• BMB Reports
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• v.38 no.6
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• pp.683-689
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• 2005

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying ahexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5% of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100% with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.

• Journal of Periodontal and Implant Science
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• v.34 no.1
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• pp.205-221
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• 2004

Periodontal ligament(PDL) cells have been known as playing an important roles in periodontal regeneration and gingival fibroblasts are also important to periodontal regeneration by forming connective tissue attachment. There were rare studies about the gene expression patterns of PDL cells and gingival fibroblasts, therefore in this study, we tried cDNA microarray-based gene expression monitoring to explain the functional differences of PDL cells and gingival fibroblasts in vivo and to confirm the characteristics of PDL cells. Total RNA were extracted from PDL cells and gingival fibroblasts of same person and same passages, and mRNA were isolated from the total RNA using Oligotex mRNA midi kit(Qiagen) and then fluorescent cDNA probe were prepared. And microarray hybridization were performed. The gene expression patterns of PDL cells and gingival fibroblasts were quite different. About 400 genes were expressed more highly in the PDL cells than gingival fibroblasts and about 300 genes were more highly expressed in the gingival fibroblasts than PDL cells. Compared growth factor- and growth factor receptor-related gene expression patterns of PDL cells with gingival fibroblasts, IGF-2, IGF-2 associated protein, nerve growth factor, placental bone morphogenic protein, neuron-specific growth- associated protein, FGF receptor, EGF receptor-related gene and PDGF receptor were more highly expressed in the PDL cells than gingival fibroblasts. The results of collagen gene expression patterns showed that collagen type I, type III, type VI and type VII were more highly expressed in the PDL cells than gingival fibroblasts, and in the gingival fibroblasts collagen type V, XII were more highly expressed than PDL cells. The results of osteoblast-related gene expression patterns showed that osteoblast specific cysteine-rich protein were more highly expressed in the PDL cells than gingival fibroblasts. The results of cytoskeletal proteins gene expression patterns showed that a-smooth muscle actin, actin binding protein, smooth muscle myosin heavy chain homolog and myosin light chain were more highly expressed in the PDL cells than gingival fibrobalsts, and ${\beta}-actin$, actin-capping protein(${\beta}$ subunit), actin- related protein Arp3(ARP) and myosin class I(myh-1c) were more highly expressed in the gingival fibroblasts than PDL cells. Osteoprotegerin/osteoclastogenesis inhibitory factor(OPG/OCIF) was more highly expressed in the PDL cells than gingival fibroblasts. According to the results of this study, PDL cells and gingival fibroblasts were quite different gene expression patterns though they are the fibroblast which have similar shape. Therefore PDL cells & gingival fibroblasts are heterogeneous populations which represent distinct characteristics. If more studies about genes that were differently expressed in each PDL cells & gingival fibroblasts would be performed in the future, it would be expected that the characteristics of PDL cells would be more clear.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.04a
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• pp.80-80
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• 2018

Chlorogenic acid is a phenolic compound found in Cudrania tricuspidata fruits. In the present study, the effect of chlorogenic acid on the inhibition of adipogenesis in 3T3-L1 adipocytes was investigated. Cells were stained with Oil red O reagent to detect lipid droplets in adipocytes. The 3T3-L1 cells were lysed and measured for intracellular triglyceride and adipokine by ELISA kit. The protein expression of adipogenesis-related gene was evaluated by Western blot analysis. Chlorogenic suppressed lipid droplet and intracellular triglyceride accumulation in a concentration manner and also decreased secretion of adipokines such as leptin and adiponectin, compared with fully differentiated adipocytes. Treatment of 3T3-L1 cells with chlorogenic acid reduced the protein levels of peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$) and, CCAAT/enhancer binding proteins alpha ($C/EBP{\alpha}$). This indicates that chlrogenic acid was effective as an anti-obesity agent by repressing the differentiation of 3T3-L1 into adipocytes and inhibiting triglyceridef formation in adipocyte and that it exerts its role mainly through the significant down-regulation of $PPAR{\gamma}$ and $C/EBP{\alpha}$.

• Ocean and Polar Research
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• v.41 no.2
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• pp.79-88
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• 2019

In this study, the inhibitory effect of Atriplex gmelinii C. A. Mey. against the activity of MMP-2 and MMP-9 secreted from phorbol-12-myristate-13-acetate (PMA)-stimulated HT-1080 cells was evaluated by gelatin zymography and enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase-chain reaction (RT-PCR), and Western blot assay. Specimens of the halophyte A. gmelinii were extracted twice for 24 hr with methylene chloride ($CH_2Cl_2$), and then twice with methanol (MeOH), in turn. Each extract significantly inhibited the enzymatic activities in gelatin zymography and MMP ELISA kit, and expression of MMP-2 and 9 in mRNA and protein levels. Two crude extracts were combined and then the combined crude extracts were fractionated into n-hexane, 85% aqueous methanol (85% aq.MeOH), n-butanol (n-BuOH), and water ($H_2O$) fractions, according to solvent polarity. Among solvent-partitioned fractions, the 85% aq.MeOH fraction showed the strongest inhibitory effect against MMP-2 and -9 in gelatin zymography and MMP ELISA kit. In RT-PCR, all solvent-partitioned fractions significantly suppressed mRNA expression of MMP-2 and -9. On the other hand, in Western blot assay, all solvent-partitioned fractions except $H_2O$ significantly reduced expression levels of protein. HT 1080 cell migration was most significantly inhibited by the n-BuOH fraction followed by the 85% aq.MeOH and $H_2O$ fractions. These results suggest that A. gmelinii could be used as a potential source to inhibit tumor cell metastasis.

• Biomedical Science Letters
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• v.13 no.2
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• pp.149-152
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• 2007

Mast cells play important roles in immune-related diseases, in particular, allergic diseases. Although 9-cis retinoic acid (9CRA) has been known as an immune regulator, its function in mast cells is not characterized well. In a previous paper, we demonstrated that 9CRA differentially decreases both CCR2 expression and the MCP-1-induced chemotactic activity of the human mast cell line, HMC-1 cells. In the present study, we examined the effects of 9CRA on the migration and expressions of inflammatory cytokines in HMC-1 cells. It was found that 9CRA significantly inhibited the migration of HMC-1 cells in response to stem cell factor (P<0.01), and it had no effect on the mRNA and protein expression of c-kit, a receptor binding to SCF. We further investigated the alternation of inflammatory cytokine expression and identified that 9CRA blocked the mRNA and protein expressions of Th2 cytokines such as interleukin (IL)-4 and IL-5. Taken together, our results demonstrate that 9CRA blocks SCF-induced cell movement and the protein secretion of IL-4 and IL-5, and this indicates that 9CRA may have anti-inflammatory effects on mast cells.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1347-1354
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• 2006

The aims of this study were to establish a simple and effective method for isolating male germline stem cells (GSCs), and to test the possibility of using these cells as a new approach for male infertility treatment. Testes obtained from neonatal and adult mice were manually decapsulated. GSCs were collected from seminiferous tubules by a two-step enzyme digestion method and plated on gelatin-coated dishes. Over 5-7 days of culture, GSCs obtained from neonates and adults gave rise to large multicellular colonies that were subsequently grown for 10 passages. During in vitro proliferation, oct-4 and two immunological markers (Integrin ${\beta}1,\;{\alpha}6$) for GSCs were highly expressed in the cell colonies. During another culture period of 6 weeks to differentiate to later stage germ cells, the expression of oct-4 mRNA decreased in GSCs and Sertoli cells encapsulated with calcium alginate, but the expression of c-kit and testis-specific histone protein 2B(TH2B) mRNA as well as the localization of c-kit protein was increased. Expression of transition protein (TP-l) and localization of peanut agglutinin were not seen until 3 weeks after culturing, and appeared by 6 weeks of culture. The putative spermatids derived from GSCs supported embryonic development up to the blastocyst stage with normal chromosomal ploidy after chemical activation. Thus, GSCs isolated from neonatal and adult mouse testes were able to be maintained and proliferated in our simple culture conditions. These GSCs have the potential to differentiate into haploid germ cells during another long-term culture.

• Herbal Formula Science
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• v.31 no.1
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• pp.11-19
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• 2023

Objectives : The purpose of this study was to examine the effects of herbal medicines on pacemaker potentials of large intestinal interstitial Cells of Cajal (ICC) in mice. Methods : We made the ICC culture in large intestine in mice and used the electrophysiological method to record pacemaker potentials. Also we used MTT assay to check cell viability and examined the ICC protein expression by western blot. Results : 1.Glycyrrhiza uralensis Fischer (GF) (50-150 ㎍/ml) induced pacemaker depolarization and decreased frequency with concentration-dependent manners. EC₅₀ is 58.95 ㎍/ml. Angelica gigas (AG) (50-200 ㎍/ml) induced pacemaker depolarization and decreased frequency with concentration-dependent manners. EC₅₀ is 77.22 ㎍/ml. Poncirus fructus (PF) (10-100 ㎍/ml) induced pacemaker depolarization and decreased frequency with concentration-dependent manners. EC₅₀ is 13.39 ㎍/ml. Citrus unshiu S. Marcov. (CU) (10-500 ㎍/ml) induced pacemaker depolarization and decreased frequency with concentration-dependent manners. EC₅₀ is 139.80 ㎍/ml. Gardenia jasminoides J. Ellis (GJ) (100-500 ㎍/ml) induced pacemaker depolarization and decreased frequency with concentration-dependent manners. EC₅₀ is 78.70 ㎍/ml. Coptis chinensis (CC) (100-1000 ㎍/ml) induced pacemaker depolarization and decreased frequency with concentration-dependent manners. EC₅₀ is 138.10 ㎍/ml. Scutellaria baicalensis (SB) (10-100 ㎍/ml) had no effects on pacemaker potentials and decreased frequency with concentration-dependent manners. IC₅₀ is 18.34 ㎍/ml. Atractylodes macrocephala koidzumi (AM) (10-100 ㎍/ml) induced pacemaker hyperpolarizations and decreased frequency with concentration-dependent manners. IC₅₀ is 18.54 ㎍/ml. 2. PF, SB and AM had no effects on cell death in large ICC. 3. PF increased the ANO1 and c-kit protein expression and SB and AM increased the c-kit protein expression in large ICC. Conclusions : These results suggest that PF, SB, and AM are likely to be the optimal combination of herbal medicines that can be used to treat diseases such as gastrointestinal motility disorders such as irritable bowel syndrome.

• Journal of Pharmacopuncture
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• v.16 no.4
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• pp.36-42
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• 2013

Objectives: Shengmaisan (SMS) is a traditional Chinese medicine prescription widely used for the treatment of diverse organs in Korea. The interstitial cells of Cajal (ICCs) are pacemaker cells that play an important role in the generation of coordinated gastrointestinal (GI) motility. We have aimed to investigate the effects of SMS in the ICCs in the mouse small intestine. Methods: To dissociate the ICCs, we used enzymatic digestions from the small intestine in a mouse. After that, the ICCs were identified immunologically by using the anti-c-kit antibody. In the ICCs, the electrophysiological whole-cell patch-clamp configuration was used to record pacemaker potentials in the cultured ICCs. Results: The ICCs generated pacemaker potentials in the mouse small intestine. SMS produced membrane depolarization with concentration-dependent manners in the current clamp mode. Pretreatment with a $Ca^{2+}$ free solution and thapsigargin, a $Ca^{2+}$-ATPase inhibitor in the endoplasmic reticulum, stopped the generation of the pacemaker potentials. In the case of $Ca^{2+}$-free solutions, SMS induced membrane depolarizations. However, when thapsigargin in a bath solution was applied, the membrane depolarization was not produced by SMS. The membrane depolarizations produced by SMS were inhibited by U-73122, an active phospholipase C (PLC) inhibitors. Furthermore, chelerythrine and calphostin C, a protein kinase C (PKC) inhibitors had no effects on SMS-induced membrane depolarizations. Conclusions: These results suggest that SMS might affect GI motility by modulating the pacemaker activity through an internal $Ca^{2+}$- and PLC-dependent and PKC-independent pathway in the ICCs.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.1
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• pp.13-23
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• 2010

Objective: The present study was carried out to induce differentiation of human embryonic stem cells (hESCs) into germ cells and to establish a culture condition for single hESCs dissociated by enzyme. Methods: Embryonic body (EB) was formed by hanging drop culture for 3 days from hESCs colony. The EBs were cultured in the medium supplemented with retionic acid (RA) or/and bone morphogenetic protein-4 (BMP4) for 14 days to differentiate into germ cells. Germ cell specific markers, c-kit and VASA were used for immunohistochemistry of EB. Human ESCs colonies were dissociated into single cells by Collagenase, Tryple and Accutase, and then colony formation rate of the single cells was examined. Rho-associated kinase inhibitor (ROCK inhibitor, Y27632) was added into the culture medium of single cells to reduce the apoptotic damage during the dissociation. Results: Single cells dissociated with Tryple or Accutase showed higher colony formation rates compared to the cells dissociated with Collagenase. Seeding of $5{\times}10^3$ cells/well (4 well dish) was efficient to obtain high colony formation rate compared to other concentrations of seeding cell. Addition of Y27632 significantly increased the colony formation rate of the single cells dissociated by Tryple. Immunohistochemistry of EB with c-kit and VASA markers showed a weak fluorescence signals compared to the signals from the testicular tissue. Conclusion: Dissociation with Tryple was useful to obtain healthy single cells and addition of Y27632 was beneficial for survival and colony formation of the single cells. Unlike other studies, we just observed a dim fluorescence staining of the germ cell markers, probably caused by the short-term culture for the differentiation of EB compared to other studies.

• Microbiology and Biotechnology Letters
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• v.14 no.3
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• pp.219-223
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• 1986

In order to preparr the hybridoma cells which produce a monoclonal antibody to human fibroblast interferon(Hu IFN-$\beta$), spleen cells from BALB/cmice immunized with the purified Hu IFN-$\beta$ were fused with NS-O cells, a myeloma cell line. Forty hybrids with high titer among 1300 hybrids Isolated by an ELISA screening method were subcloned using the soft agarose cloning and limiting dilution methods, and 11 hybrids were selected. As a result of iso-typing the hybrids using the mouse monoclonal typing kit, two hybridomas were found to produce 1gG 2a type of monoclonal an-tibodies. The ascites fluid from nude mice inoculated intraperitoneally with the above hybridomas was removed and purified using a protein A-Sepharose CL-4B. Monoclonal antibody was proven to have only the heavy and light chains on SDS-polyacrylamide gel electrophoresis.