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Immunoglobulin genes and their transcriptional control in teleosts
Hikima, J.i.,Jung, T.S.,Aoki, T. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2011 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.35 No.9
Immunoglobulin (Ig), which exists only in jawed vertebrates, is one of the most important molecules in adaptive immunity. In the last two decades, many teleost Ig genes have been identified by in silico data mining from the enormous gene and EST databases of many fish species. In this review, the organization of Ig gene segments, the expressed Ig isotypes and their transcriptional controls are discussed. The Ig heavy chain (IgH) locus in teleosts encodes the variable (V), the diversity (D), the joining (J) segments and three different isotypic constant (C) regions including Cμ, Cδ, and Cζ/τ genes, and is organized as a ''translocon'' type like the IgH loci of higher vertebrates. In contrast, the Ig light (L) chain locus is arranged in a ''multicluster'' or repeating set of VL, JL, and CL segments. The IgL chains have four isotypes; two κ L1/G and L3/F), σ (L2) and λ. The transcription of IgH genes in teleosts is regulated by a VH promoter and the Eμ3' enhancer, which both function in a B cell-specific manner. The location of the IgH locus, structure and transcriptional function of the Eμ3' enhancer are important to our understanding of the evolutional changes that have occurred in the IgH gene locus.
Hikima, J.i.,Ohtani, M.,Kondo, H.,Hirono, I.,Jung, T.S.,Aoki, T. Pergamon Press ; Elsevier Science 2011 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.35 No.3
Both PU.1 and C/EBPα transcription factors play important roles in myeloid development and inflammatory response. These transcripts were cloned from the Japanese flounder (Paralichthys olivaceus) and were highly conserved with those of other vertebrates. PU.1 mRNA was mainly expressed in lymphoid tissues while C/EBPα mRNA was widely expressed in all tissues examined. Higher levels of PU.1 mRNA were expressed in the IgM<SUP>+</SUP> cells of both PBL and KL, while C/EBPα expression was higher only in the IgM<SUP>-</SUP> cells of KL. The expression of C/EBPα mRNA was induced only in KL stimulated with LPS. Interestingly, PU.1 mRNA expression was induced by Edwardsiella tarda, whereas the expression of C/EBPα mRNA was induced by Streptococcus iniae infection. Both PU.1 and C/EBPα drove transcription from the LPS-responsive region of the Japanese flounder TNFα gene, suggesting that both PU.1 and C/EBPα induced by bacterial infection are involved in inflammation mediated through TNFα expression.
Ohtani, Maki,Hikima, Jun-ichi,Hwang, Seong Don,Morita, Takahiro,Suzuki, Yoshiaki,Kato, Goshi,Kondo, Hidehiro,Hirono, Ikuo,Jung, Tae-Sung,Aoki, Takashi Elsevier 2012 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.36 No.4
<P><B>Highlights</B></P><P>► Japanese flounder type I IFN gene was cloned and clustered with Acanthopterygii. ► The poly I:C-responsible region (−634 to −179bp) was found in IFN promoter. ► Transcriptional activity of IFN promoter was enhanced by the flounder IRF3. ► The activity of IFN promoter was induced by RLRs after poly I:C-stimulation.</P> <P><B>Abstract</B></P><P>Type I interferon (IFN) induces the antiviral response in innate immunity. The type I IFN gene cloned from Japanese flounder (<I>Paralichthys olivaceus</I>) has a length of 1189bp and consisting of 5 exons and 4 introns. In a phylogenetic tree of type I IFNs, Japanese flounder grouped with other Acanthopterygii. To gain insight into the transcriptional regulation of IFN gene, the 1.36kb 5′-upstream region including numerous canonical motifs to bind transcription factors [for example, IFN regulatory factor (IRF)] was analyzed. In HINAE cells using a luciferase reporter assay, poly I:C-responsive transcriptional activity was found in the region from −634 to −179bp. This region includes several IRF motifs. In the presence of poly I:C, overexpression of IRF3 and RLR strongly enhanced transcriptional activity. These results suggest that the transcriptional regulation of Japanese flounder type I IFN is regulated by IRF3 after triggering with dsRNA sensors.</P>
Ohtani, M.,Hikima, J.i.,Jung, T.S.,Kondo, H.,Hirono, I.,Takeyama, H.,Aoki, T. Academic Press 2013 FISH AND SHELLFISH IMMUNOLOGY Vol.34 No.2
Phage display libraries are used to screen for nucleotide sequences that encode immunoglobulin variable (V) regions that are specific for a target antigen. We previously constructed an immunoglobulin new antigen receptor (IgNAR) phage display library. Here we used this library to obtain an IgNAR V region that is specific for viral hemorrhagic septicemia virus (VHSV). A phage clone (clone 653) was found to be specific for VHSV by the biopanning method. The V region of clone 653 was used to construct a 6 x His tagged recombinant IgNAR-653 V protein (rIgNAR-653) using the Escherichia coli pET system. The rIgNAR-653 protein bound specifically to VHSV, confirming its activity.
Ohtani, M.,Hikima, J.i.,Kondo, H.,Hirono, I.,Jung, T.S.,Aoki, T. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2011 Developmental and comparative immunology Vol.35 No.5
Cytosolic pattern recognition receptors such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) play an important role in sensing viral RNAs. The receptor encoded by melanoma differentiation-associated gene 5 (MDA5), an RLR, recognizes viral RNA in the cytoplasm and enhances antiviral response in host cells. The full-length MDA5 gene in Japanese flounder, Paralichthys olivaceus was cloned and found to have 11,251 nucleotides. MDA5 transcript abundance was significantly increased in whole kidney infected with viral hemorrhagic septicemia virus (VHSV) as well as whole kidney and peripheral blood leukocytes stimulated with poly I:C in vitro. Hirame natural embryo (HINAE) cells overexpressing MDA5 showed a lower cytopathic effect (CPE) against VHSV, hirame rhabdovirus (HIRRV) and infectious pancreatic necrosis virus (IPNV) infection. When infected with VHSV, MDA5-overexpressing HINAE cells had 24-75 fold lower virus titer than normal HINAE cells. These results suggest that Japanese flounder MDA5 is involved in the induction of antiviral response.
Ohtani, Maki,Hikima, Jun-ichi,Jung, Tae Sung,Kondo, Hidehiro,Hirono, Ikuo,Aoki, Takashi Springer-Verlag New York Inc 2013 Marine biotechnology Vol.15 No.1
<P>To develop a multi-antigen-specific immunoglobulin new antigen receptor (IgNAR) variable (V) region phage display library, CDR3 in the V region of IgNAR from banded houndshark (Triakis scyllium) was artificially randomized, and clones specific for hen egg white lysozyme (HEL) were obtained by the biopanning method. The nucleotide sequence of CDR3 in the V region was randomly rearranged by PCR. Randomized CDR3-containing segments of the V region were ligated into T7 phage vector to construct a phage display library and resulted in a phage titer of 3.7??10(7) PFU/ml. Forty clones that contained randomized CDR3 inserts were sequenced and shown to have different nucleotide sequences. The HEL-specific clones were screened by biopanning using HEL-coated ELISA plates. After six rounds of screening, nine clones were identified as HEL-specific, eight of which showed a strong affinity to HEL in ELISA compared to a negative control (i.e., empty phage clone). The deduced amino acid sequences of CDR3 from the HEL-specific phage clones fell into four types (I-IV): type I contains a single cysteine residue and type II-IV contain two cysteine residues. These results indicated that the artificially randomized IgNAR library is useful for the rapid isolation of antigen-specific IgNAR V region without immunization of target antigen and showed that it is possible to isolate an antigen-specific IgNAR V region from this library.</P>
Comparative Sequence Analysis of a Multidrug-Resistant Plasmid from Aeromonas hydrophila
del Castillo, Carmelo S.,Hikima, Jun-ichi,Jang, Ho-Bin,Nho, Seong-Won,Jung, Tae-Sung,Wongtavatchai, Janenuj,Kondo, Hidehiro,Hirono, Ikuo,Takeyama, Haruko,Aoki, Takashi American Society for Microbiology 2013 Antimicrobial agents and chemotherapy Vol.57 No.1
<B>ABSTRACT</B><P>Aeromonas hydrophilais a pathogenic bacterium that has been implicated in fish, animal, and human disease. Recently, a multidrug resistance (MDR) plasmid, pR148, was isolated fromA. hydrophilaobtained from a tilapia (Oreochromis niloticus) farm in Thailand. pR148 is a 165,906-bp circular plasmid containing 147 coding regions showing highest similarity to pNDM-1_Dok1, an MDR plasmid isolated from a human pathogen. pR148 was also very similar to other IncA/C plasmids isolated from humans, animals, food, and fish. pR148 contains a mercuric resistance operon and encodes the complete set of genes for the type 4 secretion system. pR148 encodes a Tn<I>21</I>type transposon. This transposon contains the drug resistance genes<I>qacH</I>,<I>bla</I>OXA-10,<I>aadA1</I>, and<I>sul1</I>in a class 1 integron;<I>tetA</I>and<I>tetR</I>in transposon Tn<I>1721</I>; and<I>catA2</I>and a duplicate<I>sul1</I>in a locus showing 100% similarity to IncU plasmids isolated from fish. The<I>bla</I>OXA-10and<I>aadA1</I>genes showed 100% similarity to those from theAcinetobacter baumanniiAYE genome. The similarity of pR148 to a human pathogen-derived plasmid indicates that the plasmids were either transferred between different genera or that they are derived from a common origin. Previous studies have shown that IncA/C plasmids retain a conserved backbone, while the accessory region points to lateral gene transfer. These observations point out the dangers of indiscriminate use of antibiotics in humans and in animals and the necessity of understanding how drug resistance determinants are disseminated and transferred.</P>
Park, Seong Bin,Hikima, Jun-ichi,Suzuki, Yoshiaki,Ohtani, Maki,Nho, Seong Won,Cha, In Seok,Jang, Ho Bin,Kondo, Hidehiro,Hirono, Ikuo,Aoki, Takashi,Jung, Tae Sung Elsevier 2012 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.36 No.4
<P><B>Highlights</B></P><P>► The gene encoding nucleotide-binding oligomerization domain 1 (NOD1) was cloned in olive flounder. ► NOD1 was expressed in all fish tissues examined. ► NOD1 mRNA levels was elevated in fish infected with <I>Edwardsiella tarda</I>, <I>Streptococcus iniae</I>, or VHSV. ► The inhibition of <I>E. tarda</I> and the increase of IL-1β levels were observed in NOD1 over-expressing cells.</P> <P><B>Abstract</B></P><P>The gene encoding nucleotide-binding oligomerization domain 1 (NOD1) was cloned from olive flounder (<I>Paralichthys olivaceus</I>) and the role played by NOD1 during <I>Edwardsiella tarda</I> infection was evaluated. The complete open reading frame of NOD1 was 2820bp in length, encoding a 939-amino acid polypeptide. The NOD1 protein contains three conserved domain structures including C-terminal LRRs, a central NACHT motif, and an N-terminal CARD domain, which show similarities of 49–74% to those of other vertebrate counterpart proteins. NOD1 expression was observed in all fish tissues examined, and the levels increased in olive flounder infected with <I>E. tarda</I>, <I>Streptococcus iniae</I>, or viral hemorrhagic septicemia virus (VHSV). When hirame natural embryo (HINAE) cells over-expressing NOD1 were infected with <I>E. tarda</I>, bacterial growth was inhibited, and the IL-1β transcript level increased compared to that of the control. These findings imply that NOD1 plays an important role in response to <I>E. tarda</I> infection of olive flounder.</P>
Characterization and functional analysis of two PKR genes in fugu (<i>Takifugu rubripes</i>)
del Castillo, Carmelo S.,Hikima, Jun-ichi,Ohtani, Maki,Jung, Tae-Sung,Aoki, Takashi Elsevier 2012 FISH AND SHELLFISH IMMUNOLOGY Vol.32 No.1
<P><B>Abstract</B></P><P>PKR (protein kinase R) is a serine–threonine kinase that inhibits protein synthesis by the phosphorylation of the eukaryotic translation initiation factor 2-alpha (eIF2α), and activates NFκB by inducing NFκB-inducing kinase and IκB (inhibitor of NFκB) kinase. This can lead to antiviral and anti-proliferative effects. In this study, the complete sequence and organization of two fugu PKR genes (fPKRs) were determined by <I>in silico</I> analysis and conventional PCR. The full-length <I>fPKR1</I> and <I>fPKR2</I> genes were 3832 bp and 4325 bp, which encoded 523 and 492 amino acids, respectively. Both encoded two dsRNA binding domains and a Serine/Threonine protein kinase domain, and showed very high similarity to green spotted puffer PKRs. Gene expression of the two fPKRs was measured by quantitative real-time PCR on tissue samples from healthy fish and peripheral blood leukocytes stimulated with polyinosinic:polycytidylic acid (PolyI:C) or lipopolysaccharides (LPS). The fPKRs were highly expressed in the skin and fPKR2 was significantly induced in PBLs by PolyI:C but not by LPS. The fPKRs inhibited translation of a luciferase reporter gene in a dose-dependent manner and induced transcriptional activity of a mammalian NFκB luciferase reporter. These results demonstrate that two PKRs in a single species can both be independently, but not equally, functional and support the hypothesis that fish PKRs have roles in the innate immune response similar to those of mammalian PKRs.</P> <P><B>Highlights</B></P><P>► The complete sequence and structure of two PKR genes in fugu were determined. ► The fugu PKRs were highly expressed in the skin of healthy fish. ► The fugu PKRs were demonstrated to inhibit luciferase activity and induce NFκB.</P>