• KSBB Journal
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• v.23 no.6
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• pp.513-518
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• 2008

This paper described the extraction/purification of $\beta$-carotene from recombinant E.coli and evaluation of anti-wrinkle activity of purified $\beta$-carotene. No significant differences in extraction yields were observed when hexane or isobutyl acetate was used. However, extraction from wet-cell cake resulted in 2-fold higher amount of $\beta$-carotene than that from dry cells. Disruption of 5 g-wet cells by ultrasonic homogenizer, acetone dehydration, extraction with isobutyl acetate resulted in 36 mg of $\beta$-carotene corresponding to 61.2% of recovery. The formation and separation of $\beta$-carotene crystal improved the purity. 633 mg of $\beta$-carotene crystal with 93% purity was obtained from 223 g/L of wet-cell cake harvested from 2.5-L fed-batch culture broth. The cultures of normal human primary fibroblast were performed to investigate the effect of $\beta$-carotene on cytotoxicity as MTT assay and anti-wrinkle activity as collagen synthesis assays. $1.7{\mu}M$ of $\beta$-carotene was found to be optimal concentration at which 1.4-fold higher amount of collagen was synthesized than that in absence of $\beta$-carotene. This indicates that highly purified $\beta$-carotene can be obtained from recombinant E.coli by applying simple method with less toxic solvent and can be used in functional cosmetics as anti-wrinkle agent.

• Journal of Periodontal and Implant Science
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• v.42 no.3
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• pp.73-80
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• 2012

Purpose: The purpose of this study was to investigate the effects of the immunosuppressants FK506 and cyclosporin A (CsA) on the osteogenic differentiation of rat mesenchymal stem cells (MSCs). Methods: The effect of FK506 and CsA on rat MSCs was assessed in vitro. The MTT assay was used to determine the deleterious effect of immunosuppressants on stem cell proliferation at 1, 3, and 7 days. Alkaline phosphatase (ALP) activity was analyzed on days 3, 7, and 14. Alizarin red S staining was done on day 21 to check mineralization nodule formation. Real-time polymerase chain reaction (RT-PCR) was also performed to detect the expressions of bone tissue-specific genes on days 1 and 7. Results: Cell proliferation was promoted more in the FK506 groups than the control or CsA groups on days 3 and 7. The FK506 groups showed increased ALP activity compared to the other groups during the experimental period. The ALP activity of the CsA groups did not differ from the control group in any of the assessments. Mineralization nodule formation was most prominent in the FK506 groups at 21 days. RT-PCR results of the FK506 groups showed that several bone-related genes-osteopontin, osteonectin, and type I collagen (Col-I)-were expressed more than the control in the beginning, but the intensity of expression decreased over time. Runx2 and Dlx5 gene expression were up-regulated on day 7. The effects of 50 nM CsA on osteonectin and Col-I were similar to those of the FK506 groups, but in the 500 nM CsA group, most of the genes were less expressed compared to the control. Conclusions: These results suggest that FK506 enhances the osteoblastic differentiation of rat MSCs. Therefore, FK506 might have a beneficial effect on bone regeneration when immunosuppressants are needed in xenogenic or allogenic stem cell transplantation to treat bone defects.

• Journal of Periodontal and Implant Science
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• v.41 no.6
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• pp.293-301
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• 2011

Purpose: We have previously reported that tetra-cell adhesion molecule (T-CAM) markedly enhanced the differentiation of osteoblast-like cells grown on anorganic bone mineral (ABM). T-CAM comprises recombinant peptides containing the Arg- Gly-Asp (RGD) sequence in the tenth type III domain, Pro-His-Ser-Arg-Asn (PHSRN) sequence in the ninth type III domain of fibronectin (FN), and the Glu-Pro-Asp-Ilu-Met (EPDIM) and Tyr-His (YH) sequence in the fourth fas-1 domain of ${\beta}$ig-h3. Therefore, the purpose of this study was to evaluate the cellular activity of osteoblast-like cells and the new bone formation on ABM coated with T-CAM, while comparing the results with those of synthetic cell binding peptide (PepGen P-15). Methods: To analyze the cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed, andto analyze gene expression, northernblot was performed. Mineral nodule formations were evaluated using alizarin red stain. The new bone formations of each group were evaluated using histologic observation and histomorphometrc analysis. Results: Expression of alkaline phosphatase mRNA was similar in all groups on days 10 and 20. The highest expression of osteopontin mRNA was observed in the group cultured with ABM/P-15, followed by those with ABM/T-CAM and ABM on days 20 and 30. Little difference was seen in the level of expression of collagen type I mRNA on the ABM, ABM/T-CAM, and ABM/P-15 cultured on day 20. There were similar growth and proliferation patterns for the ABM/T-CAM and ABM/P-15. The halo of red stain consistent with $Ca^{2+}$ deposition was wider and denser around ABM/T-CAM and ABM/P-15 particles than around the ABM particles. The ABM/T-CAM group seemed to have bone forming bioactivity similar to that of ABM/P-15. A complete bony bridge was seen in two thirds of the defects in the ABM/T-CAM and ABM/P-15 groups. Conclusions: ABM/T-CAM, which seemed to have bone forming bioactivity similar to ABM/P-15, was considered to serve as effective tissue-engineered bone graft material.

• Herbal Formula Science
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• v.26 no.3
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• pp.207-221
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• 2018

Objectives : Present study investigated hepatoprotective effect of Haegan-jeon extract (HE) and tried to elucidate molecular mechanism involved. According to molecular mechanism, present study optimized herbal composition of HE (op-HE) and compared in vitro and in vivo hepatoprotective effects of op-HE to HE. Methods : For in vitro experiments, HepG2 cells were exposed to arachidonic acid (AA, $10{\mu}M$) and iron ($5{\mu}M$) for inducing oxidative stress. Cell viability, GSH contents, $H_2O_2$ production, mitochondrial membrane potential, immunoblot and reporter gene assay were performed to investigate cytoprotective effects and responsible molecular mechanisms. For in vivo experiments, hepatoprotective effect of HE and op-HE were assessed on $CCl_4-induced$ liver injury mice model. Results : HE pretreatment prevented AA+iron-mediated hepatocytes apoptosis. In addition, AA+iron-induced mitochondrial dysfunction, $H_2O_2$ production, glutathione depletion were reduced by HE pretreatment. In addition, nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation, antioxidant response element (ARE)-driven reporter gene activity, and antioxidant genes expression were increased by HE. Based on reporter gene and MTT assays, we found that op-HE consisting three medicinal herbs also significantly increased transactivation of Nrf2 and reduced the AA+iron-mediated cytotoxicity. Moreover, in $CCl_4-induced$ liver injury mice model, HE-op had an ability to ameliorate $CCl_4-mediated$ increases in serum alanine transferase and aspartate aminotransferase activity, hepatic degeneration, inflammatory cell infiltration, and collagen deposition. Hepatoprotective effects of op-HE were comparable to those of HE. Conclusions : Present study suggests that op-HE as well as HE exhibit hepatoprotective effect against oxidative stress-mediated liver injury via Nrf2 activation.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.1
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• pp.65-75
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• 2003

Objectives: To investigate the role of TGF (Transforming growth factor-$\beta$) involved in the paracrinic communication during decidualization between UEC (uterine epithelial cells) and USC (uterine stromal cells), we have employed a co-culture system composed of human endometrial epithelial and stromal cells in defined hormonal conditions. Design: In the co-culture, endometrial epithelial cells cultured in the matrigel-coated cell culture insert are seeded on top of the endometrial stromal cells cultured within a collagen gel. The co-culture was maintained for 48 hours under the following hormonal conditions: progesterone dominant condition (100 nM P4 and 1 nM E2) or estrogen-dominant condition (100 nM E2 and 1 nM P4). 10 ng/ ml HGF and/or 10 ng/ml TGF-$\beta$1 are added. Methods: RT-PCR is utilized to detect mRNAs quantitatively. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining are utilized to detect proteins in the tissue. Results: Prolactin mRNA is expressed in the co-cultured stromal cells under the progesterone dominant condition. TGF-$\beta$1 and its receptors are expressed in both the co-cultured epithelial and stromal cells irrespective of the steroid present, which is in contrast with no or negligible expression of TGF-$\beta$1 or its receptor in cells separately cultured. Both estrogen and progesterone significantly elevate the concentration of hepatocyte growth factor (HGF) in the conditioned medium of the co-culture with the value of 4, 325 pg/ml in E2-dominant and 2, 000 pg/ml in P4-dominant condition compare to 150 pg/ml in no hormone. In separately cultured stromal cells, administration of HGF induces the expression of TGF receptor 1 in both hormonal conditions, but induction of TGF receptor 2 is only manifest in the P4-dominant condition. Administration of TGF-$\beta$ and HGF directly induce the decidualization marker prolactin mRNA in separately cultured stromal cells. Conclusion: It is likely that steroid hormones induces prolactin mRNA indirectly by promoting the cell to cell communication between the stromal and the epithelial cells. TGF-$\beta$ and HGF are two possible paracrine mediators in the human endometrial decidualization.

• Journal of Chest Surgery
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• v.46 no.1
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• pp.1-13
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• 2013

Background: Glutaraldehyde (GA) is a widely used cross-linking agent for improving mechanical properties and resistance to enzymatic degradation of collagenous tissue, but it has several drawbacks such as calcification and cytotoxicity. The aim of this study was to find the alternative effective cross-linking methods to GA. Materials and Methods: Bovine pericardium was processed with GA with ethanol+octanol and glycine detoxification, and polyethylene glycol (PG) space filler, dimethyl 3,3'-dithiobispropionimidate (DTBP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) treatment, and the physical fixation of ultraviolet irradiation were done. The biologic material properties of variously treated pericardial tissues were assessed by biochemical, mechanical and histological tests. Treated pericardial tissues were also implanted subcutaneously or intramuscularly into the rabbit for 10 weeks to assess the xenoreactive antibody response of immunoglobulin G and M, their anti-calcification effect. Results: The biochemical and mechanical properties of EDC fixed pericardial tissues were comparable to the GA fixed tissue. The cytotoxicity was lowest in space filler treated GA fixed group. In rabbit subcutaneous or intramuscular implantation models, decellularization, space filler, EDC treatment group showed significantly lower calcium content than GA only and DTBP treatment group (p<0.05, analysis of variance). The titer of anti $Gal{\alpha}1-3Gal{\beta}1$-4GlcNAc-R antibodies did not change in the postimplantation serial enzyme-linked immunosorbent assay. Hematoxylin and eosin and von Kossa staining showed that decellularization, space filler, EDC, and ultraviolet treatment had less inflammatory cell infiltration and calcium deposits. Conclusion: The decellularization process, PG filler, and EDC treatments are good alternative cross-linking methods compared to GA only fixation and primary amine of DTBP treatment for cardiovascular xenograft preservation in terms of the collagen cross-linking stability and in vivo anti-calcification effects.

• International Journal of Oral Biology
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• v.33 no.3
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• pp.105-111
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• 2008

Thrombospondins (TSP-1, TSP-2) are secretory extracellular glycoproteins that are involved in a variety of physiological processes such as tumor cell adhesion, invasion, and metastasis. The present study was undertaken to elucidate the involvement of thrombospondins in the adhesion of osteoblast-like cells using the TSP-1 or TSP-2 antisense MG63 and MC3T3-E1 cell lines. For downregulation of TSPs expression, we prepared antisense constructs for TSP-1 and TSP-2 using the pREP4 an episomal mammalian expression vector, which be able to produce the specific antisense oligonucleotides around chromosome. MG63 and MC3T3-E1 osteoblast-like cells were transfected with the antisense constructs and nonliposomal Fugene 6, and then selected under hygromycin B (50 ${\mu}g/ml$) treatment for 2 weeks. Western blot analysis revealed that expression of the TSP proteins was downregulated in the antisense cell lines. The cell adhesion assay showed that adhesive properties of TSP-1 and TSP-2 antisense MG63 cells on the polystyrene culture plate were reduced to 17% and 21% of the control cells, respectively, and those of the TSP-1 and TSP-2 antisense MC3T3-E1 cells also decreased to 19% and 27% of control, respectively. Adhesion of TSP-1 and TSP-2 antisense MC3T3-E1 cells on Type I collagen-coated culture plate decreased to 27% and 76%, respectively. These results indicate that TSP-1 and TSP-2 proteins may have an important role in adhesion of osteoblast-like cells to extracellular matrix.

• Polymer(Korea)
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• v.27 no.5
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• pp.397-404
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• 2003

The interaction of cell adhesive protein and polypeptide with bone marrow stromal stem cells (BMSCs) grown in tissue engineered films and scaffolds were examined. Several proteins or polypeptide known as cell-adhesive were coated adsorption on poly(lactide-co-glycolide) (PLGA) films and scaffolds and adhesion and proliferation behavior of BMSC on those surfaces were compared. The protein and polypeptide used include collagen IV, fibrinogen, laminin, gelatin, fibronectin, and poly(L-lysine). The protein and polypeptide were adsorbed on the PLGA film surfaces with almost monolayer coverage except poly(L-lysine). BMSCs were cultured for 1, 2, and 4 days on the protein- or polypeptide-adsorbed PLGA films and scaffolds. The cell adhesion and proliferation behaviors were assessed by sulforho damine B assay. It was observed that the protein- or polypeptide-adsorbed surfaces showed better cell adhesion and proliferation than the control.

• Journal of Periodontal and Implant Science
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• v.35 no.1
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• pp.99-109
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• 2005

It has been focused on the importance of the host inflammatory response in periodontal pathogenesis and progression, treatment has been introduced to control the host response and the method, which diminishes production and activity of MMP by doxycycline, has been used in periodontal field. MMP is a proteolytic enzyme which plays a major role in tissue destruction and MMP-1 is secreted in the periodontally healthy tissue, while MMP-8, 9, 13, etc in the inflammatory state. Among these, MMP-13 has been discovered lately and reported to degrade primarily type II collagen. Periodontal ligament (PDL) cell plays a role in destruction of periodontal tissue. This study was to evaluate the effect of doxycycline and mefenamic acid, non-steroidal antiinflammatory drug on MMP-13 mRNA expression in the rat PDL cell. Doxycycline concentration of $1{\sim}100\;{\mu}g/ml$ was added rat PDL cell and cell activity was measured by MIT assay at day 1 and 3. MMP-13 gene expression was evaluated by RT-PCR after PDL cells were pre-treated for 1hour with doxycycline (50 ${\mu}g/ml$) alone or with mefenamic acid ($10^{-6}M$), then added $IL-1{\beta}$(1.0 ng/ml) and incubated for 16-18 hours. The results are as follows: 1. Cell activity decreased Significantly at 24 and 72 hours in 100 ${\mu}g/ml$ (p<0.05). 2. Level of MMP-13 mRNA was in 20.2% increase by $IL-1{\beta}$ and in pre-treating doxycycline group, expression of $IL-1{\beta}$ induced MMP-13 mRNA was inhibited by 31% than $IL-1{\beta}$ treated only. 3. Mefenamic acid did not inhibit on the expression of $IL-1{\beta}$ induced MMP-13 mRNA, while mefenamic acid in combination with doxycycline inhibited the expression by 41% compared to only $IL-1{\beta}$ stimulation. These results suggest that doxycycline synergistically inhibit the expression of $IL-1{\beta}$ induced MMP-13 mRNA in combination with mefenamic acid.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.81-89
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• 2004

Tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and lymphotoxin-$\alpha$ (LT-$\alpha$, TNF-$\beta$) can initiate and perpetuate human diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and insulin-dependent diabetes mellitus (IDDM). TNFs can be blocked by the use of soluble TNF receptors. However, since monomeric soluble receptors generally exhibit low affinity or function as agonists, the use of monomeric soluble receptors has been limited in the case of cytokines such as TNF-$\alpha$, TNF-$\alpha$, interleukin (IL)-1, IL-4, IL-6, and IL-13, which have adapted to a multi component receptor system. For these reasons, very high-affinity inhibitors were created for the purpose of a TNFs antagonist to bind the TNFR and trigger cellular signal by using the multistep polymerase chain reaction method. First, recombinant simple TNFR-Ig fusion proteins were constructed from the cDNA sequences encoding the extracellular domain of the human p55 TNFR (CD120a) and the human p75 TNFR (CD120b), which were linked to hinge and constant regions of human $IgG_1$ heavy chain, respectively using complementary primers (CP) encoding the complementary sequences. Then, concatameric TNFR-Ig fusion proteins were constructed using recombinant PCR and a complementary primer base of recombinant simple TNFR-Ig fusion proteins. For high level expression of recombinant fusion proteins, Chinese hamster ovary (CHO) cells were used with a retroviral expression system. The transfected cells produced the simple concatameric TNFR-Ig fusion proteins capable of binding TNF and inactivating it. These soluble versions of simple concantameric TNFR-Ig fusion proteins gave rise to multiple forms such as simple dimers and concatameric homodimers. Simple TNFR-1g fusion proteins were shown to have much more reduced TNF inhibitory activity than concatameric TNFR-Ig fusion proteins. Concatameric TNFR-Ig fusion proteins showed higher affinity than simple TNFR-Ig fusion proteins in a receptor inhibitor binding assay (RIBA). Additionally, concatameric TNFR-Ig fusion proteins were shown to have a progressive effect as a TNF inhibitor compared to the simple TNFR-Ig fusion proteins and conventional TNFR-Fc in cytotoxicity assays, and showed the same results for collagen induced arthritis (CIA) in mice in vivo.