• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.81-81
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• 2003

DNA methylation is a covalent modification of DNA that can modulate gene expression and is now recognized as a major component of the epigenome. During evolution, the dinucleotide CpG has been progressively eliminated from the genome of higher eukaryotes and is present at only 5% to 10% of its predicted frequency. Approxymately 80% of the remaining CpG sites contain methylated cytosines in most vertebrates and they are distributed in a pattern that is unique in each tissue and is inversely correlated with gene expression. The pattern of methylation is faithfully maintained during cell division by the enzyme Dnmt1, the maintenance DNA methyltransferase, which catalyzes the transfer of a methyl group from S-adenosyl-methionine to the 5'-position of the cytosine ring. We have been identified bovine Dnmt1 cDNA full-length recently (AY173048) Little is known on the functions of Dnmt1 in bovine preimplantation embryos. Thus, we analyzed the specific pattern of Dnmt1 in in vitro derived/nuclear transfer bovine and in vivo derived mouse embryos to monitor the epigenetic reprogramming process. We investigated these process by using indirect immunofluresence with an antibody to Dnmt1. According to other studies, Dnmt1 accumulates in nuclei of early growing oocytes but is sequestered in the cytoplasm of mature oocytes. In 2-cell and 4-cell embryos, Dnmt1 is cytoplasmic, but at the 8-cell stage, it is present only in the nucleus. By the blastocyst stage, Dnmt1o is again found only in the cytoplasm. Thus, nuclear localization of Dnmt1o in preimplantation embryos is limited to the 8-cell stages After implantation, Dnmt1 is localized in the nucleus in mouse. However, we have found different patterns of Dnmt1 nuclear localization. Though we used the common antibody, immune-localization data revealed that Dnmt1 antibody have been detected at the nucleus in 1-cell to blastocyst embryos. Therefore, maybe we think that the functions of Dnmt1 between bovine and mice are different. In order to Identify the mechanisms that regulate DNA methylation in bovine preimplantation embryo, we have plans on using bovine oocyte and somatic specific Dnmt1 antibodies.

• 대한약리학회지
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• 제2권2호
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• pp.23-33
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• 1966

Hexachlorophene was reported previously to have a powerful parasiticidal effects on Clonorchis sinensis in vitro and in vivo, but the mechanism of its effect was not known. In the present report it was observed that there was an influence of hexachlorophene on the oxygen consumption, the glycolysis, the glycogenesis and the protein synthesis of C. sinensis. A hundred mg. of C. sinensis collected from the biliary tracts of the infested rabbits was incubated in 2 ml of K.R.P. medium with vavious concentration of hexachlorophene, $glucose-1-C^{14}$ and $glycine-1-C^{14}$ in a 25 ml incubation flask with central well. The oxygen consumption was observed by Warburg manometer, the glycogenolysis by measurement of radioactivities of extracted glycogen and protein from C. sinensis incubated with $C^{14}-glucose$ or$C^{14}-glycine$. 1) The oxygen consumption by C. sinensis was markedly inhibited during all stages of incubation in concentration of $10^{-4}$ and $10^{-5}g/ml$ of hexachlorophene, but in $10^{-6}$, slightly increased initially and gradually decreased after 3 hours of incubation. 2) Hexachlorophene inhibited the glycolysis by C. sincnsis markedly in the concentration of $10^{-4}$, $10^{-5}$, $10^{-6}$ and $10^{-7}g/ml$. 3) The protein synthesis by C. sinensis from glycine was inhibited in the concentration of $10^{-5}$, $10^{-6}$ and $10^{-7}g/ml$ of hexachlorophene. 4. The glycogen synthesis by C. sinensis in each concentration of $10^{-4}$, $10^{-5}$ and $10^{-6}g/ml$ of hexachlorophene was inhibited markedly. The speed of inhibition was more rapid in high concentration than in low, and in low concentration even the glycogen itself which had synthesized in their stage in their body was consumed in later stage. 5) The effects of oxygen consumption, glycolysis and glycogen synthesis were not influenced in the concentration of $10^{-5}g/ml$ of chloroquine phosphate, whereas hexachlorophene and dithiazanine inhibited markedly in same concentration, and the former was more potent than the latter.

• 대한의생명과학회지
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• 제13권4호
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• pp.263-272
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• 2007

Nebulin is a giant actin binding protein (600-900 kDa) which is specific to skeletal muscle. This protein is known to regulate thin filaments length in sarcomere as a molecular template. The C-terminus of nebulin is located in the Z-disc of muscle sarcomere and is bound to other proteins such like myopalladin, titin, archvillin, and desmin. The N-terminus of nebulin binds to tropomodulin at the pointed ends of the thin filaments. In recent research, nebulin not only found in brain but also expressed in heart, stomach, and liver. So, the roles of nebulin in non-muscle tissue have been studied. However, lack of information or studies on nebulin binding proteins and nebulin function in brain are available so far. Therefore, the current study have investigated a novel binding partner of Nebulin C-terminus by using yeast two-hybrid screening with human brain cDNA library. Nebulin C-terminus, containing simple repeats, serine rich and SH3 domain, interacts with osteonectin C-terminal region. The specific interaction of nebulin and osteonectin were confirmed in vitro by using GST pull-down assay and reconfirmed in vivo by using transfected COS-7 cells with EGFP-tagged nebulin and DsRed-tagged osteonectin. Consequently, this study identified SH3 domain in nebulin C-terminus specifically binds to extracellular Ca-binding (EeC domain in osteonectin. Also, nebulin C-terminus fusion protein colocalized with osteonectin EC domain fusion protein in transfected COS-7 cells. The current study found the interaction between nebulin and osteonectin in human brain for the first time and suggested the nebulin in brain may be associated with osteonectin, as a regulator of cell cycle progression and mitosis.

• Molecules and Cells
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• 제40권2호
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• pp.123-132
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• 2017

Insulin signaling is coordinated by insulin receptor substrates (IRSs). Many insulin responses, especially for blood glucose metabolism, are mediated primarily through Irs-1 and Irs-2. Irs-1 knockout mice show growth retardation and insulin signaling defects, which can be compensated by other IRSs in vivo; however, the underlying mechanism is not clear. Here, we presented an Irs-1 truncated mutated mouse ($Irs-1^{-/-}$) with growth retardation and subcutaneous adipocyte atrophy. $Irs-1^{-/-}$ mice exhibited mild insulin resistance, as demonstrated by the insulin tolerance test. Phosphatidylinositol 3-kinase (PI3K) activity and phosphorylated Protein Kinase B (PKB/AKT) expression were elevated in liver, skeletal muscle, and subcutaneous adipocytes in Irs-1 deficiency. In addition, the expression of IRS-2 and its phosphorylated version were clearly elevated in liver and skeletal muscle. With miRNA microarray analysis, we found miR-33 was down-regulated in bone marrow stromal cells (BMSCs) of $Irs-1^{-/-}$ mice, while its target gene Irs-2 was up-regulated in vitro studies. In addition, miR-33 was down-regulated in the presence of Irs-1 and which was up-regulated in fasting status. What's more, miR-33 restored its expression in re-feeding status. Meanwhile, miR-33 levels decreased and Irs-2 levels increased in liver, skeletal muscle, and subcutaneous adipocytes of $Irs-1^{-/-}$ mice. In primary cultured liver cells transfected with an miR-33 inhibitor, the expression of IRS-2, PI3K, and phosphorylated-AKT (p-AKT) increased while the opposite results were observed in the presence of an miR-33 mimic. Therefore, decreased miR-33 levels can up-regulate IRS-2 expression, which appears to compensate for the defects of the insulin signaling pathway in Irs-1 deficient mice.

• Journal of Ginseng Research
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• 제46권3호
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• pp.418-425
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• 2022

Background: Sorafenib is effective in treating hepatoma, but most patients develop resistance to it. STAT3 signaling has been implicated in sorafenib resistance. Artesunate (ART) and 20(R)-ginsenoside Rg3 (Rg3) have anti-hepatoma effects and can inhibit STAT3 signaling in cancer cells. This study aimed to evaluate the effects of Rg3 in combination with ART (Rg3-plus-ART) in overcoming sorafenib resistance, and to examine the involvement of STAT3 signaling in these effects. Methods: Sorafenib-resistant HepG2 cells (HepG2-SR) were used to evaluate the in vitro anti-hepatoma effects of Rg3-plus-ART. A HepG2-SR hepatoma-bearing BALB/c-nu/nu mouse model was used to assess the in vivo anti-hepatoma effects of Rg3-plus-ART. CCK-8 assays and Annexin V-FITC/PI double staining were used to examine cell proliferation and apoptosis, respectively. Immunoblotting was employed to examine protein levels. ROS generation was examined by measuring DCF-DA fluorescence. Results: Rg3-plus-ART synergistically reduced viability of, and evoked apoptosis in HepG2-SR cells, and suppressed HepG2-SR tumor growth in mice. Mechanistic studies revealed that Rg3-plus-ART inhibited activation/phosphorylation of Src and STAT3 in HepG2-SR cultures and tumors. The combination also decreased the STAT3 nuclear level and induced ROS production in HepG2-SR cultures. Furthermore, overactivation of STAT3 or removal of ROS diminished the anti-proliferative effects of Rg3-plus-ART, and removal of ROS diminished Rg3-plus-ART's inhibitory effects on STAT3 activation in HepG2-SR cells. Conclusions: Rg3-plus-ART overcomes sorafenib resistance in experimental models, and inhibition of Src/STAT3 signaling and modulation of ROS/STAT3 signaling contribute to the underlying mechanisms. This study provides a pharmacological basis for developing Rg3-plus-ART into a novel modality for treating sorafenib-resistant hepatoma.

• Biomolecules & Therapeutics
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• 제9권3호
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• pp.167-175
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• 2001

Recent studies have shown that cytokines are capable of modulating cardiovascular function and that some drugs used in the treatment of heart failure variably modulate the production of cytokines. Hige- namine, a positive inotropic isoquinoline alkaloid, has been used traditionally as cardiac stimulant, and reported to reduce nitric oxide (NO) and inducible nitric oxide synthase (iNOS) expression in LPS- and/or cytokine-activated cells in vitro and in vivo. Therefore, we investigated whether higenamine modulates the production of proinflammatory cytokines in myocardial infarction. In addition, effects of higenamine on antioxidant action and antioxidant enzyme expression (MnSOD) were studied. Myocardial infarction (MI) was confirmed by measuring left ventricular (LV) pressure after occlusion of the left anterior descending coronary artery (LAD) for 5 weeks in rats. Treatment of higenamine (10 mg/kg/day) reduced infarct size about 35 %, which accompanied by reduction of production TNF-$\alpha$, IL-6, but not IFN-${\gamma}$ and IL-1$\beta$ in the myocardium. The expression of TNF-$\alpha$ mRNA in infracted myocardium was significantly reduced by higenamine. Although iNOS mRNA was not detected, nitrotyrosine staining was significantly increased in myocardium of Ml compared to higenamine-treated one, Indicating that peroxynitrite-induced damage is evident in MI. Cytochrome c oxidation by peroxynitrite was concentration-dependently reduced by higenamine, an effect which was almost compatible to glutathion. Higenamine treatment did not affect the expression of MnSOD mRNA in myocardial tissues in MI. Taken together, higenamine may be beneficial in oxidative stress conditions such as ischemic-reperfusion injury and MI due to antioxidant action as well as modulation of cytokines.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.107-107
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• 1997

Cytochrome P450 enzymes have been intensively investigated in hepatic tissues and several mammalian cell lines. Compared to most studies about cytochrome P450 isozymes in liver in vivo and hepatic, cell lines in vitro, the study of cytochrome P450IA1 in human breast cancer cells could be very important to understand the mechanism of the regulation of CYPIA1 gene expression and cell growth. MCF-7 human breast cancer cells are well characterized to study estrogen and antiestrogen action due to the fact that they contain high level of estrogen receptor and have biological markers characterized. And also MCF-7 cells express high level of arylhydrocarbon hydroxylase activity and human cytochrome P450IA1 cDNA was cloned from MCF-7 cells. Ah receptor was characterized in many breast cancer cell lines and polycyclic aromatic hydrocarbon such as 3-MC induced the expression of CYPIA1 gene and cytochrome P450- dependent monooxygenase activity. We undertook a study to examine the effect of estrogens and other chemicals on the regulation of human CYPIA1 gene expression in MCF-7 cells via RTPCR analysis, that might help us to understand the mechanism of the regulation of CYPIA1 gene expression and MCF-7 cell growth. Expression vector containing the functional 5'-regulatory region of human CYPIA1 fused to the CAT reporter gene was transfected into estrogen receptor positive MCF-T cells or estrogen receptor negative MDA-MB-231 cells. After these cells were treated with various chemicals, RTPCR was carried out to measure both CYPIA1 mRNA and CAT mRNA levels. 1nM 3-MC increased in both P450 and CAT mRNA levels over those of control by two folds in MCF-7 cells but does not in MDA-MB-231 cells. Estrogen or tamoxifen or retinoic acid or chrysin decreased in both P450 and CAT mRNA levels that were induced by 3-MC in MCF-7 when each chemical was administered with 3-MC concomitantly. These results suggested that the level of CYPIA1 gene expression is modulated with estrogen-related molecules and make it possible to speculate that ER is related to CYPIA1 gene expression and cell growth in breast cancer cells. [Supported by grants from the Korean Ministry of Education ]

• 한국식품위생안전성학회지
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• 제31권2호
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• pp.67-73
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• 2016

본 총설에서는 비-플라보노이드 폴리페놀인 레스베라트롤이 간, 골격근 및 지방조직에서 지질대사에 관계된 다양한 신호전달체계를 조절하여 지질 대사 효과를 유발시키는 과정에 관해 고찰하였다. 구체적으로 in vitro 연구에서 레스베라트롤은 지방생성을 줄여주고 apoptosis를 증가시켜 지방세포의 발달과정에 기인하며, 지방세포의 분화에 있어 중요한 전사인자인 $C/EBP{\beta}$, $C/EBP{\alpha}$, SREBP1c 및 $PPAR{\gamma}$의 활성을 감소시켜 항 비만 효과를 유발하는 효과가 있다는 것이 많은 논문들을 통해 증명되었다(Fig. 2). 또한, in vivo 연구에서 레스베라트롤은 지방 축적 과정을 억제하고 지질 분해 및 산화 경로를 자극하여 체지방 증가율을 감소시킨다는 것이 증명되었다. 최근 다양한 연구의 결과물(Table 2)들은 레스베라트롤이 지방생성, 지방분해, 열발생 및 지방산 산화에 관여하며 또한, 백색 지방을 갈색 지방으로 변화시키는 능력이 있다는 것을 증명하였다. 흥미롭게도 레스베라트롤은 비만뿐만이 아닌 심장발작 및 뇌졸중과 같은 다양한 대사질환을 예방하는데 도움이 되고, 결장암 및 간암 세포의 성장을 억제하는 효능이 있다는 사실이 밝혀지기도 하였다. 하지만 인간에 대한 레스베라트롤의 명확한 메커니즘을 알지 못하고 인간에게 나타나는 부작용에 관한 연구가 없기 때문에, 안전성을 확보하기 위해서는 다양한 실험모델을 이용한 레스베라트롤의 단기간 및 장기간에 대한 깊은 연구가 요구된다.

• 대한한방종양학회지
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• 제8권1호
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• pp.103-125
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• 2002

This experimental study was carried out to evaluate the anti-tumor and the immunomodulatory effects of Jiaweicitaowan(加未慈桃丸) against cancer. The in vitro anti-tumor effects were evaluated by MTT assay. The cytotoxicity, extension of survival days, the effect of inhibition solid tumor which was induced sarcoma 180, and the changes of body weight were evaluated for in vivo effects of anti-tumor. To evaluate the immunomodulatory effects of Jiawei- citaowan(加未慈桃丸), delayed type hypersensitivity, hemagglutinin, hemolysin titers for humoral immune response, rosette forming cells for cell-mediated immune response, natural killer cell activity, proliferation of lymphocyte, productivty of Interleukin-2, and carbon clearance were measured with methotrexate treated mice. The results were as follows; 1. In the case of existence ability of tumor cell, IC50 had an anti-tumor ativity resulted 2.52mg/ml to SNU-C4. 0.41mg/ml to SNU-396, resulted to 0.09mg/mlSNU-1. 2. The groups of Jiaweicitaowan(加未慈桃丸) 10mg/ml, 20mg/kg had no body weight loss. reduction in intake of water and feed, so these had no toxicity. 3. In the case of the effect of extention of existence. the group of 20mg/kg Jiaweicitaowan(加未慈桃丸) extract treated group was showed 250% in ILS. 4. The effect of inhibition solid tumor was significantly decreased in both 10mg/kg, 20mg/kg of Jiaweicitaowan(加未慈桃丸) extract treated groups as compared with control group S. The groups of 10mg/kg, 20mg/kg Jiaweicitaowan(加未慈桃丸) had significant effect of body weight change compared to control group. 6. Delayed type hypersensitivity was not significant in both Jiaweicitaowan(加未慈桃丸) extract treated groups as compared with control group. 7. Hemagglutinin and Hemolysin titers were significantly increased by dose-dependent. so these results showed that the humoral immume respose was activated. 8. For the effect of rosette formimg cells was not significant in hoth Jiaweicitaowan(加未慈桃丸) extract treated groups as compared with control group. 9. Natural killer cell activity was significantly increased in both Jiaweicitaowan(加未慈桃丸) extract treated groups as compared with control group in the ratio of 100: 1, 50: 1 of effector and target cells, but in the ratio of 10:1, the Jiaweicitaowan(加未慈桃丸) extract treated groups were not significant. 10. The proliferation of lymphocyte and productivty of Interleukin-2 were significantly increased by dose-dependent in both 10mg/ kg, 20mg/ kg of Jiaweicitaowan(加未慈桃丸) extract treated groups as compared with control group. 11. In the phagocytic effect, the 20mg/kg of Jiaweicitaowan(加未慈桃丸) extract treated group showed the increasing effect with significance as compared with control group. According to the results, we can suggest that Jiaweicitaowan(加未慈桃丸) has the antitumor and the immunomodulatory effects.

• Nutrition Research and Practice
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• 제11권6호
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• pp.470-478
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• 2017

BACKGROUND/OBJECTIVE: Orostachys japonicus A. Berger (Crassulaceae) has been used in traditional herbal medicines in Korea and other Asian countries to treat various diseases, including liver disorders. In the present study, the anti-fibrotic effects of O. japonicus extract (OJE) in cellular and experimental hepatofibrotic rat models were investigated. MATERIALS/METHODS: An in vitro hepatic stellate cells (HSCs) system was used to estimate cell viability, cell cycle and apoptosis by MTT assay, flow cytometry, and Annexin V-FITC/PI staining techniques, respectively. In addition, thioacetamide (TAA)-induced liver fibrosis was established in Sprague Dawley rats. Briefly, animals were divided into five groups (n = 8): Control, TAA, OJE 10 (TAA with OJE 10 mg/kg), OJE 100 (TAA with OJE 100 mg/kg) and silymarin (TAA with Silymarin 50 mg/kg). Fibrosis was induced by treatment with TAA (200 mg/kg, i.p.) twice per week for 13 weeks, while OJE and silymarin were administered orally two times per week from week 7 to 13. The fibrotic related gene expression serum biomarkers glutathione and hydroxyproline were estimated by RT-PCR and spectrophotometry, respectively, using commercial kits. RESULTS: OJE (0.5 and 0.1 mg/ mL) and silymarin (0.05 mg/mL) treatment significantly (P < 0.01 and P < 0.001) induced apoptosis (16.95% and 27.48% for OJE and 25.87% for silymarin, respectively) in HSC-T6 cells when compared with the control group (9.09%). Further, rat primary HSCs showed changes in morphology in response to OJE 0.1 mg/mL treatment. In in vivo studies, OJE (10 and 100 mg/kg) treatment significantly ameliorated TAA-induced alterations in levels of serum biomarkers, fibrotic related gene expression, glutathione, and hydroxyproline (P < 0.05-P < 0.001) and rescued the histopathological changes. CONCLUSIONS: OJE can be developed as a potential agent for the treatment of hepatofibrosis.