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At least 19 recordsLinked to original sources

PubMed2006

Detection of DNAs homologous to betanodavirus genome RNAs in barfin flounder Verasper moseri and Japanese flounder Paralichthys olivaceus.

I investigated the presence of DNA homologous to genome RNA1 and RNA2 (RNA1 DNA and RNA2 DNA) of betanodaviruses - the causative agent of viral nervous necrosis (VNN) --in eggs, sperm, ovarian cavity fluid, larvae, and juveniles of barfin flounder Verasper moseri and larvae and juveniles of Japanese flounder Paralichthys olivaceus collected at 6 sites in Hokkaido, Japan, from 1994 to 2001. RNA1 DNA and RNA2 DNA were detected by PCR in 13 and 33 % of barfin flounder samples and 0 and 69% of Japanese flounder samples, respectively. No infectious virus was detected by cell culture or by successive immunoblot against coat protein (genome RNA2 product) using an E-11 cell line, except for a virus present in 1 dead fish collected during an outbreak of VNN in 1995. Nucleotide sequence analysis showed that RNA1 DNA had a 82 to 96 % similarity to betanodavirus genome RNA1, and that RNA2 DNA had a 69 to 98 % similarity to RNA2. The detection rate of RNA2 DNA after intraperitoneal injection of betanodavirus strain HCF-1 into larvae and juveniles of the 2 flounder species was higher in samples from surviving fish than in the uninfected controls, whereas the detection rate of RNA1 DNA did not show a clear trend. Infectious virus was only detected in samples from fish that died subsequent to injection. Transfection assays of the viral genome RNAs into the barfin flounder cell line MK-1 and Japanese flounder cell lines H-1 and H-2 resulted in production of RNA2 DNA in all 3 cell lines. Quantitative measurement by ELISA revealed reverse transcriptase (RTase) activity. These results suggest that the DNA forms are produced and persist in the 2 flounder species as both clinical and subclinical infections, and do not lead to virion production.

Amino Acid Sequence↗

PubMed2004

A pore-forming protein, perforin, from a non-mammalian organism, Japanese flounder, Paralichthys olivaceus.

A perforin cDNA of Japanese flounder, Paralichthys olivaceus, was cloned from a cDNA library of kidney stimulated with ConA/PMA. The full-length cDNA is 2,157 bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3 kb. The amino acid sequence of the Japanese flounder perforin is 36% identical to that of rat perforin and 37% identical to amino acid sequences of mouse and human perforin. The Japanese flounder perforin also showed low homology to human and mouse complement components (C6, C7, C8 and C9), ranging from 19% to 24%. However, the membrane attack complex/perforin domain is conserved. A phylogenetic analysis placed the Japanese flounder perforin in the same cluster with other known mammalian perforins. RT-PCR analysis revealed that the perforin gene was expressed in the peripheral blood leukocytes, head kidney, trunk kidney, spleen, heart, gill and intestine of healthy fish. Recombinant perforin produced in insect cells using the baculovirus expression system showed calcium-dependent hemolytic activity.

Amino Acid Sequence↗

PubMed2006

Molecular cloning and characterization of Toll-like receptor 9 in Japanese flounder, Paralichthys olivaceus.

Toll-like receptor (TLR) 9 cDNA and gene were cloned from Japanese flounder, Paralichthys olivaceus. The Japanese flounder TLR9 cDNA encodes 1065 amino acids. The leucine-rich domain (LRD) and the Toll/interleukin-1 receptor (TIR) domain found in other vertebrate TLR9s were conserved in Japanese flounder TLR9. The gene is composed of three exons and two introns. The Japanese flounder tumor necrosis factor (TNF) gene promoter was activated in Japanese flounder TLR9-transformed hirame natural embryo (HINAE) cells upon stimulation with synthesized CpG oligodeoxynucleotide (ODN), but not by stimulation with GpC ODN. The Japanese flounder TLR9 gene was highly expressed in epithelial and lymphoid organs, such as the gills, intestines, kidney, spleen and stomach in an apparently healthy fish. The mRNA copy numbers of Japanese flounder TLR9 and its adapter protein, the myeloid differentiation factor 88 (MYD88) were increased in some organs including blood, gill, kidney and spleen after Edwardsiella tarda challenge. Immunohistochemical analysis revealed that TLR9 and MYD88 were expressed in the same cells of kidney. Few TLR9-expressing cells were found in gill, kidney and spleen in healthy Japanese flounder, but many were found in these organs after E. tarda challenge and were coincident with lesions that had been colonized by the bacteria.

Amino Acid Sequence↗

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↗

PubMed2006

Cloning and expression of a novel serine protease from Japanese flounder, Paralichthys olivaceus.

Two different cDNA clones of Japanese flounder (types I-1 and I-2) with lengths of 1096 and 1572bp, respectively, were found to encode the same serine protease consisting of 244 identical amino acid residues with three putative N-glycosylation sites, an 18-amino acid signal peptide and a 2-amino acid activation peptide. The amino acid sequence of the Japanese flounder serine protease shares 39-44% identity to known hematopoietic serine proteases. Genomic analysis showed that two different clones were alternatively spliced from the same gene. A phylogenetic tree analysis showed that the Japanese flounder serine protease clustered with a hypothetical fugu protein and this cluster belonged to the neutrophil serine protease family cluster, which includes myeloblastin, N-elastase, and azurocidin. Expression of the Japanese flounder serine protease gene was observed to be up-regulated in head kidney cells after infection with Hirame rhabdovirus and LPS induction. In situ hybridization indicated that cells expressing Japanese flounder serine protease are different from CD8(+) and immunoglobulin(+) cells.

Amino Acid Sequence↗

PubMed2003

Cloning and characterisation of a cDNA encoding Japanese flounder Paralichthys olivaceus IgD.

A cDNA containing the gene for Japanese flounder IgD consisted of 3240 bp encoding 998 amino acid residues. The amino acid sequence of the constant region of Japanese flounder IgD shares 38-80% identity with the sequences of previously reported teleost IgDs. The structure of the constant region of Japanese flounder IgD, which contains the micro1, delta1, delta2, delta3, delta4, delta5, delta6, delta7, and TM regions, is similar to the structures of the constant regions of the IgDs of channel catfish and Atlantic salmon. Southern blot hybridisation showed that the Japanese flounder IgD gene exists as a single locus. The Japanese flounder IgD gene was mainly detected in peripheral blood leucocytes (PBLs) and small amounts were detected in the spleen, head and trunk kidney, although IgM mRNA was detected in similar amounts in PBLs, the head kidney, and spleen. The copy number of IgM mRNA in Japanese flounder PBL was 56-fold higher than that of IgD.

Amino Acid Sequence↗

PubMed2006

Functional characterisation of the Japanese flounder, Paralichthys olivaceus, Mx promoter.

The Japanese flounder, Paralichthys olivaceus, genome appears to encode a single Mx gene based on Southern blotting and previous cDNA studies. The 5' flanking region of the Japanese flounder Mx gene was cloned and analysed for its regulatory regions. A TATA box (-24 to -30), two interferon-stimulated response elements (ISREs) (-69 to -80 and -508 to -521) and two Sp1 sites (-563 to -572 and -994 to -1003) were identified relative to the transcription start site. The effects of various stimuli, as well as the effects of various promoter mutations, were investigated in a transient expression system using Japanese flounder (hirame) natural embryo (HINAE) cells and luciferase reporter gene constructs. Although not sensitive to LPS, ConA or PMA, reporter gene expression increased more than 10-fold after stimulation by polyinosinic:polycytidilic acid (poly I:C), an established inducer of interferon. Deletion mutational analyses revealed the ISRE closest to the transcription start site to be crucial for promoter activity. The distal ISRE, despite its relatively distant location, contributed to induce maximal promoter activity, but when alone was not sufficient by itself to elicit any significant promoter activity. An electrophoretic mobility shift assay confirmed the binding of transcription factors to both ISREs. Induction of luciferase by poly I:C was inhibited by 2-Aminopurine, a protein kinase (PKR) inhibitor, in a dose-dependent (1-10 mM) manner, suggesting that PKR may be required as a signal transducer for type I IFN signaling in fish. This Mx reporter assay may be useful for quantifying the responses and elucidating the regulation pathways of IFN type I.

Animals↗

PubMed2006

Characterization of Japanese flounder (Paralichthys olivaceus) NK-lysin, an antimicrobial peptide.

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.

Amino Acid Sequence↗

PubMed2005

Characterization and expression of a CD40 homolog gene in Japanese flounder Paralichthys olivaceus.

A CD40 homolog cDNA encoding 300 amino acid residues was isolated from a Japanese flounder leukocyte cDNA library. The amino acid sequence identities of Japanese flounder CD40 and previously reported CD40s of cow, human, mouse, and chicken range from 32 to 35%. The positions of cysteine residues, CD40 ligand binding amino acid residues, and four cysteine-rich domains are well conserved in Japanese flounder CD40. The Japanese flounder CD40 gene is composed of nine exons and eight intervening introns spread over 6 kb. The nucleotide sequence of the 5'-flanking region of this gene revealed the presence of several regulatory regions, including a TATA-like box, AP-1-, CEBPB-, IRF-1-, LYF-1-, NF-kappaBp-, SP-1-, and Stat-1-like motifs. In healthy fish, reverse transcriptase-polymerase chain reaction (RT-PCR) detected constitutive expression of CD40 in all tested tissues (leukocytes, kidney, spleen, liver, intestine, brain, gill, and skin). The mRNA of CD40 was predominantly expressed in several tissues that contained lymphocytes (leukocytes, kidney, spleen, intestine, and gill). Expression of the Japanese flounder CD40 molecule was induced in peripheral blood leukocytes from 1 to 6 h following Con A (50 microg/ml)/phorbol myristate acetate (PMA) (0.35 microg/ml) stimulation, with a peak at 1 h after stimulation, and increased at 1, 3, and 6 h after induction by lipopolysaccharide (LPS) (500 microg/ml) compared with the control.

Amino Acid Sequence↗

PubMed2005

Molecular characterization of the Japanese flounder, Paralichthys olivaceus, CD3epsilon and evolution of the CD3 cluster.

A second CD3 gene, i.e. CD3epsilon, has been cloned and sequenced in Japanese flounder. The full length cDNA is 1006 bp and encodes 164 amino acids. When compared with other known CD3epsilon peptide sequences, the most conserved region of the Japanese flounder CD3epsilon chain peptide is the cytoplasmic domain and the least conserved is the extracellular domain. A phylogenetic analysis based on the deduced amino acid sequence grouped the two Japanese flounder CD3 sequences with CD3epsilon and CD3gamma(delta, respectively. The Japanese flounder CD3epsilon gene has Lyf-1, GATAs, Oct-1, CEBPs, AP-1, and NF-AT but lacks TATA and CCAAT elements in the 5' flanking region. The Japanese flounder CD3 cluster (consisting of CD3epsilon and CD3gamma(delta) spans only 10.4 kb. The two genes are oppositely transcribed only 3.8 kb apart. Both Japanese flounder CD3 genes have five exons. The two Japanese flounder CD3 genes were predominantly expressed in PBLs, kidney, spleen, and gills. A polyclonal rabbit antiserum that reacts with the CD3 marker on human T cells also reacted with Japanese flounder CD3epsilon. The epitope highly conserved between mammalian and non-mammalian CD3epsilons, this antibody bound to a single 15 kDa peptide.

Amino Acid Sequence↗

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

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↗

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↗

PubMed2003

Fine structure of soft and hard tissues involved in eye migration in metamorphosing Japanese flounder (Paralichthys olivaceus).

The body of a Japanese flounder (Paralichthys olivaceus) changes from a symmetrical to an asymmetrical form during metamorphosis. To obtain detailed information on the mechanisms of the migration of the right eye to the left side, soft and hard tissues in the head of larval flounders were examined using transmission electron microscopy (TEM). Retrorbital vesicles (Rvs) are pairs of sac-like structures under the eyes. It has been suggested that the asymmetrical development of Rvs, with the right (blind) one being bigger than the left, is the driving force behind eye migration. The present study revealed that the ultrastructure of the Rv sheath is quite similar to that of a lymphatic capillary. Thus, it is possible that the Rv is a part of the lymph system, and is probably related to the secondary vascular system in teleosts. If we assume that the Rv sheath has a high permeability to liquid, similar to lymphatic capillaries, it is not plausible that the active expansion of the Rv pushes the eyeball. On the other hand, the pseudomesial bar (Pb) is a bone that is unique to flounders and is present only on the right (blind) side. At the beginning of eye migration, an aggregation of fibroblast-like cells is observed in the dermis under the right eye, where the Pb will subsequently be formed. These cells have a well-developed rough endoplasmic reticulum (rER) and mitochondria, and are probably responsible for formation of the thick layers of collagen fibrils around them. Since it is unlikely that the active expansion of the Rv causes eye migration, the role played by the Pb and its rudiment becomes more significant in right eye migration in the Japanese flounder becomes more significant.

Animals↗

PubMed2004

Immunoanalysis of antiviral Mx protein expression in Japanese flounder (Paralichthys olivaceus) cells.

Mx proteins are interferon-inducible GTPases that possess antiviral properties in vertebrates. Japanese flounder (Paralichthys olivaceus) Mx protein has previously been shown to possess some antiviral activity against rhabdoviruses. A polyclonal antibody was generated against a purified peptide fragment of Japanese flounder Mx protein that had been produced in an Escherichia coli expression system. The PAb detected the approximately 71 kDa Mx protein from Japanese flounder (hirame) natural embryo (HINAE) cells that had been cultured with poly I:C, an interferon inducer, but not in unstimulated cells. The polyclonal antibody did not cross react with Mx protein from carp epithelial, grouper fin and zebrafish embryo cell lines that had been similarly induced or transfected with poly I:C. By immunofluorescence cytochemistry, Japanese flounder Mx protein was localized to the cell cytoplasm. Hirame rhabdovirus stimulated expression of Mx protein in the infected and surrounding HINAE cells. Within virus-infected cells, there was some indication of Mx protein colocalizing with viral proteins. Poly I:C stimulation of HINAE cells induced an early increase in Mx protein mRNA transcripts, but maximum Mx mRNA transcript and protein expression was reached after 48 h. Both Mx mRNA transcripts and protein levels were maintained till at least 72 h.

Amino Acid Sequence↗

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↗

PubMed2006

Japanese flounder (Paralichthys olivaceus) embryos are difficult to cryopreserve by vitrification.

The first successful cryopreservation of fish embryos was reported in the Japanese flounder by vitrification [Chen and Tian, Theriogenology, 63, 1207-1219, 2005]. Since very high concentrations of cryoprotectants are needed for vitrification and fish embryos have a large volume, Japanese flounder embryos must have low sensitivity to cryoprotectant toxicity and high permeability to water and cryoprotectants. So, we investigated the sensitivity and the permeability of Japanese flounder embryos. In addition, we assessed the survival of flounder embryos after vitrification with solutions containing methanol and propylene glycol, following Chen and Tian's report. The embryos were relatively insensitive to the toxicity of individual cryoprotectants at lower concentrations, especially methanol and propylene glycol as their report. Although their permeability to water and cryoprotectants could not be measured from volume changes in cryoprotectant solutions, the embryos appeared to be permeable to methanol but less permeable to DMSO, ethylene glycol, and propylene glycol. Although vitrification solutions containing methanol and propylene glycol, which were used in Chen and Tian's report, were toxic to embryos, a small proportion of embryos did survived. However, when vitrified with the vitrification solutions, no embryos survived after warming. The embryos became opaque during cooling with liquid nitrogen, indicating the formation of intracellular ice during cooling. When embryos had been kept in vitrification solutions for 60 min after being treated with the vitrification solution, some remained transparent during cooling, but became opaque during warming. This suggests that dehydration and/or permeation by cryoprotectants were insufficient for vitrification of the embryos even after they had been over-treated with the vitrification solutions. Thus, Chen and Tian's cryopreservation method lacks general application to Japanese flounder embryos.

Animals↗

PubMed2003

The analysis of immune responses of a novel CC-chemokine gene from Japanese flounder Paralichthys olivaceus.

A novel CC-chemokine gene was isolated from the Japanese flounder Paralichthys olivaceus by expressed sequence tag analysis. The function of this CC-chemokine gene was studied by DNA injection. To investigate the immune responses to the CC-chemokine, a plasmid construct containing the novel CC-chemokine and a CMV promoter was injected into the epaxial muscle of Japanese flounder. Quantification of CC-chemokine protein expressed in serum on 1, 3 and 5 days after plasmid injection were estimated by ELISA. CC-chemokine gene injection increased the migration of phagocytic cells. Macrophage functions such as production of superoxide anion and phagocytosis were also stimulated by this gene injection. Thus, this gene from Japanese flounder has functional similarities to that of a mammalian CC-chemokine gene.

Amino Acid Sequence↗