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.

A molluscan calreticulin ortholog from Haliotis discus discus: Molecular characterization and transcriptional evidence for its role in host immunity.

Calreticulin (CALR), a Ca(2+) binding chaperone of the endoplasmic reticulum (ER) and mainly involved in Ca(2+) storage and signaling. In this study, we report the molecular characterization and immune responses of CALR homolog from disk abalone (AbCALR). The full length AbCALR cDNA (1837 bp) had an ORF of 1224 bp. According to the multiple alignments analysis, N- and P-domains were highly conserved in all the selected members of CALRs. In contrast, the C-domain which terminated with the characteristic ER retrieval signal (HDEL) was relatively less conserved. The phylogenetic analysis showed that all the selected molluscan homologs clustered together. Genomic sequence of AbCALR revealed that cDNA sequence was dispersed into ten exons interconnected with nine introns. AbCALR mRNA expression shows the significant (P < 0.05) up-regulation of AbCALR transcripts in hemocytes upon bacterial (Listeria monocytogenes and Vibrio parahaemolyticus), viral (Viral haemorrhagic septicaemia virus; VHSV) and immune stimulants (LPS and poly I:C) challenges at middle and/or late phases. These results collectively implied that AbCALR is able to be stimulated by pathogenic signals and might play a potential role in host immunity.

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).

Molecular delineation of a caspase 10 homolog from black rockfish (Sebastes schlegelii) and its transcriptional regulation in response to pathogenic stress.

Caspase 10 is an initiator caspase in death cascades of death receptor mediated apoptotic signaling. We identified and molecularly characterized a novel homolog of caspase 10 from black rockfish (Sebastes schlegelii) and designated as RfCasp10. The complete coding region of RfCasp10 was found to consist of 1659 bps, encoding a 553 amino acid protein with a predicted molecular mass of 61.7 kDa. The characteristic caspase family domain architecture, including death effecter domains (DEDs), was clearly identified in RfCasp10. Moreover, the RfCasp10 gene was found to contain 13 exons. Our pairwise sequence alignment confirmed the prominent sequence similarity of RfCasp10 with its fish homologs, and phylogenetic reconstruction affirmed its homology and substantial evolutionary relationship with known caspases 10 similitudes, in particular with those of teleosts. As detected by qPCR, RfCasp10 was markedly expressed in blood tissues under physiological conditions, whereas its expression was found to be upregulated under pathogenic stress, elicited by Streptococcus iniae and polyinosinic:polycytidylic acid in blood, liver, and spleen tissues. Collectively, our study suggests the plausible elicitation of RfCasp10 mediated apoptosis in immune relevant tissues of black rockfish as a host immune response to a bacterial or viral infection.

A potential antioxidant enzyme belonging to the atypical 2-Cys peroxiredoxin subfamily characterized from rock bream, Oplegnathus fasciatus.

Peroxiredoxins (Prxs), a diverse family of antioxidant enzymes, exert their antioxidant function through which different peroxide species are detoxified. This study describes both structural and functional characterization of a mitochondrial Prx identified in rock bream, Oplegnathus fasciatus (RbPrx5). The ORF (573 bp) of RbPrx5 encoded a protein of 190 amino acids (20 kDa) containing a putative mitochondrial targeting sequence (residues 1-20) and a thioredoxin-2 motif (residues 31-190) and three conserved Cys residues. Homology assessment and phylogenetic analysis clearly disclosed relatively higher amino acid sequence similarities and a closer evolutionary position of RbPrx5 with those of other teleost homologs. The ORF of RbPrx5 was distributed among six exons as found in other vertebrates, but it possessed an additional exon in its 5'-UTR. In silico examination of RbPrx5 gene's putative promoter region revealed the presence of several cis-elements which may be important in its transcriptional regulation. Constitutive expression of RbPrx5 was detected in eleven tissues with the highest level in the heart. Modulation of RbPrx5 transcription was evidenced from varying mRNA levels in head kidney post in vivo LPS-, poly I:C-, Edwardsiella tarda bacterial- and rock bream iridoviral-challenges. The antioxidant function of RbPrx5 was investigated using recombinant RbPrx5 protein. Results of an in vitro mixed-function oxidase assay demonstrated a dose-dependent inhibition of DNA damage by rRbPrx5. A H2O2 tolerance assay showed that in vivo overexpression of rRbPrx5 increased the bacterial survival under H2O2-mediated oxidative stress condition. These findings provide an overall insight into the structural, expressional and functional aspects of RbPrx5.

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.

Insights into molecular profiles and genomic evolution of an IRAK4 homolog from rock bream (Oplegnathus fasciatus): immunogen- and pathogen-induced transcriptional expression.

As a pivotal signaling mediator of toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signaling cascades, the IL-1R-associated kinase 4 (IRAK4) is engaged in the activation of host immunity. This study investigates the molecular and expressional profiles of an IRAK4-like homolog from Oplegnathus fasciatus (OfIRAK4). The OfIRAK4 gene (8.2 kb) was structured with eleven exons and ten introns. A putative coding sequence (1395bp) was translated to the OfIRAK protein of 464 amino acids. The deduced OfIRAK4 protein featured a bipartite domain structure composed of a death domain (DD) and a kinase domain (PKc). Teleost IRAK4 appears to be distinct and divergent from that of tetrapods in terms of its exon-intron structure and evolutionary relatedness. Analysis of the sequence upstream of translation initiation site revealed the presence of putative regulatory elements, including NF-κB-binding sites, which are possibly involved in transcriptional control of OfIRAK4. Quantitative real-time PCR (qPCR) was employed to assess the transcriptional expression of OfIRAK4 in different juvenile tissues and post-injection of different immunogens and pathogens. Ubiquitous basal mRNA expression was widely detected with highest level in liver. In vivo flagellin (FLA) challenge significantly intensified its mRNA levels in intestine, liver and head kidney indicating its role in FLA-induced signaling. Meanwhile, up-regulated expression was also determined in liver and head kidney of animals challenged with potent immunogens (LPS and poly I:C) and pathogens (Edwardsiella tarda and Streptococcus iniae and rock bream iridovirus (RBIV)). Taken together, these data implicate that OfIRAK4 might be engaged in antibacterial and antiviral immunity 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.

Characterization of rock bream (Oplegnathus fasciatus) cytosolic Cu/Zn superoxide dismutase in terms of molecular structure, genomic arrangement, stress-induced mRNA expression and antioxidant function.

Superoxide dismutases (SODs) are dedicated to scavenge and dismutate the superoxide anions in order to protect the cells from oxidative stress by establishing the redox homeostasis. In this study, we describe a cytosolic Cu/ZnSOD, the second SOD member from rock bream Oplegnathus fasciatus (Of-cCu/ZnSOD) at molecular, genomic structural-, transcriptional- and functional-levels. The determination of genomic arrangement of Of-cCu/ZnSOD by means of a BAC library revealed that its primary transcript is represented by five exons and encoded a peptide of 154 amino acids. In silico investigation of Of-cCu/ZnSOD indicated the presence of several family characteristics including two Cu/ZnSOD signatures, seven metal liganding residues and eight ß-sheets forming a ß-barrel topology. Alignment and modeling studies confirmed the conservation of Cu/ZnSOD at primary and tertiary levels. While invertebrate Cu/ZnSOD members mainly demonstrate a tetraexonic structure, the vertebrate members have acquired an additional intron in the third exon resulting in a quinquepartite arrangement with class-specific exon lengths. Although, teleost Cu/ZnSOD members resembled the mammalian orthologs in their genomic organization, they shared a proximal position with molluscan members in the phylogeny. The antioxidant (AO) activity of Of-cCu/ZnSOD was affirmed by a recombinant protein which was also used to examine the biophysical and biochemical properties. The pronounced activity was detected when the rOf-cCu/ZnSOD was expressed with the Cu(2+) and Zn(2+) supplementation. The optimum activities were observed at pH10 and 25°C, and KCN strongly inhibited the activity of the rOf-cCu/ZnSOD. Furthermore, a constitutive mRNA expression of Of-cCu/ZnSOD with higher levels in blood>liver>heart and brain was observed, which was consistent with the transcriptional profile of Of-mMnSOD, suggesting important physiological role(s). This idea was further strengthened by the temporal assessment of Of-cCu/ZnSOD transcripts in animals under pathological (bacteria- or viral-induced) and physiological (H2O2-induced oxidative) stress conditions using qPCR, in which it exhibited significantly up-regulated levels. Screening of Of-cCu/ZnSOD 5'-flanking region revealed the presence of several important transcription factor binding sites that potentially govern the Cu/ZnSOD expression. These findings conjointly contribute to expand our understanding regarding the piscine Cu/ZnSODs and; in particular, the AO enzyme network of rock bream.

Functional characterization of the evolutionarily preserved mitochondrial antiviral signaling protein (MAVS) from rock bream, Oplegnathus fasciatus.

Antimicrobial immune defense is evolutionarily preserved in all organisms. Mammals have developed robust, protein-based antiviral defenses, which are under constant investigation. Studies have provided evidences for the various fish immune factors sharing similarity with those of mammals. In this study, we have identified an ortholog of mitochondrial antiviral signaling protein from rock bream, Oplegnathus fasciatus. RbMAVS cDNA possesses an open reading frame (ORF) of 1758 bp coding for a protein of 586 amino acids with molecular mass of approximately 62 kDa and isoelectric point of 4.6. In silico analysis of RbMAVS protein revealed a caspase recruitment domain (CARD), a proline rich domain and a transmembrane domain. RbMAVS protein also contains a putative TRAF2 binding motif, (319)PVQDT(323). Primary sequence comparison of RbMAVS with other orthologues revealed heterogeneity towards the C-terminus after the CARD region. RbMAVS transcripts were evident in all the examined tissues. RbMAVS expression was induced in vivo after poly I:C challenge in peripheral blood cells, liver, head kidney and spleen tissues. Over-expression of RbMAVS potently inhibited marine birnavirus (MABV) infection in rock bream heart cells and induced various cytokines and signaling molecules in vitro. Thus, RbMAVS is an antiviral protein and potentially involved in the recognition and signaling of antiviral defense mechanism in rock bream.

Molecular profile and functional characterization of the ferritin H subunit from rock bream (Oplegnathus fasciatus), revealing its putative role in host antioxidant and immune defense.

Ferritins are iron binding proteins made out of 24 subunits, involved in iron homeostasis and metabolism in cellular environments. Here, we sought to identify and functionally characterize a one type of subunits of ferritin (ferritin H-like subunit) from rock bream (Oplegnathus fasciatus; RbFerH). The complete coding sequence of RbFerH was 531 bp in length, encoding a 177-amino acid protein with a predicted molecular mass of 20.8 kDa. The deduced protein structure possessed the domain architecture characteristic of known ferritin H subunits, including metal ligands for iron binding, a ferroxidase center, and two iron-binding region signatures. As expected, the 5' untranslated region of the RbFerH cDNA sequence contained a putative iron response element region, a characteristic regulatory element involved in its translation. The RbFerH gene comprised 5 exons and 4 introns spanning a 4195 bp region. Overexpressed recombinant RbFerH protein demonstrated prominent Fe(II) ion depriving activity, bacteriostatic properties, and protective effects against oxidative double-stranded DNA damage. Using quantitative polymerase chain reaction (qPCR), we found that RbFerH was expressed ubiquitously in the majority of physiologically important tissues in rock bream. A greater abundance of the mRNA transcripts were detected in blood and liver tissues. Upon administering different microbial pathogens and pathogen-derived mitogens, RbFerH transcription was markedly elevated in the blood of rock bream. Taken together, our findings suggest that RbFerH acts as a potent iron sequestrator in rock bream and may actively participate in antimicrobial as well as antioxidative defense.

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.

Molecular characterization and expressional affirmation of the beta proteasome subunit cluster in rock bream immune defense.

Immunoproteasomes are primarily induced upon infection and formed by replacing constitutive beta subunits with inducible beta subunits which possess specific cleavage properties that aid in the release of peptides necessary for MHC class I antigen presentation. In this study, we report the molecular characterization and expression analysis of the inducible immunosubunits PSMB8, PSMB9, PSMB9-L, and PSMB10 from rock bream, Oplegnathus fasciatus. The three subunits shared common active site residues and were placed in close proximity to fish homologues in the reconstructed phylogenetic tree, in which the mammalian homologues formed separate clades, indicating a common ancestral origin. The rock bream immunosubunits possessed higher identity and similarity with the fish homologues. RbPSMB8, RbPSMB9, RbPSMB9-L, and RbPSMB10 were multi-exonic genes with 6, 6, 7 and 8 exons, respectively. These four genes were constitutively expressed in all the examined tissues. Immunostimulants such as lipopolysaccharide and poly I:C induced RbPSMB8, RbPSMB9, RbPSMB9-L, and RbPSMB10 in liver and head kidney, suggesting their possible involvement in immune defense in rock bream.

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.

Effects of electroacupuncture on oxaliplatin-induced neuropathic cold hypersensitivity in rats.

This study investigated whether and how electroacupuncture (EA) attenuates cold hypersensitivity (allodynia) in a rat model of oxaliplatin-induced neuropathic pain. Cold allodynia [evaluated by immersing the tail into cold water (4 °C) and measuring the withdrawal latency] was induced 3 days after an oxaliplatin administration (6 mg/kg, i.p.). EA stimulation (2/100 Hz, 0.3-ms pulse duration, 0.2-0.3 mA) was delivered to ST36 acupoint or non-acupoint for 20 min. Low-frequency (2 Hz) EA at ST36 relieved cold allodynia more effectively than high-frequency EA at ST36 or low-frequency EA at non-acupoint. Naloxone (opioid antagonist, 2 mg/kg, i.p.) completely blocked such EA-induced anti-allodynia, whereas phentolamine (α-adrenergic antagonist, 2 mg/kg, i.p.) did not. Moreover, plasma ß-endorphin levels significantly increased right after the end of EA and subsequently decreased. These results indicate that low-frequency EA at ST36 in rats has a marked relieving effect on oxaliplatin-induced cold allodynia that is mediated by the endogenous opioid, but not noradrenergic, system.

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.

Effect of bee venom acupuncture on oxaliplatin-induced cold allodynia in rats.

Oxaliplatin, a chemotherapy drug, often leads to neuropathic cold allodynia after a single administration. Bee venom acupuncture (BVA) has been used in Korea to relieve various pain symptoms and is shown to have a potent antiallodynic effect in nerve-injured rats. We examined whether BVA relieves oxaliplatin-induced cold allodynia and which endogenous analgesic system is implicated. The cold allodynia induced by an oxaliplatin injection (6 mg/kg, i.p.) was evaluated by immersing the rat's tail into cold water (4°C) and measuring the withdrawal latency. BVA (1.0 mg/kg, s.c.) at Yaoyangguan (GV3), Quchi (LI11), or Zusanli (ST36) acupoints significantly reduced cold allodynia with the longest effect being shown in the GV3 group. Conversely, a high dose of BVA (2.5 mg/kg) at GV3 did not show a significant antiallodynic effect. Phentolamine ( α -adrenergic antagonist, 2 mg/kg, i.p.) partially blocked the relieving effect of BVA on allodynia, whereas naloxone (opioid antagonist, 2 mg/kg, i.p.) did not. We further confirmed that an intrathecal administration of idazoxan ( α 2-adrenergic antagonist, 50 µ g) blocked the BVA-induced anti-allodynic effect. These results indicate that BVA alleviates oxaliplatin-induced cold allodynia in rats, at least partly, through activation of the noradrenergic system. Thus, BVA might be a potential therapeutic option in oxaliplatin-induced neuropathy.

Genomic characterization, expression analysis, and antimicrobial function of a glyrichin homologue from rock bream, Oplegnathus fasciatus.

Antimicrobial peptides are important innate effector molecules, playing a vital role in antimicrobial immunity in all species. Glyrichin is a transmembrane protein and an antibacterial peptide, exerting its functions against a wide range of pathogenic bacteria. In this study, cDNA and a BAC clone harboring the glyrichin gene were identified from rock bream and characterized. Genomic characterization showed that the OfGlyrichin gene exhibited a 3 exon-2 intron structure. OfGlyrichin is a 79-amino-acid protein with a transmembrane domain at (22)GFMMGFAVGMAAGAMFGTFSCLR(44). Pairwise and multiple sequence alignments showed high identity and conservation with mammalian orthologues. Phylogenetic analysis showed a close relationship with fish species. Higher levels of OfGlyrichin transcripts were detected in the liver from healthy rock bream which were induced by immunogens like lipopolysaccharide, poly I:C, rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. The synthetic peptide (pOf19) showed antibacterial activity against Escherichia coli, E. tarda, and S. iniae. Analysis of the bacterial morphological features after pOf19 peptide treatment showed breakage of the cell membrane, affirming that antibacterial function is accomplished through membrane lysis. The pOf19 peptide also showed antiviral activity against RBIV infection. The high conservation of the genomic structure and protein, together with the antimicrobial roles of OfGlyrichin, provide evidence for the evolutionary existence of this protein playing a vital role in innate immune defense in rock bream.

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.