• Journal of Gastric Cancer
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• v.3 no.4
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• pp.186-190
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• 2003

Purpose: The balance between cell proliferation and apoptosis is crucial for homeostatic maintenance in a cell population. Decreased apoptosis or uncontrolled proliferation can lead to cancer. The Fas receptor signal through a cytoplasmic death domain is very important in the apoptotic pathway. To identify the effect of the death domain of the Fas gene in the development and/or progression of gastric cancer, we examined the apoptotic potential of five known Fas mutants detected in gastric cancers. Materials and Methods: A wild-type Fas gene was cloned with cDNA from normal liver tissue and full length Fas was sequenced. Mutants of the gene were generated with sitedirected mutagenesis by using the wild-type gene and specific primers. Wild- and mutant-type genes were transfected to HEK293 cells. Forty-eight hours after transfection the cells were stained with DAPI and cell death was counted under fluorescent microscopy. Results: In wild-type Fas-transfected cells, the percentage of apoptotic cells was $85.9\pm3.6\%$, and significant cell death and classic morphologic signs of apoptosis were observed. However, the percentages of apoptotic cells transfected with N239D, E240G, D244V, and R263H of tumor-derived mutant Fas were $29.5\pm2.08\%,\;28.5\pm3.34\%,\;25.225\pm2.06\%,\;and\;36.625\pm4.49\%$, respectively. Conclusion: These results suggest that inactivation of Fas caused by mutations in the death domain of the Fas gene may be one of the possible escape mechanisms against Fas-mediated apoptosis and that inactivating mutation of the Fas may contribute to the development or progression of gastric cancers.

• BMB Reports
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• v.55 no.6
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• pp.275-280
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• 2022

The treatment of atopic dermatitis (AD) is challenging due to its complex etiology. From epidermal disruption to chronic inflammation, various cells and inflammatory pathways contribute to the progression of AD. As with immunosuppressants, general inhibition of inflammatory pathways can be effective, but this approach is not suitable for long-term treatment due to its side effects. This study aimed to identify a plant extract (PE) with anti-inflammatory effects on multiple cell types involved in AD development and provide relevant mechanistic evidence. Degranulation was measured in RBL-2H3 cells to screen 30 PEs native to South Korea. To investigate the anti-inflammatory effects of Parasenecio auriculatus var. matsumurana Nakai extract (PAE) in AD, production of cytokines and nitric oxide, activation status of FcεRI and TLR4 signaling, cell-cell junction, and cell viability were evaluated using qRT-PCR, western blotting, confocal microscopy, Griess system, and an MTT assay in RBL-2H3, HEK293, RAW264.7, and HaCaT cells. For in vivo experiments, a DNCBinduced AD mouse model was constructed, and hematoxylin and eosin, periodic acid-Schiff, toluidine blue, and F4/80-staining were performed. The chemical constituents of PAE were analyzed by HPLC-MS. By measuring the anti-degranulation effects of 30 PEs in RBL-2H3 cells, we found that Paeonia lactiflora Pall., PA, and Rehmannia glutinosa (Gaertn.) Libosch. ex Steud. show an inhibitory activity of more than 50%. Of these, PAE most dramatically and consistently suppressed cytokine expression, including IL-4, IL-9, IL-13, and TNF-α. PAE potently inhibited FcεRI signaling, which mechanistically supports its basophil-stabilizing effects, and PAE downregulated cytokines and NO production in macrophages via perturbation of toll-like receptor signaling. Moreover, PAE suppressed cytokine production in keratinocytes and upregulated the expression of tight junction molecules ZO-1 and occludin. In a DNCB-induced AD mouse model, the topical application of PAE significantly improved atopic index scores, immune cell infiltration, cytokine expression, abnormal activation of signaling molecules in FcεRI and TLR signaling, and damaged skin structure compared with dexamethasone. The anti-inflammatory effect of PAE was mainly due to integerrimine. Our findings suggest that PAE could potently inhibit multi-inflammatory cells involved in AD development, synergistically block the propagation of inflammatory responses, and thus alleviate AD symptoms.

• Journal of Physiology & Pathology in Korean Medicine
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• v.23 no.1
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• pp.57-62
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• 2009

Smilacis Chinae Radix has been used as an anti-inflammatory agent. This study was performed to anti-inflammatory and MAP kinase signaling pathway in vitro. Experimental studies were obtained by measuring the Cytotoxicity, production of NO, PGE2, TNF-$\alpha$ and protein level of catalase, SOD, MAP kinase, The results were summarized as follows: Smilacis Chinae Radix was not cytotoxic effects against Raw264.7 and HEK293 cells. Concentration of $100{\mu}g/m{\ell}$ Smilacis Chinae Radix inhibited the production of NO in the Raw264.7 cell stimulated with LPS. All concentrations of Smilacis Chinae Radix not significantly inhibited the production of PGE2 in the Raw264.7 cell stimulated with LPS. All concentrations of Smilacis Chinae Radix did not inhibit the production of TNF-$\alpha$ in the Raw264.7 cell stimulated with LPS. Smilacis Chinae Radix has not effect of blocking NF-${\kappa}B$ into nucleus in LPS-induced macrophage Raw264.7 cell. Smilacis Chinae Radix has the effect of Cytoprotection through activation of ERK and inhibition of p38 and JNK. Accordingly the results show Smilacis Chinae Radix could induce anti-inflammation and Cytoprotection effects against In vitro, but it needs more research on the precise mechanism of such effects.

• IMMUNE NETWORK
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• v.9 no.4
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• pp.138-146
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• 2009

Background: The MHC region of the chromosome contains a lot of genes involved in immune responses. Here we have investigated the mouse NG29/Cd320 gene in the centrometrically extended MHC region of chromosome 17. Methods: We cloned the NG29 gene by RT-PCR and confirmed the tissue distribution of its gene expression by northern blot hybridization. We generated the NG29 gene expression constructs and polyclonal antibody against the NG29 protein to perform the immunofluorescence, immunoprecipitation and flow cytometric analysis. Results: The murine NG29 gene and its human homologue, the CD320/8D6 gene, were similar in the gene structure and tissue expression patterns. We cloned the NG29 gene and confirmed its expression in plasma membrane and intracellular compartments by transfecting its expresssion constructs into HEK 293T cells. The immunoprecipitation studies with rabbit polyclonal antibody raised against the NG29-NusA fusion protein indicated that NG29 protein was a glycoprotein of about 45 kDa size. A flow cytometric analysis also showed the NG29 expression on the surface of Raw 264.7 macrophage cell line. Conclusion: These findings suggested that NG29 gene in mouse extended MHC class II region was the orthologue of human CD320 gene even though human CD320/8D6 gene was located in non-MHC region, chromosome 19p13.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.4
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• pp.439-447
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• 2017

Myotonia congenita (MC) is a genetic disease that displays impaired relaxation of skeletal muscle and muscle hypertrophy. This disease is mainly caused by mutations of CLCN1 that encodes human skeletal muscle chloride channel (CLC-1). CLC-1 is a voltage gated chloride channel that activates upon depolarizing potentials and play a major role in stabilization of resting membrane potentials in skeletal muscle. In this study, we report 4 unrelated Korean patients diagnosed with myotonia congenita and their clinical features. Sequence analysis of all coding regions of the patients was performed and mutation, R47W and A298T, was commonly identified. The patients commonly displayed transient muscle weakness and only one patient was diagnosed with autosomal dominant type of myotonia congenita. To investigate the pathological role of the mutation, electrophysiological analysis was also performed in HEK 293 cells transiently expressing homo-or heterodimeric mutant channels. The mutant channels displayed reduced chloride current density and altered channel gating. However, the effect of A298T on channel gating was reduced with the presence of R47W in the same allele. This analysis suggests that impaired CLC-1 channel function can cause myotonia congenita and that R47W has a protective effect on A298T in relation to channel gating. Our results provide clinical features of Korean myotonia congenita patients who have the heterozygous mutation and reveal underlying pathophyological consequences of the mutants by taking electrophysiological approach.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.2
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• pp.241-249
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• 2017

Plasma membrane hyperpolarization associated with activation of $Ca^{2+}$-activated $K^+$ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary ${\gamma}^2$-subunit, LRRC52 (leucine-rich-repeat-containing 52), is known to mediate the pH-sensitive, sperm-specific $K^+$ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical $BK_{Ca}$ activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting ${\gamma}^2$ subunit, hLRRC52. As previously reported, Slo3 $K^+$ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 $K^+$ current, and internal alkalinization and $Ca^{2+}$ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 $K^+$ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular $Ca^{2+}$. In contrast, elevation of intracellular $Ca^{2+}$ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate $Ca^{2+}$-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.3
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• pp.413-418
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• 2016

Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

• Korean Journal of Medicinal Crop Science
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• v.17 no.5
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• pp.321-327
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• 2009

Sunlight, and in particular its UV component, is the major environmental trigger that underlies the major signs of human skin and skin cancer in general. Therefore, this study was carried out to investigate the UV protection effects of R. coreanus. R. coreanus was extracted by ultra high pressure extraction process at 500 MPa and $30^{\circ}C$ for 5 and 15 minutes. The cytotoxicity of the extracts extracted by ultra high pressure process on human dermal fibroblast cell CCD-986sk, human kidney normal cell HEK293, and human lung normal cell HEL299 was measured as 17.5%, 16.5% and 14.0%, respectively in adding $1.0\;mg/m{\ell}$ of the samples, which was much lower than that from conventional water extraction method at $100^{\circ}C$ as 23.2%, 22.5%, 21.2%. The secretion of $NO^-$ from macrophage showed $15.9\;{\mu}M$ on the R. coreanus extract from this process, which was higher than others. Prostaglandin $E_2$ ($PGE_2$) production from UV-induced human skin cells was also greatly decreased down to $510\;pg/m{\ell}$, compared to the control. From the results, we considered that the extracts from R. coreanus could be potent natural materials for skin anti-inflammation agent, and could be used as a potential anti-aging for the photo-damaged skin.

• Korean Journal of Medicinal Crop Science
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• v.14 no.6
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• pp.317-321
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• 2006

This study was performed to compare effect of anticancer activities on Rhodiola sachalinensis by ultrasonifi-cation process and solvent. Compared the yield to the water extracts (WE), wafer extracts with ultrasonification (WEU) at60, loot and ethyl alcohol extracts (EE), ethyl alcohol extracts with ultrasonification (EEU) at 60 ${\circ}$C from Rhodiola sachalinensis. Experimental studies were progressed through the anticancer activities such as cell cytotoxicity, inhibition activities of cell growth. The cell cytotoxicity using human embryonic kidney cell (HEK293) was showed cytotoxicity of below 26.26%by extracts of Rhodiola sachalinensis in 1.0 mg/ml concentration, The anticancer activities were increased in over 70% by extracts of Rhodiola sachalinensis in A549, AGS, Hep3B and MCF-7 cells. From the results, the extract Rhodiola sachalinensis were showed useful anticancer activities.

• Journal of Pharmaceutical Investigation
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• v.35 no.1
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• pp.17-23
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• 2005

Polyethylenimine (PEI) has been used as cationic polymers for efficient gene transfer without the need for endosomolytic agents. Various kinds of PEIs with different molecular weight were tested in order to investigate the effects of the molecular weight of PEI on the transfection efficiency and cell cytotoxicity. The ${\beta}-galactosidase$ expression $(pCMV-{\beta}-gal)$ plasmid was used as a model DNA. Complex formation between PEI and pDNA was assessed by 1% agarose gel electrophoresis method. Particle size and zeta-potential of complexes were determined by electrophoretic light scattering spectrometer. In vitro transfection efficiency was assayed by measuring ${\beta}-galactosidase$ activity. Cell cytotoxicity was determined by MTT assay. Particle sizes of the complexes became smaller on increasing molecular weights of PEI and N/P ratios. Surface potential of complexes was increased as the molecular weight of PEI increased. Transfection efficiency of $pCMV-{\beta}-ga1$ on the HEK 293 cells was greatest with PEI 25 K system but having the lowest cell viability. PEI with high molecular weight showed higher transfection efficiency and cell viability than PEI with low molecular weight.