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Biomedical subjects

I Hirono

Publications and source records attributed to I Hirono.

At least 19 recordsLinked to original sources
PubMed2006

Immune modulation and expression of cytokine genes in rainbow trout Oncorhynchus mykiss upon probiotic feeding.

This study elucidates the immune modulation including the expression of cytokine genes following dietary administration of three selected probiotic bacteria--Lactobacillus rhamnosus, Enterococcus faecium and Bacillus subtilis to fish, rainbow trout Oncorhynchus mykiss. They were fed for 45 days on either a basal control diet or one of the three probiotic diets containing the specific bacteria in freeze-dried form at a density of 10(9)CFUgfeed-1. The non-specific immune parameters examined--superoxide anion production by the head kidney leukocytes and the alternate complement activity of serum was improved by probiotic feeding. Besides this, the relative gene expressions of interleukin-1beta1, tumor necrosis factor 1 and 2 and transforming growth factor-beta were up regulated in the spleen and the head kidney. The comparatively better performance of E. faecium could possibly be linked to their suitable ambient temperature conditions. Thus, probiotic bacteria delivered in feed exerts its influence on the immune system of fish, both at cellular and molecular levels.

Animal Feed↗
PubMed2006

Putative virulence-related genes in Vibrio anguillarum identified by random genome sequencing.

The genome of Vibrio anguillarum strain H775-3 was partially determined by a random sequencing procedure. A total of 2,300 clones, 2,100 from a plasmid library and 200 from a cosmid library, were sequenced and subjected to homology search by the BLAST algorithm. The total length of the sequenced clones is 1.5 Mbp. The nucleotide sequences were classified into 17 broad functional categories. Forty putative virulence-related genes were identified, 36 of which are novel in V. anguillarum, including a repeat in toxin gene cluster, haemolysin genes, enterobactin gene, protease genes, lipopolysaccharide biosynthesis genes, capsule biosynthesis gene, flagellar genes and pilus genes.

Bacterial Capsules↗
PubMed2005

Molecular cloning and functional analysis of Photobacterium damselae subsp. piscicida haem receptor gene.

A haem receptor gene from Photobacterium damselae subsp. piscicida (formerly known as Pasteurella piscicida) has been cloned, sequenced and analysed for its function. The gene, designated as pph, has an open reading frame consisting of 2154 bp, a predicted 718 amino acid residues and exists as a single copy. It is homologous with the haem receptors of Vibrio anguillarum hupA, V. cholerae hutA, V. mimicus mhuA and V. vulnificus hupA at 32.7, 32.7, 45.6 and 30.9%, respectively, and is highly conserved, consisting of a Phe-Arg-Ala-Pro sequence (FRAP), an iron transport related molecule (TonB) and a Asn-Pron-Asn-Leu sequence (NPNL), binding motifs associated with haem receptors. As a single gene knockout mutant P. damselae subsp. piscicida was able to bind haem in the absence of pph, suggesting that other receptors may be involved in its iron transport system. This study shows that the P. damselae subsp. piscicida pph belongs to the haem receptor family, is conserved and that its iron-binding system may involve more than one receptor.

Amino Acid Motifs↗
PubMed2005

Detection of quinolone-resistance genes in Photobacterium damselae subsp. piscicida strains by targeting-induced local lesions in genomes.

Quinolone-resistant strains of the fish-pathogenic bacterium, Photobacterium damselae subsp. piscicida are distributed widely in cultured yellowtail, Seriola quinqueradiata (Temminck & Schlegel), in Japan. The quinolone resistance-determining region (QRDR) was amplified with degenerate primers, followed by cassette ligation-mediated PCR. Open reading frames encoding proteins of 875 and 755 amino acid residues were detected in the gyrA and parC genes, respectively. Resistant strains of P. damselae subsp. piscicida carried a point mutation only in the gyrA QRDR leading to a Ser-to-Ile substitution at residue position 83. No amino acid alterations were discovered in the ParC sequence. A mutation in the gyrA gene was also detected in nalidixic acid-resistant mutants of strain SP96002 obtained from agar medium containing increased levels of quinolone. These results suggest that GyrA, as in other Gram-negative bacteria, is a target of quinolone in P. damselae subsp. piscicida. Furthermore, we attempted to detect a point mutation using targeting-induced local lesions in genomes (TILLING), which is a general strategy used for the detection of a variety of induced point mutations and naturally occurring polymorphisms. We developed a new detection method for the rapid and large-scale identification of quinolone-resistant strains of P. damselae subsp. piscicida using TILLING.

Amino Acid Sequence↗
PubMed2003

Genetic diversity and molecular markers of the tropical abalone (Haliotis asinina) in Thailand.

Genetic diversity of abalone in Thailand, Haliotis asinina, H. ovina, and H. varia, was analyzed by polymerase chain reaction (PCR) of 18S and 16S rDNAs, with randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP). Species-specific RAPD markers were found in each abalone species. Restriction analysis of 18S (nuclear) ribosomal DNA with Alu I, Taq I, and Hae III and 16S (mitochondrial) rDNA with Bam HI, Eco RI, Hae III, and Alu I gave 12 and 13 digestion patterns, respectively. A total of 49 composite haplotypes were found. A dendogram obtained by the unweighted pair-group method with arithmetic mean, constructed from divergence between pairs of composite haplotypes, revealed reproductively isolated gene pools of these abalone and indicated that H. asinina and H. ovina are genetically closer than H. varia. When H. varia was discovered owing to small sample sizes, geographic heterogeneity analysis and FST estimate indicated clear genetic differentiation between H. ovina originating from the Andaman Sea (west) and the Gulf of Thailand (east, P<0.0001), whereas partial differentiation was observed between the Philippines and the remaining H. asinina samples (P<0.0021). The amplified 16S rDNAs of individuals representing composite haplotypes found in this study were cloned and sequenced. A neighbor-joining tree constructed from sequence divergence of 16S rDNA accurately allocated those sequences according to species origins of abalone. Species-specific PCR based on 16S rDNA polymorphism was successfully developed in H. asinina and H. varia but not in H. ovina.

Animals↗
PubMed2002

Differential expression of two tumor necrosis factor genes in rainbow trout, Oncorhynchus mykiss.

A second TNF gene (TNF2) has been cloned and sequenced in rainbow trout. In common with the first TNF gene isolated (TNF1), this gene is more TNF alpha-like than TNF beta-like. The full length cDNA is 1519bp, containing a 765bp open reading frame. The gene has four exons, of 380, 49, 60 and 1030bp, respectively. Analysis of the 5' flanking regions of TNF1 and TNF2 reveals several interesting differences in identified transcriptional regulatory elements, with a CATAAA box present 26bp upstream of the transcription start in both genes. Expression analysis in LPS stimulated macrophages has shown a much stronger expression of TNF2 relative to TNF1, with expression being detected earlier and lasting longer.

Amino Acid Sequence↗
PubMed2001

Molecular cloning, expression and evolution of the Japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein.

In this study, we cloned the goose-type (g-type) lysozyme gene from the Japanese flounder genomic DNA library, the first such data in fish and only the second after the chicken g-type lysozyme gene. The Japanese flounder g-type lysozyme gene was 1252 bp in length from the transcription site to the polyadenylation site, coded for 758 bp of mRNA and 195 deduced amino acids, which contain five exons and four introns. A phylogenetic analysis based on amino acid sequences showed that the flounder gene was closer to g-type lysozyme, followed by phage-type lysozyme and then chicken-type (c-type) lysozyme. Although exon 1 of the flounder gene differs from exons 1 and 2 of the chicken g-type lysozyme gene, three catalytic residues, as well as their neighboring amino acids were conserved between the Japanese flounder and the four avian g-type lysozymes. In a Southern blot analysis using the genomic DNA of homo-cloned Japanese flounder, the flounder g-type lysozyme gene showed a simple pattern, suggesting that it is encoded by a single copy gene. A Northern blot analysis showed that this gene was expressed in all tissues of Japanese flounder that we examined in this study and showed major differences from those expressed tissues of the chicken g-type gene. Japanese flounder g-type lysozyme mRNA levels in the intestine, heart and whole blood increased after injecting the fish with Edwardsiella tarda. Recombinant flounder g-type lysozyme, which has an optimal pH and temperature of pH 6.0 and 25 degrees C, possessed lytic activity against Micrococcus lysodeikticus and several fish pathogenic bacteria. This is the first report of a g-type lysozyme gene other than for reported avian species.

Animals↗
PubMed2001

Cloning of Japanese flounder Paralichthys olivaceus CD3 cDNA and gene, and analysis of its expression.

Two distinct CD3 homologue cDNAs, CD3-1 and CD3-2, were isolated from a Japanese flounder leukocyte cDNA library. CD3-1 consisted of 961 bp encoding 178 amino acid residues, and CD3-2 consisted of 927 bp encoding 182 amino acid residues. The two deduced amino acid sequences had an identity of 95.1%, and neither had N-linked glycosylation sites. The identities between the Japanese flounder CD3s and previously reported CD3s (CD3 epsilon, CD3 gamma, or CD3 delta) of Xenopus laevis, chicken, and various mammals were approximately 25%. The Japanese flounder CD3s had an extracellular domain, a CXXCXE motif, and an immunoreceptor tyrosine-based activation motif (ITAM), each of which are important characteristics of CD3 chains. Furthermore, the positions of four cysteine residues in the extracellular domain were preserved in both of the Japanese flounder CD3s. A phylogenetic tree based on the amino acid sequences confirmed that the Japanese flounder CD3s are closer to CD3 epsilon than to CD3 gamma and CD3 delta. However, the gene structure of Japanese flounder CD3 is identical to the chicken and Xenopus CD3 gamma/delta genes and the mammalian CD3 delta gene. Southern blot hybridization and the DNA sequence of the CD3 gene of homocloned Japanese flounder indicated that the CD3 gene exists as a single copy. Southern blot hybridization also showed the presence of a polymorphic variant of Japanese flounder CD3. An RT-PCR analysis detected Japanese flounder CD3 mRNA in several organs that contained lymphocytes. The proportion of CD3-positive cells in the peripheral blood leukocytes was 34.9%.

Amino Acid Sequence↗
PubMed2001

Construction and characterization of BAC libraries for three fish species; rainbow trout, carp and tilapia.

Bacterial artificial chromosome (BAC) libraries are important tools for genomic research. We have constructed seven genomic BAC libraries from three fish species, rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio) and tilapia (Oreochromis niloticus). The two rainbow trout BAC libraries have average insert sizes of 58 and 110 kb. The average size of inserts in the carp BAC library is 160 kb. The average insert sizes of the four tilapia BAC libraries are 65, 105, 145 and 194 kb, respectively. These libraries represent good coverage of each genome (2-64 x coverage). The libraries can be screened by conventional colony hybridization and provide a starting point for the construction of high-density filtres or polymerase chain reaction (PCR) screening approaches. These BAC libraries will facilitate the positional cloning of quantitative trait loci (QTLs) for a variety of economically important traits in these species.

Animals↗
PubMed2001

Cloning and expression analysis of rainbow trout Oncorhynchus mykiss tumour necrosis factor-alpha.

A rainbow trout (Oncorhynchus mykiss) gene for tumor necrosis factor (TNF) has been cloned and sequenced. The cDNA contains an open reading frame of 738 nucleotides that translate into a 246 amino-acid putative peptide, with a 5' untranslated region (UTR) of 140 bp and a 3' UTR of 506 bp. Two potential N-linked glycosylation sites exist in the translation. The genomic sequence measures 2007 bp and contains three introns that intercept four coding exons. Expression studies using RT-PCR have shown that the trout TNF gene is constitutively expressed in the gill and kidney of unstimulated fish. Trout TNF expression could be up-regulated by stimulation of isolated head kidney leucocytes with lipopolysaccharide (LPS). Similarly, stimulation of a trout macrophage cell line (RTS11) with LPS resulted in an increased transcript level, as did incubation with recombinant trout interleukin (IL)-1 beta. The optimal timing for induction of TNF expression in trout macrophages was determined using recombinant trout IL-1 beta, where a clear induction was apparent by 2 h and peaked at 4 h. Evidence that this TNF gene is equivalent to mammalian TNF-alpha is discussed.

Amino Acid Sequence↗
PubMed2000

Assessment of DNA vaccine potential for juvenile Japanese flounder Paralichthys olivaceus, through the introduction of reporter genes by particle bombardment and histopathology.

Genetic immunisation potential, following DNA bombardment for juvenile Japanese flounder, Paralichthys olivaceus was examined. GFP plasmids bombarded at two pressures, 150 and 300psi were sampled at 1, 7, 14 and 28 days, greater immunofluorescence was observed at the higher bombardment pressure. Histopathology, at 3 h post bombardment showed considerable damage to fish epithelial and dermal tissues when bombarded at pressures greater than 200 psi, with many DNA-coated gold particles present. At 150psi there was little pathology and no DNA-coated particles. Histopathology, up to 28 days again showed little pathology at 150 psi with few DNA-coated particles, whereas at 300 psi there was significant pathology observed with many DNA-coated particles seen in conjunction with the cytoplasm of inflammatory cells. By day 28 epithelial coverage was observed with tissue damage restricted to the dermal layer. Chloramphenicol acetyltransferase (CAT) assay showed long term and stable expression of the CAT protein from day 1 to day 60. The transcription activity of two promoters; pCMV-CAT and pSV2-CAT showed greater activity in the former. It was concluded that DNA vaccination potential for juvenile flounder is a viable option.

Animals↗
PubMed2000

Molecular cloning, characterization, and expression of TNF cDNA and gene from Japanese flounder Paralychthys olivaceus.

We cloned a cDNA and the gene for Japanese flounder TNF. The TNF cDNA consisted of 1217 bp, which encoded 225 amino acid residues. The identities between Japanese flounder TNF and members of the mammalian TNF family were approximately 20-30%. The positions of cysteine residues that are important for disulfide bonds were conserved with respect to those in mammalian TNF-alpha. The Japanese flounder TNF gene has a length of approximately 2 kbp and consists of four exons and three introns. The positions of the exon-intron junction positions of Japanese flounder TNF gene are similar to those of human TNF-alpha. However, the length of the first intron of Japanese flounder is much shorter than that of the human TNF-alpha gene. There are simple CA or AT dinucleotide repeats in the 5'-upstream and 3'-downstream regions of the Japanese flounder TNF gene. Southern blot hybridization indicted that Japanese flounder TNF exists as a single copy. Expression of Japanese flounder TNF mRNA is greatly induced after stimulation of PBLs with LPS, Con A, or PMA. These results indicated that Japanese flounder TNF is more like mammalian TNF-alpha than mammalian lymphotoxin-alpha, with respect to its gene structure, length of amino acid sequence, number and position of cysteine residues, and regulation of gene expression.

Amino Acid Sequence↗
PubMed2000

Virulence properties of motile aeromonads isolated from farmed frogs Rana tigerina and R. rugulosa.

Virulence factors were compared in Aeromonas species isolated from clinically normal and septicaemic farmed frogs from Thailand. Haemolysin activities against frog erythrocytes were significantly different within the collection of aeromonads. Groups of high haemolytic activity (unspeciated Aeromonas, Au), moderate haemolytic activity (A. hydrophila), and low haemolytic activity (A. veronii biovar sobria, A. veronii biovar veronii, A. caviae, A. schubertii) were noted. DNA colony hybridisation studies revealed that Au isolates possessed a haemolysin gene (ASH1) which was not present in any of the other Thai aeromonads or type strains tested. Elastinolytic activity was demonstrated in 90% of the Au isolates, 60% of the A. hydrophila isolates and in none of the other motile aeromonads. The cytotoxic activity of the Aeromonas isolates varied according to the source of cells used in the assays. Cells from rainbow trout were extremely sensitive to Au toxins but less so to toxins produced by other species. In contrast mammalian cells showed very little sensitivity to Au toxins but were more sensitive to toxins produced by A. hydrophila. Selection of suitable assay substrates is therefore important.

Aeromonas↗
PubMed2000

Identification of viral induced genes in Ig+ leucocytes of Japanese flounder Paralichthys olivaceus, by differential hybridisation with subtracted and un-subtracted cDNA probes.

Up-regulated genes of leucocytes expressing immunoglobulin (Ig+ leucocytes) of hirame rhabdovirus (HRV)-infected Japanese flounder were identified by differential hybridisation, using subtracted and un-subtracted cDNA probes. Ig+ leucocytes were separated from apparently healthy and HRV-infected Japanese flounder by the magnetic beads antibody method using mouse anti-Japanese flounder Ig monoclonal antibody (mab). A cDNA library was constructed from HRV-infected Japanese flounder leucocytes, and was screened with subtracted cDNA probes enriched in genes up-regulated by HRV infection. Fifty cDNAs were isolated for further analysis. These included cDNAs coding for homologues of interferon-inducible 56K protein (IFI56), Stat3, CEF-10, RGS5, inducible poly(A) binding protein, prolylcarboxylpeptidase, basigin III (Ig superfamily), MUC-18 (Ig superfamily), proteasome-nexin 1 (SERPIN), herpes virus entry mediator (TNFR family), collagenase III, gelatinase-b, megakaryocyte stimulating factor, Rab8-interacting protein, IgM, IgD and 20 unknown cDNA clones. The majority of these identified genes are reported for the first time in fish. From leucocytes mRNA for homologues of IFI56, CEF-10, Stat3, SERPIN and inducible poly (A) binding protein expression was shown to increase following HRV infection.

Animals↗
PubMed2000

Genomic bacterial artificial chromosome library of the Japanese flounder Paralichthys olivaceus.

We have constructed a genomic bacterial artificial chromosome (BAC) library from homozygous cloned Japanese flounder Paralichthys olivaceus using the pBAC- lac vector. This BAC library consists of about 49,100 clones and is deposited in 128 microtiter plates with 384 wells. The average size of inserted DNA was calculated to be 165 kb. The BAC library was determined to cover 9 times the Japanese flounder haploid genome. The Japanese flounder genomic BAC library will be useful for gene isolation as well as quantitative trait loci (QTL) analysis.

Journal Article↗
PubMed2000

Molecular Cloning and Novel Repeated Sequences of a C-type Lysozyme Gene in Japanese Flounder (Paralichthys olivaceus).

A clone of a DNA fragment of approximately 10 kb that included a c-type lysozyme gene of Japanese flounder (Paralichthys olivaceus) was isolated from a Japanese flounder genomic DNA library. This clone was subcloned, and the nucleotide sequence of the c-type lysozyme gene was determined. The flounder c-type lysozyme gene, which consisted of 3,617 bp, was found to be composed of four exons and three introns. The transcription start site was determined to be 44 bp upstream of the ATG codon by primer extension analysis. The number of exons and introns and the positions of the catalytic residues of the flounder gene were the same as those of the human and chicken c-type lysozyme genes. However, introns 1 and 3 of the flounder gene were shorter, and intron 2 was longer, than those in the other reported vertebrates genes. There were no Alu repeated sequences in the flounder lysozyme gene, as determined for the human lysozyme gene, but 45 repeated sequences were found in the second intron of the flounder lysozyme gene. We suggest that the length of intron 2 in the Japanese flounder c-type lysozyme gene depends on the presence of this repeated sequence. Furthermore, Southern blot analysis revealed that the c-type lysozyme gene was probably a single copy in the genomic DNA of homo-cloned Japanese flounder.

Journal Article↗
PubMed2000

Stable Expression of a Foreign Gene, Delivered by Gene Gun, in the Muscle of Rainbow Trout Oncorhynchus mykiss.

We report the efficient delivery of a foreign gene into muscle of rainbow trout Oncorhynchus mykiss with a gene gun. The foreign gene was a reporter gene, chloramphenicol acetyltransferase (CAT). Two CAT-containing plasmids were used: pCMV-CAT, which contains cytomegalovirus immediate early promoter, and pSV2-CAT, which contains the simian virus 40 early promoter. All plasmids were introduced by particle bombardment using a gene gun. During the 90-day sampling period following bombardment, CAT was strongly and stably expressed in the muscle of all the fish bombarded with pCMV-CAT and pSV2-CAT. No CAT expression was detected in the blood samples until 90 days after introduction, when it was found in only one fish from the pCMV-CAT group and one from the pSV2-CAT group. The stable and long-term expression of plasmid DNA in muscle makes muscle an attractive target tissue for the introduction of viral DNA for the purpose of DNA vaccination.

Journal Article↗