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( Su Jin Lee ),( Youngdeuk Lee ),( Gun Hoo Park ),( Navaneethaiyer Umasuthan ),( Soo Jin Heo ),( Mahanama De Zoysa ),( Won Kyo Jung ),( Dae Won Lee ),( Hanjun Kim ),( Do Hyung Kang ),( Chulhong Oh ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.6
L-Asparaginase (E.C. 3.5.1.1) is an enzyme involved in asparagine hydrolysis and has the potential to effect leukemic cells and various other cancer cells. We identified the Lasparaginase gene (L-ASPG86) in the genus Mesoflavibacter, which consists of a 1,035 bp open reading frame encoding 344 amino acids. Following phylogenetic analysis, the deduced amino acid sequence of L-ASPG86 (L-ASPG86) was grouped as a type I asparaginase with respective homologs in Escherichia coli and Yersinia pseudotuberculosis. The L-ASPG86 gene was cloned into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells. Recombinant L-asparaginase (r-L-ASPG86) showed optimum conditions at 37-40oC, pH 9. Moreover, r-L-ASPG86 did not exhibit glutaminase activity. In the metal ions test, its enzymatic activity was highly improved upon addition of 5 mM manganese (3.97-fold) and magnesium (3.35-fold) compared with the untreated control. The specific activity of r-LASPG86 was 687.1 units/mg under optimum conditions (37°C, pH 9, and 5 mM MnSO4).
De Zoysa, Mahanama,Nikapitiya, Chamilani,Whang, Ilson,Lee, Jae-Seong,Lee, Jehee Elsevier 2009 FISH AND SHELLFISH IMMUNOLOGY Vol.27 No.5
<P><B>Abstract</B></P><P>Antimicrobial peptides (AMPs) play an important role in the immune defense against pathogenic microorganisms. In this study, a histone H2A full-length cDNA was cloned from disk abalone <I>Haliotis discus discus</I>. We identified a 40-amino acid AMP designated as abhisin from the N-terminus of the abalone histone H2A sequence. Abhisin shows the characteristic features of AMPs including net positive charge (+13), higher hydrophobic residues (27%) and 2.82 kcal/mol protein binding potential. Abhisin shares 80% amino acid identity with the buforin I peptide that is derived from Asian toad histone H2A. We synthesized the synthetic peptide of abhisin, and characterized its antimicrobial activities. Our results showed the growth inhibition of Gram positive (G+) <I>Listeria monocytogenes</I>, Gram negative (G-) <I>Vibrio ichthyoenteri</I> bacteria, and fungi (yeast) <I>Pityrosporum ovale</I> by abhisin treatment at 250 μg/mL. However, stronger activity was displayed against the G+ than G− bacteria. Additionally, scanning electron microscope (SEM) observation results confirmed that <I>P. ovale</I> cells were damaged by abhisin treatment. Interestingly, abhisin treatment (50 μg/mL) decreased the viability of THP-1 leukemia cancer cells approximately by 25% but there was no effect on the normal vero cells, suggesting that abhisin has cytotoxicity against cancer cells but not normal cells. Quantitative real time RT-PCR results revealed that histone H2A transcription was significantly induced at 3 h post-infection with bacteria in abalone gills and digestive tract. These results suggest that abhisin is a potential antimicrobial agent, and its precursor histone H2A may be involved in the innate immune defense system in abalone.</P>
De Zoysa, Mahanama,Kang, Hyun-Sil,Song, Young-Bo,Jee, Youngheun,Lee, Young-Don,Lee, Jehee Elsevier 2007 FISH AND SHELLFISH IMMUNOLOGY Vol.23 No.1
<P><B>Abstract</B></P><P>Myxovirus resistance (Mx) protein is one of the most studied antiviral proteins. It is induced by the type I interferon system (IFN α/β) in various vertebrates, but its expression has not been identified or characterized in mollusks or other multi-cellular invertebrates to date. In this study, we isolated the Mx gene from a disk abalone (<I>Haliotis discus discus</I>) normalized cDNA library. Mx cDNA was sequenced, cloned and compared to other known Mx proteins. The full-length 1664bp of abalone Mx cDNA contained a 1533-bp open reading frame that codes for 511 amino acids. Within the coding sequence of abalone Mx, characteristic features were found, such as a tripartite guanosine-5′-triphosphate (GTP)-binding motif and a dynamin family signature. In addition, leucine residues in the C-terminal region displayed a special leucine domain at L<SUB>468</SUB>, L<SUB>475</SUB>, L<SUB>489</SUB> and L<SUB>510</SUB>, suggesting that abalone Mx may have a similar oligomerization function as other leucine zipper motifs. Abalone Mx protein exhibited 44% amino acid similarity with channel catfish Mx1, rainbow trout Mx2 and Atlantic halibut Mx. Abalones were injected intramuscularly with the known IFN inducer poly I:C and RT-PCR was performed for Mx mRNA analysis. The results showed enhanced Mx expression in abalone gill and digestive tissues 24h as well as 48h after injection of poly I:C. Mx mRNA was expressed in gill, digestive gland, mantle and foot tissues in healthy abalone, suggesting that the basal level of Mx expressed is tissue-specific. There is no known Mx protein closely related to abalone Mx according to phylogenetic analysis. Abalone Mx may have diverged from a common gene ancestor of fish and mammalian Mx proteins, since abalone Mx showed high similarity in terms of conserved tripartite GTP-binding, dynamin family signature motifs and poly I:C enhancement of Mx mRNA expression.</P>
De Zoysa, Mahanama,Nikapitiya, Chamilani,Lee, Youngdeuk,Lee, Sukkyoung,Oh, Chulhong,Whang, Ilson,Yeo, Sang-Yeop,Choi, Cheol Young,Lee, Jehee Elsevier 2010 FISH AND SHELLFISH IMMUNOLOGY Vol.29 No.6
<P><B>Abstract</B></P><P>The regulation of transcriptional activation is an essential and critical point in gene expression. In this study, we describe a novel transcription factor activator protein-1 (Ap-1) gene from disk abalone <I>Haliotis discus discus</I> (AbAp-1) for the first time in mollusk. It was identified by homology screening of an abalone normalized cDNA library. The cloned AbAp-1 consists of a 945 bp coding region that encodes a putative protein containing 315 amino acids. The AbAp-1 gene is composed of a characteristic Jun transcription factor domain and a highly conserved basic leucine zipper (bZIP) signature similar to known Ap-1 genes. The AbAp-1 shares 46, 43 and, 40% amino acid identities with fish (<I>Takifugu rubripes</I>), human and insect (<I>Ixodes scapularis</I>) Ap-1, respectively.</P><P>Quantitative real time RT-PCR analysis confirmed that AbAp-1 gene expression is constitutive in all selected tissues. AbAp-1 was upregulated in gills after bacteria (<I>Vibrio alginolyticus, Vibrio parahemolyticus</I> and <I>Lysteria monocytogenes</I>) challenge; and, upregulated in hemocytes and gills by viral hemorrhagic septicemia virus (VHSV) challenge. Shell damage and tissue injury also increased the transcriptional level of Ap-1 in mantle together with other transcription factors (NF-kB, LITAF) and pro-inflammatory TNF-α. All results considered, identification and gene expression data demonstrate that abalone Ap-1 is an important regulator in innate immune response against bacteria and virus, as well as in the inflammatory response during tissue injury. In addition, stimulation of Ap-1 under different external stimuli could be useful to understand the Ap-1 biology and its downstream target genes, especially in abalone-like mollusks.</P>
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.