Involvement of Glycogen Synthase Kinase-3β in Palmitate-Induced Human Umbilical Vein Endothelial Cell Apoptosis

Choi, Sung-E.,Kang, Yup,Jang, Hyun-Ju,Shin, Ha-Chul,Kim, Hyo-Eun,Kim, Hyo-Soo,Kim, Hae Jin,Kim, Dae Jung,Lee, Kwan-Woo S. Karger 2007 Journal of vascular research Vol.44 No.5

<P><I>Background/Aims:</I> The death of endothelial cells may play a critical role in the development of various vascular diseases, including atherosclerosis. While free fatty acids (FFAs) may stimulate endothelial apoptosis, the molecular and cellular mechanisms of this effect have not been studied intensively. To elucidate the mechanisms involved in FFA-induced endothelial cell apoptosis, we investigated the effect of different pharmacological inhibitors on palmitate-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Interestingly, lithium, a glycogen synthase kinase-3 (GSK-3) inhibitor, showed a strong protective effect. <I>Methods and Results:</I> To examine the involvement of GSK-3β in palmitate-induced HUVEC apoptosis, its dephosphorylation at Ser<SUP>9</SUP> and enzymatic activation in response to palmitate treatment were monitored by immunoblotting and in vitro kinase assays, respectively. GSK-3β was dephosphorylated and its enzymatic activity increased in palmitate-treated HUVECs. In addition, pretreatment with other GSK-3β inhibitors, e.g. SB216763 or TDZD-8, as well as adenoviral transduction with a catalytically inactive GSK-3β had significant protective effects against palmitate-induced HUVEC apoptosis. <I>Conclusion:</I> These results demonstrate that the GSK-3β signalling pathway is involved in palmitate-induced HUVEC apoptosis.</P><P>Copyright © 2007 S. Karger AG, Basel</P>

A clinical drug library screen identifies clobetasol propionate as an NRF2 inhibitor with potential therapeutic efficacy in KEAP1 mutant lung cancer

Choi, E-J,Jung, B-J,Lee, S-H,Yoo, H-S,Shin, E-A,Ko, H-J,Chang, S,Kim, S-Y,Jeon, S-M Macmillan Publishers Limited, part of Springer Nat 2017 Oncogene Vol.36 No.37

<P>The Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor E2-related factor 2 (NRF2) pathway has a central role in cellular antioxidant defense. NRF2 activation due to KEAP1 or NRF2 mutations occurs frequently in many cancers, suggesting that NRF2 inhibition could be a promising therapeutic strategy. However, no potent NRF2 inhibitors are clinically available to date. To develop potent NRF2 inhibitors for therapeutic purpose, we screened similar to 4000 clinical compounds and determined clobetasol propionate (CP) as the most potent NRF2 inhibitor. Mechanistically, CP prevented nuclear accumulation and promoted beta-TrCP-dependent degradation of NRF2 in a glucocorticoid receptor-and a glycogen synthase kinase 3 (GSK3)-dependent manner. As a result, CP induced oxidative stress and strongly suppressed the anchorage-independent growth of tumors with KEAP1 mutation, but not with the wild-type KEAP1. Further, CP alone or in combination with rapamycin strongly inhibited the in vitro and in vivo growth of tumors harboring mutations in KEAP1 or both KEAP1 and LKB1 that are frequently observed in lung cancer. Thus, CP could be a repurposed therapeutic agent for cancers with high NRF2 activity. We also proposed that the use CP and rapamycin in combination could be a potential therapeutic strategy for tumors harboring both KEAP1 and LKB1 mutations.</P>

Inhibitory effects of kaurenoic acid from Aralia continentalis on LPS-induced inflammatory response in RAW264.7 macrophages

Choi, R.J.,Shin, E.M.,Jung, H.A.,Choi, J.S.,Kim, Y.S. G. Fischer 2011 Phytomedicine Vol.18 No.8

This study investigates the anti-inflammatory effects of a diterpenoid, kaurenoic acid, isolated from the root of Aralia continentalis (Araliaceae). To determine its anti-inflammatory effects, LPS-induced RAW264.7 macrophages were treated with different concentrations of kaurenoic acid and carrageenan-induced paw edema mice model was used in vivo. Kaurenoic acid (ent-kaur-16-en-19-oic acid) dose-dependently inhibited nitric oxide (NO) production, prostaglandin E<SUB>2</SUB> (PGE<SUB>2</SUB>) release, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression at micromolar concentrations in LPS-induced RAW264.7 macrophages with IC<SUB>50</SUB> (the half maximal inhibitory concentration) values of 51.73 (+/-2.42) μM and 106.09 (+/-0.27) μM in NO production and PGE<SUB>2</SUB> release, respectively. Kaurenoic acid also dose-dependently inhibited LPS-induced activation of NF-κB as assayed by electrophorectic mobility shift assay (EMSA) and it almost abolished NF-κB DNA binding affinity at 100μM. Furthermore, the in vivo anti-inflammatory effect of kaurenoic acid was examined in a carrageenan-induced paw edema model. Eight ICR mice in each group were injected with carrageenan and observed hourly, compared with the control group. Kaurenoic acid dose-dependently reduced paw swelling up to 34.4% at 5h after induction, demonstrating inhibition in an acute inflammation model. Taken together, our data suggest that kaurenoic acid, a major diterpenoid from the root of A. continentalis shows anti-inflammatory activity and the inhibition of iNOS and COX-2 expression might be one of the mechanisms responsible for its anti-inflammatory properties.

Differential Ubiquitin Binding by the Acidic Loops of Ube2g1 and Ube2r1 Enzymes Distinguishes Their Lys-48-ubiquitylation Activities

Choi, Yun-Seok,Lee, Yun-Ju,Lee, Seo-Yeon,Shi, Lei,Ha, Jung-Hye,Cheong, Hae-Kap,Cheong, Chaejoon,Cohen, Robert E.,Ryu, Kyoung-Seok American Society for Biochemistry and Molecular Bi 2015 The Journal of biological chemistry Vol.290 No.4

<P>The ubiquitin E2 enzymes, Ube2g1 and Ube2r1, are able to synthesize Lys-48-linked polyubiquitins without an E3 ligase but how that is accomplished has been unclear. Although both E2s contain essential acidic loops, only Ube2r1 requires an additional C-terminal extension (184–196) for efficient Lys-48-ubiquitylation activity. The presence of Tyr-102 and Tyr-104 in the Ube2g1 acidic loop enhanced both ubiquitin binding and Lys-48-ubiquitylation and distinguished Ube2g1 from the otherwise similar truncated Ube2r1<SUP>1–183</SUP> (Ube2r1C). Replacement of Gln-105–Ser-106–Gly-107 in the acidic loop of Ube2r1C (Ube2r1C<SUP>YGY</SUP>) by the corresponding residues from Ube2g1 (Tyr-102–Gly-103–Tyr-104) increased Lys-48-ubiquitylation activity and ubiquitin binding. Two E2∼UB thioester mimics (oxyester and disulfide) were prepared to characterize the ubiquitin binding activity of the acidic loop. The oxyester but not the disulfide derivative was found to be a functional equivalent of the E2∼UB thioester. The ubiquitin moiety of the Ube2r1C<SUP>C93S</SUP>-[<SUP>15</SUP>N]UB<SUP>K48R</SUP> oxyester displayed two-state conformational exchange, whereas the Ube2r1C<SUP>C93S/YGY</SUP>-[<SUP>15</SUP>N]UB<SUP>K48R</SUP> oxyester showed predominantly one state. Together with NMR studies that compared UB<SUP>K48R</SUP> oxyesters of the wild-type and the acidic loop mutant (Y102G/Y104G) forms of Ube2g1, <I>in vitro</I> ubiquitylation assays with various mutation forms of the E2s revealed how the intramolecular interaction between the acidic loop and the attached donor ubiquitin regulates Lys-48-ubiquitylation activity.</P>

Efficient proteolytic cleavage by insertion of oligopeptide linkers and its application to production of recombinant human interleukin-6 in Escherichia coli

Lee, E.G.,Baek, J.E.,Lee, S.H.,Kim, T.W.,Choi, J.H.,Rho, M.C.,Ahn, J.O.,Lee, H.W.,Jung, J.K. IPC Science and Technology Press ; Elsevier Scienc 2009 Enzyme and microbial technology Vol.44 No.5

Efficient expression and purification of bioactive recombinant human interleukin-6 (hIL6) was successfully achieved in Escherichia coli (E. coli) by fusion of the maltose-binding protein (MBP) with hIL6 and the insertion of oligopeptide linkers. MBP/hIL6 was over-expressed in the soluble form at a concentration of approximately 2.5g/L. For hIL6 recovery, enterokinase, factor Xa, and thrombin were employed to cleavage MBP from the fusion constructs. However, undesired and non-specific cleavage fragments as well as rhIL6 were obtained following the cleavage. The introduction of oligopeptide linkers at the C-terminal end of the fusion construct could improve the efficiency and the rate of the enzymatic cleavage reaction, and the rhIL6 purification was achieved by using MBP affinity chromatography, factor Xa cleavage, and reverse-phase chromatography, resulting in an overall yield as high as 33% (equivalent to 0.27ghIL6/L) at purity over 98%. The biological activity of the purified recombinant hIL6 was demonstrated by confirming the presence of the signal transducer and activator of transcription 3 (STAT3) signaling pathway. This study suggests that the optimized peptide linker specifically designed for both fusion partner and target molecule has a great potential for efficient recombinant protein production.

β-Galactosidase Gene of Thermus thermophilus KNOUC112 Isolated from Hot Springs of a Volcanic Area in New Zealand: Identification of the Bacteria, Cloning and Expression of the Gene in Escherichia coli

Nam, E.S.,Choi, J.W.,Lim, J.H.,Hwang, S.K.,Jung, H.J.,Kang, S.K.,Cho, K.K.,Choi, Y.J.,Ahn, J.K. Asian Australasian Association of Animal Productio 2004 Animal Bioscience Vol.17 No.11

To isolate the $\beta$-galactosidase producing thermophilic bacteria, samples of mud and water were collected from hot springs of avolcanic area near Golden Springs in New Zealand. Among eleven isolated strains, the strain of KNOUC112 produced the highest amounts of $\beta$-galactosidase at 40 h incubation time (0.013 unit). This strain was aerobic, asporogenic bacilli, immobile, gram negative, catalase positive, oxidase positive, and pigment producing. Optimum growth was at 70-72$^{\circ}C$, pH 7.0-7.2, and it could grow in the presence of 3% NaCl. The main fatty acids of cell components were iso-15:0 (30.26%), and iso-17:0 (31.31%). Based on morphological and biochemical properties and fatty acid composition, the strain could be identified as genus Thermus, and finally as Thermus thermophilus by phylogenetic analysis based on 16S rRNA sequence. So the strain is designated as Thermus thermophilus KNOUC112. A gene from Thermus thermophilus KNOUC112 encoding $\beta$-galactosidase was amplified by PCR using redundancy primers prepared based on the structure of $\beta$-galactosidase gene of Thermus sp. A4 and Thermus sp. strain T2, cloned and expressed in E. coli JM109 DE3. The gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase(KNOUC112$\beta$-gal) consisted of a 1,938 bp open reading frame, encoding a protein of 73 kDa that was composed of 645 amino acids. KNOUC112$\beta$-gal was expressed as dimer and trimer in E. coli JM109 (DE3) via pET-5b.

돼지 대장에서 분리한 E.coli의 중금속 내성에 관한 연구

조창현,정욱진,이영수,최인실,강희옥,박남규,김명화,변미경,이현숙 경상대학교 환경보전연구소 1993 環境保全硏究所報 Vol.1 No.1

돼지의 대장에서 서식하는 466개의 대장균 균주들을 분리하여, 중금속인 Ag, Cd, Cu, Hg, Ni 및Pb에 대한 저항성을 조사 하였다. 거의 대부분의 균주들이 이들 여섯가지 중금속 모두에 강한 저항성을 보였다. 이것은 우리 주위환경의 중금속 오염 정도가 심각하다는 것을 간접적으로 나타내는 것이다. Ag, Pb 및 Hg를 함유하고 있는 고체 배지에서 균주를 성장시켰을때, colony 색깔이 중금속 자체의 광택과 같은 색깔을 나타내는 점으로 보아, 이들 중금속에 대한 저항기작은 유해한 중금속 이온을 세포내 에서 무해한 금속 형태로 전환시켜 세포내에 축적시키는 기작임을 시사하였다. 그 중 가장 높은 저항성을 나타내는 isolate 385를 Ag와 Pb를 함유한 액체배지에서 각각 배양한 뒤 세포내 Ag와 Pb 축적량을 조사한 결과, 건조세포 무게당 0.72g Ag 및 0.23g Pb를 세포내에 축적하고 있었다. 따라서, 이 균주를 유전자 조작 등의 방법으로 개발 한다면 산업 폐수내에 존재하는 이 중금속들의 제거에 효율적으로 사용할 수 있을 것이라 사료된다. We isolated E.coli from porcine intestines and examined the resistances to various heavy-metals, Ag, Cd, Cu, Hg, Ni, and Pb. The 466 isolates were resistant to the heavy-metals. Among them, 72.1% was survived in 1 mM AgNO₃, 9.3% in 80 mM AgNO₃, 95.9% in 0.6 mM Cd(NO₃)₂, 5.6% in 3 mN Cd(NO₃)₂. 95.9% in Cu(NO₃)₂, 48.5% in 0.2 mM HgCl₂, 3.4% in 0.6 mM HgCl₂, 64.4% in 5 mM NiCl₂ and 67.4% in 10 mM Pb(NO₃)₂. The isolate 385 was most resistant to silver and lead ions and the MICs of the ions were 80 mM and 11 mM, respectively. These resistances were inducible by Ag^+ and Pb^2+ ions. When isolate 385 grew in LB-agar plates containing AgNO₃ or Pb(NO₃)₂, the colony colors were changed from light yellow to deep brown. This change to brown color suggests that the resistances of 385 cells to Ag^+ and Pb^2+ ions were due to the reducing mechanism which converted them into the elementary metals(Ag^0, Pb^0) after the uptake of the ions into the cells. The resistant cells accumulated 0.72gr of Ag^0 and 0.23gr of Pb^0 per cells dry wt.

Bactericidal effect of low concentration electrolyzed water on E. coli O157:H7 on lettuce

Tian Ding,Myoung-Su Park,S.M.E. Rahman,Jun Wang,Joong-Hyun Park,Fereidoun Forghani,Na-Jung Choi,Ha-Na Kim,Gwang-Hee Kim,Hyo-Jung Choi,Deog-Hwan Oh 한국식품영양과학회 2011 한국식품영양과학회 학술대회발표집 Vol.2011 No.10

20대여성의 유방자가검진에 대한 지식과 자기효능감의 관계연구

강지혜,유리나,박민아,박신영,양은주,이주은,전시은,정윤혜,최연아,홍재하,이자형,정덕유,배노연 이화여자대학교 간호과학대학 2009 이화간호학회지 Vol.- No.43

The purpose of this study was to know relationship between important variables which influence lifestyle in college women`s health promotion, prove adjustable factors and provide basic data which develops nursing intervention health promotion program. The method of this study was descriptive correlational study. The convenience sample was 206 college woman who live alone in lodgings, dormitory, a reading room or off-campus housing. Research instruments were the Multidimensional Health Locus of Control(MHLC) developed by Wallston, Wallston & Devellis(1978), Interpersonal Support Evaluation List developed by Cohern, Hoberman(1983) and The Health Promotion Lifestyle Profile(HPLP) developed by Walker, Pender & Sechrist(1978) and the data was used after Factor Analysis. The result of this study was follows. 1. It can be found that the level of the college woman`s health promotion lifestyle was 2.46±.33 with higher fulfilling level: According to general characteristic, the level of health promotion lifestyle was 5% significant statistical differences by age and period of menstruation. 2. The level of health locus of control was 3.25±.31 with higher fulfilling level. 3. The interpersonal support level was 3.05±.44 with higher fulfilling level. 4. The level of interpersonal support turned out most convincing factor in health promotion lifestyle (9%). In addition to age factor, two factors was 10% power of explanation in whole health promotion.

커피찌꺼기를 이용한 활성탄의 제조 및 흡착특성

최병혁,정진희,김진영,김한석,김명숙,최호은,성낙창 동아대학교 환경문제연구소 2008 硏究報告 Vol.30 No.1

The exhausted coffee is a kind of biomass resources. In this study Activated carbon was prepared from ex.hausted coffee by activation with potassium Hydroxide. Processes of this study were as following : rosting step and carbonizaton step and chemical activation step. The rostiong step was canied out at more than 300 °C. The carbonization and activation step was simultaneously caπied out at 500-700°C. The purpose of this study is to evaluate the feature and usability of activated cofee char through SEM photographs, Iodine absorbability, TGA analysis, and technical analysis. For the activated carbon produced under optimal condition, iodine adsorptivity was 774mg/g, specific surface area was foπned well. In case of making activated carbon using exhausted coffee, it is considered that it has value enough as an activated carbon.