Involvement of Estrogen and Its Receptors in Morphological Changes in the Eyes of the Japanese Eel, Anguilla japonica, in the Process of Artificially-Induced Maturation.

During the long migration from river habitats to the spawning ground, the Japanese eel undergoes sexual maturation. This spawning migration occurs concurrently with morphological changes, such as increases in eye size; however, the mechanisms by which sex steroids and their receptors influence these changes in peripheral tissues remain unclear. The aim of this study was to investigate changes in the eyes of female Japanese eels during sexual maturation, and our research focused on estrogen receptor (ER)α and ERß transcripts. During ovarian development, the gonadosomatic index increased and yolk-laden oocytes developed rapidly. These changes occurred in conjunction with a steady increase in plasma levels of estradiol-17ß (E2). Concomitant increases in transcript levels of ERα and ERß in eye, brain, pituitary, and ovary were also observed. Fluorescence in-situ hybridization analyses revealed that ERα and ERß transcripts were present in the choriocapillary layer and photoreceptor layer of the eyes, and the analysis also revealed that their signals in these layers became stronger in mature females compared to those observed in immature females, suggesting that under the influence of gonadotropins, morphological changes in the eyes are regulated by E2 through the activation of its receptors. In conclusion, E2 plays a crucial role in physiological adaptations that occur in peripheral tissues during the spawning migration.

Identification and molecular characterization of peroxiredoxin 6 from Japanese eel (Anguilla japonica) revealing its potent antioxidant properties and putative immune relevancy.

Peroxiredoxins (Prdx) are thiol specific antioxidant enzymes that play a pivotal role in cellular oxidative stress by reducing toxic peroxide compounds into nontoxic products. In this study, we identified and characterized a peroxiredoxin 6 counterpart from Japanese eel (Anguilla japonica) (AjPrdx6) at molecular, transcriptional and protein level. The identified full-length coding sequence of AjPrdx6 (669 bp) coded for a polypeptide of 223 aa residues (24.9 kDa). Deduced protein of AjPrdx6 showed analogy to characteristic structural features of 1-cysteine peroxiredoxin sub-family. According to the topology of the generated phylogenetic reconstruction AjPrdx6 showed closest evolutionary relationship with Salmo salar. As detected by Quantitative real time PCR (qPCR), AjPrdx6 mRNA was constitutively expressed in all the tissues examined. Upon the immune challenges with Edwardsiella tarda, lipopolysaccharides and polyinosinic:polycytidylic acid, expression of AjPrdx6 mRNA transcripts were significantly induced. The general functional properties of Prdx6 were confirmed using purified recombinant AjPrdx6 protein by deciphering its potent protective effects on cultured vero cells (kidney epithelial cell from an African green monkey) against H2O2-induced oxidative stress and protection against oxidative DNA damage elicited by mixed function oxidative (MFO) system. Altogether, our findings suggest that AjPrdx6 is a potent antioxidant protein in Japanese eels and its putative immune relevancy in pathogen stress mounted by live-bacteria or pathogen associated molecular patterns (PAMPs).

A CXC chemokine gene, CXCL12, from rock bream, Oplegnathus fasciatus: Molecular characterization and transcriptional profile.

Chemokines are small, structurally related chemotactic cytokines characterized by the presence of conserved cysteine residues. In the present study, we identified the cDNA of a CXC chemokine from Oplegnathus fasciatus, designated as OfCXCL12. An open reading frame of 297 bp encoded a 98 amino acid peptide with a putative signal peptide of 23 amino acids. The CXC family-specific small cytokine domain (SCY), which is highly conserved among vertebrates, was located between residues 29 and 87. The characteristic conserved cysteine residues in the CXC motif of OfCXCL12 were separated by tyrosine (Y). Similar to other vertebrate CXCL12 proteins, OfCXCL12 also lacked the ELR motif and hence belongs to ELR(-) subfamily. Phylogenetic analysis revealed two distinct clades, consisting of fish and tetrapod CXCL12 homologs. Constitutive expression with significantly higher levels of OfCXCL12 mRNA transcription was detected in immune-related organs, including the head kidney, spleen, and kidney. Infection with bacterial and viral agents led to significant upregulation of mRNA expression in both the head kidney and spleen, in a stimulant-specific manner. Stimulation of peripheral blood leukocytes by the mitogen concanavalin-A significantly induced OfCXCL12 transcription. Results from the present study suggest an important role for OfCXCL12 in immune defense against bacterial and viral infection in rock bream.

Two carboxypeptidase counterparts from rock bream (Oplegnathus fasciatus): molecular characterization, genomic arrangement and immune responses upon pathogenic stresses.

Carboxypeptidases (CPs) are proteases that hydrolyze C-terminal peptide bonds. They are involved in regulating the complement system of the immune system. Here, we report the molecular characterization and immune response of two carboxypeptidases, named carboxypeptidase A (Rb-CPA) and carboxypeptidase N1 (Rb-CPN1), from rock bream. The genomic sequence of Rb-CPA contains 12 exons interrupted by 11 introns, while the genomic sequence of Rb-CPN1 has 9 exons and 8 introns. The cDNA sequence of Rb-CPA encodes a 421-amino-acid (AA) polypeptide (48kDa), and the cDNA of Rb-CPN1 encodes a 448-AA polypeptide (51kDa). The amino acid sequences of Rb-CPA and Rb-CPN1 were found to harbor two characteristic Zn-binding signature domains and a peptidase-M14 Zn carboxypeptidase site. Pairwise analysis revealed that Rb-CPA and Rb-CPN1 had the highest identity with the corresponding proteins from Anoplopoma fimbria (87.6%) and Dicentrarchus labrax (96.9%), respectively. qPCR results indicated that Rb-CPA and Rb-CPN1 were constitutively expressed mainly in the kidney, heart, liver, and head kidney. Both genes were transcriptionally regulated in the liver upon challenge with pathogenic bacteria (Streptococcus iniae, Edwardsiella tarda), rock bream iridovirus (RBIV), and the immune modulators polyinosinic:polycytidylic acid and lipopolysaccharide. Taken together, our findings suggest that Rb-CPA and Rb-CPN1 have immune-related functions in rock bream.

Molecular characterization and transcriptional analysis of non-mammalian type Toll like receptor (TLR21) from rock bream (Oplegnathus fasciatus).

Toll-like receptors (TLRs) are a large family of pattern recognition receptors, which are involved in triggering host immune responses against various pathogens by detecting their evolutionarily conserved pathogen associated molecular patterns (PAMPs). TLR21 is a non-mammalian type TLR, which recognizes unmethylated CpG DNA, and is considered as a functional homolog of mammalian TLR9. In this study, we attempted to identify and characterize a novel TLR21 counterpart from rock bream (Oplegnathus fasciatus) designated as RbTLR21, at molecular level. The complete coding sequence of RbTLR21 was 2919bp in length, which encodes a polypeptide of 973 amino acids with a predicted molecular mass of 112kDa and a theoretical isoelectric point of 8.6. The structure of the deduced RbTLR21 protein is similar to that of the members of typical TLR family, and includes the ectodomain, which consists of 16 leucine rich repeats (LRRs), a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain. According to the pairwise sequence analysis data, RbTLR21 was homologous to that of the orange-spotted grouper (Epinephelus coioides) with 76.9% amino acid identity. Furthermore, our phylogenetic analysis revealed that RbTLR21 is closely related to E. coioides TLR21. The RbTLR21 was ubiquitously expressed in all the tissues tested, but the highest expression was found in spleen. Additionally, upon stimulation with Streptococcus iniae, rock bream iridovirus (RBIV), and Edwardsiella tarda, RbTLR21 mRNA was significantly up-regulated in spleen tissues. Collectively, our findings suggest that RbTLR21 is indeed an ortholog of the TLR21 family and may be important in mounting host immune responses against pathogenic infections.

Antibacterial activity and immune responses of a molluscan macrophage expressed gene-1 from disk abalone, Haliotis discus discus.

The membrane-attack complex/perforin (MACPF) domain-containing proteins play an important role in the innate immune response against invading microbial pathogens. In the current study, a member of the MACPF domain-containing proteins, macrophage expressed gene-1 (MPEG1) encoding 730 amino acids with the theoretical molecular mass of 79.6 kDa and an isoelectric point (pI) of 6.49 was characterized from disk abalone Haliotis discus discus (AbMPEG1). We found that the characteristic MACPF domain (Val(131)-Tyr(348)) and transmembrane segment (Ala(669)-Ile(691)) of AbMPEG1 are located in the N- and C-terminal ends of the protein, respectively. Ortholog comparison revealed that AbMPEG1 has the highest sequence identity with its pink abalone counterpart, while sequences identities of greater than 90% were observed with MPEG1 members from other abalone species. Likewise, the furin cleavage site KRRRK was highly conserved in all abalone species, but not in other species investigated. We identified an intron-less genomic sequence within disk abalone AbMPEG1, which was similar to other mammalian, avian, and reptilian counterparts. Transcription factor binding sites, which are important for immune responses, were identified in the 5'-flanking region of AbMPEG1. qPCR revealed AbMPEG1 transcripts are present in every tissues examined, with the highest expression level occurring in mantle tissue. Significant up-regulation of AbMPEG1 transcript levels was observed in hemocytes and gill tissues following challenges with pathogens (Vibrio parahemolyticus, Listeria monocytogenes and viral hemorrhagic septicemia virus) as well as pathogen-associated molecular patterns (PAMPs: lipopolysaccharides and poly I:C immunostimulant). Finally, the antibacterial activity of the MACPF domain was characterized against Gram-negative and -positive bacteria using a recombinant peptide. Taken together, these results indicate that the biological significance of the AbMPEG1 gene includes a role in protecting disk abalone through the ability of AbMPEG1 to initiate an innate immune response upon pathogen invasion.

Genomic characterization of interferon regulatory factor 5 from rock bream (Oplegnathus fasciatus) and its role in antiviral defense.

The interferon regulatory factor 5 (IRF5) is a key mediator of the Toll-like receptor (TLR)7 and TLR8 signaling pathways. In this study, we describe the identification of IRF5 (Rb-IRF5) from rock bream fish (Oplegnathus fasciatus) and its characteristics features at the genomic and expression levels. The full-length Rb-IRF5 sequence was identified from a cDNA library and its genomic sequence was obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The genomic sequence is comprised of 8 exons interrupted by 7 introns. The complete coding sequence of Rb-IRF5 is 1497 bp in length and encodes for 498 amino acids. The putative Rb-IRF5 protein consists of 3 important conserved domains: a DNA-binding domain (DBD) at the N-terminus, an IRF-associated domain (IAD), and a virus-activated domain (VAD) at the C-terminus. Based on pairwise sequence analysis, the highest sequence similarity/identity for Rb-IRF5 was observed with the IRF5 gene from turbot fish (>87%) and Japanese flounder (83%). Several important putative transcription factor-binding sites shared by the IRF gene family, including the NF-κB, Ap-1, IRF-1, and ICSBP/ISRE sites, were found in the 5' flanking region of Rb-IRF5. The predicted tertiary structure of the dimerized IAD and VAD of the Rb-IRF5 protein resembled that of its orthologs from humans. In healthy rock bream, the highest constitutive expression of Rb-IRF5 was detected in the liver. After iridovirus and polyinosinic-polycytidylic acid (poly(I:C)) challenge, Rb-IRF5 expression was significantly induced in the head kidney. Furthermore, rock bream recombinant type I interferon (Rb-IFN1) was also found to be an efficient inducer of Rb-IRF5 in a head kidney primary cell culture model. Upon IRF5 transfection, rock bream Mx (Rb-Mx), interferon I (Rb-IFN1) and tumor-necrosis factor α (Rb-TNFα) genes get significantly upregulated in rock bream heart cells. The findings of the present study explain the involvement of Rb-IRF5 in the induction of interferons and pro-inflammatory cytokines and thereby provide a model for how IRF5 modulates immune responses against viral infections in rock bream.

Three complement component 1q genes from rock bream, Oplegnathus fasciatus: genome characterization and potential role in immune response against bacterial and viral infections.

Complement component 1q (C1q) is a subcomponent of the C1 complex and the key protein that recognizes and binds to a broad range of immune and non-immune ligands to initiate the classical complement pathway. In the present study, we identified and characterized three novel C1q family members from rock bream, Oplegnathus fasciatus. The full-length cDNAs of C1q A-like (RbC1qAL), C1q B-like (RbC1qBL), and C1q C-like (RbC1qCL) consist of 780, 720 and 726 bp of nucleotide sequence encoding polypeptides of 260, 240 and 242 amino acids, respectively. All three RbC1qs possess a leading signal peptide and collagen-like region(s) (CLRs) in the N-terminus, and a C1q domain at the C-terminus. The C1q characteristic Gly-X-Y repeats are present in all three RbC1qs, while the CLR-associated sequence that enhances phagocytic activity is present in RbC1qAL ((49)GEKGEP(54)) and RbC1qCL ((70)GEKGEP(75)). Moreover, the coding region was distributed across six exons in RbCqAL and RbC1qCL, but only five exons in RbC1qBL. Phylogenetic analysis revealed that the three RbC1qs tightly cluster with the fish clade. All three RbC1qs are most highly expressed in the spleen and liver, as indicated by qPCR tissue profiling. In addition, all three are transcriptionally responsive to immune challenge, with liver expression being significantly up-regulated in the early phase of infection with intact, live bacteria (Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus) and in the late phase of exposure to purified endotoxin (lipopolysaccharide). These data collectively suggest that the RbC1qs may play defense roles as an innate immune response to protect the rock bream from bacterial and viral infections.

Evidences for the involvement of an invertebrate goose-type lysozyme in disk abalone immunity: cloning, expression analysis and antimicrobial activity.

Lysozymes are ubiquitously distributed enzymes with hydrolytic activity against bacterial peptidoglycan and function to protect organisms from microbial pathogens. In this study, an invertebrate goose-type lysozyme, designated as abLysG, was identified in the disk abalone, Haliotis discus discus. The full-length cDNA of abLysG was 894 bp in length with an open reading frame of 789 bp encoding a polypeptide of 263 amino acids containing a signal peptide and a characteristic soluble lytic transglycosylase domain. Six cysteine residues and two catalytic residues (Glu(142) and Asp(168)) conserved among molluscs were also identified. The 3D homology structural models of abLysG and hen egg white lysozyme had similar conformations of the active sites involved in the binding of substrate. BAC sequence data revealed that the genomic structure of disk abalone g-type lysozyme comprises 7 exons with 6 intervening introns. The deduced amino acid sequence of abLysG shared 45.2-61.6% similarity with those of other molluscs and vertebrates. The TFSEARCH server predicted a variety of transcription factor-binding sites in the 5'-flanking region of the abLysG gene, some of which are involved in transcriptional regulation of the lysozyme gene. abLysG expression was detected in multiple tissues with the highest expression in mantle. Moreover, qPCR analysis of abLysG mRNA expression demonstrated significant up-regulation in gill in response to infection by live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia) and bacterial mimics (LPS and PGN). Expression of the recombinant disk abalone g-type lysozyme in Escherichia coli BL21, demonstrated its bacteriolytic activity against several Gram-negative and Gram-positive bacterial species. Collectively these data suggest that abLysG is an antimicrobial enzyme with a potential role in the disk abalone innate immune system to protect it from bacterial and viral infections.

A bifunctional invertebrate-type lysozyme from the disk abalone, Haliotis discus discus: genome organization, transcriptional profiling and biological activities of recombinant protein.

Lysozyme is an important enzyme in the innate immune system that plays a vital role in fighting microbial infections. In the current study, we identified, cloned, and characterized a gene that encodes an invertebrate-type lysozyme from the disk abalone, Haliotis discus discus (abLysI). The full-length cDNA of abLysI consisted of 545 bp with an open reading frame of 393 bp that encodes 131 amino acids. The theoretical molecular mass of mature abLysI was 12.3 kDa with an isoelectric point of 8.03. Conserved features in other homologs, such as catalytic sites for lytic activity (Glu(30) and Asp(41)), isopeptidase activity (His(107)), and ten cysteine residues were identified in abLysI. Genomic sequence analysis with respect to its cDNA showed that abLysI was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative promoter region. Homology and phylogeny analysis of abLysI depicted high identity and closer proximity, respectively, with an annelid i-type lysozyme from Hirudo medicinalis, and indicated that abLysI is a novel molluscan i-type lysozyme. Tissue-specific expressional studies revealed that abLysI is mainly transcribed in hepatopancreas followed by mantle. In addition, abLysI mRNA expression was induced following bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and viral (viral hemorrhagic septicemia virus) challenges. Recombinantly expressed abLysI [(r)abLysI] demonstrated strong lytic activity against Micrococcus lysodeikticus, isopeptidase activity, and antibacterial activity against several Gram-positive and Gram-negative bacteria. Moreover, (r)abLysI showed optimum lytic activity at pH 4.0 and 60 °C, while exhibiting optimum isopeptidase activity at pH 7.0. Taken together, these results indicate that abLysI is potentially involved in immune responses of the disk abalone to protect it from invaders.

Characterization of MIF family proteins: MIF and DDT from rock bream, Oplegnathus fasciatus.

Macrophage migration inhibitory factor (MIF) is a pleiotropic molecule playing vital roles in various signaling cascades, including cell proliferation, and activation of immune responses against infections. It is well known as a pivotal regulator of innate immunity. In this study, we have rescued and characterized two members of the MIF family, macrophage migration inhibitory factor (OfMIF) and D-Dopachrome tautomerase (OfDDT) from rock bream, Oplegnathus fasciatus. The deduced OfMIF and OfDDT protein sequences revealed the presence of the catalytic oxidoreductase (CXXC), motif. They also possessed highly conserved proline (P(2)) and lysine residues (K(33)), responsible for their isomerase and tautomerase functions. Rock bream MIF and DDT homologues shared higher identity with fish homologues and also with mammals and occupied a distinct position in the phylogenetic tree, depicting their evolutionary conservation. The spatial expression analysis revealed the highest expression of both OfMIF and OfDDT in liver, while portraying constitutive expression in other tissues. The recombinant proteins purified using the Escherichia coli system revealed potent oxidoreductase activity against insulin with both dithiothreitol and glutathione as reducing agents. Stimulation of rock bream head kidney cells with recombinant OfMIF and OfDDT proteins induced the expression of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8) and interleukin-1ß (IL-1ß). These results together suggest their involvement in rock bream immune defense and this study on the novel MIF family member DDT from rock bream will pave the way for further studies of this homologue in other teleosts and delineate its multiple functions.

Genomic characterization and transcriptional evidence for the involvement of complement component 7 in immune response of rock bream (Oplegnathus fasciatus).

The complement component 7 (C7) is the central mediator of pathogenic attack at the membrane surface and its binding to the C5b-7 complex triggers cytolytic signaling. In this study, C7 of rock bream (Oplegnathus fasciatus) was identified (Rb-C7) and characterized at the genomic level. The Rb-C7 gene contains 18 exons and 17 introns and is composed of a 2490 bp complete open reading frame (ORF). The encoded polypeptide (830 amino acids) contains a number of well-conserved C7 signature domains. Important putative transcription factor binding sites, including those for NF-κB, SP-1, C/EBP, AP-1 and OCT-1, are present in the 5'-flanking region of Rb-C7. Phylogenetic analysis revealed a close proximity of Rb-C7 with the orthologues in tilapia and Japanese flounder. Quantitative real-time PCR (qPCR) analysis confirmed constitutive Rb-C7 expression throughout all the examined tissue of healthy rock bream, with highest expression in liver. In immune challenge experiment, Rb-C7 expression was up-regulated in head kidney and liver in response to Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide and rock bream iridovirus (RBIV). Furthermore, significant increases of both intracellular expression level and the number of Rb-C7-expressing cells were detected by in situ hybridization assay in head kidney and liver tissues upon E. tarda infection. These results suggested that Rb-C7 is lytic pathway gene in complement system and its transcriptional regulation may be an important immune response in pathogenic defense mechanism of rock bream.

Expression profile of cystatin B ortholog from Manila clam (Ruditapes philippinarum) in host pathology with respect to its structural and functional properties.

Cystatins are a well-characterized group of cysteine protease inhibitors, which play crucial roles in physiology and immunity. In the present study, an invertebrate ortholog of cystatin B was identified in Manila clam (Ruditapes philippinarum) (RpCytB) and characterized at the molecular level, demonstrating its inhibitory activity against the well-known cysteine protease, papain. The complete coding sequence of RpCytB (297 bp in length) encodes a 99 amino acid peptide with a calculated molecular mass of 11 kDa and a theoretical isoelectric point of 5.9. The derived peptide was found to harbor typical features of cystatin proteins, including the 'Q-X-V-X-G' motif, which was identified as QLVAG in RpCytB. Phylogenetic analysis of RpCytB revealed close evolutionary relationships with its invertebrate counterparts, especially those from mollusks. Recombinant RpCytB (rRpCytB) was overexpressed as a fusion with maltose binding protein (MBP) in Escherichia coli BL21 (DE3) cells. Purified rRpCytB fusion protein exhibited a detectable inhibitory activity against papain, while the control MBP showed an almost constant negligible activity. While quantitative RT-PCR detected ubiquitous RpCytB expression in all tissues examined, the expressions in hemocytes and gills were relatively higher. Upon in vivo immune challenge with lipopolysaccharide (LPS), the expression of RpCytB in gills and hemocytes was down-regulated. Similar challenges with poly I:C and intact Vibrio tapetis bacteria revealed a complicated transcriptional regulation, wherein mRNA expression levels fluctuated over time of exposure. Moreover, a precise induction of RpCytB expression after bacterial infection was detected in gills by in situ hybridization. Collectively, our findings in this study indicate that RpCytB expression is sensitive to host pathological conditions and may contribute cysteine protease inhibitory activity to modulate the immune response.

Genomic characterization and expression analysis of complement component 8α and 8ß in rock bream (Oplegnathus fasciatus).

The complement component 8α and 8ß are glycoproteins that mediate formation of the membrane attack complex (MAC) on the surface of target cells. Full-length complement C8α (Rb-C8α) and C8ß (Rb-C8ß) sequences were identified from a cDNA library of rock bream (Oplegnathus fasciatus), and their genomic sequences were obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The Rb-C8α gene contains 64bp of 5'-UTR, open reading frame (ORF) of 1794bp, which encodes a polypeptide of 598 amino acids, 212bp of 3'-UTR. The Rb-C8ß gene contains 5'-UTR of 27bp, open reading frame (ORF) of 1761bp, which encodes a polypeptide of 587 amino acids, 3'-UTR of 164bp. Rb-C8α consists of 11 exons interrupted by 10 introns and Rb-C8ß consists of 12 exons interrupted by 11 introns. Sequence analysis revealed that both Rb-C8α and Rb-C8ß contain thrombospondin type-1, a low-density lipoprotein receptor domain class A, membrane attack complex/perforin (MACPF) domain and epidermal growth factor like domain. The promoter regions of both genes contain important putative transcription factor binding sites including those for NF-κB, SP-1, C/EBP, AP-1, and OCT-1. Rb-C8α and Rb-C8ß showed the highest amino acid identity of 62% and 83% to rainbow trout C8α and Japanese flounder C8ß respectively. Quantitative real-time PCR analysis confirmed that Rb-C8α and Rb-C8ß were constitutively expressed in all examined tissues, isolated from healthy rock bream, with highest expression occurring in liver. Pathogen challenge, including Edwardsiella tarda, Streptococcus iniae, and rock bream iridovirus led to up regulation of Rb-C8α and Rb-C8ß in liver. Positive regulations upon bacterial and viral challenges, and high degree of evolutionary relationship to respective orthologues, confirmed that Rb-C8α and Rb-C8ß important immune genes, likely involved in the complement system lytic pathway of rock bream.

Interferon regulatory factors 4 and 8 in rock bream, Oplegnathus fasciatus: structural and expressional evidence for their antimicrobial role in teleosts.

The interferon regulatory factor (IRF) members IRF4 and IRF8 contribute to B-lymphocyte development and can act as regulators of immunoglobulin (Ig) light chain gene transcription. These two IRFs are closely interrelated and are expressed at high levels in the lymphoid and myeloid cells of the immune system. In this study, the complete cDNA and genomic sequences of rock bream IRF4 (RbIRF4) and IRF8 (RbIRF8) were identified by homology screening of a multi-tissue normalized cDNA library and a BAC library, respectively, which had been established using Roche 454 GS-FLX™ technology. The full-length RbIRF4 cDNA is composed of 3442 bp and encodes a polypeptide of 462 amino acids; the genomic DNA is 9262 bp in length, consisting of eight exons and seven introns. The full-length RbIRF8 cDNA is composed of 2186 bp and encodes a 422 amino acid polypeptide; the genomic DNA is 4120 bp in length, consisting of nine exons and eight introns. The deduced amino acid sequences of RbIRF4 and RbIRF8 include a conserved DNA-binding domain (DBD) encompassing a tryptophan pentad-repeat and an IRF-association domain (IAD). Several putative transcription factor binding sites were also identified in 5' flanking region of both RbIRF4 and RbIRF8, and include those of immune-related factors. Quantitative real time PCR analysis of healthy rock bream detected the highest expression levels of RbIRF4 and RbIRF8 in lymphomyeloid-rich tissues. In addition, viral (rock bream iridovirus) and bacterial (Edwardsiella tarda and Streptococcus iniae) infection stimulated RbIRF4 and RbIRF8 expressions in head kidney and spleen. These results suggest not only that RbIRF4 and RbIRF8 may have a protective function against virus and bacteria pathogen invasion in rock bream, but also that IRFs may be immunomodulatory factors of teleost fish.

Molecular cloning and functional characterization of two duplicated two-cysteine containing type I interferon genes in rock bream Oplegnathus fasciatus.

Two type I interferon (IFN) genes, designated as rbIFN1 and rbIFN2, have been cloned and characterized in rock bream. They are both comprised of 5 exons and 4 introns, and are closely linked on the rock bream chromosome in a unique head-to-head configuration. Both genes encode 183 amino acid (aa) precursor with a putative 17 aa signal peptide in the N-terminal. Only one amino acid divergence is present between two IFNs. Compared with the type I IFNs in higher vertebrates, two rock bream IFNs possess conserved alpha helical structure and share approximately 20% identity in aa sequence. The highest aa sequence homology (83.2%) was found with European seabass IFNs. Phylogenetic analysis grouped two rock bream IFNs into the subgroup-d of two-cysteine containing IFNs. The gene synteny analysis revealed that they are orthologous with the zebrafish IFNφ4 on chromosome-12 and paralogous to each other, which are likely derived from a gene duplication event followed by an inversion. A number of cis-regulatory elements associated with immune response including 15 IRF and 6 NF-κB binding sites are predicted in the shared 4.5 kb 5'-flanking region. Highest constitutive expression of two IFNs was detected in blood cells and skin. Their expression in blood cells and head kidney was up-regulated by lipopolysaccharide, poly I:C, Edwardsiella tarda, Streptococcus iniae and iridovirus. Furthermore, recombinant rbIFN1 protein produced by E. coli induced a rapid and transient expression of the interferon inducible Mx gene in head kidney cells. These results suggest that two duplicated type I IFN genes are involved in rock bream host response to both viral and bacterial pathogens.

Genomic characterization and expression analysis of complement component 9 in rock bream (Oplegnathus fasciatus).

The complement component 9 (C9) is a single-chain glycoprotein that mediates formation of the membrane attack complex (MAC) on the surface of target cells. Full-length C9 sequence was identified from a cDNA library of rock bream (Oplegnathus fasciatus), and its genomic sequence was obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The rock bream complement component 9 (Rb-C9) gene contains 11 exons and 10 introns and is composed of a 1782 bp complete open reading frame (ORF) that encodes a polypeptide of 593 amino acids. Sequence analysis revealed that the Rb-C9 protein contains two thrombospondin type-1domains, a low-density lipoprotein receptor domain class A, a membrane attack complex & perforin (MACPF) domain, and an epidermal growth factor (EGF)-like domain. Important putative transcription factor binding sites, including those for NF-κB, SP-1, C/EBP, AP-1 and OCT-1, were found in the 5' flanking region. Phylogenetic analysis revealed a close proximity of Rb-C9 with the orthologues in puffer fish, and Japanese flounder. Quantitative real-time RT-PCR analysis confirmed that Rb-C9 was constitutively expressed in all the examined tissues isolated from healthy rock bream, with highest expression occurring in liver. Pathogen challenge, including Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide endotoxin and rock bream iridovirus led to up-regulation of Rb-C9 in liver but no change in peripheral blood cells. The observed response to bacterial and viral challenges and high degree of evolutionary relationship to respective orthologues, confirmed that Rb-C9 is an important immune gene, likely involved in the complement system lytic pathway of rock bream.

Cystatin B homolog from rock bream Oplegnathus fasciatus: genomic characterization, transcriptional profiling and protease-inhibitory activity of recombinant protein.

Cysteine proteases are present in all living organisms and, in animals, function in a vast array of physiological and pathological processes. Cysteine protease inhibitors act upon the cysteine proteases to regulate their activity. The cystatin superfamily of cysteine protease inhibitors has members represented in all living organisms studied to date. Here, we report the identification of a new member of the family 1 cystatin in Oplegnathus fasciatus rock bream (denoted as RbCyt B) and the characterization at the molecular level. The complete genomic sequence of RbCyt B consists of three exons and a promoter region. The open reading frame (ORF) encodes for a 100 amino acids length polypeptide with a single cystatin-like domain and a cysteine protease inhibitor signature motif. The conserved N-terminal glycine, glutamine-valine-glycine motif, QxVxG, and a variant of the proline-tryptophan, PW, motif were identified. RbCyt B showed closest phylogenetic distance to Dicentrarchus labrax cystatin B, and shared up to 73% amino acid identity and 90% amino acid similarity with known cystatin B genes. RbCyt B mRNA expression was detected in nine different tissues and was highly expressed in liver, spleen, gill, brain, intestine, kidney, head kidney, and blood, as compared with muscle. In vivo immune stimulation with Edwardsiella tarda bacteria caused significant up-regulation of RbCyt B mRNA in head kidney and spleen at 24h post-infection (P<0.05). Recombinant RbCyt B was expressed in Escherichia coli, and the purified protein demonstrated 82% papain inhibitory activity at 500 × 10(-3) µg µL(-1) in a concentration-dependent manner. These results suggest that RbCyt B is a member of family 1 cystatin with high homology to cystatin B, and is a biologically active protein possessing papain inhibitory activity and potentially involved in immune responses against invading Gram-negative bacteria in rock bream.

Caspase 3 from rock bream (Oplegnathus fasciatus): genomic characterization and transcriptional profiling upon bacterial and viral inductions.

Caspase 3 is a prominent mediator of apoptosis and participates in the cell death signaling cascade. In this study, caspase 3 was identified (Rbcasp3) and characterized from rock bream (Oplegnathus fasciatus). The full-length cDNA of Rbcasp3 is 2683 bp and contains an open reading frame of 849 bp, which encodes a 283 amino acid protein with a calculated molecular mass of 31.2 kDa and isoelectric point of 6.31. The amino acid sequence resembles the conventional caspase 3 domain architecture, including crucial amino acid residues in the catalytic site and binding pocket. The genomic length of Rbcasp3 is 7529 bp, and encompasses six exons interrupted by five introns. Phylogenetic analysis affirmed that Rbcasp3 represents a complex group in fish that has been shaped by gene duplication and diversification. Many putative transcription factor binding sites were identified in the predicted promoter region of Rbcasp3, including immune factor- and cancer signal-inducible sites. Rbcasp3, excluding the pro-domain, was expressed in Escherichia coli. The recombinant protein showed a detectable activity against the mammalian caspase 3/7-specific substrate DEVD-pNA, indicating a functional role in physiology. Quantitative real time PCR assay detected Rbcasp3 expression in all examined tissues, but with high abundance in blood, liver and brain. Transcriptional profiling of rock bream liver tissue revealed that challenge with lipopolysaccharides (LPS) caused prolonged up-regulation of Rbcasp3 mRNA whereas, Edwardsiella tarda (E. tarda) stimulated a late-phase significant transcriptional response. Rock bream iridovirus (RBIV) up-regulated Rbcasp3 transcription significantly at late-phase, however polyinosinic-polycytidylic acid (poly(I:C)) induced Rbcasp3 significantly at early-phase. Our findings suggest that Rbcasp3 functions as a cysteine-aspartate-specific protease and contributes to immune responses against bacterial and viral infections.

Survey of contamination of estrogenic chemicals in Japanese and Korean coastal waters using the wild grey mullet (Mugil cephalus).

We monitored the contamination by environmental estrogens (EEs) of coastal areas in Korea and Japan using the wild grey mullet. The grey mullet were collected from Ansan, Jeju, Yeosu, Tongyeong, and Busan in Korea and Nagasaki, Omuta, and Fukuoka in Japan. Contamination by EEs was determined by measuring vitellogenin (VTG) levels in serum and identifying gonadal abnormalities histologically (i.e., testis-ova). In four sites in Korea (Ansan, Yeosu, Tongyeong, and Busan) and two sites in Japan (Nagasaki and Fukuoka), serum VTG in immature and male grey mullet was detected at levels greater than 1.0 microg/ml, which is considered to be an abnormal level. Although, testis-ova were observed in some individuals collected in Ansan, Tongyeong, and Busan in Korea and Omuta in Japan, there was no correlation between individuals with testis-ova and individuals with abnormal levels of VTG. Furthermore, in Japan, serum VTG levels of fish collected from Nagasaki and Fukuoka were also greater than 1.0 microg/ml. Although individuals with testis-ova were found in Omuta, these fish expressed normal levels of serum VTG. Our results suggest that the grey mullets living in these coastal areas are influenced by EEs in the environment. Furthermore, it appears that the production of VTG and the occurrence of testis-ova are caused by different mechanisms.