• IMMUNE NETWORK
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• v.20 no.5
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• pp.42.1-42.10
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• 2020

Long-lasting post-switched plasma cells (PCs) arise mainly from germinal center (GC) reactions, but little is known about the mechanism by which GC B cells differentiate into PCs. Based on our observation that the expression of the transcription factor CCAAT/enhancer binding protein β (C/EPBβ) is associated with the emergence of post-switched PCs, we enquired whether a cell-autonomous function of C/EPBβ is involved in the program for PC development. To address this, we generated C/EPBβ-deficient mice in which the Cebpb locus was specifically deleted in B cells after transcription of the Ig γ1 constant gene segment (Cγ1). In response to in vitro stimulation, B cells from these Cebpb^fl/flCγ1^Cre/+ mice had defects in the induction of B lymphocyte-induced maturation protein 1 (Blimp1) and the formation of IgG1⁺ PCs, but not in proliferation and survival. At steady state, the Cebpb^fl/flCγ1^Cre/+ mice had reduced serum IgG1 titers but normal IgG2c and IgM titers. Moreover, upon immunization with T-dependent Ag, the mice produced reduced levels of Ag-specific IgG1 Ab, and were defective in the production of Ag-specific IgG1 Ab-secreting cells. These results suggest that a cell-autonomous function of C/EPBβ is crucial for differentiation of post-switched GC B cells into PCs through a Blimp1-dependent pathway.

• Journal of Physiology & Pathology in Korean Medicine
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• v.23 no.1
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• pp.41-49
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• 2009

Soeumin-Googwihyangso-san(SGGHSS) has been used for the prevention or treatment of Soeumin-allergic rhinitis. This study was performed to demonstrate anti-allergic and anti-inflamatory effects of SGGHSS methanol extract in HMC cell lines and activated mouse B cells and CD4+ T cells. SGGHSS inhibited the production of TNF-$\alpha$ in PMA plus A23187 activated HMC-1 cells but not that of IL-6, as measured by ELISA. SGGHSS inhibited the expression of CD23 and surface IgE in B cells as determined by flowcytometry. It also inhibited secretion of IFN-$\gamma$ and IgG1, the Th1 related IgG type, but increased that of IL-4 in anti-CD40 and IL-4 treated B cells as measured by ELISA. As for Th cell differentiation, SGGHSS did not much affect IL-4 or IFN-$\gamma$. Taken together, our data showed that SGGHSS exerted an anti-inflammtory effect by inhibiting TNF-$\alpha$ in mast cells and has anti-allergic activity not via inhibition of CD4+ T cell, but via inhibition of B cells. These results suggest some evidence that SGGHSS can be applied to allergic disease.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.7-8
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• 2003

Since it was first reported in 1997, somatic cell cloning has been demonstrated in several other mammalian species. On the mouse, it can be cloned from embryonic stem (ES) cells, fetus-derived cells, and adult-derived cells, both male and female. While cloning efficiencies range from 0 to 20%, rates of just 1-2% are typical (i.e. one or two live offspring per one hundred initial embryos). Recently, abnormalities in mice cloned from somatic cells have been reported, such as abnormal gene expression in embryo (Boiani et al., 2001, Bortvin et al., 2003), abnormal placenta (Wakayama and Yanagimachi 1999), obesity (Tamashiro et ai, 2000, 2002) or early death (Ogonuki et al., 2002). Such abnormalities notwithstanding, success in generating cloned offspring has opened new avenues of investigation and provides a valuable tool that basic research scientists have employed to study complex processes such as genomic reprogramming, imprinting and embryonic development. On the other hand, mouse ES cell lines can also be generated from adult somatic cells via nuclear transfer. These 'ntES cells' are capable of differentiation into an extensive variety of cell types in vitro, as well assperm and oocytes in vivo. Interestingly, the establish rate of ntES cell line from cloned blastocyst is much higher than the success rate of cloned mouse. It is also possible to make cloned mice from ntES cell nuclei as donor, but this serial nuclear transfer method could not improved the cloning efficiency. Might be ntES cell has both character between ES cell and somatic cell. A number of potential agricultural and clinical applications are also are being explored, including the reproductive cloning of farm animals and therapeutic cloning for human cell, tissue, and organ replacement. This talk seeks to describe both the relationship between nucleus donor cell type and cloning success rate, and methods for establishing ntES cell lines. (중략)

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.103-103
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• 2003

The purpose of this study is to evaluate an efficacy of in vitro differentiated human embryonic stem (hES, MB03) cells expressing Nurr1 in relief of symptomatic motor behavior of Parkinson's disease (PD) animal models MB03 was genetically modified to express Nurr1 protein and was induced to differentiate according to 2-/4+ protocol using retinoic acid and ascorbic acid. The differentiation-induced cells were selected for 10 to 20 days thereafter in N2 medium. Upon selection, cells expressing GFAP, TH, or NF200 were 38.8%, 11%, and 20.5%, respectively. in order to examine therapeutic effects of the differentiated cells in PD animal model, rats were unilaterally lesioned by administration of 6-kydroxydopamine HCI (6-OHDA) into medial forebrain region (MFB, AP -4.4 mm, ML 1.2 mm, DV 78 mm with incision bar set at -2.4 mm), as a reference to bregma and the surface of the skull. Confirmation of successful lesion by apomorphine-induced rotational behavior, differentiated cells were transplanted into the striatum (AP 1.0, ML 3.5, DV -5.0; AP 0.6, ML 2.5, DV -4.5). Improvements of asymmetric motor behavior by the transplantation were examined every two weeks after the surgery. In two weeks, numbers of rotation by the experimental rats were $-14.8 \pm 33.9%$ (P<0.05) of the number before transplantation, however, the ratio increased slightly to $13.6 \pm 56.3%$ in six weeks. In contrast, the ratio of sham-grafted animals ranged from 112.3+8.5% to 139.2+28.9% during the examination. Immunohistochemical studies further confirmed the presence, survival, migration, and expression of TH of the transplanted human cells.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.77-83
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• 2009

Erythropoietin (EPO), a glycoprotein hormone produced from primarily cells of the peritubular capillary endothelium of the kidney, is responsible for the regulation of red blood cell production. We have been investigating the roles of glycosylation site added in the biosynthesis and function of recombinant protein. We constructed three EPO mutants ($\Delta$69, $\Delta$105 and $\Delta$69,105), containing an additional oligosaccharide chains. EPOWT and EPO$\Delta$69 were effectively expressed in transient and stably transfected CHO-K1 cell lines. But, it wasn't detected any protein in the culture medium of EPO$\Delta$105 and EPO$\Delta$69,105 mutants. The growth and differentiation of EPO-dependent human leukemic cell line (F36E) were used to measure the cytokine dependency and in vitro bioactivity of rec-hEPO. MTT assay values were increased by survival of F36E cells at 24h. To analysis biological activity in vivo, two groups of ICR-mice (7 weeks old) were injected subcutaneously with 10 IU per mice of rec-hEPO molecules on days 0 and 2. Red blood cell and hematocrit values were measured on 6 days after the first injection. The hematocrit values were remarkably increased in all treatment groups. The pharmacokinetic analysis was also affected in the mice injected with rec-hEPO molecules 2.5 IU by tail intravenous. Protein samples were detected by Western blotting. An EPO$\Delta$69 protein migrated as a broad band with an average apparent molecular and detected slightly high band. Enzymatic N-deglycosylation resulted in narrow band and was the same molecular size. The biological activity of EPO$\Delta$69 was enhanced to compare with wt-hEPO. The half-life was longer than wt-hEPO. The results suggest that hyperglycosyalted recombinant human erythropoietin (EPO$\Delta$69) may have important biological and therapeutic good points.

• Journal of Periodontal and Implant Science
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• v.25 no.2
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• pp.210-221
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• 1995

The purpose of this study was performed to investigate the mineralization and differentiation of osteobalsts for bone regeneration in vitro and the effect of rate of the composition in periodontal cells on mineralization. For this study, healthy gingival tissues were surgically obtained from the patients during 1st premolar extraction for the purposes of orthodontic treament. Gingival tissue was washed several time with Phosphate buffered saline contained high concentration of antibiotics and antifungal agent, and cultured in Dulbecco's Modified Eagle's Medium(DMEM, Gibco, U.S.A.). Every cell were cultured in state at $37^{\circ}C$, 100% of humidity, 5% of $CO_2$ incubator. Bone marrow stromal cells were isolated from 5-clay-old rat femur with using medium irrigation mathod by syringe. Cell suspension medium were centrifuged at 1500 rpm for 5 min and then cultured in the petri dish. Two kinds of cell were freezed and stocked in the liquid nitrogen tank until experiment. Cell were incubated into the 24 multi-well plate with $5{\times}10^4$cell/well of medium at $37^{\circ}C$, 100% of humidity 5% $CO_2$ incubator for 24 hours. After discarded of the supernatent of medium, O.5ml of medium were reapplied and incubated. And counted the number of cell using the hemocytometer and inverted light microscope. We have measured the number of mineralized nodule with using Alizarin red S. staining in microscope. Furthermore every cell were observed the morphological change between every rate of co-culture of the two kinds of cell. The results were as follows; The rate of proliferation of co-culture cell revealed high rate tendency compared the bone marrow stromal cell only and low growth rate to compared with gingival fibroblast only. The tendency of formation of the mineralized nodule were observed dose-depend pattern of bone marrow stromal cell. It is concluded that the gingival fibroblast may inhibit the formation of mineralized nodule in the culture of the bone marrow stromal cell.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.3
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• pp.285-296
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• 1997

Injury to brain transforms resting astrocytes to their reactive form, the hallmark of which is an increase in glial fibrillary acidic protein (GFAP), the major intermediate filament protein of their cell type. The overall glial response after brain injury is referred to as reactive gliosis. Glial-neuronal interaction is important for neuronal migration, neurite outgrowth and axonal guidance during ontogenic development. Although much attention has been given to glial regulation of neuronal development and regeneration, evidences also suggest a neuronal influence on glial cell differentiation, maturation and function. The aim of the present study was to analyze the effects of glial-hippocampal neuronal co-culture on GFAP expression in the co-cultured astrocytes. The following antibodies were used for double immunostaining chemistry; mouse monoclonal antibodies for confirm neuronal cells, rabbit anti GFAP antibodies for confirm astrocytes. Primary cultured astrocytes showed the typical flat polygonal morphology in culture and expressed strong GFAP and vimentin. Co-cultured hippocampal neurons on astrocytes had phase bright cell body and well branched neurites. About half of co-cultured astrocytes expressed negative or weak GFAP and vimentin. After 2 hour glutamate (0.5 mM) exposure of glial-neuronal co-culture, neuronal cells lost their neurites and most of astrocytes expressed strong CFAE and vimentin. In Western blot analysis, total GFAP and vimentin contents in co-cultured astrocytes were lower than those of primary cultured astrocytes. After glutamate exposure of glial-neuronal co-culture, GFAP and vimentin contents in astrocytes were increased to the level of primary cultured astrocytes. These results suggest that neuronal cell decrease GFAP expression in co-cultured astrocytes and hippocampal neuronal-glial co-culture can be used as a reactive gliosis model in vitro for studying GFAP expression of astrocytes.

• Journal of Biomedical Engineering Research
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• v.39 no.4
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• pp.168-174
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• 2018

Adipocytes affect obesity through the regulation of lipid metabolism. Physical loading is an important regulator of fat tissue. There are ongoing in vitro studies inducing mechanotransduction on 3T3-L1 preadipocytes with mechanical stimulus in order to treat obesity by inhibiting adipogenesis and provoking cell death. In this study, our goal was to suggest a new therapy for obesity by investigating whether fluid shear stress (FSS) changes transcription factors on 3T3-L1 related with adipogenesis and cell death. FSS loading was applied to 3T3-L1 preadipocytes at 1Pa and 1Hz. After loading, bright field images were taken and an immunofluorescence assay was conducted to observe actin stress fiber formation. Western blot analysis was conducted to identify the activation of the ERK pathway as well as the adipogenic factors, which including C/EBPs and $PPAR{\gamma}$. The expression of osteopontin, a protein related to inflammation in adipose tissue, and cell death related factors, Bax, Bcl-2, and Beclin, were also measured. Results showed that FSS stimulated the formation of actin stress fibers in 3T3-L1 and also that the activation of C/EBPs decreased significantly when compared with the control group. $PPAR{\gamma}$ activation in the 2 hour FSS group was lower than the 1 hour FSS group, which implied that the results were time dependent. Additionally, there were no differences in the expression of cell death factors after FSS loading. In summary, similar to other fibroblasts, the formation of actin stress fibers induced by mechanotransduction may affect the differentiation of 3T3-L1, leading to inhibition of adipogenesis and inflammation.

• Journal of Pharmacopuncture
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• v.16 no.4
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• pp.7-13
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• 2013

Objectives: Whether the ultra-highly-diluted remedies used in homeopathy can effectively bring about modulations of gene expressions through acetylation/deacetylation of histones has not been explored. Therefore, in this study, we pointedly checked if the homeopathically-diluted anti-cancer remedy Condurango 30C (ethanolic extract of Gonolobus condurango diluted $10^{-60}$ times) was capable of arresting the cell cycles in cervical cancer cells HeLa by triggering an epigenetic modification through modulation of the activity of the key enzyme histone deacetylase 2 vis-a-vis the succussed alcohol (placebo) control. Methods: We checked the activity of different signal proteins (like $p21^{WAF}$, p53, Akt, STAT3) related to deacetylation, cell growth and differentiation by western blotting and analyzed cell-cycle arrest, if any, by fluorescence activated cell sorting. After viability assays had been performed with Condurango 30C and with a placebo, the activities of histone de-acetylase (HDAC) enzymes 1 and 2 were measured colorimetrically. Results: While Condurango 30C induced cytotoxicity in HeLa cells in vitro and reduced HDAC2 activity quite strikingly, it apparently did not alter the HDAC1 enzyme; the placebo had no or negligible cytotoxicity against HeLa cells and could not alter either the HDAC 1 or 2 activity. Data on $p21^{WAF}$, p53, Akt, and STAT3 activities and a cell-cycle analysis revealed a reduction in DNA synthesis and G1-phase cell-cycle arrest when Condurango 30C was used at a 2% dose. Conclusion: Condurango 30C appeared to trigger key epigenetic events of gene modulation in effectively combating cancer cells, which the placebo was unable to do.

• Development and Reproduction
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• v.14 no.3
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• pp.155-162
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• 2010

The trophectoderm is one of the earliest cell types to differentiate in the forming placenta. It is an important for the initial implantation and placentation during pregnancy. Trophoblast stem cells (TBSCs) develop from the blastocyst and are maintained by signals emanating from the inner cell mass. However, several limitations including rarity and difficulty in isolation of trophoblast stem cells derived from blastocyst still exist. To establish a model for trophoblast differentiation, we isolated TBSCs from human term placenta ($\geq$38 weeks) and characterized. Cell cycle was analyzed by measuring DNA content by FACS analysis and phenotype of TBSCs was characterized by RT-PCR and FACS analysis. TBSCs have expressed various markers such as self-renewal markers (Nanog, Sox2), three germ layer markers (hNF68, alpha-cardiac actin, hAFP), trophoblast specific markers (CDX-2, CK7, HLA-G), and TERT gene. In FACS analysis, TBSCs isolated from term placenta showed that the majority of cells expressed CD13, CD44, CD90, CD95, CD105, HLA-ABC, cytokeratin 7, and HLA-G. Testing for CD31, CD34, CD45, CD71, vimentin and HLA-DR were negative. TBSCs were shown to decrease the growth rate when cultured in conditioned medium without FGF4/heparin as well as the morphology was changed to a characteristic giant cell with a large cytoplasm and nucleus. In invasion assay, TBSCs isolated from term placenta showed invasion activities in in vivo using nude mice and in vitro Matrigel system. Taken together, these results support that an isolation potential of TBSCs from term placenta as well as a good source for understanding of the infertility mechanism.