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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>
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>
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,Lee, Seongdo,Yang, Hyerim,Lee, Jehee ACADEMIC PRESS LTD 2019 FISH AND SHELLFISH IMMUNOLOGY Vol.91 No.-
<P><B>Abstract</B></P> <P>Representing a multifunctional complex group of proteins, glutathione S- transferases (GSTs) play a major role in the phase II detoxification process in a wide range of organisms. This study focused on the potential detoxification ability of disk abalone (<I>Haliotis discus discus</I>) GST theta (<I>AbGST-θ</I>) under different stress conditions with special reference to post immune challenges. Characterization of AbGST-θ revealed with 226 amino acids, 26.6 kDa of predicted molecular mass and 8.9 of theoretical isoelectric point. As illustrated in the multiple sequence alignment, eight glutathione binding sites (G-sites) and ten substrate binding sites (H-sites) were identified in well-distinct N-terminal and C-terminal domains of AbGST-θ, respectively. AbGST-θ exhibited its highest sequence identity with <I>Mizuhopecten yessoensis</I> (59.1%) and the phylogenetic tree clearly positioned AbGST-θ with pre-defined GST-θ molluscan homologues. The <I>AbGST-θ</I> was highly expressed in the digestive tract of un-challenged abalones. Upon administering the challenge experiment, <I>AbGST-θ</I> showed significant modulations in their transcriptional levels depending on the time and the tissue type. The optimum temperature was 37 °C and optimum pH was 7.5 for AbGST-θ. The determined enzyme kinetic parameters of AbGST-θ showed low affinity towards 1-Chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates. Nonetheless, with Cibacron blue IC<SUB>50</SUB> (half maximal inhibitory concentration) was calculated to be 0.08 μM while observing 100% inhibition with 100 μM. Furthermore, AbGST-θ resulted in significant protection ability towards H<SUB>2</SUB>O<SUB>2</SUB>, CdCl<SUB>2,</SUB> and ZnCl<SUB>2</SUB> in the disk diffusion assay. Collectively, this study provides evidences for the detoxification ability and the immunological host defensive capability of <I>AbGST-θ</I> in disk abalone<I>.</I> </P> <P><B>Highlights</B></P> <P> <UL> <LI> We identified Glutathione S-transferases theta from disk abalone (AbGST-θ). </LI> <LI> AbGST-θ revealed with 226 amino acids. </LI> <LI> <I>AbGST-θ</I> was highly expressed in the digestive tract of un-challenged abalones. </LI> <LI> <I>AbGST-θ</I> mRNA expression level significantly increased after immune challenge. </LI> <LI> Enzyme kinetics, optimum conditions and heavy metal stress responses were measured. </LI> </UL> </P>
Tharuka, M.D. Neranjan,Priyathilaka, Thanthrige Thiunuwan,Yang, Hyerim,Pavithiran, Amirthalingam,Lee, Jehee ACADEMIC PRESS LTD 2019 FISH AND SHELLFISH IMMUNOLOGY Vol.86 No.-
<P><B>Abstract</B></P> <P>Viperin is recognized as an antiviral protein that is stimulated by interferon, viral exposures, and other pathogenic molecules in vertebrate. In this study, a viperin homolog in the Big-belly seahorse (<I>Hippocampus abdominalis</I>; HaVip) was functionally characterized to determine its subcellular localization, expression pattern, and antiviral activity <I>in vitro</I>. The <I>HaVip</I> coding sequence encodes a 348 amino acid polypeptide with predicted molecular weight of 38.48 kDa. Sequence analysis revealed that HaVip comprises three main domains: the N-terminal amphipathic α-helix, a radical S-adenosyl-<SMALL>L</SMALL>-methionine (SAM) domain, and a conserved C-terminal domain. Transfected GFP-tagged HaVip protein was found to localize to the endoplasmic reticulum (ER). Overexpressed-HaVip in FHM cells was found to significantly reduce viral capsid gene expression in VHSV infection <I>in vitro</I>. Under normal physiological conditions, <I>HaVip</I> expression was ubiquitously detected in all 14 examined tissues of the seahorse, with the highest expression observed in the heart, followed by skin and blood. <I>In vivo</I> studies showed that <I>HaV</I>ip was rapidly and predominantly upregulated in blood, kidney, and intestinal tissue upon poly (I:C) stimulus. LPS and <I>Streptococus iniae</I> challenges caused a significant increase in expression of <I>HaVip</I> in all the analyzed tissues. The obtained results suggest that HaVip is involved in the immune system of the seahorse, triggering antiviral and antibacterial responses, upon viral and bacterial pathogenic infections.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Identified and characterized viperin from big-belly seahorse. </LI> <LI> The HaVip gene was ubiquitously expressed in unchallenged-fish tissues. </LI> <LI> Modulated HaVip transcription pattern revealed its contribution in the immune response. </LI> <LI> Overexpression of HaVip in FHM cells showed the antiviral activity. </LI> </UL> </P>