• Journal of Periodontal and Implant Science
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• 제32권3호
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• pp.685-695
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• 2002

황색 포도상구균은 급성 구강 감염에 있어서의 병원균이다. 그러나 그러한 황색 포도상구균의 병원성 기전은 완전히 이해되지 않았다. 이전 실험에서 황색 포도상구균의 단백질 A와 골격근의 액틴 필라멘트는 인체 상피 세포로의 황색 포도상구균의 침입에 관여한다. 구강 내 감염에 있어서의 황색 포도상구균의 병원성 기전을 조사하기 위해 인체의 치은 섬유모 세포에 대한 침입이 연구되고 있다. 급성 구강 감염을 가진 환자로부터 분리된 ATCC 25923 황색 포도상구균과 OPT 2 황색 포도상구균의 침입은 시간(0-120분)에 의존한다는 사실을 밝혀냈다. 60분을 초과하는 배양시간은 다시 배양된 균집락수 증가를 가져왔다. 배지에 접종한 세균의 숫자가 증가할 때 (100?10,000,000 cfu/ml/well), 직선적으로 증가한다. 단백질 A가 결핍된 Wood 46 황색 포도상구균의 침입은 단백질 A가 발현된 균주(ATCC 25923과 OPT 2)의 침입보다 훨씬 낮았다. 액틴 필라멘트의 합성을 방해하는 Cytochalasin D는 인체 치은 섬유모세포로 황색 포도상구균 (ATCC 25923과 OPT 2)이 침입하는 것을 방해한다. 이러한 결과는 구강내 감염을 일으키는 황색 포도상구균의 병원성 기전이 세포내 침입에 관여하고, 황색 포도상구균 단백질 A와 골격근의 액틴 필라멘트가 인체 치은 섬유모세포로의 황색 포도상구균의 침입 조절에 관여한다는 것을 보여준다.

• 대한의생명과학회지
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• 제8권4호
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• pp.211-215
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• 2002

The baculovirus/Sf9 cell expression can be employed as a powerful system for producing large amounts of the human erythrocyte glucose transporter, GLUT1 heterologously In order to exploit the system further, it is necessary to develop a convenient method for demonstrating that the transporter expressed in insect cells is biologically active. To achieve this, we have expressed the human CLUT1 in insect cells and photolabelled the expressed protein with [$^3$H] cytochalasin B, a potent inhibitor of the human erythrocyte glucose transporter. Subsequently, the labelled proteins were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Membranes labelled with [$^3$H] cytochalasln B in the presence of L-Glucose yielded a single sharp peak of labelling of apparent $M_r$ 45,000 on SDS/polyacrylamide gels. The mobility of this peak corresponded exactly to that of the band detected by anti-glucose transporter antibodies on Western blots of membranes prepared from insect cells infected with recombinant virus. In addition, the sharpness of the radioactive peak provides further evidence for the conclusion that the expressed protein is much less heavily and heterogeneously glycosylated than its erythrocyte counterpart. No peak of labelling was seen with the membranes prepared from non-infected Sf9 cells. Furthermore, the incorporation of label into this peak was completely inhibited by the presence of 500 mM-D-Glucose during tile photolabelling procedure, showing the stereoselectivity of the labelling. These evidences clearly show that human glucose transporter expressed in insect cells exhibits native-like biological activity, and that photolabelling with [$^3$H] cytochalasin B can be a convenient means for analysing the biological activity of the transport protein expressed in insect cells.

• The Korean Journal of Physiology
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• 제24권2호
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• pp.331-344
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• 1990

What makes glucose transport function sensitive to insulin in one cell type such as adipocyte, and insensitive in another such as liver cells is unresolved question at this time. Recently it is known that insulin stimulates glucose transport in adipocytes largely by redistributing transporter from the storage pool that is included in a low density microsomal fraction to plasma membrane. Therefore, insulin sensitivity may depend upon the relative distribution of gluscose transporters between the plasma membrane and in an intracellular storage compartment. In hepatocytes, the subcellular distribution of glucose transporter is less well documented. It is thus possible that the apparent insensitivity of the hepatocyte system could be either due to lack of the constitutively maintained, intracellular storage pool of glucose transporter or lack of insulin-mediated transporter translocation mechanism in this cell. In this study, I examined if any intracellular glucose transporter pool exists in hepatocytes and this pool is affected by insulin. The results obtained summarized as followings: 1) Distribution of subcellular fractions of hepatocyte showed that there are $24.9{\pm}1.3%$ of plasma membrane, $36.9{\pm}1.7%$ of nucleus-mitochondria enriched fraction, $23.5{\pm}1.2%$ of lysosomal fraction, $9.6{\pm}1.0%$ of high density microsomal fraction and $4.9{\pm}0.5%$ of low density microsomal fraction. 2) In adipocyte, there were $29.9{\pm}2.6%$ of plasma membrane, $19.4{\pm}1.9%$ of nucleus-mitochondria enriched fraction, $26.7{\pm}1.8%$ of high density microsomal fraction and $23.9{\pm}2.1%$ of low density microsomal fraction. 3) Surface labelling of sodium borohydride revealed that plasma membrane contaminated to lysosomal fraction by $26.8{\pm}2.8%$, high density microsomal fraction by $8.3{\pm}1.3%$ and low density microsomal fraction by $1.7{\pm}0.4%$ respectively. 4) Cytochalasin B bound to all of subcellular fractions with a Kd of $1.0{\times}10^{-6}M$. 5) Photolabelling of cytochalasin B to subcellular fractions occurred on 45 K dalton protein band, a putative glucose transporter and D-glucose inhibited the photolabelling. 6) Insulin didn't affect on the distribution of subcellular fractions and translocation of intracellular glucose transporters of hepatocytes. 7) HEGT reconstituted into hepatocytes was largely associated with plasma membrane and very little was found in low density microsomal fraction which equals to the native glucose transporter distribution. Insulin didn't affect on the distribution of exogeneous glucose transporter in hepatocytes. From the above results it is concluded that insulin insensitivity of hepatocyte may due to lack of intracellular storage pool of glucose transporter and thus intracellular storage pool of glucose transporter is an essential feature of the insulin action.

• Molecules and Cells
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• 제41권5호
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• pp.436-443
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• 2018

The actin cytoskeleton plays a key role in the entry of mitosis as well as in cytokinesis. In a previous study, we showed that actin disruption delays mitotic entry at G2/M by sustained activation of extracellular signal-related kinase 1/2 (ERK1/2) in primary cells but not in transformed cancer cell lines. Here, we examined the mechanism of cell cycle delay at G2/M by actin dysfunction in IMR-90 normal human fibroblasts. We observed that de-polymerization of actin with cytochalasin D (CD) constitutively activated ribosomal S6 kinase (RSK) and induced inhibitory phosphorylation of Cdc2 (Tyr 15) in IMR-90 cells. In the presence of an actin defect in IMR-90 cells, activating phosphorylation of Wee1 kinase (Ser 642) and inhibitory phosphorylation of Cdc25C (Ser 216) was also maintained. However, when kinase-dead RSK (DN-RSK) was overexpressed, we observed sustained activation of ERK1/2, but no delay in the G2/M transition, demonstrating that RSK functions downstream of ERK in cell cycle delay by actin dysfunction. In DN-RSK overexpressing IMR-90 cells treated with CD, phosphorylation of Cdc25C (Ser 216) was blocked and phosphorylation of Cdc2 (Tyr 15) was decreased, but the phosphorylation of Wee1 (Ser 642) was maintained, demonstrating that RSK directly controls phosphorylation of Cdc25C (Ser 216), but not the activity of Wee1. These results strongly suggest that actin dysfunction in primary cells activates ERK1/2 to inhibit Cdc2, delaying the cell cycle at G2/M by activating downstream RSK, which phosphorylates and blocks Cdc25C, and by directly activating Wee1.

• BMB Reports
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• 제32권5호
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• pp.429-435
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• 1999

Unlike the mammalian glucose transporter GLUT1, little is known about the nature of the endogenous sugar transporter(s) in insect cells. In order to establish the transport characteristics and other properties of the sugar transport proteins of Sf9 cells, a series of kinetic analyses was performed. A saturable transport system for hexose uptake has been revealed in the insect cells. The apparent affinity of this transport system(s) for 2-deoxy-D-glucose was relatively high, the $K_m$ for uptake being <0.5 mM. To further investigate the substrate and inhibitor recognition properties of the insect cell transporter, the ability of other sugars or drugs to inhibit 2-deoxy-D-glucose transport was examined by measuring inhibition constants ($K_j$). Transport was inhibited by D-mannose, D-glucose, and D-fructose. However, the apparent affinity of the C-4 epimer, D-galactose, for the Spodoptera transporter was relatively low, implying that the hydroxyl group at the C-4 position may play a role in the strong binding of glucose and mannose to the transporter. The results also showed that transport was stereoselective, being inhibited by D-glucose but not by L-glucose. It is therefore concluded that insect cells contain an endogenous glucose transport activity that in several aspects resembles the human erythrocyte glucose transporter. However, the mammalian and insect transporters were different in some of their kinetic properties, namely, their affinities for fructose and for cytochalasin B.

• 대한의용생체공학회:학술대회논문집
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• 대한의용생체공학회 1994년도 추계학술대회
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• pp.67-70
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• 1994

• 대한의생명과학회지
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• 제7권4호
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• pp.161-166
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• 2001

Most mammalian cells take up glucose by passive transport proteins in the plasma membranes. The best known of these proteins is the human erythrocyte glucose transporter, GLUT1. High levels of heterologous expression far the transporter are necessary for the investigation of its three-dimensional structure by crystallization. To achieve this, the baculovirus expression system has become popular choice. However, Spodoptera frugiperda Clone 9 (Sf9) cells, which are commonly employed as the host permissive cell line to support baculovirus replication and protein synthesis, grow well on TC-100 medium that contains 0.1% D-glucose as the major carbon source, suggesting the presence of endogenous glucose transporters. Furthermore, very little is known of the endogenous transporters properties of Sf9 cells. Therefore, human GLUT1 antibodies would play an important role for characterization of the GLUT1 expressed in insect cell. However, the successful use of such antibodies for characterization of GLUT1 expression m insect cells relies upon their specificity for the human protein and lack of cross-reaction with endogenous transporters. It is therefore important to determine the potential cross-reactivity of the antibodies with the endogenous insect cell glucose transporters. In the present study, the potential cross-reactivity of the human GLUT1 antibodies with the endogenous insect cell glucose transporters was examined by Western blotting. Neither the antibodies against intact GLUT1 nor those against the C-terminus labelled any band migrating in the region expected fur a protein of M$_r$ comparable to GLUT1, whereas these antibodies specifically recognized the human GLUT1. Specificity of the human GLUT1 antibodies tested was also shown by cross-reaction with the GLUT1 expressed in insect cells. In addition, the insect cell glucose transporter was found to have very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

• Journal of Applied Biological Chemistry
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• 제58권2호
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• pp.113-116
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• 2015

Silver materials may be toxic in humans because they can enter the body and accumulate, typically in the lungs. We hypothesized that the cytotoxicity of naive silver materials is affected by their size and shape. Our in vitro assays revealed that the overall toxicity was in the following order: submicro-particles>wires>micro-particles. These results contrast with previous studies, which showed that silver wires are the most toxic among the three tested materials, possibly due to differences in cell lines. Evaluations of in vivo pulmonary toxicity revealed eryptosis in the cavity lining of the lung sections. The observed eryptosis was consistent with the in vitro results. Our results indicate that silver materialinduced cytotoxicity must be measured and compared using various methods.

• Molecules and Cells
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• 제20권2호
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• pp.271-279
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• 2005

Although macrophages are an important first line of cellular defense, they are unable to effectively kill phagocytosed C. albicans. To determine the physiological basis of this inability, we investigated the alterations of macrophage proteins caused by C. albicans infection. Since the formation of C. albicans hyphae caused cell death, proteins were prepared 3 h after infection and examined by two-dimensional gel electrophoresis (2-DE). The most prominent changes were in glycolytic enzymes, which could have caused energy depletion of the infected cells. Also changed were proteins involved in maintenance of cellular integrity and NO production. Treatment of the macrophages with either cytochalasin D or taxol did not alter their inability to kill C. albicans. Our results indicate that multiple factors contribute to cell death as the pathogenic form of C. albicans becomes fully active inside macrophage cells.

• 대한의생명과학회지
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• 제11권4호
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• pp.487-492
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• 2005

The baculovirus/insect cell expression system is of great value in the study of structure-function relationships in mammalian glucose-transport proteins by site-directed mutagenesis and for the large-scale production of these proteins for mechanistic and biochemical studies. Spodoptera frugiperda Clone 21 (Sf2l) cells grow well on TC-100 medium that contains $0.1\%$ D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, very little is known about the properties of the endogenous sugar transporter(s) in Sf2l cells, although a saturable transport system for hexose uptake has been previously revealed in the Sf cells. In order to further examine the substrate and inhibitor recognition properties of the Sf2l cell transporter, the ability of hexoses to inhibit 2-deoxy-D-glucose (2dGlc) transport was investigated by measuring inhibition constants $(K_i)$. The $K_i's$ for reversible inhibitors were determined from plots of uptake versus inhibitor concentration. Transport was effectively inhibited by D-mannose and D-glucose. Of the hexoses tested, L-glucose had the least effect on 2dGlc transport in the Sf2l cells, indicating that the transport is stereoselective. Unlike the human HepG2 type glucose transport system, D-mannose had a somewhat greater affinity for the Sf2l cell transporter than D-glucose, implying that the hydroxyl group at the C-2 position is not necessary for strong binding. However, epimerization at the C-4 position of D-glucose (D-galactose) resulted in a dramatic decrease in affinity of the hexose for the Sf2l cell transporter. Such a lowering of affinity might be the result of the involvement of the C-4 hydroxyl in hydrogen bonding. It is therefore suggested that Sf2l cells were found to contain an endogenous sugar transport activity that in several aspects resembles the human HepG2 type glucose transporter, although the insect and human transporters do differ in their affinity for cytochalasin B.