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Priyathilaka, Thanthrige Thiunuwan,Bathige, S.D.N.K.,Lee, Seongdo,Lee, Jehee Elsevier 2018 Developmental and comparative immunology Vol.79 No.-
<P><B>Abstract</B></P> <P>Myeloid differentiation factor 88 (MyD88) is a crucial adaptor protein of the Toll-like receptor (TLR)- and interleukin 1 receptor-mediated signaling pathways and is involved in a diverse array of inflammatory responses via NF-κB activation. In the present study, two MyD88 variants were identified from disk abalone (<I>Haliotis discus discus</I>) and designated AbMyD88-2 and AbMyD88-X. The deduced AbMyD88-2 and AbMyD88-X comprised 433 and 354 amino acids with predicted molecular masses of 48.85 kDa and 40.17 kDa, respectively. AbMyD88-2 and AbMyD88-X possessed typical MyD88 domain structural features including an N-terminal death domain (DD) and C-terminal toll interleukin 1 receptor (TIR) domain similar to those in mammals. Expression analysis of <I>AbMyD88-2</I> and <I>AbMyD88-X</I> mRNA at different early embryonic developmental stages of abalone by qPCR revealed that their constitutive expression at all developmental stages analyzed with the considerably higher values at the 16-cell (<I>AbMyD88-</I>2) and morula stages (<I>AbMyD88-X</I>). In unchallenged disk abalones, <I>AbMyD88-2</I> was highly expressed in muscles, while <I>AbMyD88-X</I> mRNA was predominantly transcribed in hemocytes. Moreover, <I>AbMyD88-2</I> and <I>AbMyD88-X</I> mRNA were differentially modulated in abalone hemocytes after a challenge with live bacteria (<I>Vibrio parahaemolyticus</I>, <I>Listeria monocytogenes</I>), virus (viral hemorrhagic septicemia virus), and pathogen-associated molecular patterns (lipopolysaccharides and Poly I:C). Overexpression of AbMyD88-2 and AbMyD88-X in HEK293T cells induced the activation of the NF-κB promoter. AbMyD88-2 and AbMyD88-X involvement in inflammatory responses was characterized by their overexpression in RAW264.7 murine macrophage cells. These results revealed comparatively higher NO (Nitric oxide) production, induction of inflammatory mediator genes (<I>iNOS</I> and <I>COX2</I>), and proinflammatory genes (<I>IL1β, IL6</I> and <I>TNFα</I>) expression in abalone MyD88s-overexpressing cells than in mock control in the presence or absence of LPS stimulation. Altogether, these results suggest that existence of a MyD88-dependent like signaling pathway in disk abalone and that both AbMyD88-2 and AbMyD88-X might be involved in innate immune and inflammatory responses.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Two MyD88 isoforms were identified from Disk abalone (AbMyD88-2 and AbMyD88-X). </LI> <LI> AbMyD88-2 and AbMyD88-X possessed typical MyD88 domain structural features. </LI> <LI> Both MyD88 isoforms were constitutive expressed at early embryonic developmental stages of disk abalone. </LI> <LI> The mRNA expression of AbMyD88-2 and AbMyD88-X was differentially modulated upon immune stimulation in vivo. </LI> <LI> AbMyD88-2 and AbMyD88-X involved in inflammatory responses via NF-κB activation. </LI> </UL> </P>
Priyathilaka, Thanthrige Thiunuwan,Oh, Minyoung,Bathige, S.D.N.K.,De Zoysa, Mahanama,Lee, Jehee Elsevier 2017 FISH AND SHELLFISH IMMUNOLOGY Vol.65 No.-
<P><B>Abstract</B></P> <P>CXC chemokine receptor 3 (CXCR3) and 4 (CXCR4) are members of the seven transmembrane G protein coupled receptor family, involved in pivotal physiological functions. In this study, seahorse <I>CXCR3</I> and <I>CXCR4</I> (designated as <I>HaCXCR3</I> and <I>HaCXCR4</I>) cDNA sequences were identified from the transcriptome library and subsequently molecularly characterized. <I>HaCXCR3</I> and <I>HaCXCR4</I> encoded 363 and 373 amino acid long polypeptides, respectively. The HaCXCR3 and HaCXCR4 deduced proteins have typical structural features of chemokine receptors, including seven transmembrane domains and a G protein coupled receptors family 1 profile with characteristic DRY motifs. Amino acid sequence comparison and phylogenetic analysis of these two CXC chemokine receptors revealed a close relationship to their corresponding teleost counterparts. Quantitative real time PCR analysis revealed that <I>HaCXCR3</I> and <I>HaCXCR4</I> were ubiquitously expressed in all the tested tissues, with highest expression levels in blood cells. The seahorse blood cells and kidney <I>HaCXCR3</I> and <I>HaCXCR4</I> mRNA expressions were differently modulated when challenged with <I>Edwardsiella tarda</I>, <I>Streptococcus iniae</I>, lipopolysaccharide, and polyinosinic:polycytidylic acid, confirming their involvement in post immune responses.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Full length coding sequences of CXC chemokine receptor 3 and 4 from Seahorse (HaCXCR3 and HaCXCR4) were identified. </LI> <LI> The HaCXCR3 and HaCXCR4 deduced proteins have typical structural features of chemokine receptors. </LI> <LI> The <I>HaCXCR3</I> and <I>HaCXCR4</I> mRNA was significantly up-regulated upon live pathogens and PAMPs in blood and kidney. </LI> </UL> </P>
넙치 (Paralichthys olivaceus) vasa 유전자의 full-length cDNA 분리 및 생식소 특이적 발현
정형복,김유철,김효원,Thanthrige Thiunuwan Priyathilaka,이성도,Viraj Udayantha Herath Mudiyanselage,최재영,황일선,진창남,허윤성,서종표,임봉수 제주대학교 해양과환경연구소 2014 해양과환경연구소 연구논문집 Vol.38 No.-
Until recent, primordial germ cells(PGCs) are recognized only by morphological observation, such as their large size and low nucleocytoplasmic ratio. For the molecular analysis of the reproduction, it is important to identify a specific marker of germ cell development and differentiation. The VASA, which was first identified in Drosophila, is reported as a germ-line cell specific marker gene in animals. Many other researches verified its germ cell specific expression during embryogenesis and gametogenesis. VASA is a member of the DEAD(Asp-Glu-Ala-Asp) protein family of ATP-dependent RNA helicase, and plays a critical role in germ-line cell linage. Vasa is expected to be an useful molecular marker for identification of PGCs in reproduction researches of aquaculture species. In this study, we isolated the vasa cDNA, and surveyed its gonad-specific expression in Paralichthys olivaceus. The full-length cDNA of P. olivaceus vasa cDNA was isolated and deduced amino acid sequence was compared to those of the other teleosts. It was 2,461bp long, consisted in 646 amino acids in its ORF region, 175bp of 5’-UTR, and 345bp 3’-UTR. Flounder VASA contained conserved DEAD box, arginine-glycine rich region, and other domains were found. Flounder vasa expressed strongly in the testis and ovary, confirming its property as an gonad-specific marker. These result is expected to be an useful marker for flounder reproduction research and related aquaculture industry.
Sandamalika, W.M. Gayashani,Priyathilaka, Thanthrige Thiunuwan,Liyanage, D.S.,Lee, Sukkyoung,Lim, Han-Kyu,Lee, Jehee Elsevier 2018 FISH AND SHELLFISH IMMUNOLOGY Vol.77 No.-
<P><B>Abstract</B></P> <P>Glutathione S-transferase (GST; EC 2.5.1.18) isoenzymes represent a complex group of proteins that are involved in phase II detoxification in several organisms. In this study, GST kappa (GSTκ) from the disk abalone (<I>Haliotis discus discus; AbGSTκ</I>) was characterized at both the transcriptional and functional levels to determine its potential capacity to perform as a detoxification agent under conditions of different stress. The predicted AbGSTκ protein consists of 227 amino acids, with a predicted molecular weight of 25.6 kDa and a theoretical isoelectric point (pI) of 7.78. <I>In silico</I> analysis reveals that <I>AbGSTκ</I> is a disulfide bond formation protein A (DsbA), consisting of a thioredoxin domain, GSH binding sites (G-sites), and a catalytic residue. In contrast, no hydrophobic ligand binding site (H-site), or signal peptides, were detected. <I>AbGSTκ</I> showed the highest sequence identity with the orthologue from pufferfish (<I>Takifugu obscurus</I>) (60.0%). In a phylogenetic tree, AbGSTκ clustered closely together with other fish GSTκs, and was evolutionarily distanced from other cytosolic GSTs. The predicted three-dimensional structure clearly demonstrates that the dimer adopts a butterfly-like shape. A tissue distribution analysis revealed that <I>GSTκ</I> was highly expressed in the digestive tract, suggesting it has detoxification ability. Depending on the tissue and time, <I>AbGSTκ</I> showed different expression patterns, and levels of expression, following challenge of the abalone with immune stimulants. Enzyme kinetics of the purified recombinant proteins demonstrated its conjugating ability using 1-Chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates, and suggested it has a low affinity for both substrates. The optimum temperature and pH for the rAbGSTκ GSH: CDNB conjugating activity were found to be 35 °C and pH 8, respectively indicating that the abalone is well adapted to a wide range of environmental conditions. Cibacron blue (100 μM) was capable of completely inhibiting rAbGSTκ (100%) with an IC<SUB>50</SUB> (half maximal inhibitory concentration) of 0.05 μM. A disk diffusion assay revealed that rAbGSTκ could significantly protect cells from H<SUB>2</SUB>O<SUB>2</SUB>, CdCl<SUB>2,</SUB> and ZnCl<SUB>2</SUB>. Altogether, this current study suggests that <I>AbGSTκ</I> is involved in detoxification and immunological host defense mechanisms and allows abalones to overcome stresses in order for them to have an increased chance of survival.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The full-length coding sequence of <I>AbGSTκ</I> was identified in disk abalone. </LI> <LI> <I>AbGSTκ</I> is a disulfide bond formation protein A (DsbA) with a thioredoxin domain. </LI> <LI> Highest mRNA expression of AbGSTκ was observed in digestive tract. </LI> <LI> Immunological responses of AbGSTκ has evaluated. </LI> <LI> Enzyme kinetics, optimum conditions and heavy metal stress responses were measured. </LI> </UL> </P>
Elvitigala, Don Anushka Sandaruwan,Priyathilaka, Thanthrige Thiunuwan,Whang, Ilson,Nam, Bo-Hye,Lee, Jehee Elsevier 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.44 No.1
<P><B>Abstract</B></P> <P>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 (<I>Sebastes schlegelii</I>). <I>RfCat</I> 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. <I>RfCat</I> mRNA was ubiquitously expressed among different tissues examined, as detected by qPCR. In addition, <I>RfCat</I> mRNA expression was modulated in response to pathogenic stress elicited by <I>Streptococcus iniae</I> 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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Homolog of catalase was identified from black rockfish (RfCat) RfCat. </LI> <LI> RfCat resembled typical catalase domain architecture. </LI> <LI> Recombinant RfCat (rRfCat) showed detectable peroxidase activity. </LI> <LI> rRfCat could notably protect bacterial cells and DNA from oxidative damage. </LI> <LI> Transcriptional level of RfCat was modulated under pathogenic stress. </LI> </UL> </P>