• Korean Journal of Pharmacognosy
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• v.48 no.4
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• pp.298-303
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• 2017

To know the anti-oxidative effect of Epimedii Herba (Berberidaceae), the methanol extract of this plant was investigated by using a Caenorhabditis elegans model system. The methanol extract of this plant showed relatively significant DPPH radical scavenging and superoxide quenching activities. The ethyl acetate soluble fraction of Epimedii Herba (EHE), which showed the most potent DPPH radical scavenging and superoxide quenching activities, was tested on its effects on superoxide dismutase (SOD), catalase, intracellular ROS, and oxidative stress tolerance in Caenorhabditis elegans. Furthermore, in order to verify that regulation of stress-response genes is responsible for the increased stress tolerance of the EHE treated C. elegans, we checked SOD-3 expression using a transgenic strain. As a result, the EHE increased SOD and catalase activities of C. elegans, and reduced intracellular ROS accumulation in a dose-dependent manner. Besides, EHE-treated CF1553 worms showed higher SOD-3::GFP intensity than that of non-treated controls.

• The Plant Pathology Journal
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• v.29 no.1
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• pp.17-30
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• 2013

Barley stripe mosaic virus (BSMV) induces massive actin filament thickening at the infection front of infected Nicotiana benthamiana leaves. To determine the mechanisms leading to actin remodeling, fluorescent protein fusions of the BSMV triple gene block (TGB) proteins were coexpressed in cells with the actin marker DsRed: Talin. TGB ectopic expression experiments revealed that TGB3 is a major elicitor of filament thickening, that TGB2 resulted in formation of intermediate DsRed:Talin filaments, and that TGB1 alone had no obvious effects on actin filament structure. Latrunculin B (LatB) treat-ments retarded BSMV cell-to-cell movement, disrupted actin filament organization, and dramatically decreased the proportion of paired TGB3 foci appearing at the cell wall (CW). BSMV infection of transgenic plants tagged with GFP-KDEL exhibited membrane proliferation and vesicle formation that were especially evident around the nucleus. Similar membrane proliferation occurred in plants expressing TGB2 and/or TGB3, and DsRed: Talin fluorescence in these plants colocalized with the ER vesicles. TGB3 also associated with the Golgi apparatus and overlapped with cortical vesicles appearing at the cell periphery. Brefeldin A treatments disrupted Golgi and also altered vesicles at the CW, but failed to interfere with TGB CW localization. Our results indicate that actin cytoskeleton interactions are important in BSMV cell-to-cell movement and for CW localization of TGB3.

• Food Science of Animal Resources
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• v.37 no.1
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• pp.134-138
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• 2017

Salmonella enterica infects a broad range of host animals, and zoonostic infection threatens both public health and the livestock and meat processing industries. Many antimicrobials have been developed to target Salmonella envelope that performs essential bacterial functions; however, there are very few analytical methods that can be used to validate the efficacy of these antimicrobials. In this study, to develop a potential biosensor for Salmonella envelope stress, we examined the transcription of the S. enterica serovar typhimurium spy gene, the ortholog of which in Escherichia coli encodes Spy (${\underline{s}}pheroplast$ ${\underline{p}}rotein$ ${\underline{y}}$). Spy is a chaperone protein expressed and localized in the periplasm of E. coli during spheroplast formation, or by exposure to protein denaturing conditions. spy expression in S. typhimurium was examined by constructing a spy-gfp transcriptional fusion. S. typhimurium spy transcription was strongly induced during spheroplast formation, and also when exposed to membrane-disrupting agents, including ethanol and the antimicrobial peptide polymyxin B. Moreover, spy induction required the activity of regulator proteins BaeR and CpxR, which are part of the major envelope stress response systems BaeS/BaeR and CpxA/CpxR, respectively. Results suggest that monitoring spy transcription may be useful to determine whether a molecule particularly cause envelope stress in Salmonella.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.43-43
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• 2003

Large-conductance $Ca^{2+}$-actived $K^{+}$ channels ($BK_{Ca}$ channels) play a key role in setting the pace of contractile activity in muscle and are involved in the regulation of neurotransmitter release in neuron. $BK_{Ca}$ channels are activated by depolarizing membrane potential and the elevated level of intracellular calcium. Using yeast-two hybrid assay, we have identified a novel protein interacting with the cytosolic carboxyl terminus of rSlo, the brain isoform of rat large-conductance $Ca^{2+}$-activated $K^{+}$ channel $\alpha$-subunit. The novel gene encodes 51 kDa protein and is named as SIRK(rSlo-interacting RGS-like protein). SIRK is expressed in various tissues and localized in the cytosolic and the membrane fraction. Biochemical and immunological studies indicated that SIRK physically interacted with the cytosolic region of rSlo. To investigate whether SIRK can modulate the activity of rSlo, GFP-fused SIRK and rSlo were transiently transfected into COS-7 cells and the effects of SIRK was studied using electrophysiological means. We concluded that the overexpression of SIRK alters the surface expression of rSlo channel with only a limited effect on the biophysical characteristics of the channel.the channel.

• IMMUNE NETWORK
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• v.13 no.6
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• pp.264-274
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• 2013

The unrestricted population of $CD4^+Foxp3^+$ regulatory T (Treg) cells, which have been known to control the expression of autoimmune diseases and protective immunity to inflammatory reactions, has led to greater appreciation of functional plasticity. Detecting and/or isolating Ag-specific $CD4^+Foxp3^+$ Tregs at the single cell level are required to study their function and plasticity. In this study, we established and compared both MHC class II tetramer and intracellular CD154 staining, in order to detect $CD4^+Foxp3^+$ Treg specific for foreign Ag in acute and chronic infections with lymphocytic choriomeningitis virus (LCMV). Our results revealed that MHC class II tetramer staining showed a lower detection rate of LCMV $GP_{66-77}$-specific $CD4^+$ T cells because most of MHC class II tetramers were unbound and unstable when combined staining was performed with intracellular cytokines. In contrast, intracellular CD154 staining was revealed to be easier and simple for detecting LCMV $GP_{66-77}$-specific $CD4^+$ T cells, compared to MHC class II tetramer staining. Subsequently, we employed intracellular CD154 staining to detect LCMV $GP_{66-77}$-specific $CD4^+Foxp3^+$ Tregs using $Foxp3^{GFP}$ knock-in mouse, and found that LCMV $GP_{66-77}$-specific $CD4^+Foxp3^+$ Tregs and polyclonal $CD4^+Foxp3^+$ Tregs showed differential expansion in mice infected with LCMV Arms or Cl13 at acute (8 and 13 days pi) and chronic phases (35 days pi). Therefore, our results provide insight into the valuable use of intracellular CD154 staining to detect and characterize foreign Ag-specific $CD4^+Foxp3^+$ Treg in various models.

• Animal cells and systems
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• v.16 no.4
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• pp.280-288
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• 2012

Targeted degradation of proteins through ubiquitin-mediated proteolysis is an important control mechanism in various cellular processes. The process of ubiquitin conjugation is achieved by three enzyme complexes, among which the ubiquitin ligase complex (E3) is in charge of substrate specificity. The SCF (SKP1-CUL1-F-box) family portrays the largest and the most characterized member of the E3 ligases. For each SCF complex, the ubiquitination target is recognized by the F-box protein subunit, which interacts with the substrate through a unique C-terminal domain. We have characterized a novel F-box protein CFL-1 that represents a single LRR-type F-box (FBXL) in the Caenorhabditis elegans genome. CFL-1 is highly homologous to FBXL20 and FBXL2 of mammals, which are known to regulate synaptic vesicle release and cell cycle, respectively. A green fluorescence protein (GFP)-reporter gene fused to the cfl-1 promoter showed restricted expression around the amphid and the anus. Modulation of CFL-1 activity by RNAi affected the time interval between defecations. RNAi-treated worms also exhibited reduced tendency to form dauer when exposed to daumone. The potential involvement of CFL-1 in the control of defecation and pheromone response adds to the ever expanding list of cellular processes controlled by ubiquitin-mediated proteolysis in C. elegans. We suggest that CFL-1, as a single LRR-type F-box protein in C. elegans, may portray a prototype gene exerting diverse functions that are allocated among multiple FBXLs in higher organisms.

• Development and Reproduction
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• v.24 no.3
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• pp.207-214
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• 2020

Primary cell culture is a sufficient method frequently used to study the cellular properties and mechanisms of isolated cells in a controlled environment. In this study, an embryonic cell line (FGBC8) derived from the blastula stages of embryos of olive flounder Paralichthys olivaceus was developed. Furthermore, conditions for optimal long-term maintenance of this primary embryonic cell culture were investigated. Morphologically, FGBC8 cells were composed primarily of epithelial-like cells. FGBC8 cells were subcultured for >160 passages over ~830 days. The doubling time of FGBC8 cells was 73.8 h, and the modal diploid chromosome number was 48. FGBC8 cells transfected with green fluorescence protein (GFP)-expression plasmid exhibited a strong signal 48 h after transfection. Consequently, we demonstrated that fish serum is a crucial supplement for the long-term survival and maintenance of comparable morphology in these primary embryonic cells. Our results can be used as a guide for primary embryonic cell cultures for other fish species and may be useful for cell biotechnological applications.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.71-76
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• 2004

Gene delivery is one of the keen interests in animal industry as well as research on gene functions. Some of the in vivo gene delivery techniques have been successively used in various tissues for the gene therapy and transgenesis. Despite intensive efforts, it still remains to overcome problems of limited local and regional administration and low transgene expression. To improve the efficiency of gene delivery, a new procedure was tested. We injected exogenous DNA containing LacZ into the female or male gonads and then pulsed electric field. Electroporated gonads showed positive X-gal staining in many seminiferous tubules of the porcine fetal gonads. Exogenously introduced LacZ genes were also expressed in female porcine gonad. In addition, we demonstrated efficient gene delivery in gonad of adult mouse. Furthermore, we succeed to generate genetically modified germline cells showing GFP and positive X-gal signals. The results suggest that the newly developed gene delivery is an effective way of in vivo transfection in mammals. The developed gene delivery procedure should be useful in producing transgenic animals when combined with primary cell culture and nuclear transplantation.

• The Journal of Korean Obstetrics and Gynecology
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• v.18 no.1
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• pp.29-44
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• 2005

To address the ability of Herba Portulacae(HP) to induce cell death, we investigated the effect of HP on cell viability. Twenty-four hours later, loss of viability occurred following HP exposure in a dose-dependent manner. The treatment of HP, a commonly used herb formulation in Korea, Japan and China, caused a decrease in cell viability. HP also resulted in apoptotic morphology a brightly blue-fluorescent condensed nuclei by Hoechst 33258-staining, and reduction of cell volume. Our results show that 2mg/ml HP induces mitochondria membrane potential collapse. Immunoblotting data also shows that the expression of Bcl-2, antiaoptotic protein, decrease by the addition of HP. This GFP-Bax overexpression system shows that an important pro-apoptotic Bcl-2-family protein, Bax is translocated to mitochondria by the addition of 2mg/ml HP. Inerestingly, MAPK inhibitor study shows that p38 MAPK inhibitor, SB203580 inhibits HP-induced cell death and caspase-3 activation in HP-treated HeLa cells. Furthermore, HP transiently but significantly induces p38 activation. But P38 MAPK inhibitor does not have any effect on the translocation of Bax. Considering these results, HP induces apoptosis via p38 MAPK activation. But the pathway does not involve the translocation of Bax.

• Korean Journal of Pharmacognosy
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• v.48 no.2
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• pp.141-147
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• 2017

As an ongoing study about Allium hookeri (Liliaceae), this study was performed to evaluate the anti-oxidative effect of the leaves of this plant. Ethanol extract of A. hookeri leaves was successively partitioned as methylene chloride, ethyl acetate, n-butanol and $H_2O$ soluble fractions. The ethyl acetate soluble fraction showed the most potent DPPH radical scavenging and superoxide quenching activities among those fractions. To prove antioxidant activity of ethyl acetate fraction of A. hookeri leaves, we checked the activities of superoxide dismutase (SOD) and catalase, and intracellular ROS level and oxidative stress tolerance in Caenorhabditis elegans. In addition, to verify if increased stress tolerance of C. elegans by treating of ethyl acetate fraction was due to regulation of stress-response gene, we checked SOD-3 expression using transgenic strain. As a consequence, the ethyl acetate fraction increased SOD and catalase activity of C. elegans, and reduced intracellular ROS accumulation in a dose-dependent manner. Besides, the ethyl acetate fraction-treated CF1553 worms showed higher SOD-3::GFP intensity.