tyrosine recombinases. Endogenous nimavirus genomes identified from sesarmid crabs, which lack tyrosine recombinases and are flanked by a 46-bp inverted terminal repeat, integrate into (AT/TA)n microsatellites through the acquisition of a Ginger2-like cut- and-paste DDE transposase. These results suggest that endogenous nimaviruses are giant transposable elements that occupy different sequence niches through the acquisition of different integrase families.ImportanceCrustacean genomes harbor sequences originating from a family of large DNA viruses called nimaviruses, but it is unclear why they are present. We show that endogenous nimaviruses selectively insert into repetitive sequences within the host genome, and this insertion specificity was correlated with different types of integrases, which are DNA recombination enzymes encoded by the nimaviruses themselves. This suggests that endogenous nimaviruses have colonized various genomic niches through the acquisition of integrases with different insertion specificities. Our results point to a novel survival strategy of endogenous large DNA viruses colonizing the host genomes. These findings may clarify the evolution and spread of nimaviruses in crustaceans and lead to measures to control and prevent the spread of pathogenic nimaviruses in aquaculture settings.

Red sea bream iridovirus (RSIV) belonging to the genus Megalocytivirus of the family Iridoviridae is the cause of serious mass mortality of cultured marine fishes. RSIV-type megalocytiviruses show extremely high nucleotide sequence identities. Thus, epidemiological studies on this virus are limited. This study developed two primer sets amplifying the regions possessing single nucleotide polymorphism (SNP) to determine the relationships and divergence of RSIV-type megalocytiviruses isolated from cultured marine fishes in Japan. The two regions were designed according to the genome sequences of the representative RSIV genotype II of megalocytivirus members in GenBank. The SNP 1 and 2 regions have sequences homologous to hypothetical protein ORF 24 and ORF 31, respectively, of RSIV (accession no. AP017456.1). By sequencing the regions, 53 polymorphic sites were identified. The phylogenetic analysis of 25 RSIV-type megalocytivirus isolates, classified into RSIV cluster, was clustered into eight haplotypes (seven haplotypes from Oita, two haplotypes from Ehime, and one haplotype shared between Oita and Ehime). These findings suggested that SNP in the RSIV genome is a powerful application for the detection and identification of RSIV-type megalocytiviruses.

Gene expression profiles during the transition from fasting to refeeding were investigated in the gut and liver of Masu salmon Oncorhynchus masou masou. Fish were starved for 3 days and then fed at 3% of body weight. The gut and liver were resected before and after feeding, and the tissues were used for transcriptome analyses. Twenty-nine and 45 genes were more than eightfold differently expressed in the gut and liver, respectively. Genes involved in fatty acid and carbohydrate metabolism were differentially expressed in the liver tissues. Genes involved in protein folding, such as GRP78 and endoplasmin, were significantly upregulated in both organs at 6, 12, and 24 h after feeding. Furthermore, glycogen synthase kinase (GSK) 3-binding protein, a negative regulator of GSK with pivotal roles in stress response, was downregulated in both organs 6 h after feeding. These results suggest that the change in nutritional status from fasting to refeeding is accompanied by cellular stress.

Vibrio penaeicida (family Vibrionaceae ) is an important bacterial pathogen that affects Japanese shrimp aquaculture. Only two whole-genome sequences of V. penaeicida are publicly available, which has hampered our understanding of the pathogenesis of shrimp vibriosis caused by this bacterium. To gain insight into the genetic features, evolution and pathogenicity of V. penaeicida, we sequenced five V. penaeicida strains (IFO 15640T, IFO 15641, IFO 15642, TUMSAT-OK1 and TUMSAT-OK2) and performed comparative genomic analyses. Virulence factors and mobile genetic elements were detected. Furthermore, average nucleotide identities (ANIs), clusters of orthologous groups and phylogenetic relationships were evaluated. The V. penaeicida genome consists of two circular chromosomes. Chromosome I sizes ranged from 4.1 to 4.3 Mb, the GC content ranged from 43.9 to 44.1 %, and the number of predicted protein-coding sequences (CDSs) ranged from 3620 to 3782. Chromosome II sizes ranged from 2.2 to 2.4 Mb, the GC content ranged from 43.5 to 43.8 %, and the number of predicted CDSs ranged from 1992 to 2273. All strains except IFO 15641 harboured one plasmid, having sizes that ranged from 150 to 285 kb. All five genomes had typical virulence factors, including adherence, anti-phagocytosis, flagella-related proteins and toxins (repeats-in-toxin and thermolabile haemolysin). The genomes also contained factors responsible for iron uptake and the type II, IV and VI secretion systems. The genome of strain TUMSAT-OK2 tended to encode more prophage regions than the other strains, whereas the genome of strain IFO 15640T had the highest number of regions encoding genomic islands. For comparative genome analysis, we used V. penaeicida (strain CAIM 285T) as a reference strain. ANIs between strain CAIM 285T and the five V. penaeicida strains were >95 %, which indicated that these strains belong to the same species. Orthology cluster analysis showed that strains TUMSAT-OK1 and TUMSAT-OK2 had the greatest number of shared gene clusters, followed by strains CAIM 285T and IFO 15640T. These strains were also the most closely related to each other in a phylogenetic analysis. This study presents the first comparative genome analysis of V. penaeicida and these results will be useful for understanding the pathogenesis of this bacterium.

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin-producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA-like (rPirA) and PirB-like (rPirB) toxins. Whole-egg powders having IgY specific to rPirA (anti-PirA-IgY) and rPirB (anti-PirB-IgY) and IgY from non-immunized hen (control-IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirA-IgY, anti-PirB-IgY and control-IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti-PirA-IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti-PirA-IgY (87%) compared with the control (12%). These results confirm that addition of anti-PirA-IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.

報告, Reports, 学生が卒業までに修得すべき看護技術について、技術内容の評価基準を明確化したルーブリックによる質的な評価表を作成した。学生がルーブリックによる自己評価を記録し教員と共有するICT システムを作成し、臨地実習にて学生の技術到達度の確認と指導に活用した。現状の教育内容の成果と課題を明確にするために、2017 年度秋セメスターから2018 年度春セメスターに行われた臨地実習期間に入力された自己評価データを集計し、学生の技術修得状況を確認した。結果からは、臨地実習や学内演習での繰り返しの実践によって高い到達度評価が得られている技術項目が存在する一方で、臨地実習での実践や経験の機会が限られる項目に関しては、評価のレベルが上がらない傾向がみられた。また臨地実習を進めながらの142 項目の技術評価は学生・教員とも負担が大きく、自己評価の更新頻度を向上させ学生の実態を客観的に把握するためには技術項目の精選による絞り込みが必要と考えられる。

紀要委員会企画, Special Articles, 本稿は、本学看護学部の2019 年度教育課程改定における活動内容の概要を記した。2012 年に教育課程変更を行って５年が経過したことや、教育職員免許法の改正により養護教諭課程の再課程認定に伴い、2019 年度に向けて看護学部教育課程の改定を行うことになった。2017 年度からカリキュラム検討委員会を中心に教育課程の検討を行い、２年間に渡って教育課程改定に向けて活動した内容をまとめた。2019 年度新教育課程としては、地域包括ケアシステムの推進に基づく社会の変遷にあわせた教育課程へと発展させるための学修内容の追加、本学の強みである充実した実習環境をもとに行われている臨地看護学実習を通して「生命の尊厳と隣人愛」に基づく教育理念を継続的に意識づけられるような教育課程を策定することができた。指定規則改正に伴う次の教育課程の改定に向けて、今回のプロセスが参考となることを期待する。

Here, we present the genome sequence of lymphocystis disease virus 2 LCDV-JP_Oita_2018 (genus Lymphocystivirus , family Iridoviridae ), which was isolated from a diseased Japanese flounder ( Paralichthys olivaceus ) in Japan. The LCDV-JP_Oita_2018 genome was assembled into a circular contig of 186,627 bp, with 140 predicted protein-coding genes and a GC content of 27%.

The Gram-negative bacterium Vibrio nigripulchritudo is an important shrimp pathogen. Here, we present the genome sequence of Vibrio nigripulchritudo TUMSAT-TG-2018, which was isolated from a diseased pacific white shrimp ( Litopenaeus vannamei ). The assembly totaled 6.8 Mbp, consisting of two chromosomes and four plasmids.

Polymorphic simple sequence repeat (SSR) markers of Ecklonia radicosa were developed using Illumina MiSeq next-generation sequencing (NGS) analysis. In total, 3112 SSRs (di- to hexanucleotide motifs repeated more than six times) were identified from 61.5-Mb assembled genomic DNA using MISA perl script. Among the SSRs, di- (853 loci, 27.4%) and trinucleotides (1813 loci, 58.3%) were dominant, while tetra- (172 loci, 5.5%), penta- (175 loci, 5.6%), and hexanucleotides (99 loci, 3.2%) were not common. After specific amplification and polymorphic tests of 75 selected loci (tri- to hexanucleotide motifs repeated more than eight times), 11 SSR markers (Eradic01–11) were successfully developed. The range of the number of alleles, observed heterozygosity, and expected heterozygosity in the 11 markers was 3–13, 0.200–0.683, and 0.258–0.864, respectively. Polymorphic information content (PIC) analysis indicated that eight markers (Eradic01, 02, 04, 05, 06, 08, 09, and 11) were highly informative (PIC > 0.5) and the other three were reasonably informative (0.5

Vibrio atypicus is a Gram-negative bacterium associated with the digestive tract of penaeid shrimp. Here, we present the draft genome sequences of two V. atypicus strains, DSM 25292T and TUMSAT1. Both assemblies were approximately 4.8 Mbp long, with GC contents of around 44%.

Aims To investigate the virulence of the Vp_PirAB-like genes in Vibrio parahaemolyticus-AHPND causing strain and factors that associate with the virulence level. Methods and Results The virulence of Vp_PirAB-like were examined using a non-virulent strain FP11 of V. parahaemolyticus transformed with a plasmid harboring Vp_PirAB-like genes and then used it to challenge shrimp Litopenaeus vannamei and Marsupenaeus japonicus. Both species experienced 100% mortality 10 days post-infection. Analysis of a mutant strain (E1M), that was originally identified as virulent strain (E1) but lost its virulence to L. vannamei, revealed that it lacked a part of the Vp_PirA-like gene and all of Vp_PirB-like gene. The copy numbers of Vp_PirA-like and Vp_PirB-like genes varied among virulent strains and were not correlated with their virulence. In Western blotting, Vp_PirA-like and Vp_PirB-like proteins were detected in both cell lysate and culture supernatant. The strongest intensity of detecting band in culture supernatant could be observed in the strain that caused the highest mortality. V. parahaemolyticus AHPND-causing strain, unlike the human tdh-positive strain, did not show any enterotoxicity. Conclusion V. parahaemolyticus AHPND-causing strains secret the Vp_PirA-like and Vp_PirB-like proteins during growing phase. The amount of secreted proteins affects the shrimp mortality. Significance and Impact of the Study The secreted proteins of Vp_PirAB-like are key factors of virulence in V. parahaemolyticus AHPND-causing strain, but not gene copy. This article is protected by copyright. All rights reserved.

Continuous shrimp cell cultures offer opportunities for studying shrimp pathogens at the cellular and molecular level and developing diagnostic tools. However, no continuous shrimp cell lines have yet been successfully established. This might be due to the lack of information on the molecular mechanisms that control shrimp cell proliferation and cell arrest under culture conditions. In this study, differentially expressed genes (DEGs) in shrimp primary culture cells were determined by comparison with normal tissue (in vivo). This study aimed to comprehensively identify key regulator genes that control cell proliferation and cell cycle arrest in culture conditions. The primary shrimp cells were derived from testicular tissue and cultured in vitro. RNA sequencing (RNA-Seq) was performed to investigate change in gene expression level across the entire transcriptome between shrimp primary cells and normal tissue (in vivo). RNA-seq results revealed over 100 genes with distinct gene expression patterns between primary cells and normal tissue. The DEG results and functional gene analysis showed a clear difference in gene expression patterns of cell cycle-related genes between primary cells and normal tissue. LRWD1, TMEM127, CDCA3, PPP2R1A, and GOLGA2, which are cell cycle-related genes, were downregulated in shrimp primary cells. These genes are required for G1/S phase transition, entry into mitosis, and cell proliferation control. However, a gene that induces cell cycle arrest, ARAF, was upregulated in culture conditions. The results demonstrated that these genes might play essential roles in cell proliferation and cell arrest under culture conditions. These candidate genes may be alternative potential targets for genetic manipulation to maintain cell proliferation and establishment of a shrimp continuous cell line.

Vibrio penaeicida is a bacterial pathogen of cultured shrimp. The draft genome sequence of V. penaeicida strain TUMSAT-NU1 consists of 100 scaffolds with a total of 6.41 Mbp. We identified possible virulence factors, and we found that V. penaeicida and Vibrio nigripulchritudo are closely related.

Partial sequences of cytokine genes IFNγ, IL-1β, IL-8, IL-10, and TNFα were identified from next-generation sequencing data for three amberjack species: Seriola quinqueradiata, S. lalandi and S. dumerili. The cytokine genes had more than 90 % nucleotide identity among the three species. Quantitative PCR (qPCR) primers were designed from the consensus sequences to detect cytokine genes. The specificities of consensus qPCR primers were investigated in the head kidney of three amberjack species treated with formalin-killed cells (FKC) of Vibrio anguillarum and polyinosinic-polycytidylic acid [poly(I:C)] at 3 h post injection (hpi) using qPCR. The primers detected up-regulation of certain cytokine genes after FKC of V. anguillarum and poly(I:C) stimulation in all species examined. Consequently, the primers are suitable for studying and detecting cytokine genes after vaccination and pathogen infection in these three amberjack species.

Here, we present the complete mitochondrial genome of the sakura shrimp, Sergia lucens (Crustacea, Decapoda, Sergestidae). The circular genome is 16,087 base pairs in length and contains 13 protein-coding genes, 22 tRNA genes, and two rRNA genes. The nucleotide composition of the S. lucens mitogenome is biased towards A + T (69.9%). The gene arrangement was identical to penaeid shrimps. Maximum likelihood phylogenetic analysis using the nucleotide sequences of 13 protein-coding genes placed S. lucens next to Acetes chinensis, supporting the conventional taxonomic relationship of Sergia and Acetes.

DNA vaccination is one method to protect farmed fish from viral and bacterial diseases. Chimeric antigens encoded by DNA vaccines have been shown to increase the resistance to viral diseases. Here, we sequenced the gene encoding lysosome-associated membrane protein-1 from Japanese flounder, Paralichthys olivaceus, (JfLAMP-1) and assessed its use in a chimeric DNA vaccine fused with the major capsule protein (MCP) from red seabream iridovirus (RSIV). JfLAMP-1 cDNA has a length of 1248 bp encoding 415 aa, which contains transmembrane and cytoplasmic domains. JfLAMP-1 is constitutively expressed in several tissues and its expression in spleen was upregulated following injection of formalin-killed cells (FKC) of Edwardsiella tarda. Immunofluorescence analysis showed that JfLAMP-1 is distributed in the small and large granules in the cytoplasm and groups close to the nucleus. The DNA encoding the luminal domain of JfLAMP-1 was replaced with the gene for the RSIV MCP, and the construct was cloned in an expression vector (pCIneo). Fish vaccinated with pCLAMP-MCP had significantly higher antibody levels than fish vaccinated with pCIneo vector harboring the MCP gene (p < 0.05) at day 30 post-vaccination.

Gene in the interleukin 1 family plays a central role in the regulation of immune and inflammatory responses. Here, we describe two novel IL-1-like gene in Japanese flounder Paralichthys olivaceus (jfIL1-L1 and jfIL1-L2). jfIL1β-L1 was homologous to Nile Tilapia IL-1β-like gene and Arctic char IL-1, and jfIL1β-L2 showed homology to hypothetical protein LOC100699119 of Nile Tilapia and rainbow trout Oncorhynchus mykiss IL-1 receptor agonist (RA). The deduced amino acids sequences of these IL-1β-like genes showed very low identities to the Japanese flounder IL-1 (jfIL-1). Phylogenetic analysis confirmed that jfIL1β-L1 and -L2 were distinct from jfIL-1. The gene encoding the predicted ORF of jfIL1β-L1 and -L2 is divided into 6 exons and 7 exons, respectively. Transcripts of jfIL1β-L1 were detected in gills, intestine, kidney and spleen, and those of jfIL1β-L2 were detected in gills, intestine and spleen. The mRNA levels of jfIL1β-L1 and -L2 were not effect or slightly decreased by treatment with LPS and the formalin-killed cells of Edwardsiella tarda whilst mRNA levels of jfIL1β were significantly increased in the kidney and spleen at 6 h by these treatments.

Hemocytes in the circulating hemolymph play important roles for immune responses in shrimp. Previous studies on immune responses by hemocytes in penaeid shrimp were based on gene expression analyses of the entire population of hemocytes and thus may have missed different immune responses of different hemocyte sub-populations. In this study, we separated hemocytes into two sub-populations by Percoll gradient centrifugation, morphological characteristics of each population were then analyzed by May-Giemsa staining, flow cytometry, and FACSCalibur. Results showed hemocytes were divided into an upper layer basophilic, and lower layer of eosinophilic hemocytes. Basophilic hemocytes were larger in size compared to eosinophilic hemocytes, which were more granulated than the basophilic hemocytes. Transcriptome analysis was then conducted through RNA-seq analysis by Miseq, which revealed 16 differentially-expressed transcripts between the two sub-populations. In the upper-layer, the highly expressed transcripts that were homologous to immune-related genes that suggest hemocytes from this layer may play as the regulator of immune system and control the action of other cells to eliminate pathogen. On the other hand, transcripts that were highly expressed in the lower-layer were homologous to the antimicrobial peptide (AMP) crustin, which supports that hemocytes on this layer have granules as crustins are normally secreted from hemocyte granules. The high expression of crustin in the lower-layer also provides insight on the mechanism of the anti-microbial function, where hemocytes produce and store AMPs in its granules. These differentially expressed genes are potential hemocyte molecular markers, and among them we identified one of the highly expressed genes in the hemocytes from the upper-layer (c11736_g1) to be a promising candidate molecular marker predicted to be a surface molecule, which is a common characteristic for molecular markers.

Heat shock proteins (HSPs) are proteins that are expressed more strongly when the cells are exposed to physiological and stressful conditions. In this study, the full-length cDNAs of heat shock proteins 40 (MjHSP40), 70 (MjHSP70) and 90 (MjHSP90) were cloned from kuruma shrimp Marsupenaeus japonicus. The open reading frames (ORFs) of the cDNA clones have lengths of 1,191, 1,959 and 2,172 bp and encode 396, 652 and 723 amino acid residues, respectively. The predicted MjHSP40 amino acid sequence contains a J domain, a glycine/phenylalanine-rich region, and a central domain containing four repeats of a CxxCxGxG motif, indicating that it is a type I HSP40 homolog. The signature sequences of the HSP70 and HSP90 gene families are conserved in the MjHSP70 and MjHSP90 amino acid sequences. The deduced amino acid sequences of MjHSP70 and MjHSP90 share high identity with previously reported shrimp HSP70s and HSP90s, respectively. The expression of MjHSP90 mRNA increased at 32°C. Additionally, the expressions of MjHSP40, MjHSP70 and MjHSP90 mRNAs increased in defense-related tissues (i.e., hemocytes and lymphoid organ) when the shrimp were challenged with white spot syndrome virus.

Expressed sequence tag (EST) analyses were performed with the aim of identifying enzyme genes in the liver of the Bleeker’s squid Loligo bleekeri. Of the 768 ESTs identified and sequenced, 669 were grouped into 324 clusters. Of these clusters, 123 comprising 245 ESTs were found to be homologous to genes reported to date. Among these, 43 clusters were annotated as enzymes according to the Enzyme Commission (EC) numbering system. Two EC groups, oxidoreductases and hydrolases, possessed a large number of ESTs. A cluster homologous to the glutathione peroxidase, an enzyme in the oxidoreductase group, contained 16 ESTs, which accounted for 2.4% of the total ESTs sequenced. There are three serine proteases, three cathepsins, two triacylgricerol lipases, and two chitinases among the clusters homologous to the enzymes in the hydrolase group. Since the squid liver functions in the digestive process, these enzymes would be involved in food digestion. Our data provide information on the various types of enzymes expressed in the squid liver and may provide a useful basis for further characterization of these enzymes.

The NK-lysin cDNA of Japanese flounder, Paralichthys olivaceus, consists of 657bp, containing an open reading frame (ORF) of 444bp, which encodes 147 amino acid residues. The amino acid sequence of Japanese flounder NK-lysin has 21% identity to porcine NK-lysin and bovine NK-lysin, 23% to equine NK-lysin, and 46% to zebrafish NK-lysin-like protein. Multiple alignments of Japanese flounder NK-lysin and other known saposin-like proteins revealed that the six cysteine residues important for structural folding are completely conserved. The Japanese flounder NK-lysin gene is approximately 2kb and consists of five exons and four introns. Japanese flounder NK-lysin mRNA constitutive expression was mainly detected in gills, heart, head kidney, intestines, peripheral blood leukocytes (PBLs), spleen and trunk kidney, and was detected at low levels in liver, muscle and ovary. However, expression was not detected in brain, skin and stomach of apparently healthy Japanese flounder. Gene expression of Japanese flounder NK-lysin was not inducible by lipopolysaccharide (LPS) treatment. A synthesized NK-lysin peptide, consisting of 27 amino acid residues, showed antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Photobacterium damselae subsp. piscicida.

The cysteine-rich peptide hepcidin is known to be an antimicrobial peptide and iron transport regulator that has been found in both fish and mammals. Recently, we found two different types (designated Hep-JF1 and Hep-JF2) of hepcidin cDNA in the Japanese flounder, Paralichthys olivaceus, by expressed sequence tag analysis. The identity of amino acid sequences between Hep-JF1 and Hep-JF2 was 51%. The Hep-JF1 and Hep-JF2 genes both consist of three exons and two introns, and both exist as single copies in the genome. The predicted mature regions of Hep-JF1 and Hep-JF2 have six and eight Cys residues, respectively. The first Cys residue of Hep-JF1 was deleted and the second was replaced with Gly. The number and positions of Cys residues in Hep-JF2 are the same as they are in human Hep. Hep-JF1 is specifically expressed in liver while the expression of Hep-JF2 was detected from gill, liver, heart, kidney, peripheral blood leucocytes, spleen and stomach. Gene expression of Hep-JF1 in liver decreased during experimental iron (iron-dextran) overload. Expression of Hep-JF1 in liver was decreased by injecting fish with iron-dextran and increased by injecting lipopolysaccharide. Iron overload did not significantly affect expression of Hep-JF2 in liver but it did increase expression of Hep-JF2 in kidney. Lipopolysaccharide injection increased expression of Hep-JF2 in both liver and kidney. In liver, some cells expressed both Hep-JF1 and Hep-JF2 while some other cells expressed just one of them. Synthesized Hep-JF2 peptide showed antimicrobial activity, while synthesized Hep-JF1 peptide did not against several bacteria including fish-pathogenic bacteria used in this study.

「アルツハイマー病者からみた世界-私はだれになって行くの-」の著者クリスティーン・ボーデンは、その著書で痴呆の初期段階に、より信頼できる診断がより早期になされると同時に、患者の機能をより長く維持させるための抗痴呆薬を利用し、前向きな生活態度を維持することが重要であることを主張している。1)これに対し我が国の現実は、早期に適切な診断を受け治療をしている患者よりも、痴呆を疑われながらも診断未確定の高齢者が多いことが指摘されている。この現状をもたらしている要因とその弊害について概観した。

Immunization by an antigen-encoding DNA (DNA vaccines) has shown promising results in rendering protection against some bacterial and viral diseases in fish. However, some studies showed that DNA vaccines induce variable levels of protection compared with the other types of vaccination. These results are driving the research interest against these infectious diseases to improve the efficacy of DNA vaccines. Recently, chimeric antigens in DNA vaccines have been reported to increase the protection against certain diseases by enhancing specific antibody responses. In this study, we assessed the use of a chimeric DNA vaccine by using lysosomeassociated membrane protein-1 (LAMP1) fused with the major capsule protein (MCP) from red seabream iridovirus. First, Japanese flounder (Paralichthys olivaceus) LAMP-1 (jfLAMP1) gene was cloned and characterized through tissue distribution and expression analysis. The ORF for jfLAMP1 gene has a length of 1248 bp that encodes for 415 aa (about 44 KDa) and shows highly conserved transmembrane and cytoplasmic domains as those of higher vertebrates. Then, jfLAMP1 was used as a carrier of MCP gene by replacing the intra-lysosomal domain, and subsequent inserting into an expression vector, pCI-neo. In the vaccination trial, juveniles of Japanese flounder were distributed into four groups: pCMCP (MCP in pCI-neo vector), pCLAMP1-MCP (chimeric DNA of jfLAMP1 and MCP in pCI-neo), rMCP (recombinant MCP) and pCI-neo (empty vector as a control). At 30 days post vaccination, blood samples were taken and the serum antibody titers were measured by ELISA. The 3 vaccinated groups showed higher antibody titers than pCI-neo group. The pCLAMP1-MCP vaccinated fish showed antibody levels significantly higher than pCMCP (p

老年看護学実習の中心的課題は、加齢がもたらす高齢者(家族)の健康と看護問題の特徴に関する理解を深め、個別性をとらえた看護実践について学ぶことである。今回、入院治療が必要な高齢者を対象とする臨地看護実習を体験した学生7名を研究対象として、彼らの実習記録を分析し、加齢現象を特徴とする高齢者の対象理解及びその傾向について、実習での学びの過程を調査した。結果、(A)身体的側面及び(B)精神心理的側面に関する顕在化している現象は、実習の初期段階で、学生自らの力で注目し理解できているが、(C)社会的側面(殊に生活や家族との関係)に注目したり、(A)(B)(C)いずれも潜在的問題に注目することは顕著に希薄である。しかし、実習の進度に伴って看護過程の展開方式を学習する段階になると、情報と情報の関連づけや統合することを学び、高齢者の特徴について多面的に捉える学習へと発展している。これらのことから実習指導へのいくつかの示唆を得たので報告する。

Chemokines are small cytokines secreted by various cell types. They not only function in cell activation, differentiation and trafficking, but they also have influences on many biological processes. In this study, three novel CC chemokine genes Paol-SCYA105, 106 and 107 in Japanese flounder (Paralichthys olivaceus) were cloned and characterized. Paol-SCYA105 was mainly detected in gill, kidney and spleen, Paol-SCYA106 was detected in all tissues examined and Paol-SCYA107 was mainly detected in the spleen and kidney. Paol-SCYA105 and Paol-SCYA106 gene expressions peaked in kidney at day 3 after viral hemorrhagic septicemia virus infection and decreased at day 6, but Paol-SCYA106 still remained at a high level at day 6. Paol-SCYA107 gene expression was significantly up-regulated in kidney at day 6 after viral hemorrhagic septicemia virus infection. In response to infection by Gram-negative Edwardsiella tarda and Gram-positive Streptococcus iniae in kidney, only Paol-SCYA106 gene expression significantly increased. Together, these results indicate that these three novel CC chemokines are involved in the immune response against pathogen infections.

Some strains of Vibrio parahaemolyticus cause acute hepatopancreatic necrosis disease (AHPND) in shrimp. We sequenced 3 AHPND and 3 non-AHPND strains and found that all of them lacked the pathogenicity island relevant to human infection. A unique sequence encoding a type IV pilus/type IV secretion system was found in 3 AHPND strains.

Although energy metabolism in mammals is critically regulated by adipokines from adipocytes, it is unclear whether this is the case in fish as well. In this study, over 30,000 expressed sequence tags (ESTs) were obtained from adipose tissue in rainbow trout Oncorhynchus mykiss peritoneal cavity and searched for genes possibly related to lipid metabolism. Large numbers of ESTs encoded digestive enzymes and hormones usually found in the pancreas in higher vertebrates, consistent with the fact that pancreatic cells are dispersed in the adipose tissue. Many ESTs encoded apolipoprotein C-I, fatty acid-binding proteins and lymphocyte G0/G1 switch protein 2, which function in lipid transport, fatty acid accumulation and adipocyte differentiation, respectively. None of the ESTs encoded adipokines. We therefore obtained a cDNA encoding adiponectin, an adipokine that regulates oxidation of glucose and lipids in peripheral tissues, using rainbow trout ESTs in the public database. Real-time RT-PCR analyses revealed that its transcript levels were high in muscle and quite low in adipose tissue. These results strongly suggest that adipocytes of rainbow trout and possibly other fish species, unlike those of mammals, are not involved in the production of adipokines.