Cheol Young Choi

Activity of antioxidant enzymes and physiological responses in ark shell, Scapharca broughtonii, exposed to thermal and osmotic stress: effects on hemolymph and biochemical parameters.

Changes in water temperature and salinity are responsible for a variety of physiological stress responses in aquatic organisms. Stress induced by these factors was recently associated with enhanced reactive oxygen species (ROS) generation, which caused oxidative damage. In the present study, we investigated the time-related effects of changes in water temperature and salinity on mRNA expression and the activities of antioxidant enzymes (SOD and CAT) and lipid peroxidation (LPO) in the gills and digestive glands of the ark shell, Scapharca broughtonii. To investigate physiological responses, hydrogen peroxide (H(2)O(2)), lysozyme activity, aspartate aminotransferase (AspAT), and alanine aminotransferase (AlaAT) were measured in the hemolymph. Water temperature and salinity changes significantly increased antioxidant enzyme mRNA expression and activity in the digestive glands and gills in a time-dependent manner. H(2)O(2) concentrations increased significantly in the high-temperature and hyposalinity treatments. LPO, AspAT and AlaAT levels also increased significantly in a time-dependent manner, while lysozyme activity decreased. These results suggest that antioxidant enzymes play important roles in reducing oxidative stress in ark shells exposed to changes in water temperature and salinity.

Characterization and expression analysis of a goose-type lysozyme from the rock bream Oplegnathus fasciatus, and antimicrobial activity of its recombinant protein

Lysozyme (muramidase) represents an important defense molecule of the fish innate immune system. Known for its bactericidal properties, lysozyme catalyzes the hydrolysis of β-(1,4)-glycosidic bonds between the N-acetyl glucosamine and N-acetyl muramic acid in the peptidoglycan layer of bacterial cell walls. In this study, the complete coding sequence of a g-type lysozyme (RBgLyz) was identified in the Oplegnathus fasciatus rock bream fish genome by means of multi-tissue normalized cDNA pyrosequencing using Roche 454 GS-FLX™ technology. RBgLyz is composed of 669 bp, with a 567 bp open reading frame that encodes 188 amino acids. Protein motif searches indicated that RBgLyz contains the soluble lytic transglycosylase domain involved in maintaining cell wall integrity. Furthermore, RBgLyz shares significant identity (81.4%) with Chinese perch Siniperca chuatsi. Quantitative real-time RT-PCR analysis results showed that RBgLyz transcripts are constitutively expressed in various tissues from healthy rock breams. In order to determine RBgLyz function in immunity, its expression was analyzed in head kidney following exposure to known immune stimulants or pathogens. RBgLyz transcripts were significantly up-regulated in response to challenge with lipopolysaccharide (LPS) and Edwardsiella tarda, as compared to non-injected control fish. Polyinosinic:polycytidylic acid (poly I:C) dsRNA stimulated a moderate expression of RBgLyz, as did Streptococcus iniae but to a lesser extent. There were no specific time-dependent effects on RBgLyz mRNA expression observed in response to rock bream iridovirus (RBIV) infection. Taken together, the gene expression results indicated that g-type lysozyme plays a role in the innate immune response to LPS, poly I:C, E. tarda and S. iniae in rock bream. Thus, we generated recombinant RBgLyz in an Escherichia coli expression system and characterized its antimicrobial activity. Our results indicated that recombinant RBgLyz had lytic activity against Gram-negative Vibrio salmonicida, Gram-positive Listeria monocytogenes, S. iniae and Micrococcus lysodeikticus. In addition, observations by scanning electron microscope (SEM) confirmed that the cell morphology of M. lysodeikticus was altered in the presence of recombinant RBgLyz.

Molecular evidence for the existence of lipopolysaccharide-induced TNF-α factor (LITAF) and Rel/NF-kB pathways in disk abalone (Haliotis discus discus)

The lipopolysaccharide-induced TNF-alpha factor (LITAF) and Rel family nuclear factor kappaB (Rel/NF-kB) are two important transcription factors which play major roles in the regulating inflammatory cytokine, apoptosis and immune related genes. Here, we report the discovery of disk abalone LITAF (AbLITAF) and Rel/NF-kB (AbRel/NF-kB) homologues and their immune responses. Full-length cDNA of AbLITAF consists of 441 bp open reading frame (ORF) that translates into putative peptide of 147 aa. Analysis of AbLITAF sequence showed it has characteristic LITAF (Zn(+2)) binding domain with two CXXC motifs. Phylogenetic analysis results further revealed that AbLITAF is a member of LITAF family. AbRel/NF-kB is 584 aa protein that contains several characteristic motifs including Rel homology domain (RHD), Rel protein signature, DNA binding motif, nuclear localization signal (NLS) and transcription factor immunoglobulin - like fold (TIG) similar to their invertebrate and vertebrate counterparts. Tissue specific analysis results showed that both AbLITAF and AbRel/NF-kB mRNA was expressed ubiquitously in all selected tissues in constitutive manner. However, constitutive expression of AbLITAF was higher than AbRel/NF-kB in all tissues except mantle. Upon immune challenge by bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes) and viral hemoragic septicemia virus (VHSV), AbLITAF showed the significant up-regulation in gills while AbRel/NF-kB transcription was not change significantly. Based on transcriptional response against immune challenge, we could suggest that regulation of TNF-alpha expression may have occurred mainly by LITAF activation rather than NF-kB in disk abalone. The cumulative data from other molluscs and our data with reference to TNF-alpha, LITAF and Rel/NF-kB from disk abalone provide strong evidence that LITAF and NF-kB are independent pathways likely to occur throughout the Phylum mollusca.

A novel molluscan sigma-like glutathione S-transferase from Manila clam, Ruditapes philippinarum: Cloning, characterization and transcriptional profiling

Glutathione S-transferases (GSTs) are versatile enzymes, act as primary intracellular detoxifiers and contribute to a broad range of physiological processes including cellular defense. In this study, a full-length cDNA representing a novel sigma-like GST was identified from Manila clam, Ruditapes philippinarum (RpGSTσ). RpGSTσ (884 bp) was found to possess an open reading frame of 609 bp. The encoded polypeptide (203 amino acids) had a predicted molecular mass of 23.21 kDa and an isoelectric point of 7.64. Sequence analysis revealed two conserved GST domain profiles in N- and C-termini. Alignment studies revealed that the identity between deduced peptides of RpGSTσ and known GSTσ members was relatively low (<35%), except a previously identified Manila clam GSTσ isoform (87.2%). Phylogenetic analysis indicated that RpGSTσ clustered together with molluscan GSTσ homologs, which were closely related to insect GSTσs. The RpGSTσ was subsequently cloned and expressed as recombinant protein, in order to characterize its biological activity. The recombinant RpGSTσ exhibited characteristic glutathione conjugating catalytic activity toward 1-chloro-2,4-dinitrobenzene, 3,4-dichloronitrobenzene and ethacrynic acid. It had an optimal pH and temperature of 8.0 and 35 °C, respectively. Expression profiles under normal conditions and in response to lipopolysaccharide-, poly I:C- and Vibrio tapetis-challenges were also investigated. RpGSTσ demonstrated a differential tissue distribution with robust transcription in gills of normal animals. We explored potential association of GSTσ in cellular defense during bacterial infection and found that in challenged clams, RpGSTσ gene was significantly induced in internal and external tissues, in conjunction with manganese- as well as copper-zinc superoxide dismutase (MnSOD and CuZnSOD) genes. Moreover, the induction was remarkably higher in hemocytes than in gill. Collectively, our findings suggested that RpGSTσ could play a significant role in cellular defense against oxidative stress caused by bacteria, in conjunction with other antioxidant enzymes, such as SODs.

Effects of LED light spectra on oxidative stress and the protective role of melatonin in relation to the daily rhythm of the yellowtail clownfish, Amphiprion clarkii

The present study aimed to test the effects of melatonin on oxidative stress in the yellowtail clownfish, Amphiprion clarkii, as produced by light emitting diodes (LEDs): red, green, and blue. We investigated the effects of the different LEDs on oxidative stress by measuring the mRNA expression of arylalkylamine N-acetyltransferase (AANAT2), the expression and activities of antioxidant enzymes (superoxide dismutase, SOD (EC 1.15.1.1); and catalase, CAT (EC 1.11.1.6)), and plasma H2O2 and plasma melatonin levels. In red light, the expression of AANAT2, SOD, and CAT mRNA was significantly higher than those under the other light spectra. SOD and CAT activities and plasma H2O2 and melatonin levels were also significantly higher for the red spectra than those for the other light spectra. These results indicate that red light induces oxidative stress. To investigate the effects of melatonin on oxidative stress, we injected melatonin into live fish (in vivo) or treated cultured pineal organ (in vitro) with melatonin. We found that AANAT2, SOD, and CAT mRNA expression levels, SOD and CAT activities, and plasma H2O2, lipid peroxidation (LPO) and melatonin levels were significantly lower than those for the controls. Therefore, our results indicate that red light induces oxidative stress and melatonin plays the role of a strong antioxidant in yellowtail clownfish.

A manganese superoxide dismutase (MnSOD) from Ruditapes philippinarum: Comparative structural- and expressional-analysis with copper/zinc superoxide dismutase (Cu/ZnSOD) and biochemical analysis of its antioxidant activities

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 O(2)(•-). 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 Mn(2+)- and Cu/Zn(2+)-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 20xa0°C, under alkaline condition (pH 9) with a specific activity of 3299xa0Uxa0mg(-1). These outcomes suggested that RpSODs were constitutively expressing inducible proteins that might play crucial role(s) in innate immunity of Manila clam.

Allograft inflammatory factor-1 in disk abalone (Haliotis discus discus): molecular cloning, transcriptional regulation against immune challenge and tissue injury.

Here, we report the identification and characterization of allograft inflammatory factor-1 (AIF-1) from disk abalone Haliotis discus discus that was denoted as AbAIF-1. The full-length cDNA of AbAIF-1 consists of a coding region (453 bp) for 151 amino acids with a 17 kDa molecular mass. Analysis of AbAIF-1 sequence showed that it shares characteristic two EF hand Ca(+2)-binding motifs. Results from phylogenetic analysis further confirm that AbAIF-1 is a member of the AIF-1 family similar to invertebrate and vertebrate counterparts suggesting it has high evolutional conservation. Tissue-specific expression and transcriptional regulation of AbAIF-1 were analyzed after bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes), viral hemorrhagic septicemia virus (VHSV) immune challenge and during tissue injury by quantitative real-time PCR. It is shown that the expression of AbAIF-1 mRNA was expressed ubiquitously in all selected tissues in constitutive manner showing the highest level in hemocytes. Upon bacteria and VHSV challenge, AbAIF-1 showed the significant up-regulation in hemocytes than gills. After the tissue injury in shell and mantle, AbAIF-1 and antioxidant selenium-dependant glutathione peroxidase (SeGPx) transcripts were significantly upregulated in abalone hemocytes. Taken together, these findings suggest that AIF-1 could response against the pathogenic challenge or tissue injury in abalone like mollusks. Also, AbAIF-1 may involve in wound healing and shell repair after the tissue injury of abalone.

Validation of housekeeping genes as internal controls for studying biomarkers of endocrine-disrupting chemicals in disk abalone by real-time PCR

Our experiments were designed to identify suitable housekeeping genes (HKGs) in disk abalone as internal controls to quantify biomarker expression following endocrine disrupting chemicals (EDCs). Relative expression levels of twelve candidate HKGs were examined by real-time reverse transcription PCR (qRT-PCR) in gill and hepatopancreas of abalone following a 7-day challenge with either tributyltin chloride (TBT) or 17β-estradiol (E2). The expression levels of several conventional HKGs, such as 18s rRNA, glyceraldehyde-3-phosphate dehydrogenase and β-actin, were significantly altered by the challenges, indicating that they might not be suitable internal controls. Instead, the geNorm analysis pinpointed ribosomal protein L-5/ elongation factor 1 and ribosomal protein L-5/ succinate dehydrogenase as the most stable HKGs under TBT and E2 challenges, respectively. Moreover, these three HKGs also showed the highest stabilities overall amongst different tissues, genders and EDC challenges. The expression of a biomarker gene, cytochrome P450 4B (CYP4), was also investigated and exhibited a significant increase after the challenges. Importantly, when unsuitable HKGs were used for normalization, the influence of two EDCs on CYP4 expression was imprecisely overestimated or underestimated, which strongly emphasized the importance of selecting appropriately validated HKGs as internal controls in biomarker studies.

Rock bream (Oplegnathus fasciatus) serpin, protease nexin-1: transcriptional analysis and characterization of its antiprotease and anticoagulant activities.

Protease nexin-1 (PN-1) is a serine protease inhibitor (SERPIN) protein with functional roles in growth, development, patho-physiology and injury. Here, we report our work to clone, analyze the expression profile and characterize the properties of the PN-1 gene in rock bream (Rb), Oplegnathus fasciatus. RbPN-1 encodes a peptide of 397 amino acids (AA) with a predicted molecular mass of 44 kDa and a 23 AA signal peptide. RbPN-1 protein was found to harbor a characteristic SERPIN domain comprised of a SERPIN signature and having sequence homology to vertebrate PN-1s. The greatest identity (85%) was observed with PN-1 from the three-spined stickleback fish, Gasterosteus aculeatus. The functional domains, including a heparin binding site and reactive centre loop were conserved between RbPN-1 and other fish PN-1s; in particular, they were found to correspond to components of the human plasminogen activator inhibitor 1, PAI-1. Phylogenetic analysis indicated that RbPN-1 was closer to homologues of green spotted pufferfish and Japanese pufferfish. Recombinant RbPN-1 demonstrated antiprotease activity against trypsin (48%) and thrombin (89%) in a dose-dependent manner, and its antithrombotic activity was potentiated by heparin. The anticoagulant function prolonged clotting time by 3.7-fold, as compared to the control in an activated partial thromboplastin time assay. Quantitative real-time PCR results indicated that RbPN-1 is transcribed in many endogenous tissues at different levels. Lipopolysaccharide (LPS) stimulated a prolonged transcriptional response in hematic cells, and Rb iridovirus up-regulated the RbPN-1 mRNA level in hematic cells to a maximum of 3.4-fold at 12 h post-infection. Interestingly, LPS and Edwardsiella tarda significantly induced the RbPN-1 transcription at the late phase of infection. In vivo studies indicated that injury response caused a temporal suppression in RbPN-1 transcription, in conjunction with that of another SERPIN, rock bream heparin cofactor II, RbHCII. Taken together, our findings suggest that PN-1 functions as an antiprotease and anticoagulant and that SERPINs (PN-1 and HCII) are likely to contribute to immunity and post-injury responses.

Profiles of antioxidant gene expression and physiological changes by thermal and hypoosmotic stresses in black porgy (Acanthopagrus schlegeli).

We determined oxidative stress by measuring the expression and activity of 3 antioxidant enzymes [Cu/Zn-superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPX)] in black porgy exposed to thermal (20 degrees C-->30 degrees C) and hypoosmotic (35 psu-->10 psu and 0 psu) stresses. The expression and activity of antioxidant enzymes were significantly higher after exposure to 30 degrees C, 10 psu, and 0psu. Furthermore, we measured H(2)O(2) and lipid peroxidation (LPO) levels. As a result, H(2)O(2) and LPO levels were significantly increased after exposure to thermal (20 degrees C-->30 degrees C) and hypoosmotic stress (35 psu-->10 psu and 0 psu) stress. These results indicate that thermal and hypoosmotic stress induces oxidative stress in black porgy. Additionally, we investigated the changes due to thermal and hypoosmotic stress by measuring plasma cortisol and ion (Na(+) and Cl(-)) levels. Plasma cortisol levels increased at 30 degrees C and at 10 psu and then decreased at 0 psu. However, plasma Na(+) and Cl(-) levels did not change after exposure to thermal stress (30 degrees C), and decreased at 10 psu and 0 psu. In conclusion, thermal and hypoosmotic environments increase oxidative stress, thereby these results may be indicators of oxidative stress in black porgy.