Two different types of hepcidins from the Japanese flounder Paralichthys olivaceus Ikuo Hirono, Jee-Youn Hwang, Yasuhito Ono, Tomofumi Kurobe, Tsuyoshi Ohira, Reiko Nozaki and Takashi Aoki Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Japan Antimicrobial peptides are important molecules in the innate immune system. Hepcidin is an antimicrobial peptide that acts against Gram-negative and Gram- positive bacteria and yeast [1–3]. Hepcidin is a 20- or 25-residue peptide containing four disulfide bonds that is expressed in the liver [1,2]. Recently, hepcidin has been proposed to also act as a central regulation of intestinal iron absorption and iron recycling by macro- phage [4,5]. Hepcidin inhibits the iron absorption in the small intestine, the release of recycled iron from macrophages, and the transport of iron across the pla- centa. Expression of the hepcidin gene in mice is enhanced by iron overload, interleukin 6 and lipopoly- saccharide (LPS) [6–9] Recently, several fish hepcidin cDNAs and genes were cloned and sequenced [3,10,11]. Previous reports of fish hepcidin were mostly concerned with sequencing and expression of some genes using RT-PCR. The only fish hepcidin whose antimicrobial activity has been characterized so far is bass, Morone chrysops hepcidin Keywords antimicrobial peptide; gene expression; hepcidin; Japanese flounder Paralichthys olivaceus Correspondence Takashi Aoki, Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo 108–8477, Japan Fax: +81 3 54630690 Tel: +81 3 54630556 E-mail: aoki@s.kaiyodai.ac.jp (Received 17 May 2005, revised 13 July 2005, accepted 18 August 2005) doi:10.1111/j.1742-4658.2005.04922.x The cysteine-rich peptide hepcidin is known to be an antimicrobial peptide and iron transport regulator that has been found in both fish and mam- mals. Recently, we found two different types (designated Hep-JF1 and Hep-JF2) of hepcidin cDNA in the Japanese flounder, Paralichthys oliva- ceus, 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 expres- sion 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 signifi- cantly 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. Syn- thesized Hep-JF2 peptide showed antimicrobial activity, while synthesized Hep-JF1 peptide did not against several bacteria including fish-pathogenic bacteria used in this study. Abbreviations HIB, heart infusion broth; LPS, lipopolysaccharide; PBL, peripheral blood leukocyte; TFA, trifluoroacetic acid. FEBS Journal 272 (2005) 5257–5264 ª 2005 FEBS 5257 [12]. Interestingly, winter flounder, Pseudopleuronectes americanus has three different types of hepcidin [11]. The gene expressions of three types of hepcidins in dif- ferent organs and tissues were different. From the results of previous report, it is speculated that hepci- dins homologues may have a variety of roles in fish. In this study, we cloned two different hepcidin genes and characterized their expressions in several organs. We also examined how these genes might be regulated, identified hepcidin-expressing cells and measured the antimicrobial activities of the synthesized peptides. Results Hepcidin genes of Japanese flounder The Hep-JF1 (AB198060) and Hep-JF2 (AB198061) cDNAs encoded 81 and 89 amino acid residues, respectively. The lengths of the predicted mature pep- tide regions of Hep-JF1 and Hep-JF2 were 19 and 26 residues (Fig. 1). The numbers of Cys residues of Hep- JF1 and Hep-JF2 were six and eight, respectively (Fig. 1). The positions from the third to the ninth amino acid residues in Hep-JF2 were deleted in Hep- JF1. The second Cys residue of Hep-JF1 was replaced with Gly. The positions of all Cys residues in Hep-JF2 and six Cys residues in Hep-JF1 were identical to that of human. The Hep-JF1 (AB198062) and Hep-JF2 (AB198063) genes were both approximately 0.7 kb. The results of Southern blot hybridization indicated that both hepci- din genes were present as single copies in the genome because the hybridized DNA bands were the same size in both the genomic DNA and the BAC clones (Fig. 2A). The hybridized DNA fragments of BAC clones were sequenced. Both genes, as for previously reported hepcidin genes, consisted of three exons and Hep-JF1 1 MKTFSAAVTVAVVLVFICIQQSSAT-SPEVQELEEAVSSDNAAAEHQEQSADSWMMPQ 57 Hep-JF2 1 A I A.TL A.V D IPFQG GGN.TPV.A MM.ME ESP 58 W. flounder-1 1 A I A.TL A.V C VPFQG GGN.TPV VM.ME ENP 58 W. flounder-3 1 V S-F A N TPV Y 57 W. flounder-2 1 V F N TPV G N 57 Bass 1 V A A L.E VPVT PM.NE Y MPVE K Y 53 Seabream 1 V A T L.E ASFT PM.NGSPV.ADE.M.EE K Y 58 Salmon 1 MKAFS.A.VL.IACMF.LE.T.VPFS RTE.VGSFDSPVGEHQ.PGGESMHLPEP 56 Zebrafish 1 LSNVFLAAV.I.TCV.VF.IT.VPFIQQVQD.HH.E.EELQENQHLTE.EHRLTDPLV 60 Human 1 MALSSQIWAA.LLLLLLLA.LTSGSVFPQQTGQL.ELQP.DRAGARASWMPMF 53 Hep-JF1 58 NRQKRDVK CGFCCKD-GGCGVCCNF- 81 Hep-JF2 59 V HISHISMCRW.CN A-K P K 89 W. flounder-1 59 T HISHISLCRW.CN ANK F K 90 W. flounder-3 58 S FKCKF.CG RA.V L K 84 W. flounder-2 58 S AD.WP NQ-N T KV- 81 Bass 54 .NRHKRHSSPGGCRF.CN PNMS R 85 Seabream 59 ASR RWRCRF.CR PRMR L QRR 85 Salmon 57 -FRFKRQIHLSLCGL.CN HNI F K 86 Zebrafish 61 LFRTKRQSHLSLCRF.CK RNK Y K 91 Human 54 Q.RR.RDTHFPICIF.CG HRSK M KT- 84 # # ## ## ## Fig. 1. Alignment of amino acid sequences of Japanese flounder and other reported hepcidins. The box indicates the mature peptide region. Hashes below the alignment indicate the positions of cysteine residues. Dashes indicate the gap for maximum matching of amino acid sequences of the three hepcidins. Dots indicate the identical amino acid residues with Hep-JF1. A B Fig. 2. Southern blot hybridization analysis and genomic organiza- tion of Japanese flounder hepcidin genes. (A) Genomic and BAC clone DNAs were digested with EcoRI (E) and PstI (P). (B) Sche- matic representation of exon–intron organizations of Hep-JF1 and Hep-JF2. Hepcidins of Japanese flounder I. Hirono et al. 5258 FEBS Journal 272 (2005) 5257–5264 ª 2005 FEBS two introns (Fig. 2B). The positions of exon–intron junctions of Hep-JF1 and Hep-JF2 are similar to those of mammals and bass. However, the second exons of Hep-JF1 and Hep-JF2 are only 1 ⁄ 10–1 ⁄ 20 as long as the second exon of mammals. Tissue-specific expressions of Japanese flounder hepcidin genes The Hep-JF1 gene was specifically detected in liver while Hep-JF2 expression was observed in gill, liver, heart, kidney, peripheral blood leucocytes, spleen and stomach (Fig. 3). Hep-JF2 gene expression was un- detectable in brain, eye, intestine, muscle, ovary or skin by RT-PCR. Iron regulation of hepcidin mRNA expression The gene expression of Hep-JF1 in liver was dramatic- ally decreased during experimental iron (iron-dextran) overloading (Figs 4 and 5). The gene expression of Hep-JF2 in liver was not significantly distinct during iron overloading. The gene expression of Hep-JF2 in kidney was undetectable when only 20 PCR cycles were used. However, the gene expression of Hep-JF2 in kidney increased after injection of iron-dextran and during iron overloading. Up-regulation of hepcidin gene expression by LPS The gene expressions of Hep-JF1 and Hep-JF2 in liver were strongly enhanced by LPS injection (Figs 4 and 5). The amounts of Hep-JF1 and Hep-JF2 mRNAs in liver 6 h after LPS injection increased to more than 10 4 and 10 times, respectively, which is than higher than the amount 3 h after LPS treatment (Fig. 5). The gene expression of Hep-JF2 in kidney was also strongly up-regulated by LPS injection (Figs 4 and 5). The amount of Hep-JF2 mRNA in kidney after 24 h LPS injection was more than 10 2 times higher than the amount after the 3 h LPS injection (Fig. 5). The gene expression of Hep-JF1 was undetectable in kidney both before and after LPS injection by RT-PCR and real- time PCR. Hepcidins genes-expressing cells in liver Some liver cells expressed both Hep-JF1 and Hep-JF2 or only one of them (Fig. 6). Figures 6(A–C) indicated the Hep-JF1 negative ⁄ Hep-JF2 positive cell, Hep-JF1 positive ⁄ Hep-JF2 negative cell and both positive cells, respectively. Antimicrobial and hemolytic activity of hepcidin Both Hep-JF2pep and Hepc20 have antimicrobial activ- ities against Gram-negative Escherichia coli, and Gram- positive Staphylococcus aureus, Latococcus garvieae, and Streptococcus iniae (Fig. 7). Hep-JF2pep has anti- microbial activity against Gram-negative Pasteurella damselae ssp. piscicida although Hepc20 did not show any activity (Fig. 7). As far as this study, Hep-JF1pep did not have antimicrobial activity against any of the bacteria. None of the three hepcidin peptides showed antimicrobial activity against Gram-negative Edward- siella tarda. Over 90% killing of E. coli, P. damselae ssp. piscicida, S. aureus, L. garvieae, and S. iniae were achieved with 25, 50, 50, 100 and 12.5 lm concentra- tions of Hep-JF2pep, respectively (Fig. 7). Similarly, over 90% killing of E. coli and S. aureus were also achieved with 25 and 100 lm concentrations of Hepc20, respectively (Fig. 7). Over 50% killing of L. garvieae, and S. iniae were achieved with 50 and 100 lm concen- trations of Hepc20, respectively (Fig. 7). No hemolysis against the Japanese flounder and rab- bit red blood cells was detected at any of the concen- trations of synthesized hepcidins. Fig. 3. Detection of Japanese flounder hepcidin mRNAs by RT-PCR. Lanes 1, brain; 2, eye; 3, gill; 4, head kidney; 5, heart; 6, intestine; 7, liver; 8, muscle; 9, ovary; 10, PBLs; 11, skin; 12, spleen; 13, stomach; 14, post kidney. M, size marker (100 bp ladder). Fig. 4. Effect of iron overloading and LPS on the expressions of hepcidin mRNAs. RNA was isolated from liver and kidney of five fish (average weight 2 g) at 1 (1w), 2 (2w) and 3 weeks (3w) after iron-dextran or NaCl ⁄ P i (control) injection. Five fish were killed after 3 (3 h), 6 (6 h) and 24 h (24 h) of lipopolysaccharide (LPS) injection. One microgram of RNA was purified from each of five fish and the five samples were pooled for PCR. The left lane is a size marker (100 bp ladder). I. Hirono et al. Hepcidins of Japanese flounder FEBS Journal 272 (2005) 5257–5264 ª 2005 FEBS 5259 Discussion The mature forms of human hepcidin [13] and bass hep- cidin [12] have eight cysteine residues. A previous study indicated that all eight cystein residues formed intramo- lecular bonds. Types I and III hepcidin of winter floun- der also have eight cysteine residues, while type II has six [11]. In the latter type, the first cysteine residue is deleted and the third cysteine residue is replaced with a glycine residue. The number and positions of cysteine residues in Hep-JF2 of Japanese flounder are the same as those of human hepcidin, while the number and posi- tions of cysteine residues in Hep-JF1 are the same as those in type II hepcidin of winter flounder. Both genes exist as single copies in the Japanese flounder genome. The structures, and therefore the functions, of Hep-JF1 and type II hepcidin of winter flounder might be differ- ent from those of human and bass hepcidins. Thus, Hep-JF2 might have the same biological function as human hepcidin, while Hep-JF1 might have a different function or be nonfunctional. Fig. 5. Amounts of Hep-JF1 and Hep-JF2 transcripts in liver and kidney after treat- ment with iron overloading or LPS by real- time PCR analysis. Y axis indicate the copy number of mRNAs. Abbreviations are the same as these in Fig. 4. A B C Fig. 6. Detection of Hep-JF1- and Hep-JF2-expressing cells in liver by in situ hybridization. (A) Hep-JF1 negative ⁄ Hep-JF2 positive cell; (B) Hep-JF1 positive ⁄ Hep-JF2 negative cell; (C) both positive cells. Hepcidins of Japanese flounder I. Hirono et al. 5260 FEBS Journal 272 (2005) 5257–5264 ª 2005 FEBS The human and mouse hepcidin genes are predomin- antly expressed in the liver [1,2]. Expression of mam- malian hepcidins is induced by interleukin-1, interleukin-6, LPS and iron overloading [6–8]. Mouse has two distinct hepcidins. Both genes are predomin- antly expressed in the liver but one is also strongly expressed in the pancreas. LPS induces the expression of type 1 mouse hepcidin but not type 2 [14]. In winter flounder, type I hepcidin is expressed in the liver and cardiac stomach, type II is not expressed in any organs, and type III is expressed in the liver, cardiac stomach and esophagus [11]. Using RT-PCR, we detected Hep-JF1 expression in the liver but not in any of the other organs and tissues examined in this study (Fig. 3). This expression pattern is similar to that of human and mouse hepcidins. It is also similar to that of type I hepcidin of winter flounder even though the amino acid sequence and number and position of cys- teine residues of Hep-JF1 are similar to those of type II winter flounder hepcidin. The gene expression of Japanese flounder Hep-JF2 was detected in several organs including gill, liver, heart, kidney, peripheral blood leucocytes, spleen and stomach (Fig. 3). This expression pattern is different from that of human, mouse and other reported fish hepcidins. In liver, some cells expressed both types of hepcidins, while other cells expressed only one of them or neither of them (Fig. 6). These results indicate that the regulation of gene expression of fish hepcidins is highly diverged in different species. Interestingly, iron (iron-dextran) overloading decreased the gene expression of Hep-JF1 in liver by more than 99% (Fig. 5). In contrast, up- regulation of the gene expression of Hep-JF2 in liver was observed (Fig. 5). Iron overloading did increase the gene expression of Hep-JF2 in kidney. Iron over- loading also induces hepcidin gene expression in mouse liver [6]. In mouse, hepcidin is thought to act as a regulator of iron stores [15]. It is not clear whether Fig. 7. Dose-dependent antibacterial activity of synthesized hepcidins. Open diamonds, Hep-JF1; open box, Hep-JF2; open triangle, Hepc20. I. Hirono et al. Hepcidins of Japanese flounder FEBS Journal 272 (2005) 5257–5264 ª 2005 FEBS 5261 Japanese flounder hepcidins have a similar function. The expression of Hep-JF1 in liver is strongly induced (> 10 000-fold) by LPS (Fig. 5). The expressions of Hep-JF2 in liver and kidney are also induced (10-fold and > 500-fold) by LPS (Fig. 5). The effect of LPS on the expressions of Hep-JF1 and Hep-JF2 is similar to its effect on the expression of mouse type 1 hepcidin. These results raise the possibility that the Japanese flounder hepcidins is involved in the innate immune system same as they are in mammals. However, at pre- sent, whether Japanese flounder hepcidins have a role in iron metabolism is not clear. Using synthetic peptides, we examined the antibacte- rial activities of Japanese flounder hepcidins Hep-JF1- pep and -JF2pep and human hepcidin Hepc20 [2]. The antimicrobial activity of synthesized human hepcidin Hepc20 was similar to that of a previous report [2]. This result suggests that all these synthesized peptide are functional of antimicrobial activity. Hep-JF2pep and Hepc20 were active against a broad spectrum of Gram-positive and Gram-negative bacteria (Fig. 7). Synthetic bass hepcidin was found to have antibacte- rial activity against only Gram-negative bacteria [12]. In our study, Hep-JF2pep had antibacterial activity against P. damselae ssp. piscicida but Hepc20 did not show any activity (Fig. 7). In addition, antimicrobial activity of Hep-JF2pep was higher than that of Hepc20 (Fig. 7). These results suggest that fish hepci- din has higher anitimicrobial activity against some fish pathogenic bacteria compared to that of human. Hep- JF1pep has no antibacterial activity against the bac- teria used in this study (Fig. 7). However, it is possible that the negative antimicrobial activity of Hep-JF1 is due to incorrect folding although the other two syn- thesized hepcidin peptides have antimicrobial activi- tites, because we did not confirm the structure of synthesized peptides. These results suggest that at least Hep-JF2 acts as an antibacterial agent in Japanese flounder. Together, the results in this study suggest that Hep- JF1 and -JF2 are involved in the Japanese flounder immune system and iron-metabolism. Further studies are needed to understand why the gene expression pat- terns and different antibacterial activities of Hep-JF1 are different from those of Hep-JF2. Experimental procedures Cloning of hepcidin genes from Japanese flounder We found two different partial cDNA sequences, LA6(10) (GenBank accession number C23432) and LC4(7) (Gen- Bank accession number C23298) that had identities to human hepcidin by our previous EST analysis [16]. In this study, we used these cDNA clones as DNA probes to screen a Japanese flounder liver cDNA library in E. coli XL-1 Blue [16] for the full length of Japanese flounder hepcidin cDNAs. cDNA clones were sequenced using ThermoSequenase (Amersham-Pharmacia, Piscata- way, NJ, USA) with M13 forward and ⁄ or M13 reverse primers and an automated DNA sequencer LC4200 (Li-Cor, Licol, NE, USA). Each determined sequence was compared with all sequences available in DDBJ ⁄ EMBL ⁄ GenBank using the blast program ver.2.0 (http://www. ncbi.nlm.nih.gov). The arrayed genomic BAC clones of Japanese flounder [17] were screened by using the Japanese flounder hepcidin cDNAs as DNA probes. The probes were hybridized as previously reported [18]. The genomic clones were sequenced as described above. Southern blot hybridization Genomic DNA of homocloned Japanese flounder [19] was isolated and digested with EcoRI and PstI as previously reported [17]. The probes were the full lengths of Japanese flounder hepcidin cDNA fragments and were labeled with [ 32 P]dCTP[a P] using a random primer labeling kit (Takara, Kyoto, Japan). Southern blot hybridizations were carried out as described previously [18]. Detection of transcript of hepcidin genes Japanese flounder peripheral blood leukocytes (PBLs) were isolated as reported previously [18]. Total RNA was extrac- ted from healthy Japanese flounder brain, eye, gill, head kidney, heart, intestine, liver, muscle, ovary, PBLs, skin, spleen, stomach, and trunk kidney using TRIZOL (Life Technologies, Rockville, MD, USA). All measures were taken to minimize pain and discomfort of animals. The procedures were conducted in accordance with the guide- lines of Tokyo University of Marine Science and Tech- nology. The purified total RNA (10 lg) was reverse transcribed into cDNA using the AMV reverse transcrip- tase first-strand cDNA synthesis kit (Life Sciences, St Petersburg, FL, USA). The final volume of the cDNA syn- thesis reaction was 25 lL. The reverse-transcribed sample (1 lL) was used in 50 lL of PCR reaction mixture. The PCR primers of Japanese flounder hepcidins and b-actin used in this study are listed in Table 1. The b-actin primer set was used for a positive control of RT-PCR [20]. PCR was performed with an initial denaturation step of 2 min at 95 °C, and then 25 cycles were run as follows: 30 s of dena- turation at 95 °C, 30 s of annealing at 55 °C, and 30 s of extension at 72 ° C. The reacted products were electrophore- sed on a 2.0% agarose gel. Hepcidins of Japanese flounder I. Hirono et al. 5262 FEBS Journal 272 (2005) 5257–5264 ª 2005 FEBS Iron overloading and LPS treatment Fish were given a single injection of iron-dextran (Sigma, St Louis, MO, USA) corresponding to a dose of 1 mgÆg )1 body weight. Other fish were given a single injection of NaCl ⁄ P i for use as a negative control. Five fish (average weight: 2 g) were killed after anesthesia at 1, 2 and 3 weeks after iron or NaCl ⁄ P i injection. LPS from Escherichia coli 0111:B4 (Sigma) was injected intraperitoneally at 0.5 lgÆg )1 body weight. Five fish (average weight: 2 g) were killed after 3, 6 and 24 h post application. One microgram of RNA was purified from each of five fish and the five sam- ples were pooled for PCR. The procedures were conducted in accordance with the guidelines of Tokyo University of Marine Science and Technology. Real-time PCR Total RNA was extracted from livers and kidney of five Japanese flounder that had been injected with iron-dextran or LPS. cDNAs were synthesized as indicated above. The real-time PCR assay was carried out with the SYBR Green PCR master mix (PE Biosystems, Norwalk, CT, USA) fol- lowing a previous report [21]. The primers used for the quantitative real-time PCR analysis are listed in Table 1. The results of real-time PCR were normalized with the copy number of b-actin of each sample. In situ hybridization Digoxigenin-labeled sense and antisense RNA probes were generated with a T7 and Sp6 Dig RNA labeling kit (Boeh- ringer Mannheim, Germany) with digoxigenin-UTP and Biotin-labeled sense and antisense RNA probes, generated with a T7 and SP6 biotin RNA labeling kit (Boehringer Mannheim). In situ hybridization was carried out using a commercial kit (Nippon Gene, Tokyo, Japan) with a fluor- escence method [22]. Peptide synthesis Human hepcidin (Hepc20) [2] and Japanese flounder hepci- dins (Hep-JF1pep and Hep-JF2pep) were chemically synthes- ized by Genenet Corporation (Fukuoka, Japan). The amino acid sequences of synthesized Hepc20, Hep-JF1pep and Hep-JF2pep were DTHFPICIFCCGCCHRSKCGMCCKT, DVKCGFCCKDGGCGVCCNF and HISHISMCRWCC NCCKAKGCGPCCKF, respectively. The sequences of Hep-JF1pep and Hep-JF2pep were putative mature region of peptides which were predicted based on a comparison with amino acid sequences previously reported mammalian hepci- dins. The synthetic peptides were suspended in a buffer (6 m guanidine ⁄ HCl, 20 mm EDTA, and 0.5 m Tris ⁄ HCl pH 8.0) to a final concentration of 0.5 mgÆmL )1 (w ⁄ v) and subse- quently incubated in a 50 °C sonicating water bath for 30 min. The peptides were reduced by adding dithiothreitol to a final concentration of 10 mm, overlaying with N 2 gas, and then incubating in a 50 °C water bath overnight. The reduced peptides were loaded into a C18 September-Pak cartridge (Waters, Milford, MA, USA) equilibrated with 0.1% (v⁄ v) trifluoroacetic acid (TFA) in milliQ water, washed with 15% acetonitrile and 0.1% (v ⁄ v) TFA in milliQ water, and eluted with 40% acetonitrile and 0.1% (v ⁄ v) TFA in milliQ water. Bacterial killing assay E. coli (strain JC-2) and S. aureus (strain JC-1; both National Institute of Health, Tokyo, Japan) were grown in heart infusion broth (HIB) at 37 °C. E. tarda (strain MZ8901 isolated from Japanese flounder in Japan) was grown in HIB at 25 ° C. P. damselae ssp. piscicida (strain P97-008 isolated from yellowtail Seriola quinqueradiata in Japan) was grown in HIB containing 2% (w ⁄ v) NaCl (2HIB) at 25 °C. S. iniae (strain TUMST1 isolated from Japanese flounder in Japan) and L. garvieae (strain SA8201 isolated from yellowtail S. quinqueradiata in Japan) were grown in Todd–Hewitt broth at 25 °C. These bacteria were collected in the mid-log phase and washed in 10 mm sodium phosphate, pH 7.4, supplemented with 0.03% of the appropriate medium. Various concentrations of hep- cidin peptides (0.1–100 lm) with 100 lLof10 5 )10 6 bac- teriaÆmL )1 were incubated at the appropriate temperature for each bacterium. After 3 h incubation, 10-fold dilutions were prepared and plated on the appropriate agar plates and incubated for 24 h. Hemolytic activity of synthesized Japanese floun- der hepcidins The hemolytic activity of the synthetic Japanese flounder hepcidins was measured by using microtiter plate twofold dilution method [23]. The synthetic peptides were serially diluted twofold (50–0.1 lm) in a 96 microtiter plate. Rabbit Table 1. PCR primers used in this study. Gene Primer Sequence Purpose Hep-JF1 Hep-JF1-F1 5¢-gaaggcattcagcattgcag-3¢ RT-PCR Hep-JF1-R1 5¢-ttgaggttgttgcgggaatc-3¢ Hep-JF1-F2 5¢-ctcgcctttgtttgcattcagg-3¢ Real-time PCR Hep-JF1-R2 5¢-tgcctgacgggactctccatcc-3¢ Hep-JF2 Hep-JF2-F1 5¢-tgacaagagtcaccagcaga-3¢ RT-PCR Hep-JF2-R1 5¢-cctccagctcttgtacctca-3 ¢ Hep-JF2-F2 5¢-tcaaagatgaagacattcagtgc-3¢ Real-time PCR Hep-JF2-R2 5¢-agagctctgctggatgcaaa-3¢ b-Actin Jfactin-F 5¢-tttccctccattgttggtcg-3¢ RT-PCR, real-time PCR Jfactin-R 5¢-ggtttaaagtctcaaagtgc-3¢ I. Hirono et al. Hepcidins of Japanese flounder FEBS Journal 272 (2005) 5257–5264 ª 2005 FEBS 5263 or Japanese flounder red blood cells were added to each well (final concentration 2%). Samples were incubated at 37 or 25 °C for 12 h, respectively. After incubation, the plate was centrifuged and hemolytic activity was observed. 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Hepcidins of Japanese flounder I. Hirono et al. 5264 FEBS Journal 272 (2005) 5257–5264 ª 2005 FEBS . is different from that of human, mouse and other reported fish hepcidins. In liver, some cells expressed both types of hepcidins, while other cells expressed only one of them or neither of them (Fig winter flounder, Pseudopleuronectes americanus has three different types of hepcidin [11]. The gene expressions of three types of hepcidins in dif- ferent organs and tissues were different. From the results. Two different types of hepcidins from the Japanese flounder Paralichthys olivaceus Ikuo Hirono, Jee-Youn Hwang, Yasuhito Ono, Tomofumi Kurobe, Tsuyoshi Ohira, Reiko