Expression, purification and structural characterization of recombinant hepcidin, an antimicrobial peptide identified in Japanese flounder, Paralichthys olivaceus

The cysteine-rich peptide hepcidin is an antimicrobial peptide and iron transport regulator that has been found in vertebrates including birds, fish and mammals. To elucidate the structure and biological function of fish hepcidin, which is difficult to produce synthetically, we have cloned several plasmid constructs encoding hepcidin from Japanese flounder, Paralichthys olivaceus, and tested expression of recombinant peptides, each with an N-terminal hexahistidine (6xHis) tag, in inclusion bodies or the periplasmic space of Escherichia coli. Hepcidin expressed in inclusion bodies was reduced, and subsequently refolded using a dilution technique with a cysteine redox system. The oxidized His-hepcidin monomer was separated from protein multimers and mass spectrometry analysis showed that the peptide was of the predicted size and contained four disulfide bonds. Removal of the 6xHis tag was attempted using enzymatic cleavage by Factor Xa and tobacco etch virus (TEV) protease or chemical cleavage by hydroxylamine. The Factor Xa cleavage was unsuccessful and hydroxylamine cleavage resulted in aggregation of cleaved peptide. TEV protease cleavage was successful but immediately resulted in hexamer formation despite varying reaction conditions (redox, non-redox, pH, temperature, target protein concentration, type of buffer). However, the recombinant His-hepcidin fusion peptide monomer showed considerable antimicrobial activity. NMR-based studies showed that hepcidin contained a rare vicinal disulfide linkage at the top of a loop structure and a short beta-sheet structure encompassing residues 7-13 and 19-25 that is stabilized by three disulfide bonds.     

Larval morphometry of the Japanese flounder,Paralichthys olivaceus

The larval development of the Japanese flounder,Paralichthys olivaceus, was surveyed using two types of morphometric analyses, modified allometry and polar coordinate analysis by principal component analysis (PCA). In the former, centroid size was used as a growth index instead of total length (TL), such enabling the determination of more detailed changes in each character than ordinary allometry based upon TL. Polar coordinate analysis disclosed two remarkable inflexions during the larval development ofP. olivaceus. Postlarvae ofP. olivaceus were found to undergo four developmental phases. From the point of view of metamorphosis, the phases were named drifting larva, premetamorphic larva, metamorphic larva and postmetamorphic larva, respectively. These phases were also tested by other characters related to flounder metamorphosis.     

Cloning and expression of partial Japanese flounder (Paralichthys olivaceus) IgD

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7 d Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.     

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.     

牙鲆变态过程中的细胞凋亡

利用整体的原位TUNEL方法检测了牙鲆(Paralichthysolivaceus)变态过程中身体各器官细胞凋亡的分布及变化情况。结果如下:(1)与眼睛移动相关的脑颅骨骼的细胞凋亡右侧眼睛移动开始之后,在额骨、中筛软骨和犁骨软骨中出现细胞凋亡,并保持到眼睛移动结束;(2)中枢神经和感觉器官的细胞凋亡在眼睛移动开始之前,脊髓和脊髓鞘出现细胞凋亡,在眼睛移动开始之后,脊髓和脊髓鞘细胞凋亡停止,而在脑、眼睛和内耳出现细胞凋亡,并一直持续到眼睛移动结束;(3)与游泳、捕食和消化等功能相关的器官的细胞凋亡在眼睛移动开始后,冠状幼鳍的基部出现凋亡;在变态中后期,尾鳍基部出现细胞凋亡;下颌骨、鳃弓以及肝脏在眼睛移动开始之后,出现细胞凋亡,也一直持续到眼睛移动结束。细胞凋亡通过有序地去除多余的细胞来参与器官形态建立和重组,本研究的结果表明,在牙鲆器官功能变化过程中,细胞凋亡在与其相适应的的器官形态重塑中起着重要作用[动物学报52(2):355-361,2006]。     

Digestive capacity, growth and social stress in newly-metamorphosed Japanese flounder (Paralichthys olivaceus)

Hierarchies readily appear when rearing flounders through metamorphosis in space-limited conditions. In this experiment, subordinate fish were stressed, as suggested by their elevated cortisol level compared to dominant fish. Subordinate fish, although of smaller size than the dominant fish, showed no suppressed digestive capacity. By separating the two hierarchical groups into different tanks at low density, the cortisol level of the subordinate fish substantially decreased. The removal of the stressor also resulted in an increase in digestive function and improved coloration. Also, compensatory growth in length but not in weight was observed in the subordinate group, suggesting that the subordinate fish devoted their energy to increase their length. These results imply that the climax of flounder metamorphosis (i.e. the settlement stage) is a highly sensitive period, where social interactions may induce high levels of stress. However, Japanese flounder early juveniles prove to have a high recovery capacity from stress and show to allocate energy preferentially to growth in length. This seems to be an adaptation to diminish the probability of death by predation.     

Neuromast formation in the prehatching embryos of the Japanese flounder (Paralichthys olivaceus)

The present paper clarifies the initial development of the lateral line organs in the embryonic Japanese flounder, Paralichthys olivaceus. The first appearances of lateral line primordia, and the proliferation, distribution and morphological development of the free neuromasts, including nerve ending formation: establishment of hair cell innervations via the formation of synapses, were examined by light microscopy, scanning and transmission electron microscopy. The first pair of neuromast primordia appeared in the otic region ≈ 30 h prior to hatching and subsequently differentiated into free neuromasts, otic neuromasts, after ≈ 8 h. At hatching, a pair of free neuromasts and three pairs of neuromast primordia were present on the head, and three pairs of neuromast primordia were present on the trunk. The hair cell polarity of the otic neuromast until just prior to hatching was radial, but not bi‐directional. The typical afferent and efferent nerve endings in the otic neuromasts had formed by the time of hatching, suggesting that the otic neuromasts are functional prior to hatching. The three neuromast primordia located on each side of the trunk were derived from a long, narrow ectodermal cell cluster and erupted through the epidermis after hatching.     

Molecular cloning of multiple chitinase genes in Japanese flounder, Paralichthys olivaceus

Three different chitinase genes (fChi1, fChi2 and fChi3) were identified from Japanese flounder, Paralichthys olivaceus. The deduced amino-acid sequences of flounder chitinases revealed a typical chitinase structure containing a catalytic glyco-18 domain, a hinge region and a chitin binding domain type 2. The fChi1 and fChi2 mRNAs were predominantly expressed in the gastric glands of stomach. In contrast, expression of fChi3 was found in spleen, pancreas, stomach, intestine, liver, kidney and gonads of adult flounder by RT-PCR. The expression level of fChi3 in the adult tissues was below the detection limit of in situ hybridization (ISH) analysis; however, ISH signals were detected in the liver of flounder larvae. These results suggest that fChi1 and fChi2 are acidic chitinases that digest dietary chitin and that fChi3 probably is a macrophage specific chitinase (chitotriosidase) for biodefense and has an important unknown role in the liver during larval stages.     

Cryopreservation of flounder (Paralichthys olivaceus) embryos by vitrification

Conventional cryopreservation of complex teleost embryos has been unsuccessful, possibly because their large size (1-7 mm diameter), multi-compartmental structure and low water permeability lead to intracellular ice formation and chilling injury. To overcome these obstacles, we have developed a vitrification procedure for cryopreservation of flounder (Paralichthys olivaceus) embryos. In initial toxicity tests, propylene glycol (PG) and methanol (MeOH) were less toxic to embryos than dimethylformamide (DMF) or dimethyl sulfoxide (Me2SO), whereas ethylene glycol (EG) and glycerol (Gly) were toxic to all tested embryos. Embryos between four-somite and tail bud stages were more tolerant to vitrifying solutions than embryos in other developmental stages. Four vitrifying solutions (FVS1-FVS4) were prepared by combining a basic saline solution (BS2) and cryoprotectants PG and MeOH in different proportions (FVS1: 67, 20 and 13%; FVS2: 60, 24 and 16%; FVS3: 55, 27 and 18%; FVS4: 50, 30 and 20% of BS2, PG and MeOH, respectively). Their impact on flounder embryos was then compared. FVS1 produced the highest survival rate; whereas deformation rate was highest for FVS4. Five-step equilibration of embryos in FVS2 resulted in higher survival rates than equilibration in 4, 3, 2 or 1 steps. Flounder embryos varying from the 14-somite to the pre-hatching stage were cryopreserved in the four vitrifying solutions in liquid nitrogen for 1-7 h. From eight experiments, 20 viable thawed embryos were recovered from 292 cryopreserved embryos. Fourteen larvae with normal morphology hatched successfully from the 20 surviving frozen-thawed embryos from five experiments. Embryos at the tail bud stage exhibited greater tolerance to vitrification than embryos at other stages. These results establish that cryopreservation of flounder embryos by vitrification is possible. The technology has many potential applications in teleost germplasm resource conservation.     

Production, characterisation and applicability of monoclonal antibodies to immunoglobulin of Japanese flounder (Paralichthys olivaceus)

Immunoglobulin (Ig) of Japanese flounder (Paralichthys olivaceus) was purified by a combination of salting-out and DEAE Sepharose Column chromatography. The purified immunoglobulin had an apparent molecular weight of 74 kDa (heavy chain) and 24 kDa (light chain) in SDS-PAGE. Eighteen hybridomas secreting monoclonal antibodies (MAbs) against Japanese flounder Ig were obtained by immunisation of Balb/C mice with purified Ig preparations, which were selected on the basis of the double indirect enzyme-linked immunosorbent assay (D-ELISA). Two of them designated as 2D8 and 2H1 were cloned by limiting dilution and characterised with western blotting, indirect immunofluorescence assay test (IIFAT) and fluorescence-activated cell sorter (FACS) analysis. Under reducing conditions in western blotting, both MAb 2D8 and MAb 2H1 were specific for the heavy chain of Japanese flounder Ig. MAb 2D8 was used to identify surface Ig-positive lymphocytes in the peripheral blood, spleen and pronephros of healthy Japanese flounder by flow cytometry. FACS analysis revealed that 40.48% of lymphocytes in the peripheral blood, 17.32% in the spleen and 9.67% in the pronephros were reactive to 2D8.     

Isolation and characterization of new microsatellite markers from the Japanese flounder (Paralichthys olivaceus)

The isolation and characterization of eight polymorphic microsatellite loci from a Japanese flounder partial genomic library are reported. The eight markers isolated in this study were highly polymorphic and their positions on the linkage genome map of the Japanese flounder were determined. Therefore, they are useful for ecological studies of wild populations. These markers are more effective than other markers with no information of chromosomal locations.     

Identification and differential expression of microRNAs during metamorphosis of the Japanese flounder (Paralichthys olivaceus)

Background

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of 20–25 nucleotides that play a key role in diverse biological processes. Japanese flounder undergo dramatic metamorphosis in their early development. The metamorphosis is characterized by morphological transformation from a bilaterally symmetrical to an asymmetrical body shape concomitant with extensive morphological and physiological remodeling of organs. So far, only a few miRNAs have been identified in fish and there are very few reports about the Japanese flounder miRNA.

Methodology/Principal Findings

Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the metamorphic period of Japanese flounder. Subsequently, aligning these sequencing data with metazoan known miRNAs, we characterized 140 conserved miRNAs and 57 miRNA: miRNA* pairs from the small RNA library. Among these 57 miRNA: miRNA* pairs, twenty flounder miRNA precursors were amplified from genomic DNA. We also demonstrated evolutionary conservation of Japanese flounder miRNAs and miRNA* in the animal evolution process. Using miRNA microarrays, we identified 66 differentially expressed miRNAs at two metamorphic stages (17 and 29 days post hatching) of Japanese flounder. The results show that miRNAs might play a key role in regulating gene expression during Japanese flounder metamorphosis.

Conclusions/Significance

We identified a large number of miRNAs during flounder metamorphosis, some of which are differentially expressed at two different metamorphic stages. The study provides an opportunity for further understanding of miRNA function in the regulation of flounder metamorphosis and gives us clues for further studies of the mechanisms of metamorphosis in Japanese flounder.     

Experimental horizontal transmission of viral hemorrhagic septicemia virus (VHSV) in Japanese flounder Paralichthys olivaceus

Infection by viral hemorrhagic septicemia virus (VHSV) has recently occurred among wild and farmed Japanese flounder Paralichthys olivaceus in Japan. In the present study, horizontal transmission of VHSV among Japanese flounder was experimentally demonstrated by immersion challenge. Exposure to a flounder isolate (Obama25) of VHSV revealed a dose-response, with higher mortality (81 and 70%) at the 2 higher exposure levels (6.0 and 4.0 log10 TCID50 ml(-1)). In a second experiment, high titers of VHSV were expressed from moribund and dead flounder based on virus detection in holding-tank waters 2 to 3 d prior to death of the fish and 1 d after death. The virus could not be detected in tank waters 2 d after death. Finally, a third cohabitation experiment in small tanks demonstrated horizontal transmission of VHSV from experimentally infected to uninfected fish.     

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.     

Two different types of hepcidins from the Japanese flounder Paralichthys olivaceus

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.