• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.119-124
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• 2006

The present study compared the developmental potential of cloned porcine embryos with mesenchymal stem cells (MSCs), fetal fibroblasts (FFs) and cumulus cells (CCs) by assessing the cleavage and blastocyst rate, total cell number, inner cell mass (ICM) ratio and apoptosis. MSCs were isolated by ficoll gradients from femur of -6 month old female pig, and maintained for primary cultures. FFs from a female fetus at ${\sim}30$ day of gestation were established, and CCs were obtained from cumulus oocyte complexes (COCs) aspirated from $3{\sim}6$ mm follicles in diameter. Donor cells at $3{\sim}4$ passage were employed for nuclear transfer (NT). COCs were matured and fertilized in vitro(IVF) as control. Cleavage rate was significantly (P<0.05) higher in IVF than in NT embryos with MSCs, FFs and CCs ($82.7{\pm}8.9%\;vs\;70.6{\pm}5.4,\;68.7{\pm}5.1\;and\;63.4{\pm}5.6%$, respectively). However, blastocyst rates in IVF and NT embryos derived from MSCs ($24.5{\pm}2.8\;and\;20.4{\pm}8.3%$) did not differ, but were significantly (P<0.05) higher than NT derived from FFs and CCs ($10.6{\pm}2.7\;and\;9.8{\pm}2.1%$). Total cell number and the ratio of ICM to total cells among blastocysts cloned from MSCs ($35.4{\pm}5.2\;and\;0.40{\pm}0.09%$, respectively) were significantly (P<0.05) higher than those from FFs and CCs ($24.9{\pm}6.2%\;vs\;0.19{\pm}0.16,\;23.6{\pm}5.5\;and\;0.17{\pm}0.16%$, respectively). Proportions of TUNEL positive cells in NT embryos from FFs and CCs ($6.9{\pm}1.5\;and\;7.4{\pm}1.7%$, respectively) were significantly (P<0.05) higher than in MSCs ($4.8{\pm}1.4%$) and IVF ($2.3{\pm}0.9%$). The results demonstrate that MSCs have a greater potential as donor cells than FFs and CCs in achieving enhanced production of cloned porcine embryos.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.2
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• pp.201-211
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• 2016

Although the antioxidant and cardioprotective effects of NecroX-5 on various in vitro and in vivo models have been demonstrated, the action of this compound on the mitochondrial oxidative phosphorylation system remains unclear. Here we verify the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity during hypoxia-reoxygenation (HR). Necrox-5 treatment ($10{\mu}M$) and non-treatment were employed on isolated rat hearts during hypoxia/reoxygenation treatment using an ex vivo Langendorff system. Proteomic analysis was performed using liquid chromatography-mass spectrometry (LC-MS) and non-labeling peptide count protein quantification. Real-time PCR, western blot, citrate synthases and mitochondrial complex activity assays were then performed to assess heart function. Treatment with NecroX-5 during hypoxia significantly preserved electron transport chain proteins involved in oxidative phosphorylation and metabolic functions. NecroX-5 also improved mitochondrial complex I, II, and V function. Additionally, markedly higher peroxisome proliferator-activated receptor-gamma coactivator-$1{\alpha}$ ($PGC1{\alpha}$) expression levels were observed in NecroX-5-treated rat hearts. These novel results provide convincing evidence for the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity and in preserving $PGC1{\alpha}$ during cardiac HR injuries.

• BMB Reports
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• v.44 no.7
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• pp.490-495
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• 2011

Transcription factor AP-$2{\alpha}$ involves in the process of mammalian embryonic development and tumorigenesis. Many studies have shown that AP-$2{\alpha}$ functions in association with other interacting proteins. In a two-hybrid screening, the regulatory subunit ${\beta}$ of protein casein kinase 2 ($CK2{\beta}$) was identified as an interacting protein of AP-$2{\alpha}$; we confirmed this interaction using in-vitro GST pull-down and in-vivo co-immunoprecipitation assays; in an endogenous co-immunoprecipitation experiment, we further found the catalytic subunit ${\alpha}$ of protein casein kinase 2 ($CK2{\alpha}$) also exists in the complex. Phosphorylation analysis revealed that AP-$2{\alpha}$ was phosphorylated by CK2 kinase majorly at the site of Ser429, and such phosphorylation could be blocked by CK2 specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in a dose-dependent manner. Luciferase assays demonstrated that both $CK2{\alpha}$ and $CK2{\beta}$ enhanced the transcription activity of AP-$2{\alpha}$; moreover, $CK2{\beta}$ increased the stability of AP-$2{\alpha}$. Our data suggest a novel cellular function of CK-2 as a transcriptional co-activator of AP-$2{\alpha}$.

• BMB Reports
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• v.51 no.5
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• pp.255-260
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• 2018

Wntless/GPR177 functions as WNT ligand carrier protein and activator of $WNT/{\beta}$-catenin signaling, however, its molecular role in gastric cancer (GC) has remained elusive. We investigated the role of GPR177 in gastric tumorigenesis and provided the therapeutic potential of a clinical development of anti-GPR177 monoclonal antibodies. GPR177 mRNA expression was assessed in GC transcriptome data sets (GSE15459, n = 184; GSE66229, n = 300); protein expression was assessed in independent patient tumor tissues (Yonsei TMA, n = 909). GPR177 expression were associated with unfavorable prognosis [log-rank test, GSE15459 (P = 0.00736), GSE66229 (P = 0.0142), and Yonsei TMA (P = 0.0334)] and identified as an independent risk predictor of clinical outcomes: GSE15459 [hazard ratio (HR) 1.731 (95% confidence interval; CI; 1.103-2.715), P = 0.017], GSE66229 [HR 1.54 (95% CI, 1.10-2.151), P = 0.011], and Yonsei TMA [HR 1.254 (95% CI, 1.049-1.500), P = 0.013]. Either antibody treatment or GPR177 knockdown suppressed proliferation of GC cells and sensitized cells to apoptosis. And also inhibition of GPR177 suppresses in vitro and in vivo tumorogenesis in GC cells and inhibits $WNT/{\beta}$-catenin signaling. Finally, targeting and inhibition of GPR177 with antibody suppressed tumorigenesis in PDX model. Together, these results suggest GPR177 as a novel candidate for prognostic marker as well as a promising target for treatment of GC patients.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.5
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• pp.587-594
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• 2000

At present embryonic stem (ES) cells with confirmed pluripotential properties are only available in the mouse. Recently, we were able to isolate, culture and genetically transform primordial germ cell (PGC)-derived cells from pig embryos and demonstrate their ability to contribute to chimera development in the pig. In order to determine whether the system we developed could be used to isolate embryonic germ (EG) cells from other mammalian species, we placed isolated PGCs from cattle, goats, rabbits and rats in culture. Briefly, PGCs were isolated from fetuses of cow (day 30-50), goat (day 25), rabbit (day 15-18) and rat (day 11-12), and plated on STO feeder cells in Dulbecco's modified Eagle's medium (DMEM): Ham's F10 medium (1:1) supplemented with 0.01 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM $\beta$ - mercaptoethnol, soluble recombinant human stem cell factor (SCF; 40ng/ml), human basic fibroblast growth factor (bFGF; 20ng/ml) and human leukemia inhibitory factor (LIF; 20ng/ml). For maintenance of the cells, colonies were passed to fresh feeders every 7-10 days. In all species tested, we were able to obtain and maintain colonies with ES-like morphology. Their developmental potential was tested by alkaline phosphatase (AP) staining and in vitro differentiation assay. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. GFP-expressing colonies were detected in cattle, rabbits and rats. These results suggest that PGC-derived cells from cattle, goats, rabbits and rats can be isolated, cultured, and genetically transformed, and provide the basis for analyzing their developmental potential and their possible use for the precise genetic modification of these species.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.9
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• pp.4983-4988
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• 2013

Objectives: To establish a taxol-resistant cell line of human ovarian carcinoma (A2780/Taxol) and investigate its biological features. Methods: The drug-resistant cell line (A2780/Taxol) was established by continuous stepwise selection with increasing concentrations of Taxol. Cell morphology was assessed by microscopy and growth curves were generated with in vitro and in vivo tumor xenograft models. With rhodamine123 (Rh123) assays, cell cycle distribution and the apoptotic rate were analyzed by flow cytometry (FCM). Drug resistance-related and signal associated proteins, including P-gp, MRPs, caveolin-1, PKC-${\alpha}$, Akt, ERK1/2, were detected by Western blotting. Results: A2780/Taxol cells were established with stable resistance to taxol. The drug resistance index (RI) was 430.7. Cross-resistance to other drugs was also shown, but there was no significant change to radioresistance. Compared with parental cells, A2780/Taxol cells were significantly heteromorphous, with a significant delay in population doubling time and reduced uptake of Rh123 (p<0.01). In vivo, tumor take by A2780 cells was 80%, and tumor volume increased gradually. In contrast, with A2780/Taxol cells in xenograft models there was no tumor development. FCM analysis revealed that A2780/Taxol cells had a higher percentage of G0/G1 and lower S phase, but no changes of G2 phase and the apoptosis rate. Expression of P-gp, MRP1, MRP2, BCRP, LRP, caveolin-1, PKC-${\alpha}$, Phospho-ERK1/2 and Phospho-JNK protein was significantly up-regulated, while Akt and p38 MARK protein expression was not changed in A2780/Taxol cells. Conclusion: The A2780/Taxol cell line is an ideal model to investigate the mechanism of muti-drug resistance related to overexpression of drug-resistance associated proteins and activation of the PKC-${\alpha}/ERK$ (JNK) signaling pathway.

• Journal of Bio-Environment Control
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• v.21 no.2
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• pp.140-146
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• 2012

The commercial product "pyto-patch" prepared as nano sized silver particle less than 5 nm, has effective antifungal activity against Collectotrichum gloeosporioides, Botrytis cinerea, Sclerotinia sclerotiorum in vitro. As a fungal growth inhibiton mechanism, it can reduce spore germination rate and mycelial growth. As s promising fungicide, Phyto-patch can control anthracnose effectively. The spore of C. gloeosporioides dipped in 5 ppm phyto-patch dilute suppressed germination rate to 13.2%, and mycelial growth stopped for 15 days at 10 ppm. The spore postinoculated on 10 ppm phyto-path smeared PDA surface could not germinate for 3 days and prohibit pathogen infection effectively. In field test, the anthracnose development of 4 ppm phyto-patch treated plot was less than 7% after 21 days compaired to 40% of it in untreated plot. In heavy rainfall season, pepper anthracnose effectivly contrrolled by regular 10 ppm phytopatch spraying every 7 days. The diseased pepper fruit decreased to 5.8% compaired to 94.6% in untreated plot. During drying period, the diseased pepper fruit havested in phyto-patch treated plot was 24.2%, but pepper fruit havested in untreated plot destroyed to 100% within 3 days. The nano silver particle coated on multching textile prevented late blight of pepper effectively and disease occurance delayed about month.

• Korean Journal of Medicinal Crop Science
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• v.11 no.2
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• pp.161-166
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• 2003

Korean ginseng(Panax gmseng C.A. Meyer) is very difficult to obtain stable production of qualified ginseng roots because of variable stresses in soil environments. In environment stresses, soil condition is the most important factor, among which nutrients, especially inorganic materials such as N, P, K, Ca, Mg, Fe, etc., influence greatly on the ginseng growth. However, present ginseng field soils in Korea contain so much amount of such inorganic materials that a variety of remarkable disorders were noted in many ginseng plantations, resulting in decrease of qualitative ginseng production. Therefore, it is required to search for genetic resources and genes tolerant to salt stress for the development of ginseng cultivars. Selection of stress-tolerant ginseng lines in fields is very difficult because it is almost impossible to control properly the environmental conditions of soil. On the contrary, it can be studied with ease to search for stress-tolerant ginseng lines through in vitro culture because of easy manipulation of stress conditions. Murashige & Skoog(MS) media with 2.5 folds of $KNO_3,\;NH_4NO_3,\;MgSO_4\;7H_2O,\;KH_2PO_4,\;and\;CaCl_2\;2H_2O$ was established for the selection of ginseng lines tolerant to salt stress under the embryo culture.

• Journal of the korean academy of Pediatric Dentistry
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• v.34 no.1
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• pp.53-61
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• 2007

In case of missing tooth caused by dental caries or periodontal disease, it can be restored by various methods, and there has been much interest in implant and tooth transplantation. The success of tooth transplantation is going to be attained through the knowledge of growth, development and calcification of tooth. Tooth transplantation has been experimented in vivo and in vitro. Many animals such as rats, mice, cats and dogs are used for tooth transplantation experiment in vivo. In most experiments, tooth was transplanted into the extraoral site, but rare into the intraoral site In this study, to observe the capacity of formation and mineralization of tooth germ, first molar of a matured white rat was extracted and the cap stage tooth germ of a 13.5 Embryonic day embryo rat was transplanted into the extracted socket. The rats were killed 6 months later and the radiographical and histological results are as followings. 1. Tooth germ transplanted for 2 and 6 months are developing calcified tooth material such as dentin, cementum, pulp tissue, and epithelium around enamel space in the maxilla was seen. 2. The epithelium around enamel space was located beneath the oral epithelium and contained connective tissue and periodontal ligament. 3. Tooth formation was progressed as transplantation period but the size of newly formed tooth was small and the shape of tooth was incomplete.

• Journal of Periodontal and Implant Science
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• v.31 no.2
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• pp.371-388
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• 2001

A novel glucanhydrolase(DXAMase) from a mutant of Lipomyces starkeyi(KSM 22) has been shown effective in hydrolysis of mutan, reduction of mutan formation by Streptococcus mutans and removal pre-formed sucrose-dependentadherent microbial film and DXAMase has been strongly bound to hydroxyapatitie. These in vitro properties of Lipomyces starkeyi DXAMase are desirable for its application as a dental plaque control agent. This study was performed to determine the adjunctive oral hygiene benefits and safety of dextranase(Lipomyces starkeyi KSM 22 DXAMase)-containing mouthwash when used alongside normal tooth-brushing. This 6-month clinical trial was placebo-controlled double-blind design evaluating 1U/ml dextranase mouthwash and 0.12% chlorhexidine mouthwash. A total 39 systemically healthy subjects, who had moderate levels of plaque and gingivitis were included. At baseline, 1, 3 and 6 months, subjects were scored for plaque accumulation(Turesky modification of Quingley-Hein's plaque index), gingivitis status($L\ddot{o}e$ and Silness gingival index), and tooth stain(Area and severity index system by Lang et al). Additionally, oral mucosal examinations were performed and subjects questioned for adverse symptoms. Two weeks after pre-experiment examinations and a professional prophylaxis, the subjects provided with allocated mousewash and instructed to use 20-ml volumes for 30s twice daily after toothbrushing. All the groups showed significant increase in plaque accumulation since 1 month of experiment. During 6 months' period, the Dextranase mouthwash group showed the least increase in plaque accumulation, compared to the Chlorhexidine mouthwash and placebo groups. As for gingival inflammation, all the groups showed significant increase during 6 months of experiment. The Experimental group(Dextranase mouthwash) also showed the least increase in gingival index score, compared to the Positive control(Chlorhexidine mouthwash)as well as the Negative control(placebo)groups. Whereas the tooth stain was increased significantly in the Positive control group, compared to the baseline score and the Negative controlgroup since 3 months of mouthrinsing. It was significantly increased after 6 months in the Experimental group, still less severe than the Positive control group. As for the oral side effect, the Experimental group showed less tongue accumulation, bad taste, compared to the Positive control group. From these results, mouthrinsing with Lipomyces starkeyi KSM 22 dextranase provided adjunctive benefits to toothbrushing, comparable to 0.12% chlorhexidine mouthwash in inhibition of plaque accumulation and gingival inflammation and local side effects were if anything less frequent and less intense than chlorhexidine, with long-term use of the mouthwash. All data had provided positive evidence for Lipomyces starkeyi KSM 22 dextranase as an antiplaque agent and suggested that further development of dextranase formulations for plaque control are warranted.