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Single Cell Array of Biotinylated Cells Using Surface Functionalization and Microcontact Printing
Lee, Zee-Won,Lee, Kyung-Bok,Hong, Jang-Hee,Kim, Jae-Hong,Choi, Inpyo,Choi, Insung S. Chemical Society of Japan 2005 Chemistry letters Vol.34 No.5
<P>This paper describes a versatile method for generating single cell arrays on a glass substrate, which could be applicable to any arbitrary cell types, by a combination of surface functionalization, biotinylation of cells, and microcontact printing (μCP).</P>
Lee, Hye-Mi,Shin, Dong-Min,Choi, Dae-Kyoung,Lee, Zee-Won,Kim, Ki-Hye,Yuk, Jae-Min,Kim, Chang Deok,Lee, Jeung-Hoon,Jo, Eun-Kyeong Blackwell Publishing Ltd 2009 Cellular microbiology Vol.11 No.4
<P>Summary</P><P><I>Mycobacterium ulcerans</I> (MU), an environmental pathogen, causes Buruli ulcer, a severe skin disease. We hypothesized that epidermal keratinocytes might not be a simple barrier, but play a role during MU infection through pattern-recognition receptors expressed in keratinocytes. We found that keratinocyte Toll-like receptors (TLRs) 2 and 4 and Dectin-1 actively participate in the innate immune response to MU, which includes the internalization of bacteria, the production of reactive oxygen species (ROS), and the expression of chemokines and LL-37. Human keratinocytes constitutively expressed TLRs 2 and 4 and induced Dectin-1 in response to MU. Exposing keratinocytes to MU resulted in rapid ROS production, which in turn contributed to the mRNA and protein expression of LL-37. In addition, TLR2, Dectin-1 and, to an extent, TLR4 are essential for the MU-mediated expression of CXCL8, CCL2 and LL-37 in keratinocytes. Furthermore, confocal analysis showed that the Dectin-1 is necessary for keratinocytes to internalize bacilli. Importantly, blockade of ROS and LL-37 significantly increased the intracellular MU growth in keratinocytes, suggesting an important role of these mediators for cutaneous innate immune responses. Our results demonstrate that TLR2, TLR4 and Dectin-1 actively sense, internalize and respond in an innate way to MU in human epidermal keratinocytes.</P>
Lee, Sang Kwang,Kim, Jae Ho,Kim, Sung-Soo,Kang, Taewook,Park, Nam Hyun,Kwon, Kyung-Hoon,Lee, Sang Sook,Lee, Zee Won,Suh-Kim, Hae young,Cho, Kun,Yun, Su Yeoung,Han, Ji Young,Yoo, Jong Shin,An, Hyun Joo Springer-Verlag 2013 Analytical and bioanalytical chemistry Vol.405 No.16
<P>Mulitpotent mesenchymal stem cells (MSCs) derived from human bone marrow are promising candidates for the development of cell therapeutic strategies. MSC surface protein profiles provide novel biological knowledge concerning the proliferation and differentiation of these cells, including the potential for identifying therapeutic targets. Basic fibroblast growth factor (bFGF) affects cell surface proteins, which are associated with increased growth rate, differentiation potential, as well as morphological changes of MSCs in vitro. Cell surface proteins were isolated using a biotinylation-mediated method and identified using a combination of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. The resulting gel lines were cut into 20 bands and digested with trypsin. Each tryptic fragment was analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. Proteins were identified using the Mascot search program and the International Protein Index human database. Noble MSC surface proteins (n = 1,001) were identified from cells cultured either with (n = 857) or without (n = 667) bFGF-containing medium in three independent experiments. The proteins were classified using FatiGO to elucidate their function. We also confirmed the proteomics results using Western blotting and immunofluorescence microscopic analysis. The nature of the proteins identified makes it clear that MSCs express a wide variety of signaling molecules, including those related to cell differentiation. Among the latter proteins, four Ras-related Rab proteins, laminin-R, and three 14-3-3 proteins that were fractionated from MSCs cultured on bFGF-containing medium are implicated in bFGF-induced signal transduction of MSCs. Consequently, these finding provide insight into the understanding of the surface proteome of human MSCs.</P>
Lee, Sang Kwang,Kim, Yongtae,Kim, Sung-Soo,Lee, Jeong Hwa,Cho, Kun,Lee, Sang Sook,Lee, Zee-Won,Kwon, Kyung-Hoon,Kim, Young Hye,Suh-Kim, Haeyoung,Yoo, Jong Shin,Park, Young Mok WILEY-VCH Verlag 2009 Proteomics Vol.9 No.18
<P>Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi-lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2-DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin–avidin affinity column were separated by 2-DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole-time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin-related proteins, F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF-induced morphological change of MSCs.</P>
Hwang, Jeong Won,Oh, Jung Han,Yoo, Hwa-Seung,Lee, Yeon-Weol,Cho, Chong-Kwan,Kwon, Ki-Rok,Yoon, Jung-Ho,Park, Junsoo,Her, Song,Lee, Zee-Won,Jang, Ik-Soon,Choi, Jong-Soon Institute for Advanced Research in Asian Science a 2012 The American journal of Chinese medicine Vol.40 No.1
<P>Administration of mountain ginseng (MG) extract can restore advanced cancer to a normal state. To elucidate the mechanism by which MG extract prevents the progression of lung cancer, the processes of proliferation and death of lung cancer cells (A549) were examined after treatment with MG extract. Butanol-extracted MG (BX-MG) showed a high inhibitory effect (IC(50) = 2 mg/ml) by attenuating proliferation and inducing apoptosis in lung cancer cells. By HPLC-UV analysis of BX-MG, ginsenosides, Rb1 was identified as the most abundant ginsenoside, followed by Rg1, Re, Rc and Rb2. BX-MG induced caspase-3 dependent apoptosis by inhibiting NF-관B. In addition, BX-MG activated p53 and p21, resulting in the attenuated proliferation of A549 cells. Reduced activity of the NF-관B promoter and increased activity of the p53 promoter indicate that BX-MG regulates apoptosis at the level of transcription in lung cancer cells. Furthermore, BX-MG blocked the nuclear translocation of RelA and the associated reduction in surviving. These results suggest that BX-MG inhibits lung cancer cell growth by activating tumor suppressors and inhibiting nuclear translocation of NF-관B.</P>
Park, Jong Pil,Lee, Kyung-Bok,Lee, Seok Jae,Park, Tae Jung,Kim, Min Gon,Chung, Bong Hyun,Lee, Zee-Won,Choi, Insung S.,Lee, Sang Yup John Wiley & Sons 2005 Biotechnology and Bioengineering Vol.92 No.2
<P>A novel strategy for micropatterning proteins on the surface of polyhydroxyalkanoate (PHA) biopolymer by microcontact printing (µCP) is described. The substrate binding domain (SBD) of the Pseudomonas stutzeri PHA depolymerase was used as a fusion partner for specifically immobilizing proteins on PHA substrate. Enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) fused to the SBD could be specifically immobilized on the micropatterns of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Laser scanning confocal microscopic studies suggested that two fusion proteins were micropatterned in their functionally active forms. Also, antibody binding assay by surface plasmon resonance suggested that protein–protein interaction studies could be carried out using this system. © 2005 Wiley Periodicals, Inc.</P>
Zhang, Tiejun,Li, Yuwen,Park, Kyeong Ah,Byun, Hee Sun,Won, Minho,Jeon, Juhee,Lee, Yoonjung,Seok, Jeong Ho,Choi, Seung-Won,Lee, Sang-Hee,Man Kim, Jin,Lee, Ji Hoon,Son, Chang Gue,Lee, Zee-Won,Shen, Han- Landes Bioscience 2012 AUTOPHAGY Vol.8 No.4
<P>Targeted disruption of STAT3 function has proven to be a useful cancer therapeutic approach by inducing apoptotic cell death. Cucurbitacin is currently under development as a small molecule of STAT3 inhibitor to trigger cell death in many cancers. Here, we systematically studied the molecular mechanisms underlying cucurbitacin-induced cell death, in particular the involvement of autophagy. Treatment with cucurbitacin resulted in non-apoptotic cell death in a caspase-independent manner. Notably, cucurbitacin enhanced excessive conversion of lipidated LC3 (LC3-II) and accumulation of autophagosomes in many cell types. Such autophagy and cell death induced by cucurbitacin were independent of its ability to inhibit STAT3 function, but mainly mediated by enhanced production of mitochondrial-derived reactive oxygen species (ROS), and subsequently activation of extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK). Interestingly, both the autophagy inhibitor wortmannin and knockdown of Atg5 or Beclin 1 failed to rescue the cells from cucurbitacin-induced cell death, as suppression of autophagy induced the mode of cell death to shift from autophagic cell death to caspase-dependent apoptosis. Thus the present study provides new insights into the molecular mechanisms underlying cucurbitacin-mediated cell death and supports cucurbitacin as a potential anti-cancer drug through modulating the balance between autophagic and apoptotic modes of cell death.</P>
Effect of Botulinum Toxin Type A on Morphology of Salivary Glands in Patients with Cerebral Palsy
Zee-Ihn Lee,Dong-Hyun Cho,Won-Duck Choi,박동휘,Seung-Deuk Byun 대한재활의학회 2011 Annals of Rehabilitation Medicine Vol.35 No.5
Objective To investigate the eff ect of botulinum toxin type A (BTXA) on drooling and the morphologic change of the salivary gland in patients with cerebral palsy. Method Eight cerebral palsy patients suffering from severe drooling participated in this study. BTXA was injected into both submandibular and parotid glands under intravenous sedation and with ultrasound guidance (1 unit/gland/kg: maximum 100 units) in an outpatient or inpatient procedure. The severity of drooling was measured before injection and 3 weeks after injection using the Teacher Drooling Scale, the Drooling Score-severity,frequency and the Visual Analog Scale. To investigate the morphologic change of the salivary glands, the size of salivary glands were measured before injection and 3 weeks after injection using computed tomography of the neck. The measurement values were analyzed by Wilcoxon signed rank test. Results Statistically significant improvements were shown in all three parameters for assessing the severity of drooling after BTXA injections (p<0.05). Size of the salivary glands were signifi cantly decreased at 3 weeks after BTXA injection (p<0.05). Conclusion Salivary gland injection with BTXA could be a useful treatment method to reduce drooling in patients with cerebral palsy and decreased size of salivary glands may partially explain the mechanism.