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Nikapitiya, Chamilani,Oh, Chul-Hong,Lee, Young-Deuk,Lee, Suk-Kyoung,Whang, Il-Son,Lee, Je-Hee The Korean Society of Fisheries and Aquatic Scienc 2010 Fisheries and Aquatic Sciences Vol.13 No.1
An agar-degrading Agarivorans sp. AG17 strain was isolated from the red seaweed Grateloupia filicina collected from Jeju Island. A beta-agarase gene from Agarivorans sp. AG17 was cloned and designated as agrA. agrA has a 2,985 bp coding region encoding 995 amino acids and was classified into the glycoside hydrolase family (GHF)-50. Predicted molecular mass of the mature protein was 105 kDa. His-tagged agrA was overexpressed in Escherichia coli and purified as a fusion protein. The enzyme showed 158.8 unit/mg specific activity (optimum temperature at $65^{\circ}C$ and pH 5.5 in acetate buffer) with unique biochemical properties (high thermal and pH stabilities). Enzyme produced neoagarohexaose, neoagarotetraose and neoagarobiose by degrading agar, and hydrolyzed neoagaro-oligosaccharides were biologically active. Hence the purified enzyme has potential for use in industrial applications such as the development of cosmetics and pharmaceuticals.
Nikapitiya, C.,Kim, W.S.,Park, K.,Kwak, I.S. Academic Press 2014 FISH AND SHELLFISH IMMUNOLOGY Vol.41 No.2
Macrophthalmus japonicus is an intertidal mud crab is an ecologically important species in Korea, can tolerate a wide range of natural and anthropogenic stressors. Environmental changes especially salinity cause physiological stress to the marine habitats. Differential gene transcription of M. japonicus tissues provided information about tissue specific responses against salinity. Five potential genes were identified and their transcription levels were determined quantitatively comparison to seawater (SW: 31 +/- 1 psu) in M. japonicus gills and hepatopancreas after exposed them to different salinities. Ecdysteroid receptor (Mj-EcR), trypsin (Mj-Tryp), arginine kinase (Mj-AK), lipopolysaccharide and β-1,3-glucan binding protein (Mj-LGBP) and peroxinectin (Mj-Prx) in hepatopancreas up-regulated against different salinities. In contrast, the gills, Mj-EcR, Mj-Tryp and Mj-AK showed late up-regulated responses to 40 psu compared to SW. All genes except Mj-LGBP showed up regulation in the gills as time dependent manner. These genes can be considered as potential markers to assess responses in salinity changes. This study suggests hepatopancreas is a suitable tissue for transcriptional, biochemical and physiological responses analysis on M. japonicus in low and high salinity stress.
Nikapitiya Chamilani,Zoysa Mahanama De,Ekanayake Prashani Mudika,Park Ho-Jin,Lee Je-Hee The Korean Society of Fisheries and Aquatic Scienc 2006 韓國養殖學會誌 Vol.19 No.1
Anticoagulant activities of a fermented edible brown alga, Laminaria ochotensis was investigated. L. ochotensis was fermented with 15% sugar (w/v) at $25^{\circ}C$ for 10 weeks. Anticoagulant activity was measured from the supernatant of algal mixture at biweekly intervals up to $10^{th}$ week by activated partial thromboplastin (APTT), prothrombin time (PT) and thrombin time (TT) assay using citrated human plasma. Sample having high APTT activity $(6^{th}\;week)$ was filtered, ethanol precipitated and freeze-dried. The polysaccharide compound having anticoagulant activity was purified by DEAE ion exchange chromatography followed by Sepharose-4B gel filtration chromatography. Anticoagulant activity, polysaccharide concentration, and heparin like activity were determined for the collected fractions by APTT, $phenol-H_2SO_4$, and glycosaminoglycan assay, respectively. The anticoagulant activity assay showed that the activity was increased up to $6^{th}$ week, and decreased thereafter. The concentration of our purified compound was $31.0{\mu}g/ml$ and showed higher APTT activity than commercial heparin. At the same concentration of $31.0{\mu}g/ml$, the heparin showed 186.5 sec activity while our purified compound showed an activity of 386 sec. Single spot on agarose gel electrophoresis showed that the compound was purified and polyacrylamide gel electrophoresis (PAGE) results revealed that the molecular mass of the purified polysaccharide compound was between 60 and 500 kDa. Therapeutic interest of the algal polysaccharide as an anticoagulant has recently been in highlighted. This purified anticoagulant compound from fermented L. ochotensis can be used as a model for anticoagulant agent or could be developed as an anticoagulant agent. This study can be extended to identify the structure and chemical composition of the purified polysaccharide, and to establish a relationship between structure and the function of the identified anticoagulant compounds.
Nikapitiya, Chamilani,Dananjaya, S.H.S.,De Silva, B.C.J.,Heo, Gang-Joon,Oh, Chulhong,De Zoysa, Mahanama,Lee, Jehee ACADEMIC PRESS LTD 2018 FISH AND SHELLFISH IMMUNOLOGY Vol.76 No.-
<P><B>Abstract</B></P> <P>Chitosan nanoparticles (CNPs) were synthesized by ionic gelation method and its immunomodulatory properties were investigated in zebrafish larvae. Average particle size and zeta potential were 181.2 nm and +37.2 mv, respectively. Initially, toxicity profile was tested in zebrafish embryo at 96 h post fertilization (hpf) stage using medium molecular weight chitosan (MMW-C) and CNPs. At 5 μg/mL, the hatching rate was almost similar in both treatments, however, the survival rate was lower in MMW-C compared to CNPs exposure, suggesting that toxicity effect of CNPs in hatched larvae was minimal at 5 μg/mL compared to MMW-C. Quantitative real time PCR results showed that in CNPs exposed larvae at 5 days post fertilization (5 dpf) stage, immune related (<I>il-1β</I>, <I>tnf-α</I>, <I>il-6</I>, <I>il-10</I>, <I>cxcl-18b</I>, <I>ccl34a.4</I>, <I>cxcl-8a</I>, <I>lyz-c</I>, <I>defβl-1</I>, <I>irf-1a</I>, <I>irf-3</I>, <I>MxA</I>) and stress response (<I>hsp-70</I>) genes were induced. In contrast, basal or down regulated expression of antioxidant genes (<I>gstp-1</I>, <I>cat</I>, <I>sod-1</I>, <I>prdx-4</I>, <I>txndr-1</I>) were observed. Moreover, zebrafish larvae (at 5 dpf stage) exposed to CNPs (5 μg/mL) showed higher survival rate at 72 h post infection stage against pathogenic <I>Aeromonas hydrophila</I> challenge compared to controls. These results suggest that although CNPs can have toxic effects to the larvae at higher doses, CNPs exposure at 5 μg/mL could enhance the immune responses and develop the disease resistance against <I>A. hydrophila</I>, which could be attributed to its strong immune modulatory properties.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Synthesized CNPs at 5 μg/L exposure was non-toxic to zebrafish at 96 hpf stage. </LI> <LI> CNPs showed immune modulatory and disease resistant against <I>A. hydrophila</I> in 5 dpf stage zebrafish after non-toxic exposure. </LI> <LI> Biodegradable CNPs could be a potential immunostimulant for larval aquaculture. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>
Nikapitiya, C.,De Zoysa, M.,Whang, I.,Kim, C.G.,Lee, Y.H.,Kim, S.J.,Lee, J. Academic Press 2009 FISH AND SHELLFISH IMMUNOLOGY Vol.27 No.2
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 H<SUB>2</SUB>O<SUB>2</SUB> in human leukemia THP-1 cells and provided protection against H<SUB>2</SUB>O<SUB>2</SUB>-induced apoptosis.
Nikapitiya, C.,De Zoysa, M.,Whang, I.,Kim, S.J.,Choi, C.Y.,Lee, J.S.,Lee, J. Academic Press 2010 FISH AND SHELLFISH IMMUNOLOGY Vol.29 No.2
The complete amino acid sequence of a calcium-regulatory gene (denoted as Ab-CaReg I) was identified from the disk abalone Haliotis discus discus cDNA library. The Ab-CaReg I is composed of 176 amino acids and the calculated molecular mass and isoelectric point were 20 and 4.2, respectively. The sequence homology of Ab-CaReg I was 28-30 and 18-27% of known calmodulin and troponin C, respectively. Four characteristic calcium-binding EF hand motifs with some modifications at conserved positions of known homologous calmodulin genes were observed in the sequence. The tissue-specific transcription analysis and variation of mRNA transcription level of Ab-CaReg I in gills and mantle after animals were immersed in seawater containing 2000 ppm CaCl<SUB>2</SUB>was quantified by SYBR Green real-time PCR analysis. Transcription variation of Ab-CaReg I in hemocytes and gills followed by bacteria challenge (Vibrio alginolyticus, Vibrio parahaemolyticus and Listeria monocytogenes) was used to investigate Ab-CaReg I in immune responses. Transcripts of Ab-CaReg I mRNA were mainly detected in hemocytes, mantle, muscle, gills, digestive tract and hepatopancreas with highest expression in hemocytes. The CaCl<SUB>2</SUB>immersion significantly altered the Ab-CaReg I mRNA transcription level by 3 h, compared to animals in normal seawater (control). The mRNA expression of Ab-CaReg I in gills and hemocytes was upregulated significantly to 11-fold and 4-fold in 3 h compared to control (uninfected), respectively, in bacteria-challenged abalones. The results suggest that Ab-CaReg I could be effectively induced to maintain internal Ca<SUP>2+</SUP>homeostasis of the animal due to influx of Ca<SUP>2+</SUP>in the cells by external stimuli such as a high dose of Ca<SUP>2+</SUP>and pathogens like bacteria.
Nikapitiya, C.,De Zoysa, M.,Oh, C.,Lee, Y.,Ekanayake, P.M.,Whang, I.,Choi, C.Y.,Lee, J.S.,Lee, J. Academic Press 2010 Fish & Shellfish Immunology Vol.28 No.4
A novel antistasin-like cDNA homologue named as Ab-Antistasin was isolated from the disk abalone Haliotis discus discus normalized cDNA library. The Ab-Antistasin (1398-bp) consisted of an 1185-bp open reading frame encoding 395 amino acid (aa) residues. The predicted molecular mass and isoelectric point of Ab-Antistasin was 44 kDa and 8.5, respectively, and showed highest identity (23.1%) to Hydra magnipapillata antistasin. The most striking feature of Ab-Antistasin is the 12-fold internal repeats (IR) of an antistasin-like domain. Ten of the 12 IR domains (26-27 aa) are highly conserved, with 6 cysteines and 1 glycine. Ab-Antistasin was comprised of three Bowman-Birk serine protease inhibitor family motifs. The recombinant Ab-Antistasin (rAb-Antistasin) was over-expressed in Escherichia coli and purified using a pMAL system. rAb-Antistasin (10 μM) was able to inhibit trypsin activity by 66% in a dose-dependent manner. Moreover, it exhibited low prolongation activity for coagulation in an APTT assay (86.0 s compared to control 42.0 s) with human blood. Endogenous Ab-Antistasin mRNA was found to be expressed in digestive tract, hepatopancreas, hemocytes, abductor muscle and mantle, with highest expression levels in digestive tract followed by hepatopancreas and hemocytes. Quantitative real time PCR results revealed that Ab-Antistasin transcription was significantly induced at 3 h post-infection (p.i.) after challenged by a mixture of bacteria (Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes) in the abalone digestive tract; in the hemocytes, induction occurred at 6 and 12 h. The results indicated that Ab-Antistasin could play an important role in the immune responses of mollusks.