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Purified porcine gastric mucin (PGM) is an alternative biomaterial to native mucin which displays multifunctional properties for exploring a wide range of biomedical applications. The present study evaluated the in vitro (RAW 264.7 macrophage cells) and in vivo (zebrafish embryos and larvae) bioactivities of PGM. The median lethal concentration (LC 50 ) of PGM was 197.9 µg/mL for embryos, while it was non-toxic to RAW 264.7 cells, even at 500 µg/mL. Following PGM exposure (100 µg/mL), a higher embryo hatching rate (59.9%) was observed at 48 h post fertilization, compared to the control (30.6%). Protective effects of PGM from pathogenic Aeromonas hydrophila were demonstrated by high larvae survival rates of 85.0% and 94.0% at 50 and 100 μg/mL of PGM exposure, respectively. Heat tolerance effect of PGM (50 and 100 µg/mL) on larvae (40 °C for 48 h) was confirmed by 75% and 100% of survival rates, respectively. Additionally, PGM reduced the A. hydrophila -induced reactive oxygen species (ROS) generation in larvae. The qRT-PCR results in PGM exposed larvae exhibited induction of immune-related genes ( tlr5a and tlr5b , myd88 , c-rel , il1β, tnf-α , il6, il10 , cxcl18b , ccl34a.4 , defbl1 , hamp , ctsd, muc2.1 , muc5.1 , muc5.2 , and muc5.3 ), stress response ( hsp70 , hsp90aa1.1 , and hsp90ab1 ), and antioxidant genes ( cat and sod1 ). Moreover, our results revealed that PGM involved in the regulation of transcriptional gene induction increases Hsp90 protein in the zebrafish larvae. Furthermore, upregulation of Il6, Il10, Tnfα, Ccl3, Defa-rs2, Defa21 and Camp and antioxidant genes ( Sod2 and Cat ) were observed in PGM-exposed RAW 264.7 cells. Overall findings confirmed the activation of immune responses, disease resistance against pathogenic bacteria, heat tolerance, and ROS-scavenging properties by PGM, which may provide insights into new applications for PGM as a multifunctional immunomodulator.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ferritins are biological iron chelators that can sequestrate excess iron to maintain iron homeostasis in the body. Ferritins basically consist of 2 types of subunits, designated as H and L. However, another new subunit, ferritin "M" which possesses characteristic features of both the H and L subunits, was recently identified in lower vertebrates, mostly in fish. In this study, a ferritin M-like subunit from rock bream (Oplegnathus fasciatus) (RbFerM) was characterized at the molecular level, and its transcriptional profile was analyzed in healthy fish, as well as in pathogen- and mitogen-stimulated fish. Furthermore, its functional properties were evaluated using the recombinant protein. The complete coding sequence of RbFerM was 528 bp in length, encoding a 176-amino acid peptide with a calculated molecular mass of 20 kDa. In silico analysis of RbFerM revealed that it has features similar to both the mammalian ferritin subunits, H and L. Phylogenetic analysis depicted the higher evolutionary proximity of RbFerM with its fish counterparts. Quantitative real time polymerase chain reaction (PCR) analysis detected a ubiquitous transcriptional profile of RbFerM in selected tissues of rock bream, in which more pronounced expression was observed in blood and liver tissues. Significant transcriptional inductions of RbFerM were detected in liver tissues upon lipopolysaccharides (LPS), Edwardsiella tarda, Streptococcus iniae, and rock bream irido virus (RBIV) exposures in time-course immune-challenge experiments. The purified recombinant protein of RbFerM demonstrated detectable iron chelating activity that varied with the temperature. Moreover, the recombinant RbFerM rendered a detectable protection effect against iron (II) and H2O2-mediated DNA damage., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Catalases are well known antioxidant enzymes that can mainly dismutate hydrogen peroxide into water and oxygen in order to prevent oxidative stress. The complete genomic DNA (gDNA) sequence of the catalase gene from rock bream (Oplegnathus fasciatus) was identified from our custom-constructed BAC genomic DNA library and designated as RbCat. RbCat consists of 13 exons, separated by 12 introns, within a 13,722-bp gDNA sequence. The complete cDNA sequence (3303 bp) of RbCat is comprised of a 1581-bp coding region, encoding a peptide of 527 amino acids (aa) in length, with a predicted molecular mass of 60 kDa and a theoretical isoelectric point of 8.34. The anticipated promoter region of RbCat contains several transcription factor-binding sites, including sites that bind with immune- and antioxidant-responsive signaling molecules, suggesting its substantial transcriptional regulation. RbCat resembles the typical catalase family signature, i.e., it is composed of the catalase proximal active site motif along with a catalase proximal heme-ligand signature motif and shares great homology with its fish counterparts. According to multiple sequence alignment, functionally important amino acids present in RbCat were thoroughly conserved among its vertebrate counterparts. Phylogenetic analysis revealed that RbCat evolved from a vertebrate origin, and further positioned it in the fish clade. Recombinant RbCat had noticeable peroxidase activity against its substrate, hydrogen peroxide, in a dose-dependent manner. However, it demonstrated substantial peroxidase activity within a broad range of temperatures and pH values. Constitutive RbCat mRNA expression of different magnitudes was detected in a tissue-specific manner, suggesting its diverse role in physiology with respect to the tissue type. Moreover, immune challenge experiments using Edwardsiella tarda and rock bream iridovirus (RBIV) as live pathogens and polyinosinic:polycytidylic acid and lipopolysaccharide as mitogens revealed that the transcription of RbCat can be modulated by immune stimulation. Collectively, the results obtained in this study suggest that RbCat can function as a potent antioxidant enzyme in rock bream and may play a role in post-immune responses with respect to its peroxidase activity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Diverse antioxidant enzymes are essential for marine organisms to overcome oxidative stress as well as for the fine-tuning of immune reactions through activating different signal transduction pathways. This study describes the transcriptional analysis of antioxidant enzymes of disk abalone by challenging with bacteria (Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes) and viral hemorrhagic septicemia virus (VHSV). Upon bacteria and VHSV challenge, Manganese superoxide dismutase (MnSOD), Copper, Zinc superoxide dismutase (CuZnSOD), catalase, thioredoxin peroxidase (TPx), Selenium-dependent glutathione peroxidase (SeGPx), and thioredoxin-2 (TRx-2) expression levels were altered in gills, and hemocytes at different magnitudes. In gills, only MnSOD, catalase, and SeGPx genes were completely upregulated by post-challenge of bacterial and VHSV. Among them, SeGPx demonstrated strong upregulation by 16-fold (bacteria) and 2-fold (VHSV) in gills, and 5-fold (bacteria) and 3.0-fold (VHSV) in hemocytes. None of the genes examined were downregulated (in gills and hemocytes) by bacteria challenge even though CuZnSOD and TPx showed downregulation (completely) in hemocytes by VHSV. In general, abalone hemocytes had lower potential to induce antioxidant enzyme transcripts upon bacteria and VHSV challenge than gills. Based upon these results, we suggest that abalones induce oxidative stress in tissues during the bacteria and VHSV challenge, and the identified response of antioxidant enzymes could be supported for maintaining a low-level of reactive oxygen species (ROS) that may serve as a signal for activating immune reactions against pathogenic conditions., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This study describes the transcriptional analysis of antioxidant and immune defense genes in gills tissue of disk abalone exposed to thermal, salinity and hypoxia-related stress, using quantitative real-time PCR. Results showed that manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD), catalase (CAT), thioredoxin peroxidase (TPx), selenium dependant glutathione peroxidase (SeGPx), and thioredoxin-2 (TRx-2) transcripts were expressed differently in gills, and that they respond collectively as a classical enzymatic antioxidant defense system in abalone. Comparative analysis of expression profiles indicated that CAT, TPx and SeGPx transcripts were significantly up-regulated (p<0.05) by all three physical stress conditions - thermal (28 degrees C), salinity (25 per thousand) and hypoxia - relative to levels in respective controls. In contrast, CuZnSOD and TRx-2 transcription were down-regulated in response to thermal stress. Interestingly, all the antioxidant transcripts exhibited significant up-regulation in response to salinity-related stress. Meanwhile, hypoxia caused up-regulation of the MnSOD, CAT, TPx and SeGPx, but not the CuZnSOD and TRx-2 transcripts. One of the most significant outcomes was the more than 10-fold induction of SeGPx, suggesting that SeGPx is a potential biomarker gene among antioxidant enzymes, under conditions of physical stress. The effects of physical stress on the transcriptional responses of immune functional genes namely suppressor of cytokine signaling-2 (SOCS-2) and myxovirus resistance (Mx) were investigated. We observed that salinity and hypoxia increase both the SOCS-2 and Mx transcripts, while thermal stress at 28 degrees C exerts less of an effect, producing mixed transcriptional responses (both up and down regulation). Based upon these results, we postulate that abalones utilize antioxidant and immune defense mechanisms together to overcome physical stresses.

Peroxiredoxins (Prxs) play an important role against various oxidative stresses and intra-cellular signal transduction. Peroxiredoxin 6 (PrxVI) was identified from the disk abalone Haliotis discus discus cDNA library and named HdPrxVI. The full length cDNA of HdPrxVI was 1457 bp with a 654 bp open reading frame (ORF) encoding 218 amino acids. The predicted molecular mass and estimated isoelectric point (pI) of HdPrxVI were 24 kDa and 7.3, respectively. The deduced amino acid sequence demonstrated the greatest degree (72.4%) of identity with Crassostrea gigas PrxVI. The conserved peroxidase catalytic center (42PVCTTE47) with a conserved cysteine residue (Cys44) and a catalytic center for PLA2 activity (27GGSWA31) were observed in the sequence, indicating that it is a member of 1-Cys Prx. Real time PCR results revealed that HdPrxVI mRNA is constitutively expressed in all tissues in a tissue-specific manner. During exposure to haemorrhagic septicaemia virus (VHSV), HdPrxVI mRNA transcription was down-regulated in the gill, suggesting that the abalone responded to the viral infection quickly, and HdPrxVI played a physiological role against virus-induced oxidative stress. The purified recombinant HdPrxVI, together with dithiothreitol (DTT), was shown to scavenge H2O2 in human leukemia THP-1 cells and provided protection against H2O2-induced apoptosis.

Thioredoxin peroxidase (TPx), also named peroxiredoxin (Prx), is an important peroxidase, which can protect organisms against various oxidative stresses. Two TPxs were isolated from a disk abalone (Haliotis discus discus) cDNA library, named as AbTPx1 and AbTPx2, respectively. AbTPx1 and AbTPx2 consist of 1315 and 1045 bp full-length cDNA with 753 and 597 bp open reading frames encoding 251 and 199 amino acids, respectively. The TPx signature motif 1 (FYPLDFTFVCPTEI) and motif 2 (GEVCPA) were conserved in both AbTPx1 and AbTPx2 amino acid sequences. Purified recombinant abalone TPx fusion proteins catalyzed the reduction of H2O2 and butyl hydroperoxide in peroxidase assays. Furthermore, both AbTPx fusion proteins were shown to protect super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Escherichia coli cells transformed with AbTPx1 and AbTPx2 coding sequences in pMAL-c2x showed resistance to H2O2 at 0.8 mM concentration by in vivo H2O2 tolerance assay. AbTPx1 and AbTPx2 mRNA were constitutively expressed in gill, mantle, abductor muscle and digestive tract in a tissue specific manner. Additionally, both TPxs mRNA were up-regulated in gill and digestive tract tissues against H2O2 at 3h post injection. The results indicate that AbTPx1 and AbTPx2 gene expressions are induced by oxidative stress and their respective proteins function in the detoxification of different ROS molecules to maintain efficient antioxidant defense in disk abalone.

The thioredoxin (Trx) system plays a significant role in cellular antioxidative defense by dismutating the surpluses of reactive oxygen species. Thus, the role of thioredoxin reductase (TrxR) cannot be ignored, owing to its participation in initiating the Trx enzyme cascade. Here, we report the identification and molecular characterization of a teleostean TrxR (RbTrxR-3) ortholog that showed high similarity with the TrxR-3 isoforms of other vertebrates. The complete RbTrxR-3 coding sequence comprised 1800 nucleotides, encoding a 600-amino acid protein with a predicted molecular mass of ~ 66 kDa. RbTrxR-3 consisted of 16 exons separated by 15 introns and had a total length of 12,658 bp. In silico analysis of the RbTrxR-3 protein sequence revealed that it possesses typical TrxR domain architecture. Moreover, using multiple sequence alignment and pairwise sequence alignment strategies, we showed that RbTrxR-3 has high overall sequence similarity to other teleostean TrxR-3 proteins, including highly conserved active site residues. Phylogenetic reconstruction of RbTrxR-3 affirmed its close evolutionary relationship with fish TrxR-3 orthologs, as indicated by its clustering pattern. RbTrxR-3 transcriptional analysis, performed using quantitative polymerase chain reaction (qPCR), showed that RbTrxR-3 was ubiquitously distributed, with the highest level of mRNA expression in the blood, followed by the gill, and liver. Live bacterial and viral stimuli triggered the modulation of RbTrxR-3 basal transcription in liver tissues that correlated temporally with that of its putative substrate, rock bream thioredoxin1 under the same conditions of pathogenic stress. Finally, resembling the typical function of TrxR protein, purified recombinant RbTrxR-3 showed detectable dose-dependent thiol reductase activity against 5,5′-dithiobis (2-nitrobenzoic) acid. Taken together, these results suggest that RbTrxR-3 plays a role in the host Trx system under conditions of oxidative and pathogenic stress. [ABSTRACT FROM AUTHOR]

Antioxidative defense renders a significant protection against environmental stress in organisms and maintains the correct redox balance in cells, thereby supporting proper immune function. Catalase is an indispensable antioxidant in organisms that detoxifies hydrogen peroxides produced in cellular environments. In this study, we sought to molecularly characterize a homolog of catalase (RfCat), identified from black rockfish ( Sebastes schlegelii ). RfCat consists of a 1581 bp coding region for a protein of 527 amino acids, with a predicted molecular weight of 60 kD. The protein sequence of RfCat harbored similar domain architecture to known catalases, containing a proximal active site signature and proximal heme ligand signature, and further sharing prominent homology with its teleostan counterparts. As affirmed by multiple sequence alignments, most of the functionally important residues were well conserved in RfCat. Furthermore, our phylogenetic analysis indicates its common vertebrate ancestral origin and a close evolutionary relationship with teleostan catalases. Recombinantly expressed RfCat demonstrated prominent peroxidase activity that varied with different substrate and protein concentrations, and protected against DNA damage. RfCat mRNA was ubiquitously expressed among different tissues examined, as detected by qPCR. In addition, RfCat mRNA expression was modulated in response to pathogenic stress elicited by Streptococcus iniae and poly I:C in blood and spleen tissues. Collectively, our findings indicate that RfCat may play an indispensable role in host response to oxidative stress and maintain a correct redox balance after a pathogen invasion. [ABSTRACT FROM AUTHOR]

Glutathione peroxidase (GPx) is an essential member of the antioxidant systems of living organisms and may be involved in immune defense against pathogenic invasion. In the current study, two selenium-dependent glutathione peroxidases ( AbSeGPx s) that shared 54.3% identity were identified from the disk abalone Haliotis discus discus . The open reading frames (ORFs) of AbSeGPx-a and AbSeGPx-b coded for 222 and 220 amino acids, respectively, with a characteristic selenocysteine residue encoded by an opal stop codon (TGA). The conserved selenocysteine insertion sequence (SECIS) element was predicted in the 3′ untranslated region (UTR) of both isoforms, and they were found to form two stem-loop structures. Amino acid comparison and phylogenetic studies revealed that the AbSeGPxs were closely related to those in other mollusk species and were evolutionarily distinct from those of other taxonomic groups. The SYBR Green qPCR was employed in investigating the transcripts of AbSeGPx s. The expression of AbSeGPx s mRNA was examined in different embryonic developmental stages and differential expression patterns for AbSeGPx-a and AbSeGPx-b were noted. Meanwhile, the highest expression of AbSeGPx s was detected in the hepatopancreas of healthy adult animals. Next, transcriptional levels were profiled in hemocytes of adults to determine the immune responses of AbSeGPx s to microbial infections. The results revealed the significant up-regulation of AbSeGPx-a in a time-dependent manner after bacterial ( Listeria monocytogenes and Vibrio parahaemolyticus ) and viral (viral hemorrhagic septicemia virus) infections. Consequently, these findings indicate that AbSeGPx-a and AbSeGPx-b might be involved in the embryonic development of disk abalone and the regulation of immune defense system of adult animals. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Superoxide dismutases (SODs), antioxidant metalloenzymes, represent the first line of defense in biological systems against oxidative stress caused by excessive reactive oxygen species (ROS), in particular . Two distinct members of SOD family were identified from Manila clam Ruditapes philippinarum (abbreviated as RpMnSOD and RpCu/ZnSOD). The structural analysis revealed all common characteristics of SOD family in both RpSODs from primary to tertiary levels, including three MnSOD signatures and two Cu/ZnSOD signatures as well as invariant Mn2+- and Cu/Zn2+-binding sites in RpMnSOD and RpCu/ZnSOD, respectively. Putative RpMnSOD and RpCu/ZnSOD proteins were predicted to be localized in mitochondrial matrix and cytosol, respectively. They shared 65.2% and 63.9% of identity with human MnSOD and Cu/ZnSOD, respectively. Phylogentic evidences indicated the emergence of RpSODs within molluscan monophyletic clade. The analogous spatial expression profiles of RpSODs demonstrated their higher mRNA levels in hemocytes and gills. The experimental challenges with poly I:C, lipopolysaccharide and Vibrio tapetis illustrated the time-dependent dynamic expression of RpSODs in hemocytes and gills. The recombinant RpMnSOD was expressed in a prokaryotic system and its antioxidant property was studied. The rRpMnSOD exhibited its optimum activity at 20 °C, under alkaline condition (pH 9) with a specific activity of 3299 U mg−1. These outcomes suggested that RpSODs were constitutively expressing inducible proteins that might play crucial role(s) in innate immunity of Manila clam. [Copyright &y& Elsevier]

Abstract: Glutathione S-Transferases (GSTs) are multifunctional cytosolic isoenzymes, distinctly known as phase II detoxification enzymes. GSTs play a significant role in cellular defense against toxicity and have been identified in nearly all organisms studied to date, from bacteria to mammals. In this study, we have identified a full-length cDNA of the theta class GST from Ruditapes philippinarum (RpGSTθ), an important commercial edible molluscan species. RpGSTθ was cloned and the recombinant protein expressed, in order to study its biochemical characteristics and determine its physiological activities. The cDNA comprised an ORF of 693bp, encoding 231 amino acids with a predicted molecular mass of 27kDa and an isoelectric point of 8.2. Sequence analysis revealed that RpGSTθ possessed characteristic conserved domains of the GST_N family, Class Theta subfamily (PSSM: cd03050) and GST_C_family Super family (PSSM: cl02776). Phylogenetic analysis showed that RpGSTθ evolutionarily linked with other theta class homologues. The recombinant protein was expressed in Escherichia coli BL21(DE3) cells and the purified enzyme showed high activity with GST substrates like CDNB and 4-NBC. Glutathione dependent peroxidase activity of GST, investigated with cumene hydroperoxide as substrate affirmed the antioxidant property of rRpGSTθ. By quantitative PCR, RpGSTθ was found to be ubiquitously expressed in all tissues examined, with the highest levels occurring in gills, mantle, and hemocytes. Since GSTs may act as detoxification enzymes to mediate immune defense, the effects of pathogen associated molecular pattern, lipopolysaccharide and intact Vibrio tapetis bacteria challenge on RpGSTθ gene transcription were studied. Furthermore, the RpGSTθ expression changes induced by immune challenges were similar to those of the antioxidant defense enzyme manganese superoxide dismutase (RpMnSOD). To our knowledge, RpGSTθ is the first molluscan theta class GST reported, and its immune-related role in Manila clam may provide insights into potential therapeutic targets for protecting this important aquaculture species. [Copyright &y& Elsevier]

Abstract: Thioredoxin (TRx) is a ubiquitous protein involved in the regulation of multiple biological processes. The TRx-2 isoform is exclusively expressed in mitochondria, where it contributes to mitochondrial redox state maintenance. In the present study, a novel thioredoxin-2 gene was identified in the Manila clam, Ruditapes philippinarum. The full-length sequence of RpTRx-2 (1561bp) consists of a 498bp coding region encoding a 166 amino acid protein. The N-terminal region of RpTRx-2 harbors a mitochondrial localization signal (56 amino acids), while the C-terminal portion contains the characteristic 89WCGPC93 catalytic active site. Phylogenetic analysis revealed that RpTRx-2 is closest to its ortholog from abalone. The broad distribution pattern of RpTRx-2 mRNA in healthy animal tissues implicates a generally significant function in normal clam physiology. The transcription level of RpTRx-2, however, is highest in hemocytes. Lipopolysaccharide and Vibrio tapetis bacterium caused up-regulation of the RpTrx-2 transcript levels in gill and hemocytes. Interestingly, clam manganese superoxide dismutase (MnSOD) mRNA levels in hemocytes elicited a corresponding response to these immune challenges. RpTRx-2 was recombinantly expressed in Escherichia coli BL21 (DE3) and used in insulin disulfide reduction assay as well as metal-catalyzed oxidation assay to elucidate its antioxidant property by reducing substrate and protecting super-coiled DNA from oxidative damage through free radical scavenging, respectively. Collectively, our data indicated that RpTRx-2, a mitochondrial TRx-2 family member, is an antioxidant enzyme that may be involved in antibacterial defense of clams. [Copyright &y& Elsevier]

Abstract: In this study, we investigated the gene expression profiling of disk abalone, Haliotis discus discus challenged by a mixture of three pathogenic bacteria Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes using a cDNA microarray. Upon bacteria challenge, 68 (1.6%) and 112 (2.7%) gene transcripts changed their expression levels ≥2 or ≤2 -fold in gills and digestive tract, respectively. There were 46 tissue-specific transcripts that up-regulated specifically in the digestive tract. In contrast, only 13 transcripts showed gill-specific up-regulation. Quantitative real-time PCR was performed to verify microarray data and results revealed that candidate genes namely Krüppell-like factor (KLF), lachesin, muscle lim protein, thioredoxin-2 (TRx-2), nuclear factor interleukin 3 (NFIL-3) and abalone protein 38 were up-regulated. Also, our results further indicated that bacteria challenge may activate the transcription factors or their activators (Krüppell-like factor, inhibitor of NF-κB or Ik-B), inflammatory cytokines (IL-3 regulated protein, allograft inflammatory factor), other cytokines (IFN-44-like protein, SOCS-2), antioxidant enzymes (glutathione-S-transferase, thioredoxin-2 and thioredoxin peroxidase), and apoptosis-related proteins (TNF-α, archeron) in abalone. The identification of immune and stress response genes and their expression profiles in this microarray will permit detailed investigation of the stress and immune responses of abalone genes. [Copyright &y& Elsevier]