• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.1
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• pp.42-51
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• 2006

Schwann cell, one of important components of peripheral nervous system, interact with neurons to mutually support the growth and replication of embryonal nerves and to maintain the different functions of adult nerves. The Ara-C, known as an antimitotic agent, have been used to have high effectiveness in eliminating fibroblasts during Schwann cell culture period. This enrichment effect is also known to be cummulative with each successive pulse of Ara-C applied and is due to a progressive loss of fibroblasts. But the cytotoxicity by Ara-C is also cummulative and noticeable over the period. To determine the most effective application time and interval of Ara-C in the Schwann cell culture, we observed the Schwann cell purity and density with the Ara-C treatment in plain and three-dimensional culture from dorsal root ganglion of new born rat. By culturing dispersed dorsal root ganglia, we can repeatedly generate homogenous Schwann cells, and cellular morphology and cell count with mean percentages were evaluated in the plain culture dishes and in the immunostainings of S-100 and GFAP in the three-dimensional culture. The Ara-C treated cultures showed a higher Schwann cell percentage (31.0%${\pm}$8.09% in P4 group to 65.5%${\pm}$24.08% in P2 group), compared with that obtained in the abscence of Ara-C (17.6%${\pm}$6.03%) in the plain culture after 2 weeks. And in the three-dimensional culture, S-100 positive cells increased to 56.22%${\pm}$0.67% and GFAP positive cells to 66.46%${\pm}$1.83% in G2 group (p<0.05), higher yield than other groups with Ara-C application. Therefore, we concluded that the Ara-C treatment is effective for the proliferation of Schwann cells contrast to the fibroblasts in vitro culture, and the first application after 24 hours from cell harvesting and subsequent 2 pulse treatment (P2 group in plain culture and G2 group in three-dimensional culture) was more effective than other application protocols.

• Korean Chemical Engineering Research
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• v.46 no.6
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• pp.1100-1106
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• 2008

In this study, we presented rapid and simple fabrication method of functionalized surface on various substrates as a universal platform for the selective immobilization of cells. The functionalized surface was achieved by using deposition of polyelectrolyte such as poly(allyamine hydrochloride) (PAH), poly(diallyldimethyl ammonium chloride) (PDAC), poly(4-ammonium styrene sulfonic acid) (PSS), poly(acrylic acid) (PAA) and fabrication of poly(ethylene glycol) (PEG) microstructure through micro-molding in capillaries (MIMIC) technique on each glass, poly(methyl methacrylate) (PMMA), polystyrene (PS) and poly(dimethyl siloxane) (PDMS) substrate. The polyelectrolyte multilayer provides adhesion force via strong electrostatic attraction between cell and surface. On the other hand, PEG microstructures also lead to prevent non-specific binding of cells because of physical and biological barrier. The characteristic of each modified surface was examined by using static contact angle measurement. The modified surface onto several substrates provides appropriate environment for cellular adhesion, which is essential technology for cell patterning with high yield and viability in the micropatterning technology. The proposed method is reproducible, convenient and rapid. In addition, the fabrication process is environmentally friendly process due to the no use of harsh solvent. It can be applied to the fabrication of biological sensor, biomolecules patterning, microelectronics devices, screening system, and study of cell-surface interaction.

• Advances in Traditional Medicine
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• v.5 no.3
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• pp.216-230
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• 2005

Although the field of study in immune enhancing compounds is relatively new, natural products from plants represent a rich and promising source of novel molecules with immunomodulating properties, Microglial cells, the main immune effector cells of the brain, usually display a ramified morphology and low expression levels of immunologically relevant antigens such as MHC class I and class II. Since any compound which participates in activation of phagocytic cells contributes to the production of potentially toxic factors, the search for convenient in vitro test-systems and study of mechanisms of action of these agents are of great interest. Human blood polymorphonuclear (PMN) cells and primary microglial cells isolated from Sprague-Dawley rats were used as cellular screening tests for study of phagocytosis-stimulating action of immunomodulating agents. Numbers of phagocytic activity were evaluated by the phagocyte ingestion of yeast cells and NO-synthase activity, nitrite production, and nitroblue tetrazolium test were determined after phagocyte stimulation. It was possible to demonstrate that indexes of phagocytic activity can be used as quantitative indicators for measurement immunomodulating activity. As a positive control, Zymosan A-induced phagocytosis in both PMN cells and primary microglial cells was used. $IFN-{\gamma}$ (0.1 -1 U/ml) stimulated phagocytosis in PMN cells 1.2 times after 2 - 3 h incubation, although at higher concentrations (10 - 100 U/ml) it strongly inhibited phagocytosis. In a similar way, at higher concentrations, $IFN-{\gamma}$ (100 - 500 U/ml) suppressed phagocytosis in zymosan-A stimulated microglial cells. When Polypodium leucotomus, cambricum and vulgare extracts were tested alone, increased levels of phagocytosis were observed in PMN. In addition, microglial cells showed both increased phagocytosis and MHC class-II antigen expressions. Surprisingly, when PMN and microglia were treated with a combination of Polypodium and $IFN-{\gamma}$, phagocytosis was not inhibited. We did not find changes in NO-synthase activity and nitrite production in both microglia and PMN cells activated by different immunomodulating agents. These results indicate that primary microglial cell cultures as well as human PMN cells can provide reproducible quantitative results in screening phagocytic activity of different immunoactive compounds. Furthermore, both inhibitory or activation mechanisms might be studied using these in vitro experimental approaches.

• The Korean Journal of Zoology
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• v.33 no.3
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• pp.310-321
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• 1990

To establish the in vitro culture system and quantitation for chondrogenesis, and to investigate the relationship between cell aggregation and chondrogenesis, chick limb bud mesenchymal cells of Hamburger-Hamilton stage 23/24 were micromass cultured in various cell densities. The chondrogenesis was assayed based on checking the alcian blue-stained nodule numbers, the amount of alcian blue extraded, the change in cell numbers, the rate of [35 S] sulfate incorporation and expression of type II collagen. Mesenchymal cells plated with an initial density of high (1 x 107 cells/ml)- and intermediates (5. $\times$ 106 cells/ml)-density were differentiated into cartilage. On the other hand, the cells of low density (2 x 106 cells/mi, 5 $\times$ 105 cells/ml) of stage 23/24 cells and the stage 18/19 cells in three kinds of cell density did not differentiate into cartilage even though the cells formed an aggregated core at the center of cultured mass. From these results and others obtained in this study, it can be stated that the stage 23/24 mesenchymal cells are likely to pass over the aggregation step and have the potentiality to differentiate into chondrocytes. Thus chondrogenesis in vitro can be observed when mesenchymal cells are plated over the threshold density of 5 $\times$ 106 cells/ml. Hyaluronidase (HAase) activity was relatively constant throughout the culture, suggesting that the role of HAase may not be important for the cells of stage 23/24.

• Journal of Ginseng Research
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• v.40 no.4
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• pp.423-430
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• 2016

Background: The induction of cellular defensive genes such as phase II detoxifying and antioxidant enzymes is a highly effective strategy for protection against carcinogenesis as well as slowing cancer development. Transcription factor Nrf2 (nuclear factor E2-related factor 2) is responsible for activation of phase II enzymes induced by natural chemopreventive compounds. Methods: Red ginseng oil (RGO) was extracted using a supercritical $CO_2$ extraction system and chemical profile of RGO was investigated by GC/MS. Effects of RGO on regulation of the Nrf2/antioxidant response element (ARE) pathway were determined by ARE-luciferase assay, western blotting, and confocal microscopy. Results: The predominant components of RGO were 9,12-octadecadienoic acid (31.48%), bicyclo[10.1.0] tridec-1-ene (22.54%), and 22,23-dihydrostigmasterol (16.90%). RGO treatment significantly increased nuclear translocation of Nrf2 as well as ARE reporter gene activity, leading to upregulation of heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1. Phosphorylation of the upstream kinases such as apoptosis signal-regulating kinase (ASK)1, mitogen-activated protein kinase (MAPK) kinase (MKK)4/7, c-Jun N-terminal kinase (JNK), and p38 MAPK were enhanced by treatment with RGO. In addition, RGO-mediated Nrf2 expression and nuclear translocation was attenuated by JNK inhibitor SP600125 and p38 MAPK inhibitor SB202190. Conclusion: RGO could be used as a potential chemopreventive agent, possibly by induction of Nrf2/ARE-mediated phase II enzymes via ASK1-MKK4/7-JNK and p38 MAPK signaling pathways.

• Weed & Turfgrass Science
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• v.4 no.4
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• pp.308-314
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• 2015

This study was conducted to establish a fluorescence assay system for high efficient mass screening of the herbicides causing rapid membrane peroxidation, based on the fact that peroxide in cellular leakage could be fluorometrically determined through the fuorescent compounds formed after reacting with homovanillic acid (HVA) and peroxidase (HRP). The assay procesure established in this study was as follows. Only single disc (4 mm diameter) excised from cucumber cotyledon is placed on the well containing test solution ($200{\mu}L$) with 96-well microplate. The plate is shaking-incubated for 8 h under light condition. Then after removing the cucumber disc, HVA and HRP are supplied in the medium buffer and incubated for 5 min at room temperature. Fluorescence values are determined at Ex 320 nm/Ex 425 nm. The higher fluorescence values are obtained in the treatment of chemical having higher herbicidal activity. Using this assay with 96-well microplates, a large number of herbicides inducing rapid membrane peroxidation seemed to be screened more efficiently than spectrophotometric microtiter assay reported previously.

• Journal of Life Science
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• v.26 no.9
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• pp.1027-1032
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• 2016

Antigen is substance causing disease derived from pathogen. Living organism has the immune system in terms of defense mechanism against antigen. Antigen is processed through several pathways such as phagocytosis, antibody action, complement activation, and cytotoxins by NK or cytotoxic T lymphocyte via MHC molecule. Lymph node (LN) is comprised of the complicated 3 dimensional network and several stromal cells. Fibroblastic reticular cells (FRC) are distributed in T zone for interaction with T cells. FRC produces the extra cellular matrix (ECM) into LN for ECM reorganization against pathogen infections and secretes homing chemokines. However, it has not so much been known about the involvement of the antigen process of FRC. The present report is for the function of FRC on antigen process. For this, FRC was positioned with several infected situations such as co-culture with macrophage, T cell, lipopolysaccharide (LPS) and TNFα stimulation. When co-culture between FRC with macrophage and T cells was performed, morphological change of FRC was observed and empty space between FRCs was made by morphological change. The matrix metallo-proteinase (MMP) activity was up-regulated by Y27632 and T cells onto FRC. Furthermore, inflammatory cytokine, TNFα regulated the expression of adhesion molecules and MHC I antigen transporter in FRC by gene chip assay. NO production was elevated by FRC monolayer co-cultured with macrophage stimulated by LPS. GFP antigen was up-taken by macrophage co-cultured with FRC. Collectively, it suggests that FRC assists of the facilitation of antigen process and LN stroma is implicated into antigen process pathway.

• Molecules and Cells
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• v.25 no.3
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• pp.390-396
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• 2008

Calreticulin (CRT) is one of the major $Ca^{2+}$ binding chaperone proteins of the endoplasmic reticulum (ER) and an unusual luminal ER protein. Postnatally elevated expression of CRT leads to impaired development of the cardiac conductive system and may be responsible for the pathology of complete heart block. In this study, the molecular mechanisms that affect $Ca^{2+}$-dependent signal cascades were investigated using CRT-overexpressing cardiomyocytes. In particular, we asked whether calreticulin plays a critical role in the activation of $Ca^{2+}$-dependent apoptosis. In the cells overexpressing CRT, the intracellular calcium concentration was significantly increased and the activity of PKC and level of SECAR2a mRNA were reduced. Phosphorylation of Akt and ERKs decreased compared to control. In addition the activity of the anti-apoptotic factor, Bcl-2, was decreased and the activities of pro-apoptotic factor, Bax, p53 and caspase 8 were increased, leading to a dramatic augmentation of caspase 3 activity. Our results suggest that enhanced CRT expression in mature cardiomyocytes disrupts intracellular calcium regulation, leading to calcium-dependent apoptosis.

• Molecules and Cells
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• v.38 no.11
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• pp.998-1006
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• 2015

Retinols are metabolized into retinoic acids by alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (Raldh). However, their roles have yet to be clarified in hepatitis despite enriched retinols in hepatic stellate cells (HSCs). Therefore, we investigated the effects of retinols on Concanavalin A (Con A)-mediated hepatitis. Con A was injected into wild type (WT), Raldh1 knockout ($Raldh1^{-/-}$), $CCL2^{-/-}$ and $CCR2^{-/-}$ mice. For migration study of regulatory T cells (Tregs), we used in vivo and ex vivo adoptive transfer systems. Blockade of retinol metabolism in mice given 4-methylpyrazole, an inhibitor of ADH, and ablated Raldh1 gene manifested increased migration of Tregs, eventually protected against Con A-mediated hepatitis by decreasing interferon-${\gamma}$ in T cells. Moreover, interferon-${\gamma}$ treatment increased the expression of ADH3 and Raldh1, but it suppressed that of CCL2 and IL-6 in HSCs. However, the expression of CCL2 and IL-6 was inversely increased upon the pharmacologic or genetic ablation of ADH3 and Raldh1 in HSCs. Indeed, IL-6 treatment increased CCR2 expression of Tregs. In migration assay, ablated CCR2 in Tregs showed reduced migration to HSCs. In adoptive transfer of Tregs in vivo and ex vivo, Raldh1-deficient mice showed more increased migration of Tregs than WT mice. Furthermore, inhibited retinol metabolism increased survival rate (75%) compared with that of the controls (25%) in Con A-induced hepatitis. These results suggest that blockade of retinol metabolism protects against acute liver injury by increased Treg migration, and it may represent a novel therapeutic strategy to control T cell-mediated acute hepatitis.

• The Korean Journal of Pharmacology
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• v.29 no.1
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• pp.73-83
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• 1993

In order to examine the effect of ${\beta}-estradiol$ on the cell growth, using a primary rabbit kidney poximal tubule cell culture system. We investigated the effect of ${\beta}-estradiol$ on alpha 1 (IV) collagen and ${\beta}-actin$ mRNA levels from primary rabbit kidney cell cultures, and also the effects of 3 growth factors and ${\beta}-estradiol$ supplementation on the growth of primary rabbit kidney proximal tubule cells in the serum-free medium. 1 nM of ${\beta}-estradiol$ showed a sizable potentiation effect on the growth of the proximal tubule cell in serum-free medium, but higher concentration (> 10 nM) of estradiol indeed inhibited the growth. In the absence of hydrocortisone as a growth supplement in serum-free medium, ${\beta}-estradiol$ caused to potentiate the growth of the cell. In the presence of hydrocortisone, ${\beta}-estradiol$ also potentiated the growth of the proximal tubule cells. According to the Northern analysis, ${\beta}-estradiol$ increased the level of ${\beta}-actin$ mRNA, although mRNA level of the alpha I(IV) collagen was not changed significantly.