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Kulatunga, D.C.M.,Dananjaya, S.H.S.,Nikapitiya, Chamilani,Godahewa, G.I.,Cho, Jongki,Kim, Cheol-Hee,Lee, Jehee,De Zoysa, Mahanama Elsevier 2019 Fish & Shellfish Immunology Vol.84 No.-
<P><B>Abstract</B></P> <P>Proteins with dithiol-disulfide oxidoreductase catalytic domain are well known for their capacity in the cellular redox homeostasis. In this study, we characterized the zebrafish thioredoxin domain containing 12 (Zf<I>txndc12</I>) gene, analyzed the transcriptional responses and studied the functional properties of its recombinant protein. Full-length cDNA of Zf<I>txndc12</I> consists 519 bp coding region encoding 172 amino acids (AA) including the signal peptide. Highly consensus active motif (<SUP>65</SUP>WCGAC<SUP>69</SUP>) and probable ER retrieval motif (<SUP>169</SUP>GDEL<SUP>172</SUP>) were identified. Ubiquitous expression of Zf<I>txndc1</I>2 mRNA was observed from one cell to juvenile stage as well as different organs of adult zebrafish. Moreover, whole mount <I>in situ</I> hybridization (WISH) results showed a higher expression of Zf<I>txndc12</I> in primordial gills, central nerves system and eye. The tissue specific expression analysis (by qRT-PCR) also showed the highest expression in gills followed by brain in adult zebrafish. In larvae, up-regulated Zf<I>txndc1</I>2 mRNA expression upon exposure to H<SUB>2</SUB>O<SUB>2,</SUB> <I>Edwardsiella tarda</I> and <I>Saprolegnia parasitica</I> suggests that it may involve in both stress and immune responses. Moreover, transcriptional expression of Zf<I>txndc12</I> was up-regulated upon <I>Streptococcus iniae</I> challenge in gills of adult zebrafish<I>.</I> The recombinant ZfTxndc12 (rZfTxndc12) was overexpressed, purified and tested for its biological activities. Results revealed that rZfTxndc12 is able to reduce the DNA damage and detoxify the H<SUB>2</SUB>O<SUB>2</SUB> toxicity in concentration dependent manner. Overall results suggest that Zf<I>txndc12</I> is important antioxidant and immune functional member of the host defense system in zebrafish.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Txndc12 is important for oxidative stress defense and redox regulation. </LI> <LI> Constitutive expression of Zftxndc12 is ubiquitous in embryonic developmental stages and adult zebrafish. </LI> <LI> Immune challenge and oxidative stress induce the Zftxndc12 transcription in zebrafish larvae and adult's gills. </LI> <LI> >Recombinant Zftxndc12 shows <I>in vitro</I> ROS scavenging and cytoprotective activity. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>
( Sangyeop Shin ),( D. C. M. Kulatunga ),( S. H. S. Dananjaya ),( Chamilani Nikapitiya ),( Jehee Lee ),( Mahanama De Zoysa ) 한국균학회 2017 Mycobiology Vol.45 No.4
Saprolegniasis is one of the most devastating oomycete diseases in freshwater fish which is caused by species in the genus Saprolegnia including Saprolegnia parasitica. In this study, we isolated the strain of S. parasitica from diseased rainbow trout in Korea. Morphological and molecular based identification confirmed that isolated oomycete belongs to the member of S. parasitica, supported by its typical features including cotton-like mycelium, zoospores and phylogenetic analysis with internal transcribed spacer region. Pathogenicity of isolated S. parasitica was developed in embryo, juvenile, and adult zebrafish as a disease model. Host-pathogen interaction in adult zebrafish was investigated at transcriptional level. Upon infection with S. parasitica, pathogen/antigen recognition and signaling (TLR2, TLR4b, TLR5b, NOD1, and major histocompatibility complex class I), pro/anti-inflammatory cytokines (interleukin [IL]-1β, tumor necrosis factor α, IL-6, IL-8, interferon γ, IL-12, and IL-10), matrix metalloproteinase (MMP9 and MMP13), cell surface molecules (CD8<sup>+</sup> and CD4<sup>+</sup>) and antioxidant enzymes (superoxide dismutase, catalase) related genes were differentially modulated at 3- and 12-hr post infection. As an anti-Saprolegnia agent, plant based lawsone was applied to investigate on the susceptibility of S. parasitica showing the minimum inhibitory concentration and percentage inhibition of radial growth as 200 μg/mL and 31.8%, respectively. Moreover, natural lawsone changed the membrane permeability of S. parasitica mycelium and caused irreversible damage and disintegration to the cellular membranes of S. parasitica. Transcriptional responses of the genes of S. parasitica mycelium exposed to lawsone were altered, indicating that lawsone could be a potential anti-S. parasitica agent for controlling S. parasitica infection.
THE RESTORATION ON THE INFERTILITY DOG OF KOREAN NATURAL MONUMENT USING SCNT
K.S. Park,J.H. Lee,K.B. Ji,D.E. Kim,E.J. Lee,D.C.M. Kulatunga,K.Y. Kim,B.S. Kim,E.Y. Kim,M.K. Kim 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11
To preserve the superior genetic resources and restore the endangered species, Somatic cell nuclear transfer (SCNT) has been used widely. In Korea, the research of dog cloning has made outstanding achievements including the production of the world`s first cloned dog. Sapsaree (Sapsalgae), the representative dog of Gyeongsan-si was designated as a Korea natural monument (No. 368). This male dog used in this study has azoospermia due to unknown cause. In this study, the aim was to confirm the cause of infertility in the cell donor dog and to evaluate the reproduction potential of dog cloning using infertile male dog by SCNT. First, to confirm the infertility of the cell donor dog, the reproductive history and the testis were evaluated. The breeding histology was not recorded in individual document. In histopathology, the Sertoli cell tumor was confirmed in biopsy of the cell donor dog after death. But, these tumors are predominantly in older dogs. Second, we produced the cloned dogs with the somatic cells of the infertile dog and the appearance was similar with the cell donor dog. Also, microsatellite analysis confirmed the genetic relationship between the cell donor and clone dogs. Third, the potential breeding capacity of the cloned dog was confirmed. In T4 assay, the normal dog (same age with cloned dogs), cell donor dog, and cloned dogs was investigated. The cell donor dog with azoospermia had very low T4 level, and cloned dogs showed higher level of T4 than normal dogs. In CASA, There was no significant difference in sperm motor ability between normal dogs and cloned dogs. As a result, cloned dogs produced by SCNT had no problem regarding the reproductive function of the testis. In AI experiment, the semen of clone dogs was used to fertilize a natural female bitch and was diagnosed pregnancy by ultrasonography. In total, 7 puppies were born by normal delivery (male: 3, female: 4). In conclusion, this study confirmed that the reproduction problem of non-genetic infertility can generate a normal descendant by SCNT. Also, the first successful research to restore infertile dogs was completed. Furthermore, SCNT would be useful for the restoration of endangered species and application of superior traits.