• Proceedings of the Korean Society of Life Science Conference
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• 2005.04a
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• pp.23-36
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• 2005

Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces cell death when expressed In yeast. To investigate whether .Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various orgarusms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs In detail. PBIl is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorhodamine123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Bax-induced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower lovels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. H$_{2O2}$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of H2O2 treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased In the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MBP phosphorylation activity of AtMPK3 i'n vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation In situ. Thus, AtNDPK2 appears to play a novel regulatory role in H2O2-mediated MAPK signaling in plants.

• Journal of Ginseng Research
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• v.41 no.4
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• pp.608-614
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• 2017

Background: Ginsenoside Rg1 belongs to protopanaxatriol-type ginsenosides and has diverse pharmacological activities. In this report, we investigated whether Rg1 could upregulate muscular stem cell differentiation and muscle growth. Methods: C2C12 myoblasts, MyoD-transfected 10T1/2 embryonic fibroblasts, and HEK293T cells were treated with Rg1 and differentiated for 2 d, subjected to immunoblotting, immunocytochemistry, or immunoprecipitation. Results: Rg1 activated promyogenic kinases, p38MAPK (mitogen-activated protein kinase) and Akt signaling, that in turn promote the heterodimerization with MyoD and E proteins, resulting in enhancing myogenic differentiation. Through the activation of Akt/mammalian target of rapamycin pathway, Rg1 induced myotube growth and prevented dexamethasone-induced myotube atrophy. Furthermore, Rg1 increased MyoD-dependent myogenic conversion of fibroblast. Conclusion: Rg1 upregulates promyogenic kinases, especially Akt, resulting in improvement of myoblast differentiation and myotube growth.

• Molecules and Cells
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• v.40 no.7
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• pp.495-502
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• 2017

The $5-HT_6R$ has been considered as an attractive therapeutic target in the brain due to its exclusive expression in the brain. However, the mechanistic linkage between $5-HT_6Rs$ and brain functions remains poorly understood. Here, we examined the effects of $5-HT_6R$-mediated cell morphological changes using immunocytochemistry, Western blot, and live-cell imaging assays. Our results showed that the activation of $5-HT_6Rs$ caused morphological changes and increased cell surface area in HEK293 cells expressing $5-HT_6Rs$. Treatment with 5-HT specifically increased RhoA-GTP activity without affecting other Rho family proteins, such as Rac1 and Cdc42. Furthermore, live-cell imaging in hippocampal neurons revealed that activation of $5-HT_6Rs$ using a selective agonist, ST1936, increased the density and size of dendritic protrusions along with the activation of RhoA-GTP activity and that both effects were blocked by pretreatment with a selective $5-HT_6R$ antagonist, SB258585. Taken together, our results show that $5-HT_6R$ plays an important role in the regulation of cell morphology via a RhoA-dependent pathway in mammalian cell lines and primary neurons.

• Development and Reproduction
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• v.21 no.4
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• pp.425-434
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• 2017

Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.

• Molecules and Cells
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• v.37 no.1
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• pp.31-35
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• 2014

Induced pluripotent stem cells (iPSCs) are capable of unlimited self-renewal and can give rise to all three germ layers, thereby providing a new platform with which to study mammalian development and epigenetic reprogramming. However, iPSC generation may result in subtle epigenetic variations, such as the aberrant methylation of the Dlk1-Dio3 locus, among the clones, and this heterogeneity constitutes a major drawback to harnessing the full potential of iPSCs. Vitamin C has recently emerged as a safeguard to ensure the normal imprinting of the Dlk1-Dio3 locus during reprogramming. Here, we show that vitamin C exerts its effect in a manner that is independent of the reprogramming kinetics. Moreover, we demonstrate that reprogramming cells under 2i conditions leads to the early upregulation of Prdm14, which in turn results in a highly homogeneous population of authentic pluripotent colonies and prevents the abnormal silencing of the Dlk1-Dio3 locus.

• Journal of Plant Biotechnology
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• v.46 no.3
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• pp.217-227
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• 2019

Production of therapeutic monoclonal antibodies (mAbs) using a plant platform has been considered an alternative to the mammalian cell-based production system. A plant-derived mAb CO17-1AK ($mAb^P$ COK) can specifically bind to various types of cancer cell lines. The target protein of $mAb^P$ COK is the epithelial cell adhesion molecule (EpCAM) highly expressed in human epithelial cancer cells, including breast and colorectal cancer cells. It has been hypothesized that its overexpression supports tumor growth and metastasis. A ganglioside is extended well beyond the surfaces of the various cell membranes and has roles in cell growth, inflammation, differentiation, and carcinogenesis. However, the regulation of EpCAM gene expression in breast cancers and the role of gangliosides in oncogenesis are unclear. Here, the purpose of this study was to determine the effects of $mAb^P$ COK on human breast cancer cell proliferation, apoptosis, and ganglioside expression patterns. Our results show that treatment with $mAb^P$ COK suppressed the growth of breast cancer cells and induced apoptotic cell death. It also upregulated the expression of metastasis-related gangliosides in breast cancer cells. Thus, treatment with $mAb^P$ COK may have chemo-preventive therapeutic effects against human breast cancer.

• KSBB Journal
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• v.7 no.2
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• pp.92-95
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• 1992

A model for the role of serum was proposed to develop a low serum medium for the large scale cu1ture of mammalian cell. The strategy of medium development adopted in this study facilitated the understanding of the role being carried out by the serum in the culture of hybridoma KAl12 cell line. In this model, the serum components were divided into two main groups : the first group encompasses the nutrient factors that determine the maximum cell density and the second group includes the growth factors that regulate the cell growth rate, The model prediction was compared with the experimental results. The model enabled us to find out several useful aspects of medium composition for cell growth. 1) One particular component in the basal medium became limiting factor when serum concentration level was more than 7%. 2) The growth regulating factors and nutrient factors limited the cell growth at 3% and 5% serum concentration levels respectively.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.121-121
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• 2003

The standard protocol for the production of transgenic mouse from ES-injected embryo has to process via chimera producing and several times breeding steps, In contrast, tetraploid-ES cell complementation method allows the immediate generation of targeted murine mutants from genetically modified ES cell clones. The advantage of this advanced technique is a simple and efficient without chimeric intermediates. Recently, this method has been significantly improved through the discovery that ES cells derived from hybrid strains support the development of viable ES mice more efficiently than inbred ES cells do. Therefore, the objective of this study was to generate transgenic mice overexpressing human resistin gene by using tetrapioid-ES cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR and cloned into pCR 2.1 TOPO T-vector and constructed in pCMV-Tag4C vector. Human resistin mammalian expression plasmid was transfected into D3-GL ES cells by lipofectamine 2000, and then after 8~10 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec. (fusion rate : 93.5%) and cultured upto the blastocyst stage (development rate : 94.6%). The 15~20 previously G418-selected ES cells were injected into tetraploid blastocysts, and then transferred into the uterus of E2.5d pseudopregnant recipient mice. To investigate the gestation progress, two El9.5d fetus were recovered by Casarean section and one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, this finding demonstrates that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mouse for the rapid analysis of gene function in vivo.

• Parasites, Hosts and Diseases
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• v.52 no.6
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• pp.595-603
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• 2014

Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time-and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite.

• Molecular & Cellular Toxicology
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• v.1 no.2
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• pp.111-115
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• 2005

Ultraviolet (UV) radiation to mammalian skin is known to alter cellular function via generation of Reactive Oxygen Species (ROS), DNA damage and DNA lesions, such as pyrimidine dimmers and photoproducts, which could lead to DNA mutation if they are not repaired. In this study, we have investigated the reduction of DNA damage and of apoptosis with a particular attention to genetic effect of paeoniflorin in Normal Human Epidermal Keratinocytes (NHEK). After UVB irradiation from $10\;to\;500mJ/cm^{2}$ to NHEK, Mean Tail Moments (MTM) were increased with UVB dose increase. The greatest amount of strand breaks was induced at $500mJ/cm^{2}$ of UVB. Even at the lowest dose of UVB ($10mJ/cm^{2}$), change in MTM was detected (P<0.0001). Pretreated cell with 0.1% paeoniflorin maximally reduced the level of DNA damage to about 21.3%, compared to untreated cell. In the lower concentrations less than 0.01% of paeoniflorin, MTM had a small increase but paeoniflorin still had reductive effects of DNA damage. We measured the apoptosis suppression of paeoniflorin with annexin V flous staining kit. As we observed under the fluorescence microscopy to detect apoptosis in the irradiated cell, the fluorescence intensity was clearly increased in the untreated cell, but decreased in treated cells with paeoniflorin. These results suggest that paeoniflorin reduces the alteration of cell membranes and prevents DNA damage. Therefore, the use of paeoniflorin as a free radical scavenger to reduce the harmful effects of UV lights such as chronic skin damage, wrinkling and skin cancer can be useful to prevent the formation of photooxidants that result in radical damage.