• The Plant Pathology Journal
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• v.39 no.4
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• pp.361-373
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• 2023

In plant-pathogen interactions, Magnaporthe oryzae causes blast disease on more than 50 species of 14 monocot plants, including important crops such as rice, millet, and most 15 recently wheat. M. oryzae is a model fungus for studying plant-microbe interaction, and the main source for fungal pathogenesis in the field. Here we report that MoJMJD6 is required for conidium germination and appressorium formation in M. oryzae. We obtained MoJMJD6 mutants (ΔMojmjd6) using a target gene replacement strategy. The MoJMD6 deletion mutants were delayed for conidium germination, glycogen, and lipid droplets utilization and consequently had decreased virulence. In the ΔMojmjd6 null mutants, global histone methyltransferase modifications (H3K4me3, H3K9me3, H3K27me3, and H3K36me2/3) of the genome were unaffected. Taken together, our results indicated that MoJMJD6 function as a nuclear protein which plays an important role in conidium germination and appressorium formation in the M. oryzae. Our work provides insights into MoJMJD6-mediated regulation in the early stage of pathogenesis in plant fungi.

• IMMUNE NETWORK
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• v.20 no.5
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• pp.40.1-40.15
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• 2020

The protein encoded by the Gene Associated with Retinoid-Interferon-Induced Mortality-19 (GRIM-19) is located in the mitochondrial inner membrane and is homologous to the NADH dehydrogenase 1-alpha subcomplex subunit 13 of the electron transport chain. Multiple sclerosis (MS) is a demyelinating disease that damages the brain and spinal cord. Although both the cause and mechanism of MS progression remain unclear, it is accepted that an immune disorder is involved. We explored whether GRIM-19 ameliorated MS by increasing the levels of inflammatory cytokines and immune cells; we used a mouse model of experimental autoimmune encephalomyelitis (EAE) to this end. Six-to-eight-week-old male C57BL/6, IFNγ-knockout (KO), and GRIM-19 transgenic mice were used; EAE was induced in all strains. A GRIM-19 overexpression vector (GRIM19 OVN) was electrophoretically injected intravenously. The levels of Th1 and Th17 cells were measured via flow cytometry, immunofluorescence, and immunohistochemical analysis. IL-17A and IFNγ expression levels were assessed via ELISA and quantitative PCR. IL-17A expression decreased and IFNγ expression increased in EAE mice that received injections of the GRIM-19 OVN. GRIM19 transgenic mice expressed more IFNγ than did wild-type mice; this inhibited EAE development. However, the effect of GRIM-19 overexpression on the EAE of IFNγ-KO mice did not differ from that of the empty vector. GRIM-19 expression was therapeutic for EAE mice, elevating the IFNγ level. GRIM-19 regulated the Th17/Treg cell balance.

• Journal of Life Science
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• v.25 no.10
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• pp.1091-1097
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• 2015

We have isolated and characterized an ascorbate peroxidase (APx) gene, OsAPx1 from rice. Northern and Western blot analyses indicated that at young seedling stage, OsAPx1 mRNA was expressed highly in root, shoot apical meristem (SAM) and leaf sheath than leaf. In mature plant, OsAPx1 gene expressed highly in root, stem and flower but weakly in leaf. OsAPx1 gene and protein expression level was induced in leaves inoculated with Magnaporthe oryzae (M. oryzae) and Xanthomonas oryzae pv. oryzae (Xoo). Phytohormones treatment showed that OsAPx1 was up-regulated by jasmonic acid (JA), but was down regulated by ABA and SA co-treatments with JA, resulting that they have antagonistic effect on pathogen responsive OsAPx1 expression. Phylogenetic analysis illustrated that Arabidopsis AtAPx1 has a close relationship with OsAPx1. In AtAPx1 knock out lines, the accumulation of O₂^- and H₂O₂ are all highly detected than wild type, revealing that the high concentration of exogenous H₂O₂ cause the intercellular superoxide anion and hydrogen peroxide accumulation in AtAPx1 knockout plant. These results suggested that OsAPx1 gene may be associated with the pathogen defense cascades as the mediator for balancing redox state by acting ROS scavenger and is associated with response to the pathogen defense via Jasmonic acid signaling pathway.

• Korean Journal of Acupuncture
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• v.24 no.2
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• pp.129-139
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• 2007

목적 : Signal transducers and activators of transcription 6 (Stat 6) 유전자는 면역세포의 발달에 있어서 중요한 유전인자이며, IL-4와 같은 사이토카인에 의해 유전자 발현이 조절된다. 본 연구에서는 Stat 6 유전자 제거 생쥐와 정상 (wild type, W/T) 생쥐에 carrageenan으로 염증을 유도한 후 족삼리에 침치료를 시행하여 시상하부에서의 유전자 발현 양상을 분석하고자 하였다. 방 법 : BALB/c (W/T, n=12) and BALB/c-Stat 6 유전자 제거 생쥐 (n=12)의 발뒤꿈치 표피에 1% carrageenan을 30 ul 주사하여 염증을 유도하였다. 침은 염증 유도 30분 후에 족삼리(ST36)에 시침하였으며, 염증유도에 의한 부종 증가율을 매 시간마다 측정하여 총 5시간동안 측정하였다. 마이크로에러이는 Stat 6 유전자 제거 생쥐를 염증 유발 군과 염증유발 후 침을 처치한 군으로 나누고, 시상하부를 적출하여 RNA를 분리한뒤 마이크로어레이 프로파일을 분석하였다. 결 과 : 염증에 의한 부종증가율을 비교한 결과, Stat 6 유전자 제거 생쥐 그룹의 부종증가율이 W/T 생쥐의 부종 증가율보다 약 50 % 정도 감소하였으며, 각 3, 4, 5시간째에 유의한 차이를 나타내었다. (각 p<0.05). W.T생쥐군과 Stat 6 유전자 제거 생쥐군 모두에서, 침 처치군이 염증 유발 군에 비해, 염증 유발 2시간 후부터 유의한 감소를 나타내었다. 시상하부의 유전자 발현을 관찰한 결과, 39개의 유전자가 3배 이상 감소하였으며, 19개의 유전자는 3배 이상 증가하였다. 결 론 : W/T 생쥐군과 Stat 6 유전자 제거 생쥐 모두에서 침의 진통효과는 나타나며, 이의 기전에는 시상하부에서의 침 치료에 의한 염증관련 유전자들의 감소와, 항염증과 관련된 유전자들이 증가가 관여하는 것으로 보인다., 10, 11), 내측전완피신경(TE5, 6, 7, 8, 9, 10, 11), 후상완피신경(TE12, 13), 상외측상완피신경(TE13), 외측쇄골상신경(TE14, 15),대이개신경(TE16, 17, 18, 19), 소후두신경(TE19, 20), 이개측두신경(TE20, 21, 22), 안면신경측두지(TE22, 23), 관골측두신경(TE23), 중층에 견갑상신경(TE15), 견갑배신경(TE15), 경상설골근신경(TE17), 후이개신경(TE18, 19, 20), 안면신경측두지(TE20, 21, 22), 심층에 후골간신경(TE5, 6, 7), 요골신경심지(TE8, 9, 12, 13), 견갑상신경(TE14), 액와신경가지(TE14), 부신경(TE16), 안면신경과 부신경가지(TE17), 설인신경(TE17), 설하신경(TE17), 경신경고리(TE17), 미주신경(TE17), 안면신경 (TE18). 3) 혈(血) 관(管) : 천층에 척측정맥배측지(TE1, 2), 고유수장지동맥배측지(TE1), 배측중수골동맥배측지(TE2), 배측중수골정맥(TE3), 척측피정맥(TE4, 5, 6, 7, 8, 9, 10, 11), 배측정맥궁(TE4), 부요측피정맥(TE6, 8, 9),요측피정맥(TE10, 11), 후견봉정맥가지(TE13, 14), 후이개동 ${\cdot}$ 정맥(TE16, 17, 18, 19, 20), 전이개동 ${\cdot}$ 정맥(TE20), 천측두동 ${\cdot}$ 정맥(TE22, 23), 중층에 후상완회선동맥(TE14), 견갑배동맥(TE15), 견갑상동맥(TE15),천측두동 ${\cdot}$ 정맥(TE21), 관골측두동 ${\cdot}$ 정맥(TE23), 심층에 배측중수골동맥(TE3), 배측수근동맥궁(TE4), 후골간동맥(TE4, 5, 6, 7, 8, 9), 전골간동맥(TE6, 7, 9)

• Analytical Science and Technology
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• v.31 no.5
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• pp.208-218
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• 2018

Autophagy is a cellular process whereby cytosolic materials or organelles are taken up in a double-membrane vesicle structure known as an autophagosome and transported into a lysosome for degradation. Although autophagy has been studied at the genetic, cellular, or biochemical level, systematic ultrastructural quantitative analysis of autophagosomes during the autophagy process by using transmission electron microscopy (TEM) has not yet been reported. In this study, we performed ultrastructural analysis of autophagosomes in wild-type (WT) mouse embryonic fibroblasts (MEFs) and autophagy essential gene (atg5) knockout (KO) MEFs. First, we performed ultrastructural analysis of autophagosomes in WT MEFs compared to atg5 KO MEFs in basal autophagy or starvation-induced autophagy. Although we observed phagopore, early, late autophagosomes, or autolysosomes in WT MEFs, atg5 KO MEFs had immature autophagosomes that showed incomplete closure. Upon starvation, late autophagosomes accumulated in WT MEFs while the number of immature autophagosomes significantly increased in atg5 KO MEF indicating that atg5 plays an important role in the maturation of autophagosomes. Next, we examined autophagosomes in the cell model expressing polyQ-expanded N-terminal fragment of huntingtin. Our TEM analysis indicates that the number of late autophagosomes was significantly increased in the cells expressing the mutant huntingtin, indicating that improving the fusion of autophagosome with lysosome may be effective to enhance autophagy for the treatment of Huntington's disease. Taken together, the results of our study indicate that ultrastructural and quantitative analysis of autophagosomes using TEM can be applied to various human cellular disease models, and that they will provide an important insight for cellular pathogenesis of human diseases associated with autophagy.

• Journal of Plant Biotechnology
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• v.38 no.2
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• pp.169-175
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• 2011

Calcium ($Ca^{2+}$) signals have been implicated in regulating plant development and responses to the environmental stresses including a programmed cell death pathway. In animals and plants, cytosolic $Ca^{2+}$ signals have been involved in the activation of programmed cell death (PCD). Recently, we reported that disruption of Arabidopsis vacuolar $\b{A}$utoinhibited $\underline{C}a^{2+}$-$\b{A}$TPases (ACAs), ACA4 and ACA11, resulted in the activation of a salicylic acid-dependent programmed cell death pathway. Although extensive studies have revealed various components of a PCD in plants, executors to directly induce PCD are well unknown. Here, we provide that the vacuolar processing enzymes (VPEs) are involved in a PCD induced by the double knockout mutant of vacuolar $Ca^{2+}$-ATPases in Arabidopsis. The gene expression of VPE was rapidly up-regulated and the enzyme activity of VPE was increased in the double mutant plants. We also generated aca4/aca11/avpe, aca4/aca11/${\gamma}$vpe and aca4/aca11/avpe/${\gamma}$vpe mutant plants. Although cell death phenotype of the double mutant plants was not completely disappeared in the triple and quadruple mutant plants, the triple and quadruple mutant plants showed to significantly delay cell death phenotype of the double mutant plants. These results suggest that the VPE is involved in the HR-like cell death in the double mutant of vacuolar $Ca^{2+}$-ATPases in Arabidopsis.

• Development and Reproduction
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• v.12 no.1
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• pp.77-86
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• 2008

This study was carried out to investigate the effect of leukemia inhibitory factor (LIF) on the derivation of mouse ES cells from isolated blastomeres. Two-cell stage mouse embryos were obtained from superovulated BDF1 female mice. Collected embryos were cultured to blastocyst stage in culture medium supplemented with 0, 1,000, 2,500 or 5,000 U/mL of LIF. Cultured blastocysts were examined by counting the number of cells in the inner cell mass (ICM) and trophectoderm (TE) using differential staining method. When 2-cell embryos were cultured with 2,500 U/ml of LIF, the cell numbers of ICM significantly increased in comparing with those of the control($21.0{\pm}4.0$ vs. $15.9{\pm}5.0$, P<0.01) and 1,000 U/mL of LIF-containing group ($21.0{\pm}4.0$ vs. $16.6{\pm}4.9$, P<0.05). We used an ES cell establishment medium with 20% Knockout Serum Replacement and 0.01 mg/mL ACTH instead of fetal bovine serum. Establishing efficacy of ES cell lines were the highest in 2,500 U/mL of LIF-containing group as 36.7% (11/30). This culture medium was applied to the culture of isolated blastomeres and to derivate ES cell lines. Three ES cell lines (21.4%) from isolated blastomeres of 2-cell stage embryos were established. In further experiments, we could establish one ES cell line (4.0%) from single blastomere of 4-cell stage embryo. The subcultured ES cells and their embryoid bodies were characterized by analyzing gene expression for undifferentiation and differentiation marker gene using immunocytochemistry and RT-PCR. In conclusion, LIF supplementation in culture medium could increase the cell number in ICM of blastocysts and support derivation of ES cell lines from isolated blastomeres.

• Journal of Life Science
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• v.19 no.4
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• pp.520-525
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• 2009

Early onset of Batten disease (EBD), one of the most lethal neurodegenerative storage disorders of childhood, is caused by inactivating mutations in the Ceroid Lipofuscinosis, Neuronal (CLN1) gene. Neurogranin, a calmodulin-binding protein, is expressed in the brain and participates in the protein kinase C (PKC) signaling pathway. While oxidative stress is the suggested cause of neurodegeneration in EBD, its molecular mechanism(s) remains obscure. In this research, we examined the levels of neurogranin in the brain mRNA of wild-type (WT) mice and EBD knockout (KO) mice, as well as the proteins. We also performed neuronal cultures to measure the expression levels of neurgranin and phosphorylated-neurogranin with or without oxidative stress inducers and anti-oxidants. Results showed that neurogranin in both EBD KO mice brain mRNA and protein extracts decreased in an age dependent manner. However, high amounts of phosphorylated-neurogranin were detected in the 6-month brain. This pattern was also confirmed by cultured neurospheres samples. Moreover, neurospheres treated with $H_2O_2$, an oxidative stress inducer, showed increased phosphorylated-neurogranin patterns. Interestingly, this pattern returned to normal status when treated with N-acetyl-L-cystein, an anti-oxidant, after $H_2O_2$ treatment was performed. Our results suggest that the phosphorylation of neurogranin is affected by oxidative stress status in EBD, and appropriate anti-oxidant treatment will relieve hyper-phosphorylation of neurogranin.

• Journal of Life Science
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• v.21 no.10
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• pp.1370-1376
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• 2011

To study the functional genomic analysis of a crenachaeon Sulfolobus acidocaldarius, we have constructed an auxotrophic mutant based on pyrEF, which encodes the pyrimidine biosynthetic enzymes orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. S. acidocaldarius was shown to be sensitive to 5-fluoroorotic acid (5-FOA), which can be selected for mutations in pyrEF genes within a pyrimidine biosynthesis cluster. Spontaneous 5-FOA-resistant mutants by ultraviolet, KH1U and KH2U, were found to contain two point mutations and a frame shift mutation in pyrE, respectively. Mutations at these sites from KH1U and KH2U decreased the activity of orotate phosphoribosyltransferase encoded by the pyrE gene and blocked the degradation of 5-FOA into toxic 5-FOMP and 5-FUMP that kill the cells. Therefore, KH1U and KH2U were uracil auxotrophs. Transformation of Sulfolobus-Escherichia coli shuttle vector pC bearing pyrEF genes from S. solfataricus P2 into S. acidocaldarius mutant KH2U restored 5-FOA sensitivity and overcame the uracil auxotrophy. This study establishes an efficient genetic strategy towards the systematic knockout of genes in S. acidocaldarius.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.161-168
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• 2016

Nucleotide metabolism in endothelium is variable between different species. Recent studies demonstrated that this variability could contribute coagulation dysfunction, even though organs of the alpha 1,3-galactosyltransferase gene knockout pig were transplanted into the primate. CD73 (ecto-5'-nucelotidase) is an enzyme at cell surface catalyzing the hydrolysis of adenosine triphosphate to adenosine, which plays role on a substance for anti-inflammatory and anti-coagulant. Thus, overexpression of CD73 in endothelial cells of the pig is considered as an approach to reduce coagulopathy. In this study, we constructed a human CD73 expression vector under control of porcine Icam2 promoter (pIcam2-hCD73), which is expressed specifically at endothelial cells, and of CMV promoter as a control (CMV-CD73). First, we transfected the CMV-CD73 vector into HEK293 cells, and then confirmed CD73 expression at cell surface by flow cytometry analysis. Next, we transfected the pIcma2-CD73 and CMV-CD73 vectors into primary porcine fibroblasts and endothelial cells. Consequence was that the pIcma2-CD73 vector was expressed only at the porcine endothelial cells, meaning that the pIcam2 promoter lead to endothelial cell-specific expression of CD73 in vitro. Finally, we nucleofected the pIcam2-hCD73 vector into passage 3 fibroblasts, and enforced hygromycin selection of 400mg/ml. We were able to obtain forty three colonies harboring pIcam2-CD73 to provide donor cells for transgenic cloned porcine production.