Molecular characterization and bioactivity of a CXCL13 chemokine in large yellow croaker Pseudosciaena crocea

A CXCL13-like chemokine cDNA was isolated from large yellow croaker (Pseudosciaena crocea) by expressed sequence tag (EST) analysis (LycCXCL13). The full-length cDNA of LycCXCL13 is 796 nucleotides (nt) encoding a protein of 97 amino acids (aa), with a putative molecular weight of 10.7 kDa. The deduced LycCXCL13 contains a 24-aa signal peptide and a 73-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines (C²⁵, C²⁷, C⁵² and C⁶⁸). It shares 35, 36 and 39% aa sequence identities to green puffer CXCL13-like, Atlantic salmon CXCL13 and Japanese flounder CXCL13 chemokines, and 24–29% identities to CXCL13 chemokines in mammals, respectively. Phylogenetic analysis showed that LycCXCL13 is more closely related to the CXCL13 subgroup than to any other CXC chemokine subgroups. LycCXCL13 gene was constitutively expressed in all tissues examined, except for intestine. Upon induction with poly(I:C) or inactivated trivalent bacterial vaccine, LycCXCL13 gene expression was significantly up-regulated in spleen, head kidney, heart and gills at 24 h post-injection. Real-time PCR results showed that LycCXCL13 gene expression reached peak level in spleen and head kidney at 12 h after induction by poly(I:C), while its expression increased to the highest level in head kidney at 24 h or in spleen at 48 h by bacterial vaccine. Recombinant LycCXCL13 protein produced in E. coli BL21 exhibited obvious chemotaxis to the peripheral blood leucocytes (PBLs) from large yellow croaker. These results suggest that LycCXCL13 may be involved in inflammatory responses as well as homeostatic processes in large yellow croaker.     

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.     

Identification of a novel Japanese flounder (<Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>) CC chemokine gene and an analysis of its function

A cDNA of Japanese flounder (Paralichthys olivaceus) CC chemokine designated as Paol-SCYA104 was cloned and sequenced. The cDNA contains an opening reading frame of 315 nucleotides encoding 104 amino acid residues. The full gene was cloned and sequenced from a BAC library. It has a length of approximately 750 bp from the start codon to the stop codon and is composed of four exons and three introns. Four cysteine residues are conserved in the same positions as those of mammalian and fish CC chemokines. Paol-SCYA104 gene was expressed in several organs, including peripheral blood leukocytes (PBLs), head kidney, trunk kidney, and spleen. The recombinant Paol-SCYA104 was expressed in Escherichia coli and the expressed protein was partially purified. The recombinant Paol-SCYA104 was able to attract Japanese flounder PBLs in a microchemotaxis chamber. On the other hand, a negative control, the fraction of the control cells carrying an expression vector lacking the Paol-SCYA104 cDNA, did not show chemotactic activity. These results indicate that Paol-SCYA104 probably acts as a CC chemokine.     

Cloning, characterization and expression analysis of a novel CXC chemokine from turbot (Scophthalmus maximus)

Chemokines represent a superfamily of chemotactic cytokines playing an important role in leucocyte chemotaxis. Here we report a novel turbot CXC chemokine screened from a turbot spleen cDNA library. The complete cDNA of the turbot CXC chemokine contains an 81bp 5' UTR, a 414bp open reading frame (ORF) encoding 137 amino acids and a 449bp 3' UTR. Four exons and three introns are identified in the turbot CXC chemokine gene. Phylogenetic analysis showed that the turbot CXC chemokine clustered apart from all other CXC chemokines. RT-PCR demonstrated that turbot CXC chemokine was expressed highly in spleen and head kidney. During the early stages of embryo development after fertilization, it appears that low expression level of turbot CXC chemokine was firstly observed at somites stage. Interestingly, the turbot chemokine was highly and rapidly (5h) induced in liver, spleen and head kidney of turbot after challenge with Vibrio anguillarum. Furthermore, the expression of CXC chemokine was also dramatically increased after challenge in turbot embryonic cells (TECs). These results indicated that the turbot CXC chemokine played an important role in turbot immune response.     

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%.     

Molecular cloning, characterization and expression analysis of a miiuy croaker (Miichthys miiuy) CXC chemokine gene resembling the CXCL9/CXCL10/CXCL11

Chemokines are a large family of chemotactic cytokines playing crucial roles in the innate immune response. In the present study, we report the cloning of a CXC chemokine gene resembling the closely related CXCL9/CXCL10/CXCL11 from the miiuy croaker Miichthys miiuy (MimiCXC). Both 5'-RACE and 3'-RACE were carried out in order to obtain the complete cDNA, which consists of a 73 bp 5'-UTR, a 369 bp open reading frame encoding 122 amino acids and a 715 bp 3'-UTR. The deduced MimiCXC contains a 19-aa signal peptide and a 103-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines. It shares 4.8%-65.6% sequence identities to mammalian CXC chemokines and the highest sequence identity of 65.6% is between MimiCXC and CXCL10 chemokine. Three exons and two introns were identified in MimiCXC gene. The MimiCXC gene was constitutively expressed in all tissues tested, although at different levels. Upon induction with Vibrio anguillarum, MimiCXC gene expression was up-regulated in kidney and spleen, however, down-regulated in liver. These results indicate that MimiCXC may be involved in immune responses as well as homeostatic processes in miiuy croaker.     

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.     

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.     

Cloning and characterization of the IkappaBalpha gene from Japanese flounder, Paralichthys olivaceus

We identified and characterized the Japanese flounder (Paralichthys olivaceus) inhibitor kappa B alpha (JFIKBA) cDNA. The JFIKBA cDNA contains an open reading frame of 960bp encoding 320 amino acid residues. JFIKBA contains 6 ankyrin repeats in the central coding region. Expression studies by RT-PCR showed constitutive expression of the JFIKBA gene in several Japanese flounder tissues (brain, muscle, gill, heart, kidney, liver, spleen and intestine). Moreover, expression of JFIKBA mRNA was induced in kidney by LPS stimulation. To investigate the role of JFIKBA, we constructed a recombinant plasmid expressing the JFIKBA coding region under the control of the cytomegalovirus (CMV) promoter. Over-expression of the JFIKBA gene in the Japanese flounder cultured cell line derived from kidney, suppressed the expression of the TNF alpha gene with lipopolysaccharide stimulation. These results indicated that JFIKBA has an important role in the innate immune system, especially in the signaling of the cytokine network.     

Molecular cloning, expression, and functional analysis of caspase-10 from Japanese flounder Paralichthys olivaceus

We isolated and sequenced caspase-10 cDNA and gene from Japanese flounder, Paralichthys olivaceus. The Japanese flounder (JF)-caspase-10 cDNA consisted of 2282 bp and encoded 495 amino acid residues. The characteristic death effector domains (DEDs) of caspases were observed in JF-caspase-10 as well as the three aspartic acid residues (D-186, -382 and -392), which are potential cleavage sites for the large and small subunit structures. The amino acid residue (His-325) and pentapeptide (QACQG), which are involved in catalytic activity, were absolutely conserved in Japanese flounder-caspase-10. JF-caspase-10 gene has a length of 6.6 kb and consists of 11 exons and 10 introns similar to that of human. The strong expression of JF-caspase-10 mRNA was detected in the gills, peripheral blood leukocytes, spleen and posterior kidney, while the weak expression was observed in the head kidney, heart, intestine, skin and stomach. The over-expression analysis of JF-caspase-10 in Japanese flounder cell line HINAE was shown to induce apoptosis 24h post-transfection using TUNEL assay.     

Generation and functional significance of CXC chemokines for neutrophil-induced liver injury during endotoxemia

The hypothesis that the neutrophil chemoattractant CXC chemokines KC and macrophage inflammatory protein-2 (MIP-2) are involved in neutrophil transmigration and liver injury was tested in C3Heb/FeJ mice treated with galactosamine (Gal, 700 mg/kg), endotoxin (ET, 100 microg/kg), or Gal + ET (Gal/ET). Hepatic KC and MIP-2 mRNA levels and plasma CXC chemokine concentrations were dramatically increased 1.5 h after Gal/ET or ET alone and gradually declined up to 7 h. Murine recombinant cytokines (TNF-alpha, IL-1 alpha, and IL-1 beta), but not Gal/ET, induced CXC chemokine formation in the ET-resistant C3H/HeJ strain. To assess the functional importance of KC and MIP-2, C3Heb/FeJ mice were treated with Gal/ET and control IgG or a combination of anti-KC and anti-MIP-2 antibodies. Anti-CXC chemokine antibodies did not attenuate hepatocellular apoptosis, sinusoidal neutrophil sequestration and extravasation, or liver injury at 7 h. Furthermore, there was no difference in liver injury between BALB/cJ wild-type and CXC receptor-2 gene knockout (CXCR2-/-) mice treated with Gal/ET. The higher neutrophil count in livers of CXCR2-/- than in wild-type mice after Gal/ET was caused by the elevated number of neutrophils located in sinusoids of untreated CXCR2-/- animals. The pancaspase inhibitor Z-Val-Ala-Asp-fluoromethylketone eliminated Gal/ET-induced apoptosis and neutrophil extravasation and injury but not CXC chemokine formation. Thus Gal/ET induced massive, cytokine-dependent CXC chemokine formation in the liver. However, neutrophil extravasation and injury occurred in response to apoptotic cell injury at 6-7 h and was independent of CXC chemokine formation.     

Evaluation of housekeeping genes as references for quantitative real time RT-PCR analysis of gene expression in Japanese flounder (Paralichthys olivaceus)

Japanese flounder (Paralichthys olivaceus) is an important economic fish species cultured worldwide. In this report, we compared the potentials of ten housekeeping genes as quantitative real time RT-PCR (qRT-PCR) references for the study of gene expression in Japanese flounder under normal physiological conditions and during bacterial infection. For this purpose, the expression of the ten genes in eight flounder tissues (liver, spleen, kidney, heart, muscle, brain, gill, and intestine) was determined by qRT-PCR before and after bacterial infection. The expression levels of the housekeeping genes were then compared and evaluated with geNorm and NormFinder algorithms. The results showed that before bacterial infection, the tested genes exhibited tissue-specific expressions to various degrees, with β-actin and ubiquitin-conjugating enzyme being ranked as the most stable genes across tissue types. Following bacterial challenge, all the tested genes varied in expression levels in tissue-dependent manners and no cross-all-tissue type reference gene was identified among the examined panel of housekeeping genes; however, α-tubulin was recognized as the most stable gene in four (spleen, heart, muscle, and gill) of the eight examined tissues. These results indicate that for qRT-PCR analysis of gene expression in Japanese flounder as a function of bacterial infection, the choice of reference genes should be made according to tissue type.     

A pore-forming protein,perforin, from a non-mammalian organism,Japanese flounder,<Emphasis Type="Italic"> Paralichthys olivaceus</Emphasis>

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.     

Gene expression of leucocytes in vaccinated Japanese flounder (Paralichthys olivaceus) during the course of experimental infection with Edwardsiella tarda

In this paper, we focused on the detection of differentially expressed genes in peripheral blood leucocytes (PBL) during the course of Edwardsiella tarda infection in vaccinated and non-vaccinated Japanese flounder (Paralichthys olivaceus). cDNA microarray analysis was performed to compare the gene expression patterns of the PBL between the vaccinated and non-vaccinated fish in response to E. tarda inoculation. Fish were vaccinated twice, at a two-week interval and experimentally challenged with E. tarda two weeks after the second vaccination. Among the 1187 analyzed genes, 42 genes were up-regulated during the course of infection either in vaccinated or non-vaccinated fish. These genes included immune-related genes, such as MMP-9, MMP-13, CXC chemokine, CD20 receptor and hepcidin. Some immune-related genes were down-regulated after the E. tarda challenge, i.e. interferon inducible Mx protein, MHC class II-associated invariant chain, MHC class II alpha and MHC class II beta encoding genes, immunoglobulin light chain precursor, immunoglobulin light chain and IgM. These responses are thought to be a common reaction of Japanese flounder PBL in the course of edwardsiellosis, irrespective of immunized condition. Ten genes were significantly up-regulated only in vaccinated fish, and 11 genes were significantly up-regulated only in non-vaccinated fish. These genes may have a correlation with the efficacy of vaccination, although we have no evidence to link the different gene expression patterns and the efficacy of vaccination at present.