-1Contents Division of Leading Research 3 Systemic Proteomics Research Center C-1 Protein tyrosine...

Contents

Division of Leading Research

3 Systemic Proteomics Research Center

C-1 Protein tyrosine phosphatases for targeted proteomics research

4 Stem Cell Research Center

B-8 Osteopontin promotes the development of natural killer cells from hematopoietic stem cells

B-9 Characterization of Annexin II as a tumor associated marker of hepatocellular carcinoma

B-10 Hypoxia-induced IL-18 increases hypoxia-inducible factor-1alpha expression trough a Rac1-dependent

NFkappaB pathway

B-11 Cystatin SN(CST1), a cystein protease inhibitor, was upregulated in gastric tumors

B-12 Upregulation of the Cyclin-dependent kinase subunit CKS2 in gastric cancer

6 Bionanotechnology Research Center

D-3 Paramagnetic gold nanostructures for dual modal bioimaging and phototherapy of cancer cells

D-4 Toxicity of gold nanoparticles functionlized with cationic, neutral and anionic side chains

D-5 Non-invasive imaging of dendritic cell migration into lymph nodes using near-infrared fluorescent

semiconductor nanocrystals

D-6 Mixed self-assembly of polydiacetylenes for highly specific and sensitive strip biosensors

D-7 Peptide antibody transducer

D-8 Kinetic characterization of artificial cleavable caspase-3

D-9 Development of cascade enzyme-linked immunosorbent assay(CELISA)

D-10 Immunization with HPV16L1 VLP expressed in Lactobacillus induces systemic and mucosal

neutralizing antibosies in Balb/C mice

D-11 Oral administration of poly-gamma-glutamic acid improves dermatitis in NC/Nga mice, a model of

human atopic dermatitis

D-12 Poly( -glutamic acid)-chitosan nanoparticles induces antigen specific humoral and cellular immunity

D-13 Nobel peptides with a specific binding to the Fc domain of immunoglobulin G

D-14 Label-free detection of p53 point mutation using field effect transistor

11 Therapeutic Antibody Research Center

A-78 Generation and characterization of a large human Fab library

A-79 Isolation and characterization of human antibodies that recognize both hL1cam and mL1cam

A-80 Panning strategies for screening of anti-L1CAM human monoclonal antibodies by phage display

A-81 TMPRSS4 promotes invasion, migration and metastasis of human tumor cells by facilitating an

epithelial-mesenchymal transition

A-82 PAUF is a metastasis factor of human pancreatic cancer

A-83 Therapeutic potential of a monoclonal antibody against PAUF in human pancreatic cancer

A-84 CTHRC1 promotes adhesion and motility of pancreatic cancer cells

14 Metabolic Syndrome Research Center

C-2 Sophora flavescens attenuates atherosclerosis and hyperlipidemia in LDL receptor-deficient mice

C-3 Antioxidant property of eupatilin attenuates atherosclerosis and improves adipokine profiles in LDL

receptor-deficient mice

C-4 The ethanolic extracts of Rhodiola rosea prevent atherosclerosis and obesity in LDLr-/- mice via

2nd KRIBB Poster Festival | Abstracts |

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stimulation of lipid decomposition pathway

C-5 Ethanolic extracts of Brassica campestris spp rapa roots prevent high-fat diet-induced obesity via

beta3-adrenergic regulation of white adipocyte lipolytic activity

Division of Biointegration Research

19 Medical Genomics Research Center

D-18 Functional and clinical evidence for NDRG2 as a candidate suppressor of liver cancer metastasis

D-19 The genome-wide effect of calsequestrin 2 (Casq2) knockdown on mouse myoblast differentiation

D-20 Genome-wide analysis of DNA methylation patterns in gastric cancer cells using genomic tiling

microarrays

D-21 Aberrant promoter methylation profile in gastric carcinoma by pyrosequencing of sample pools

D-22 Copy number variations (CNVs) identified in normal Korean individuals

D-23 LRRC3B, encoding a leucine-rich repeat-containing protein, is a putative tumor suppressor gene in

gastric cancer

D-24 Stroke web portal based on Korean oriental medicine

D-25 Genome deletion project in S. pombe

D-26 Fission yeast-based screening to identify putative HDAC inhibitors using a telomeric reporter strain

23 Disease Model Research Center

B-26 Activation of PI2-K and p38 MAPK signal pathways is required for lipopolysaccharide-induced

microglial phagocytosis

B-27 Antihypertensive effect of MB12066 compound associated with e modulation in spontaneously

hypertensive rats

B-28 Acceleration of K-rasG12D-driven tumor progression by deleted mutation of Peroxiredoxin I in mouse

embryonic fibroblasts

25 Regenerative Medicine Research Center

C-12 Expression patterns of protein kinase A subunits during meiotic maturation periods in porcine oocytes

C-13 Porcine XBP-1 is a transcription factor involved in UPR response

C-14 Characterizations of seven Drosophila insulin-like peptide genes

C-15 Isolation of differentially expressed genes in rosette-enriched neuroectodermal spheres derived from

human embryonic stem cells

27 Omics and Integration Research Center

A-65 Reconstruction of sulfur metabolism pathway in Hansenula polymorpha based on transcriptome and

metabolite analysis

A-66 Analysis of the unfolded protein response pathway in the methylotrophic yeast Hansenula polymorpha

A-67 Functional characterization of Yarrowia lipolitica homologues to Saccaromyces cerevisiae MNN4p and

MNN6p.

A-68 Global gene expression profiles of the capnophilic rumen bacterium Mannheimia succiniciproducens

grown under anaerobic conditions

A-69 Genome-wide transcriptional responses of different Escherichia coli strains to recombinant protein

overproduction

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A-70 Characterization of the YgiXY two-component signal transduction system of the capnophilic rumen

bacterium Mannheimia succiniciproducens

A-71 Tomato chromosome 2: BAC assemble and gene annotation

31 Translational Research Center

A-72 Comparative proteomic analysis of peripheral blood mononuclear cells from atopic dermatitis patients

and healthy donors

A-73 Identification of proteins involved in hepatocellular carcinogenesis using HBx transgenic mouse

A-74 Role of autophagy in oxidative stress-induced cytotoxicity in HT22 cells

A-75 A proteome profiling of differentiating human embryonic neural stem cells

A-76 Comparative proteome analysis of 3T3-L1 cell differentiation into adipocytes

A-77 Role of Hsp70 Co-chaperones in the degradation of neurodegenerative proteins

Division of Molecular Therapeutics

37 Molecular Cancer Research Center

D-15 Characterization of tailoring genes involved in the modification of geldanamycin polyketide in

Streptomyces hygroscopicus JCM4427

D-16 Cryptotanshinone inhibits constitutive STAT3 function through blocking the dimerization in DU145

prostate cancer cells

D-17 YJ-1 induces apoptosis through inhibition of HSF1 in human cancer cells

38 Natural Medicine Research Center

C-16 Diacylglycerol acyltransferase inhibitory effect by sesquiterpenoids isolated from the flower buds of

Tussilago farfara

C-17 Synthesis and biological evaluation of benzoimidazole derivatives as inhibitors of DGAT

C-18 ACAT inhibitors from the fruits of Piper longum and Piper nigrum

C-19 Tiarellic acid and randianin induce apoptosis in clone 15 HL-60 cells via caspase-3- and mitochondria-

mediated pathway

C-20 ERK1/2 signaling pathway is involved in verproside-mediated anti-inflammation in a mouse model of

allergic asthma

C-21 Nicotine attenuates iNOS expression and contributes to neuroprotection in a compressive model of spinal

cord injury

C-22 Anti-influenzavirus acitivity of quercetin 7-rhamnoside isolated from Korean medicinal plants

41 Functional Metabolite Research Center

A-5 New type of FabI-directed antibacterial agent from microbial metabolites

A-6 New anti-MRSA agent from Penicillium sp. FR0011

A-7 Cordyformamide and chrysophanol, phenolic antioxidative metabolites from the fungus Eupenicillium

shearii F80695

A-8 New lactone compounds from the Basidiomycete Stereum ostrea

A-9 Chemical and biological properties of the pine bark extract of Pinus densiflora Siebold et Zuccarini

A-10 Endoplasmic reticulum stress induction by honokiol isolated from Magnolia obovata

A-11 Repression of E-cadherin by oncogenic K-ras involving DNA methyltransferase activity

A-12 DNA methylation and moderately decreased CBR1 gene expression in K-ras overexpressed prostate

cancer cell lines

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A-13 Isolation and identification of Micromonospora sp. showing nematocidal activity against Pine wood

nematode

A-14 Diversity of culturable prokaryotes in Bigeum Island, established by phylogenetic approach

A-15 Clitocybin A, a new antioxidative compound from the culture broth of Clitocybe aurantiaca

A-16 Etoposide-resistant HT-29 human colon carcinoma cells during glucose deprivation are sensitive to

Piericidin A, a GRP78 down-regulator

Division of Biomaterials Science

49 Plant Genome Research Center

C-6 Repression of BiP, an ER-resident protein, prevents R-gene-mediated hypersensitive response (HR) but

enhances non-host pathogen-induced necrosis in N. benthamiana.

C-7 In silico screening and promoter analysis of pathogen-induced genes in pepper

C-8 Silencing of an NbLytB gene involved in the plastid nonmevalonate pathway compromised hypersensitive

response

C-9 Increased salinity tolerance by carbon metabolite production in chloroplast transgenic Nicotiana tabacum

C-10 Expression of RNAi suppressor codon-optimized for Chlamydomoans reinhardtii

C-11 Idenification and characterization of the PAMPs and effectors of Burkholderia glumae, the causative

agent of bacterial grain rot of rice

52 Systems Microbiology Research Center

A-43 Spectrum of proton irradiation-induced mutations in the tonB gene of Escherichia coli

A-44 Genome sequence of the probiotic bacterium Bifidobacterium bifidum BGN4

A-45 Identification of a new gene cluster for type II secretion in Escherichia coli BL21(DE3)

A-46 Genome sequence analysis of the marine microbe Donghaeana dokdonensis DSW-6

A-47 Multidimensional comparative omics analysis of Escherichia coli B and K-12

A-48 Enzyme platform based on yeast TFP technology for the production of cellulosic bioethanol

A-49 Development of FRET-based biosensors for rapid and quantitative analysis of sugars in biological

samples

A-50 Rapid cloning and expression of target genes by homologous recombination system, pRMT-iTGR

A-51 Catalytic protein particles based on cellulose-binding domain fusion

A-52 Protein engineering supported by homology modeling and docking simulation

A-53 Novel cold-adapted alkaline lipase from intertidal flat metagenome and proposal of a new family of

bacterial lipase

A-54 Mycotoxins, a group of fungal secondary metabolites, can induce motility of bacterial cells

A-55 Functional analysis of the fusaricidin biosynthetic gene of Paenibacillus polymyxa E681 and a possible

role of fusaricidin in biofilm formation

A-56 Airborne induction and priming of plant defenses against a bacterial pathogen

A-57 Insight into plant defense signalings against Ralstonia solanacearum in Nicotiana benthamiana ---71

A-58 Plant innate immunity by bacterial genetic materials

A-59 Role of priming resistance genes on bacilli-elicited induced systemic resistance

A-60 Improved thermostability and acetic acid tolerance of Escherichia coli by directed evolution of MetA

A-61 Autoinduction expression system in Escherichia coli

A-62 Crystal structure of fully oxidized human thioredoxin1 containing disulfide between Cys62 and Cys69

A-63 Genomic basis of the two E. coli workhorse strains for their improved capacity to synthesize membrane

proteins at high levels

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A-64 Characterization and molecular modeling of a thermophilic and alkaliphilic galactokinase from Thermus

caldophilus GK24

61 Environmental Biotechnology Research Center

B-13 Transgenic crops with enhanced tolerance to multiple environmental stresses for sustainable agriculture

B-14 Expression of sweetpotato IbMYB1 transcription factor gene induced anthocyanin accumulation in

plants

B-15 Characterization of sweetpotato orange gene involved in carotenoid biosynthesis

B-16 Enhanced tolerance of transgenic potato plants expressing 2-Cys peroxiredoxin in chloroplasts against

oxidative stress and high temperature

B-17 Transgenic sweetpotato plants expressing a cold-inducible zinc finger protein, SCOF-1

B-18 Transgenic sweetpotato plants overexpressing nucleoside diphosphate kinase 2 showed increases of

antioxidant enzymes activities and enhanced tolerance to multiple environmental stresses

B-19 Differential response of thirteen sweetpotato peroxidase genes under various abiotic stresses

B-20 Transgenic sweetpotato plants expressing spike protein of porcine epidemic diarrhea virus

B-21 Comparison of prediction models for cyanobacterial bloom in a eutrophic reservoir (Korea)

B-22 Cloning, sequencing, and expression of reductive dehalogenase from the anaerobic PCE to VC

dechlorinating enrichment culture

B-23 A study on quorum sensing system for the conservation of biodiversity in a eutrophic water

B-24 Selection of microalgae for lipid production under high-level carbon dioxide

B-25 Comparison of several methods for effective lipid extraction from microalgae

69 Division of Biotechnology R&BD

A-17 Study on cleavage by various proteases and their applications to purification of a fusion-tagged protein

A-18 Signal enhancement of surface plasmon resonance using carbon nanotube-antibody complex

A-19 Purification of recombinant human erythropoietin in milk of transgenic pigs

70 Biological Resource Center

B-1 Pontibaca methylaminivorans gen. nov., sp. nov., a novel member of the family Rhodobacteraceae

B-2 Candida alkalitolerans sp. nov., a novel alkalitolerant yeast from industrial waste water

B-3 Candida nymphaea sp. nov., a novel yeast in the Kodamaea clade from water

B-4 Gordonia kroppenstedtii sp. nov., a phenol-degrading actinomycete isolated from a polluted stream

B-5 Solirhabdus panacisegetis gen. nov., sp. nov., isolated from soil of a ginseng field

B-6 Patulibacter ginsengiterrae sp. nov., isolated from soil of a ginseng field

B-7 A polyphasic investigation on some novel Halomonas species isolated from a renal care center

73 Korean BioInformation Center(KOBIC)

D-1 SNP@WEB: a web-based catalog of SNP (Single Nucleotide Polymorphism) databases and tools

D-2 In silico identification of gene-related patents

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74 Daejeon-KRIBB-FHCRC Research Cooperation Center

D-27 Functional proteomic study reveals that GnT-V reinforces the invasive/metastatic potential of colon

cancer through aberrant glycosylation on TIMP-1

D-28 Glycomics-based biomarker discovery for liver & colon cancer

D-29 Profiling of tumor associated antigens directed autoantibodies of cancer patient

Division of Ochang R&D

79 National Primate Research Center

A-1 In silico analysis and experimental validation of recently exonized Alu in Macaca fascicularis

A-2 Identification of novel retromer complexes in the mouse testis

A-3 Pig ADAM3 disintegrin domain has an important rolein the sperm-egginteraction

A-4 Identification of a binding domain in non-human primate (Macaca fascicularis) ADAM2 for hetero

complex

81 Bio-Evaluation Center

A-20 In vitro absorption and metabolism characteristics of 2'-benzoyloxycinnamaldehyde, an antitumor drug

candidate

A-21 Transport and metabolism of JAC106, an NO Donor-containing tubulin binding agent, in Caco-2 cells

A-22 Plasma pharmacokinetics of 2'-benzoyloxycinnamaledhyde, an antitumor drug candidate, in rats

A-23 Gene expression profiling of KBH-A42 in human leukemia and bladder cell lines

A-24 KBH-A42 overcomes multi-drug resistance and induces cell death in P-glycoprotein expressing cells

A-25 The effect of inserted cucumber mosaic virus-coat protein gene chili pepper ((CMV-cp (line 7)) on the

non-target insects, green peach aphids (Myzus persicae Sulzer, Homoptera)

A-26 The soybean mosaic virus resistance Locus, Rsv3, is cosegregating with a cluster of genes that encode a

nucleotide binding site and a leucine-rich repeat region

A-27 Towards confirmation of a major quantitative trait loci for isoflavones content in soybean seed

A-28 Genetic analysis and event-specific detection of a CGMMV-CP transgenic watermelon rootstock line

A-29 Agronomic evaluation of transgenic Capsicum annuum L.resistant to anthracnose fungus

Jeonbuk Branch Institute

89 Bioindustry Research Center

A-39 Analysis of -(1 3)(1 6)-glucan produced by Aureobasidium pullulans IMS-822

A-40 Real-time assays for the detection of Bacillus cereus strains in fermented foods

A-41 Neuraminidase inhibitory activities of flavonoids from Rhodiola rosea

A-42 The strain development of Aureobasidium pullulans for pure beta-glucan elaboration

90 Molecular Bioprocess Research Center

A-30 Molecular cloning, nucleotide sequence and expression of levansucrase from Bacillus amyloliquefaciens

type 1 in Escherichia coli

A-31 Heterologous expression and characterization of L-Amino acid deaminases from Proteus mirabilis in

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Escherichia coli

A-32 Enhanced production of eicosapentaenoic acid by reengineering of EPA biosynthesis gene cluster in

Escherichia coli

A-33 Screening and characterization of a novel metalloprotease from a metagenomic library

A-34 Isolation and identification of microorganisms for cellulytic activity from soil

A-35 Screening and PCR amplification of glucansucrases from soil microorganisms

A-36 Optimization of culture conditions for 1,3-propanediol production from crude glycerol by Klebsiella

pneumoniase using response surface methodology

A-37 Expression and purification of the human papillomavirus type 11 L1 capsid protein in Escherichia coli

A-38 Isolation and identification of anti-microbial agent from volatile oils

94 Poster Board Number Index

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Division of Leading Research

Systemic Proteomics Research Center

Stem Cell Research Center

Bionanotechnology Research Center

Genome Research Center

Therapeutic Antibody Research Center

Metabolic Syndrome Research Center

2 n d K R I B B P o s t e r F e s t i v a l

Systemic Proteomics Research Center

C-1Protein tyrosine phosphatases for targetedproteomics research

Tae-Sung Yoon1, Dae-Gwin Jeong1, Seung Jun Kim1, Suk-Kyeong Jung1, Jae Hoon Kim2, Sang Jeon Chung1, Seung-Ho Kim1, HwangSeo Park3, and Seong-Eon Ryu1

1Systemic Proteomics Research Center, KRIBB2College of Applied Life Science, Cheju National University3Department of Bioscience and Biotechnology, SejongUniversity

Protein tyrosine phosphatases (PTPs) are an importantfamily of signal transduction proteins, together with proteintyrosine kinases, controlling tyrosine phosphorylation whichplays a major role in many cellular functions including cellsurvival, proliferation, and differentiation. There are atleast 107 genes coding for PTPs in the human genome.Recently, about half of all PTP genes have been implicated inhuman disease and recognized as potential drug targets.PTPs have a conservedcatalytic domain and show somesubtle structural differences in the active-site residues. Thedetailed structural information of PTPs will enable us toverify the interaction between substrates and the active-siteresidues and provide a solid foundation for rational PTPinhibitor design. To this end, the catalytic domains of 56human PTPs (RPTPalpha, RPTPbeta, LAR, SAP1, DEP1,RPTPkappa, RPTPmu, IA-2, IA-2beta, PTP-U2, PCPTP,RPTPsigma, PTPRT, PTP1B, TCPTP, STEP, SHP1,HePTP, SHP2, PTP-BAS, PTP36, PTPD1, MKP-1, MKP-2, hVH3/B23, PYST1, PYST2, hVH5, MKP-4, MKP-5,MKP-7, VHR, PIR1, HYVH1, BEDP, TMDP, VHY,DUSP20, DUSP17, DUSP21, VHX, MOSP, MGC1136,VHZ, MCE, SSH1, SSH2, PRL1, PRL2, PRL3, CDC14B,LMPTP, CDC25A, CDC25B, CDC25C, Eya2) were wellexpressed in E. coli and subjected to crystallization screening.Among them, more than 16PTPs were successfully crystallizedand some of their structural analyses have been reported.Especially with our recent structural report of SSH2,comparative analysis between seven sub-families (MKPs,atypical DSPs, SSHs, PRLs, CDC14s, PTENs, MTMs) ofDSPs or VH1-like PTPs (61 genes) would be possible.With these results, our targeted proteomics research effortsinvolving mass spectrometric analysis will be also discussed.(IUCr 2008, XXI congress of the International Union ofCrystallography : Osaka (Japan), 2008)

Keywords : crystal structure, protein tyrosine phosphatase,proteomics

Members

DirectorSeong-Eon Ryu ([email protected])

Principal ResearcherHyun-Jun Lee ([email protected])

Senior Researcher Tae-Sung Yoon ([email protected])Jeong-Hee Moon ([email protected]) Dae-Gwin Jeong ([email protected])

Researcher Young-Jae Bahn ([email protected])Kyeong-ah Lee ([email protected])Dong-Hwa Lee ([email protected])Chun-Hua Wei ([email protected])Soo-Hee Choi ([email protected])You-Me Kim ([email protected]) Seong-Kuk Jeon ([email protected])Oh-Hyung Kwon ([email protected])Na-Young Lee ([email protected])Eun-Mi Kang ([email protected])

Korea Research Institute of Bioscience and Biotechnology 3

4 2nd KRIBB Poster Festival

Stem Cell Research Center

B-8Osteopontin promotes the development ofnatural killer cells from hematopoietic stemcells

Mi Sun Kim, Zheng-Hao Piao, Mira Jeong, So HyeonYoon, Seok Hyeong Lee, Suk Ran Yoon, Tae Don Kim,Jin Woong Chung, and Inpyo Choi

Stem Cell Research Center, KRIBB

The detailed mechanisms driving the development of naturalkiller (NK) cells from hematopoietic stem cells remain tobe clearly elucidated. Here, we show that osteopontin (OPN)is a key factor for NK development. OPN-deficient miceevidenced severe impairments of NK development in bonemarrow (BM) and spleen in which the NK populationsthat express CD122 and NK cell receptors were reduced.However, the absence of intrinsic OPN expression did notaffect the NK development while the absence of OPN inmicroenvironment exhibited significant reduction in NKpopulation. The expression of OPN was induced by IL-15in BM stromal cells, and the defect in NK differentiation inIL-15-/- hematopoietic precursor cells (HPC) were recoveredby addition of recombinant OPN, suggesting that themicroenvironmental OPN may be a key factor in IL-15-mediated NK differentiation. In addition, OPN-driven NKmaturation was reduced in T-bet-deficient HPC, suggestingthat T-bet is required for OPN-mediated NK development.Collectively, these results show that paracrine OPN signalingdrives NK-lineage commitment, thus ultimately promotingNK cell development. (The 62th Annual Meeting of theKorean Association of Biological Sciences : Seoul,2008)

Keywords : differentiation, NK cells, osteopontin, stemcells

B-9Characterization of Annexin II as a tumorassociated marker of hepatocellular carcinoma

Mi-Young Park1, Na Young Ji1, Hee Gu Lee1, Jae Wha Kim1,Suk Ran Yoon1, Jong Wan Kim2, Pyung-Keun Myung3,Dae Ghon Kim4, and Eun Young Song1*

1Stem Cell Research Center, KRIBB2Department of Laboratory Medicine, Dankook MedicalSchool

3College of Pharmacy and Research Center for TransgenicCloned Pigs, Chungnam National University

4Department of Internal Medicine, Chonbuk NationalUniversity Medical School and Hospital

Annexin II is a calcium-dependent, phospholipid-binding

protein. It has several biological roles that involved in theanti-inflammatory effect of glucocorticoids, immune response.Recently, Annexin II was identified as an up-regulated genein hepatocellular carcinoma tissue by cDNA microarray.In this study, we have been characterized Annexin II as ahepatocellular carcinoma markerand determined AnnexinII in human serum. Overexpression of Annexin II mRNAand Annexin II protein in human liver tissues and hepatomacell lines was confirmed by RT-PCR and Immunologicalmethods. For quantitative analysis of Annexin II in humanserum, we have developed a solid-phase sandwich assay.Detection range was 0 5.0 g/ml and sensitivity was 40 ng/ml.Annexin II levels in the serum of patients with hepatocellularcarcinoma were significantly increased as compared withthose in the normal serum. These results suggest thatexpression of Annexin II may be increased in liver cancerpatients and may play an important role in liver cancer. Thenew ELISA method can be used as a tool to detect AnnexinII in human serum, particularly for cancer diagnostics.(Frontiers in Immunology Research 2008 InternationalConference : Florence (Italy), 2008)

Keywords : Annexin II, cDNA microarrary, ELISA,hepatocellular carcinoma

B-10Hypoxia-induced IL-18 increases hypoxia-induciblefactor-1alpha expression trough a Rac1-dependentNFkappaB pathway

Yan Shao, Hyun Woo Suh, Ji-Won Lee, Suk Ran Yoon, TaeDon Kim, Jin Woong Chung, and Inpyo Choi

Stem Cell Research Center, KRIBB

Interleukin-18 (IL-18) plays pivotal roles in linking inflammatoryimmune responses and tumor progression and metastasis,yet the manner in which this occurs remains to be sufficientlyclarified. Here we report that hypoxia induces the transcriptionand secretion of IL-18, which subsequently induces theexpression of hypoxia-inducible factor-1a (HIF-1a).Mechanistically, IL-18 induces HIF-1a through theactivity of the GTPase Rac1, which inducibly associateswith the IL-18 receptor (IL-18R ) subunit, via a PI3K-AKT-NFk-B-dependent pathway. Importantly, the knockdownof the IL-18R subunit inhibited IL-18-driven tumor cellmetastasis. Collectively, these findings demonstrate a feed-forward pathway in HIF-1a-mediated tumor progression,in which the induction of IL-18 by hypoxia or inflammatorycells augments the expression of both HIF-1 and tumorcell metastasis. (The 62th Annual Meeting of the KoreanAssociation of Biological Sciences : Seoul, 2008)

Keywords : HIF-1a, hypoxia-inducible factor-1a, IL-18,IL-18R , interleukin-18, Rac1

B-11Cystatin SN(CST1), a cystein protease inhibitor,was upregulated in gastric tumors

Eun Hwa Choi1, Jong-Tae Kim1, Hyo Ran Yoon1, JooHeon Kim2, Jong Wan Kim3, Young Il Yeom1, Eun YoungSong1, and Hee Gu Lee1,*

1Stem Cell Research Center, KRIBB2Department of Pathology, Eulji University School ofMedicine

3Department of Laboratory Medicine, Dankook UniversityCollege of Medicine

Cysteine proteases(CPs) are widely distributed proteolyticenzymes and form tight equimolar complex with the inhibitorof CP, cystatin, occluding the active site. Cystatin1(CST1)is a member of type2 cystatin family that detected in a varietyof fluids and secretions, including plasma, tears, and saliva.Using DNA microarray (Genechip) and gene expressiondata-mining, we searched the genes that was differentiallyexpressed in gastric cancers. Here, we showed, using gastriccancer tissues, that CST1 was upregulated in the cancerousregion compared with non-cancerous region. The increasedlevels of mRNA of CST1 were showed by reverse transcriptasepolymerase chain reaction(RT-PCR), and tumor-specificstaining of CST1 was detected in gastric cancer tissues.Collectively, we suggest that CST1 might be involved in theprogression of gastric cancers. (Frontiers in ImmunologyResearch 2008 International Conference : Florence(Italy), 2008)

Keywords : CST1, cystatin SN, gastric cancer, gastrictumor, p53, proliferation

B-12Upregulation of the Cyclin-dependent kinasesubunit CKS2 in gastric cancer

Min Ah Kang1, Jong-Tae Kim1, Joo Heon Kim2, Soo-YoungKim2, Young Ho Kim3, Young Il Yeom1, Young Hee Lee4,and Hee Gu Lee1,*

1Stem Cell Research Center, KRIBB2Department of Pathology and Preventive Medicine, EuljiUniversity School of Medicine

3Department of Internal Medicine, Samsung Medical Center 4Department of Biochemistry, Chungbuk National University

The CKS2, cyclin-dependent kinase (CDK) regulatorysubunit, is a component of maturation promoting factorcomposed of cyclinB1 and CDK1, which is the primarykinase complex that regulates meiosis. Here, we show,using RT-PCR analyses, that the mRNA transcripts ofCKS2 were significantly upregulated in gastric cancerscompared with normal tissues. Immunohistochemical and

clinicopathological analyses of CKS2 expression in gastriccancer tissues showed that a high level of CKS2 washighly correlated with histologic tumor differentiation andpTNM stage. GFP-CKS2-transfected cells revealed thedownregulation of tumor suppressors p53 and p21cip1 andshowed an increased cell proliferation rate relative to GFP-cells. Also, CKS2-suppressed cells treated with CKS2-siRNA showed growth retardation and a moderate upregulationof p53. Thus, we suggest that the cell cycle regulator CKS2might be deeply involved in gastric tumorigenesis. (Frontiersin Immunology Research 2008 International Conference: Florence (Italy), 2008)

Keywords : CDK1, CKS2, cyclin-dependent kinase, gastriccancer, gastric tumor, p21cip1, p53

Members

DirectorInpyo Choi ([email protected])

Principal ResearcherIn Seong Choe ([email protected])Hee Gu Lee ([email protected])Eun Young Song ([email protected])Jae Wha Kim ([email protected])

Senior Researcher Suk Ran Yoon ([email protected]) Tae Don Kim ([email protected])Jin Woong Chung ([email protected])Kee Nyung Lee ([email protected])

Post-DocHyun Woo Suh ([email protected])Hu-Nan Sun ([email protected])Yan Shao ([email protected])Jong-Tae Kim ([email protected])

Researcher Jung-Il LeeJu-Yeong Park ([email protected]) Min-Ah Lee ([email protected])Ji-Won Lee ([email protected])Mi-Sun Kim ([email protected]) So-Hyun Yun Mi-Ra Jeong ([email protected]) Swok-Hyung Lee ([email protected]) JI-Hye Park ([email protected])Zheng-Hao Piao ([email protected])Mi-Jung KimMin-Ah Kang ([email protected])Eun-Hwa ChoiHyo-Ran YoonDong-Hee Shin ([email protected]) Seung-Hee YangKyoung-Hee AnSeong-Jin Cho


Hyang-Ran Yoon ([email protected])Ji Hyun Sim ([email protected])Hye Eun KownJeoung-Hyeon SeoJin Yoe ([email protected]) Na Young JiNan-Young ParkJung-Eun Ahn ([email protected])

D-3Paramagnetic gold nanostructures for dual modalbioimaging and phototherapy of cancer cells

Yong Taik Lim, Mi young Cho, Bang Sil, Jung Min Lee,and Bong Hyun Chung

Bionanotechnology Research Center, KRIBB

Paramagnetic gold nanostructures were synthesized bycombining the paramagnetism of gadolinium with theplasmonic properties of gold nanoparticles and used fordual modal (MRI and optical) imaging and phototherapyof breast cancer cells. (Under preparation)

Keywords : gold, imaging, nanomaterial, nanoparticle,nanostructure, phototherapy

D-4Toxicity of gold nanoparticles functionlized withcationic, neutral and anionic side chains

Jina Kwon, Yong Taik Lim, Mi Young Cho, and Bong HyunChung


The development of nanotechnology offers tremendouspotential for the future of biomedical imaging and therapy.However, unexpected side effects of some nanomaterialshave been also reported and systematic toxicological studyof nanomaterials is highly required. In this presentation,we suggest gold nanoparticles as possible standardmaterials for the toxicity study of nanomaterials. To studythe interaction between nanomaterials and cells orbiological tissues, the development of well-defined (orcharacterized) nanomaterials is essential. We designedgold nanoparticles functionalized cationic, anionic andneutral side chains. The particle formation and degree ofaggregation were checked by means of UV spectroscopy,while particle size and distribution were analyzed bydynamic light scattering (DLS) and transmission electronmicroscopy (TEM). The structure of the gold core andalkanethiol components provide information relevant tothe toxicity due to the particle charge. These studies showthat cationic coated gold nanoparticles are moderatelytoxic, whereas anionic coated gold nanoparticles are quitenontoxic. (Under preparation)

Keywords : anionic, cationic, nanomaterial, nanoparticle,nanostructure


Bionanotechnology Research Center

D-5Non-invasive imaging of dendritic cell migrationinto lymph nodes using near-infrared fluorescentsemiconductor nanocrystals

Young-Woock Noh, Mi Young Cho, Yong Taik Lim*, andBong Hyun Chung*


Effective tracking of immunotherapeutic cells is essentialfor monitoring the migration of injected cells to the targettissue. Here we report the use of near infrared (NIR)-emitting fluorescent semiconductor nanocrystals, calledquantum dots (QDs), for non-invasive in vivo tracking ofdendritic cell (DC) migration into lymph nodes. Theimpact of QDs on DC viability and maturation wassystematically investigated using MTT assays and FACSanalysis. We found that the labeling of DCs with QDs hadno effect on DC phenotype or maturation potential.Cytokine and migration assays revealed that there were nosignificant changes in either cytokine production orchemokine-dependent migration of QDs-labeled DCsrelative to unlabeled cells; in both labeled and unlabeledcells, cytokine production and migratory capacity wasincreased by stimulation with lipopolysacch-aride (LPS).Furthermore, QDs did not influence allogenic naïve T cellactivation or uptake of exogenous antigens. Importantly,we also demonstrated that it was possible to track QDs-labeled DCs injected into the footpad into popliteal andinguinal lymph nodes using NIR fluorescence. Takentogether, our protocols establish the potential of non-invasive in vivo imaging of NIR-emitting QDs fortracking immunotherapeutic cells. (Under preparation)

Keywords : dendritic cells, in vivo imaging, lymph node,quantum dots

D-6Mixed self-assembly of polydiacetylenes forhighly specific and sensitive strip biosensors

Hyun Kyu Park1,2, Sang J. Chung1, Moonil Kim1, HyunGyu Park2, Jae-Hyoung Cho3, and Bong Hyun Chung1,*

1Bionanotechnology Research Center, KRIBB2Department of Chemical and Biomolecular Engineering,KAIST

3Department of Endocrinology College of Medicine,Catholic University

Polydiacetylenes (PDAs), which possess unique propertiesthat allow them to change color in response to environmentalchanges such as variations in pH, temperature, and molecularbinding, have been widely investigated as signal transducers

in biosensor applications. Most PDA-based sensors reportedto date have been evaluated largely on the basis of theirability to detect purified samples, however, and their specificityhas rarely been tested. In this study, novel PDAs fabricatedon polyvinylidene fluoride (PVDF) strips by photoreactionof composite diacetylene self-assemblies were developed asbiosensors, and nonspecific binding to off-target biomoleculeswas assessed. A mixed PDA surface containing biotin andethanolamide bound the target, i.e., streptavidin, morespecifically than did biotin alone. The optimized PDAbiosensor exhibited approximately 2,850-fold higher selectivityfor streptavidin relative to bovine serum albumin controls.What exactly was further prepared and showed distinctivesignals for the urine of diabetic patients compared to urinesamples from healthy/non-diabetic persons due to theconcentration of microalbuminuria. To our knowledge,this is the first strip-type biosensor fabricated with PDAs andthe first PDA-based biosensor that can effectively overcomethe problem of nonspecific binding. (The 102th FallMeeting of the Korean Chemical Society : Jeju, 2008)

Keywords : biosensor, PDAs, polydiacetylenes

D-7Peptide antibody transducer

Hyo Jin Kang1,2, Sun Hee Yeon1, and Sang J. Chung1,2

1Bionanotechnology Research Center, KRIBB2Nanobiotechnology Division, University of Science andTechnology(UST)

Recently, we have reported a novel strategy to immobilizeantibodies on solid surfaces by using a novel peptideconjugate, FcBP (Fc binding peptide) which specificallybind to immunoglobulin G (IgG) Fc domain. As an extensionof the research, we have fused FcBP with cell penetratingpeptides (CPPs) to develop antibody transducer. CPPshave been employed for the cellular delivery of proteins,DNA or nanopaticles as their complexes, in order to testthe cellular functions of the biomolecules or nanoparticles.There have been reported numbers of CPPs such penetratin,Arg8, HIV-1 TAT, MPS, etc. In this study, FcBP fusion peptideswith various CPPs were constructed and tested for transductionof fluorescence-labeled Abs. When the Abs were monitoredby fluorescence microscopy they were specifically deliveredinto living cells only in the presence of the fusion peptides.This represents the first peptide reagents, enabling Abtransduction into living cells without any covalent modificationof antibodies, detergents, or electroporation. (The 102th

Fall Meeting of the Korean Chemical Society : Jeju, 2008)

Keywords : cell, FcBP, Fc binding peptide, nanomaterial,nanoparticle, nanostructure


D-8Kinetic characterization of artificial cleavablecaspase-3

Hyo Jin Kang1,2, Young-mi Lee2, and Sang J. Chung1,2

1Bionanotechnology Research Center, KRIBB2Nanobiotechnology Division, University of Science andTechnology (UST)

Caspase-3 which is also known as CED-3, murine ICE anda protease resembling ICE/CPP32 in human is the first reportedapoptotic effector as well as the main downstream effectorthat cleaves the majority of cellular substrates in apoptoticcells. A large scale preparation of caspase-3 is required forscreening chemical library, structural study and kineticanalysis. Since expression of the full-length caspase-3 genein E.coli. resulted in a fully activated enzyme with a low yield,probably due to the cytotoxicity of the active caspase-3,the present method includes a separated expression of N-and C-terminal domains of caspase-3 in E.coli and subsequentrefolding of the two domains for activation. Reported hereis a novel method for a high level expression and purificationof active caspase-3 in E.coli. The method includes engineeringthe cleavage (activation) sites of the precursor protein to asequence susceptible to thrombin hydrolysis. The engineeredcaspase-3 precursors were highly overexpressed in E.coliand successfully purified by affinity column chromatography,resulting in 10~15 mg of proteins from 1 L culture. Bythrombin digestion, the purified 30 kDa precursors wereeasily converted to active proteins, consisting of 12 and 17kDa peptides on PAGE analysis. As expected, the digestionproducts showed roughly two orders of magnitudes-increasedcatalytic activities compared to those of the correspondingprecursors. (The 102th Fall Meeting of the KoreanChemical Society : Jeju, 2008)

Keywords : apoptotic effector, caspase-3, CED-3, cell,cytotoxicity, ICE/CPP32

D-9Development of cascade enzyme-linkedimmunosorbent assay(CELISA)

Young-mi Lee1, Yu-Jin Jeong1,2, Kang Hyo Jin1,2, and SangJ. Chung1,2,*

1Bionanotechnology Research Center, KRIBB 2Nanobiotechnology Division, University of Science andTechnology (UST)

Sandwich ELISA has been widely employed for diseasediagnosis and biochemical detection of target biomolecules.

Despite of its high specificity, Sandwich ELISA has beenlimited its applicability because of the low sensitivity comparedto other immunoassays. Hence, numerous approaches suchas biobarcode, immuno-PCR, and immuno-RCA have beendeveloped to improve the detection limit of sandwich-typeimmunoassays via signal amplification. Here we report a novel approach to improve the detectionlimit of immuno assay by combining ELISA with enzyme-cascading system, so called CELISA. The first example ofCELISA which consisted of trypsinogen-enterokinase systemas a cascading enzyme system was applied to detect alpha-fetoprotein (AFP), a liver cancer marker and prostate specificantigen (PSA). The CELISA system could detect PSA andAFP with 10 pM limit-of-detection. The details will bepresented. (The 102th Fall Meeting of the Korean ChemicalSociety : Jeju, 2008)

Keywords : biomolecule, ELISA, enzyme-cascading system,immuno assay

D-10Immunization with HPV16L1 VLP expressedin Lactobacillus induces systemic and mucosalneutralizing antibosies in Balb/C mice

Sun-Woo Yoon1, Il-Han Lee2, Moon-Hee Sung2,3, andHaryoung Poo1

1Bionanotechnology Research Center, KRIBB2BioLeaders Corporation3Department of Bio & Nano Chemistry, Kookmin University

The Human papillomavirus (HPV) high risk type to causecervical cancer most is HPV16. The major capsid proteinL1 is the only protein that sufficient for self-assembly ofthe virus like particles. Lactobacillus is an attractive livevaccine candidate for eliciting mucosal as well as systemicimmune responses because of its safety and intrinsicimmunogenicity. In this study, we investigated whether theexpression of HPV16 L1 protein in Lactobaciilus casei (L.casei) could affect mucosal and systemic neutralizing activity.We have constructed a lactose-inducible system using lactoseoperon promoter of Lactobaciilus casei, lacT, (LacT), andhave expressed the L1 major capsid proteins of HPV16.For immunization, BALB/c mice were administrated withequal amount of the L. casei-HPV16L1 by the oral, nasal,and vaginal routes. Anti-VLP immunoglobulin A (IgA)and IgG were also detected in serum, intestine, lung, andvagina. These antibodies effectively neutralized HPV16pseudotyped virions in an in vitro and in vivo infectivityassay. (Under preparation)

Keywords : HPV, human papillomavirus, Lactobaciilus,


mucosal immunity, neutralization activity,virus-like particle

D-11Oral administration of poly-gamma-glutamicacid improves dermatitis in NC/Nga mice, amodel of human atopic dermatitis

Tae-young Lee1, Chul-Joong Kim2, Moon-Hee Sung3, andHaryoung Poo1

1Bionanotechnology Research Center, KRIBB2National Research Laboratory, Chungnam NationalUniversity

3Department of Bio & Nanochemistry, Kookmin University

Atopic dermatitis (AD) is a chronic inflammatory skindisease characterized by pruritic and eczematous lesions,along with increased total IgE level in plasma, inflammatorycell infiltration, and the expression of T helper 2 (Th2)cytokines. NC/Nga mice kept in conventional conditionsare known to develop skin lesion resembling human AD.Poly-gamma-glutamic acid (g-PGA) is an ediblebiomaterial naturally synthesized by Bacillus subtilis(chungookjang). We examined whether high molecularweight g-PGA could prevent the development of the skinlesions in NC/Nga mice. To examine the effect of g-PGAon NC/Nga, the g-PGA was administered orally once aday for 15 weeks, starting at 6 weeks old. g-PGA remarkablychanged the immune response from type Th2 and Th1 asdetermined from cytokine level. The serum IgE and IgG1level was decreased and the expression of IgG2a wasdownregulated, whereas that of IFN-g and IL-12 wasincreased. Furthermore, histological analysis of the skinrevealed that administration of g-PGA significantlyinhibited the thickening of epidermis compared to controlmice. These results suggest that high molecular weight g-PGA is effective for immunotherapy agents for thetreatment of AD. (Under preparation)

Keywords : AD, atopic dermatitis, chronic inflammatory,g-PGA, NC/Nga, poly-gamma-glutamic acid

D-12Poly(g-glutamic acid)-chitosan nanoparticlesinduces antigen specific humoral and cellularimmunity

Kyong Soon Lee1, Moon-Hee Sung2,3, and Ha Ryoung Poo1

1Bionanotechnology Research Center, KRIBB2BioLeaders Corporation3Department of Bio & Nano Chemistry, KookminUniversity,

Nanoparticles are considered to be efficient tools forinducing potent immune responses by an Ag carrier. Inthis study, we examined the effect of Ag-carring biodegradablepoly(g-glutamic acid)(g-PGA) chitosan nanopartles on theinduction of immune responses in mice. g-PGA chitosannanoparticles strongly induced up-regulation of costimulatorymolecules, and the enhancement of T cell stimulatorycapacity in DCs. The immunization of mice with OVA-carrying nanoparticles induced Ag-specific CTL activityand Ag-specific production of IFN-g in splenocytes as well aspotent production of Ag-specific IgG Abs in serum. Theseresults suggest that Ag-carrying g-PGA chitosan nanoparticlesare capable of inducing strong cellular and humoral immuneresponses and might be potentially useful as effectivevaccine adjuvants for the therapy of infectious diseases.(Under preparation)

Keywords : g-PGA, gamma-PGA, nanomaterial,nanoparticle, nanostructure, poly(g-glutamicacid) chitosan

D-13Nobel peptides with a specific binding to the Fcdomain of immunoglobulin G

Yu-Jin Jeong2, Hyo Jin Kang2, Youngmi Lee1, Sang J.Chung1,2,*, and Bong Hyun Chung1,2,*

1Bionanotechnology Research Center, KRIBB2Nanobiotechnology Division, University of Science andTechnology (UST)

As the commercial use of antibodies is growing fast due toincreasing markets of immunosensors as well as antibodyitself for therapeutics and/or drug-targeting the purificationof antibody is becoming an industrial issue. Currently,purification of the antibody is mainly depending onaffinity-based methodologies using natural antibody-binding ligands. However, this method has drawbackssuch as requiring a harsh conditions for antibody elution,low capacity, low cost effectiveness, a limited scalability,and toxicity from leaking. As alternatives to overcome thedisadvantages of natural antibody-binding ligands such asProtein A/G, synthetic small affinity ligands have beensearched for the purification of antibody. As a part of oureffort to develop such ligands, we have established a novelmethod to discover peptides which specifically bind andrelease Immunoglobulin Gs depending in mild pH ranges.


It includes bio-synthesis, screening, and characterizationof a cyclic peptide library, consisting of general formulaCys-X9-Cys where X9 is the randomly insertednonapeptide. Employing this method, we have identifiednovel peptides for the desired purpose. In order tocharacterize their ability as affinity chromatographicmaterials, binding-releasing property of the peptides toIgGs has been fully analyzed by dot-blot assay and SPRstudy. (The 102th Fall Meeting of the Korean ChemicalSociety : Jeju, 2008)

Keywords : Immunoglobulin G, immunosensor, syntheticpeptide

D-14Label-free detection of p53 point mutation usingfield effect transistor

Sang Hee Han, Sang Kyu Kim, and Moonil Kim*


We report a new method for detecting a point mutation ofpro-apoptotic protein 53 (p53) using MOSFET. Mostmutation points of p53 are distributed in DNA bindingdomain, with the consequence that mutant p53 has adifficulty in binding DNA. Wild type human p53 andmutated human p53 R248W were employed and comparedin this study. When p53 samples applied to it's bindingDNA immobilized on gold electrode of MOSFET,different electrical properties between wild type andmutant were shown. This new method is suitable forprotein-DNA binding assay and furthermore, detectingp53 point mutation in cancer diagnosis. (The 102th FallMeeting of the Korean Chemical Society : Jeju, 2008)

Keywords : DNA, MOSFET, p53, pro-apoptotic protein

Members

Director Bong Hyun Chung ([email protected])

Principal Researcher Min-Gon Kim ([email protected])? Haryoung Poo ([email protected])Yong Beom Shin ([email protected])

Senior Researcher Sang Jeon Chung ([email protected])Chang Su Lee ([email protected])Tae Hwan Ha ([email protected])Yong Taik Lim ([email protected])Im Sik Chung ([email protected])Moon Il Kim ([email protected])Yong Won Jung ([email protected])

Post-DocHyun Kyu Park ([email protected])Joong Hyun Kim ([email protected])Sang-Im Park ([email protected])Dong Hwan Choi ([email protected])

Researcher Joo Oak Keem ([email protected])Kyoung Sook Park ([email protected])Sun Hee Yeon ([email protected]) Seok Ki Lee ([email protected])So Yeon Lee ([email protected])Young Mi Lee ([email protected])Jung Hyun Han ([email protected])Yo Han Choi ([email protected])Hyo-Jin Kang ([email protected])Sang Kyu Kim ([email protected])Young Woock Noh ([email protected])Jun Hyoung Ahn ([email protected])Jin Young Jeong ([email protected])Sun Woo Yoon ([email protected])Tae-Young Lee ([email protected])Yang Hyun Kim ([email protected])Nae-Rym Lee ([email protected])Ji-Na Kwon ([email protected])Jung Won Yun ([email protected])Tae-Han Lee ([email protected])Hyun Kyung Jeong ([email protected])Hyun Min Cho ([email protected])Sang Hee Han ([email protected])Kyong Soon Lee ([email protected])


A-78Generation and characterization of a large humanFab library

Insoo Park, Jinhong Kim, Eungsuk Lee, Youngwoo Park,and Hyo Jeong Hong

Therapeutic Antibody Research Center, KRIBB

We constructed a large (5 x 1010 recombinants) humannaïve Fab combinatorial library using the phage displaytechnology in order to isolate new high specific and highaffinity antibodies that can be used as diagnostic andtherapeutic tools. B cells were obtained from peripheralblood, bone marrow, lymph node, and spleen. We firstgenerated a light chain library, then the light chain librarywas used as vectors for the generation of the KRIBB-Fabcombinatorial library. Evaluation of the library was performedusing three different strategies: DNA sequencing of individualclones, ELISA using antibodies against tagged peptides,and selections against purified protein antigens. 100 kappachains, 100 lambda chains and 192 heavy chains wereselected and nucleotide sequences were determined. Theneach sequence was compared with the human Ig germlinesequences using IMGT's online software program(http://imgt.cines.fr). The results indicated that the librarypossesses almost all functional alleles. Analysis of solubleexpression of randomly selected clones in E.coli by ELISAindicated that more than 70% of the library clones wereexpressed as a Fab form. Selections against four differentpurified antigens allowed us to isolate a variety of high affinityantibodies (>nM ranges). (The 18th Annual InternationalConference Antibody Engineering & The 5th AnnualInternational Conference Antibody Therapeutics : SanDiego (USA), 2007)

Keywords : human Fab library, panning, phage display

A-79Isolation and characterization of humanantibodies that recognize both hL1cam andmL1cam

Insoo Park, Seulki Cho, Moonsick Jung, Jinhong Kim,Eungsuk Lee, Miwha Whang, and Hyo Jeong Hong


Monoclonal antibodies are attractive potential therapeuticagents against cancer due to their long half life, low toxicity, highaffinity and high specificity. Eight anticancer mAbs havebeen approved by the Food and Drug Administration (FDA), andmany more are in clinical development. Among these onlyone was a fully human antibody and others were derivedfrom hybridoma cells or chimeric antibodies. We were

interested in isolating fully human antibodies against L1cell adhesion molecule (L1cam or CD171) which is a 200-220kDa type I transmembrane glycoprotein of the immunoglobulin(Ig) superfamily. L1cam was initially identified as a neuralcell adhesion molecule involved in important roles in thenervous system including neural cell adhesion and migration,neurite outgrowth, fasciculation, learn and memory formation.However, recent studies have shown that L1cam is alsooverexpressed in a variety of human cancers such as ovariancarcinoma, uterine carcinoma, endometrial carcinoma,melanoma, pancreatic ductal carcinoma and cholangiocarcinoma.In addition, L1cam induces cancer cell migration andmetastasis and its overexpression is associated with badprognosis. We isolated and characterized several anti L1camantibodies that recognize both human L1cam (hL1cam)and mouse L1cam (mL1cam) from previously constructeda large, naïve human Fab combinatorial library (5 x 1010recombinants). Ab4, which showed the highest affinitywith both hL1cam (2.65 nM) and mL1cam (1.05 nM), wasselected as a lead for further development of antibody-basedcancer therapeutics. (The 18th Annual InternationalConference Antibody Engineering & The 5th AnnualInternational Conference Antibody Therapeutics : San Diego(USA), 2007)

Keywords : Ab4, cancer, hL1cam, mL1cam, monoclonalantibodies

A-80Panning strategies for screening of anti-L1CAMhuman monoclonal antibodies by phage display

Jooeun Lee1, Mi-Jin Sohn1, Moon Sik Jeong1, Eung Sueklee1, Jinhoung Kim1, Insoo Park1, Jae Myun Lee2, Joen SooShin2, and Hyo Jeong Houng1

1Therapeutic Antibody Research Center, KRIBB2Department of Microbiology Institute for Immunolgy andImmunological Diseases, Yonsei University

In the previous study, we reported that anti-L1CAM Monoclonalantibody has a potential as an anticancer agent forcholangiocarcinoma therapy. The goal of this study was tofind out whether other panning strategies generate variousantibodies compared to the conventional method. In thisstudy, we used the phage displayed Fab library from KRIBBNaïve library with a size of 5x1010. In order to generatemore various Fab antibodies, we have used four approachesof panning and screened 150 individual clones for eachpanning method. Finally, we selected 35 clones havingbinding activities to L1CAM and then analyzed theseantibodies. Their specificities and affinities were verifiedby sequence analysis, epitope specificity, binding assayagainst human L1CAM, and competitive inhibition ELISA.As a result, we found that each Fab antibody selected from


Therapeutic Antibody Research Center

each different panning method has a different characteristic.Our study suggests that the new panning strategy usingdifferent surface chemistries has a potential to search forhuman monoclonal antibodies with diverse and uniquesepuences. (The 9th International Congress on CellBiology : Seoul, 2008)

Keywords : antibody library, human monoclonal antibody,L1CAM, panning, phage display

A-81TMPRSS4 promotes invasion, migration andmetastasis of human tumor cells by facilitatingan epithelial mesenchymal transition

Heekyung Jung1, Kwang Pyo Lee1, So Jeong Park1, JiHyun Park1, Geum-Ran Hong2, Young-Kyu Park2, HyoJeong Hong1, Young Woo Park1, and Semi Kim1

1Therapeutic Antibody Research Center, KRIBB2Chonnam National University Medical School

TMPRSS4 is a novel type II transmembrane serine proteasefound at the cell surface that is highly expressed in pancreatic,colon and gastric cancer tissues. However, the biologicalfunctions of TMPRSS4 in cancer are unknown. Here weshow, using RT-PCR, that TMPRSS4 is highly elevated inlung cancer tissues compared with normal tissues and isalso broadly expressed in a variety of human cancer celllines. Knockdown of TMPRSS4 by siRNA treatment inlung and colon cancer cell lines was associated with reductionof cell invasion and cell-matrix adhesion as well as modulationof cell proliferation. Conversely, the invasiveness, motilityand adhesiveness of SW480 colon carcinoma cells weresignificantly enhanced by TMPRSS4 overexpression. Enhancedinvasion might be associated with serine proteolytic activityof TMPRSS4. Furthermore, overexpression of TMPRSS4induced loss of E-cadherin-mediated cell-cell adhesion,concomitant with the induction of SIP1/ZEB2, an E-cadherintranscriptional repressor, and led to epithelial-mesenchymaltransition events, including morphological changes, actinreorganization and upregulation of mesenchymal merkers,which were via MAPK signaling pathway. TMPRSS4-overexpressing cells also displayed markedly increasedmetastasis to the liver in nude mice upon intrasplenic injection.Taken together, these studies suggest that TMPRSS4 controlsthe invasive and metastatic potential of human cancer cellsby facilitating an epithelial-mesenchymal transition; TMPRSS4may be a potential therapeutic target for cancer treatment.(American Association for Cancer Research AnnualMeeting : San Diego (USA), 2008)

Keywords : EMT, invasion, metastasis, serine protease,TMPRSS4

A-82PAUF is a metastasis factor of human pancreaticcancer

Yangsoon Lee1, Su Jin Kim1, Sun Bok Jeon2, Jin ManKim3, and Sang Seok Koh1

1Therapeutic Antibody Research Center, KRIBB2LG Life Sciences, Ltd. 3Department of Pathology School of Medicine, ChungnamNational University

A novel pancreatic adenocarcinoma associated gene, PAUF,was identified to be over-expressed in human pancreaticcancer cells. To investigate whether and how PAUF influencestumorigenesis of pancreatic cancer, we established pancreaticcancer cell lines which modulate PAUF expression. Cellsover-expressing the gene exhibited increased growth rate,motility and invasiveness invitro, which were translatedinto enhanced metastasis invivo. On the other hand, knock-down of PAUF expression showed reduced cell growth,motility and invasiveness invitro, and reduced metastasisinvivo. Genome-wide expression analysis revealed thatCXCR4 expression was correlated with the level of PAUFexpressed in pancreatic cancer cells, which was verified byimmunohistochemical analysis of clinical tissue samples.We further demonstrated that PAUF-induced cell motilitywas inhibited by a CXCR4 antagonist. Collectively, ourresults suggest that PAUF potentiates metastasis of pancreaticcancer cells via, at least in part, CXCR4 and that PAUFcan be targeted for therapeutic intervention of humanpancreatic cancer. (The 9th International Congress on CellBiology : Seoul, 2008)

Keywords : CXCR4, metastasis, pancreatic adenocarcinoma,PAUF

A-83Therapeutic potential of a monoclonal antibodyagainst PAUF in human pancreatic cancer

Su Jin Kim, Yangsoon Lee, Eun Hye Park, Kyung-SookYoo, and Sang Seok Koh


Pancreatic cancer is an extremely malignant and lethaldisease with high mortality rate due to its late clinicalpresentations, rapid progress of remote metastasis andhigh resistance to chemo- and radiotherapy. For thatreason, development of effective and improved therapeuticagentsfor pancreatic cancer has been highly required. Werecently identified the growth factor PAUF which is over-expressed in pancreatic cancer and promotes tumor growth


and metastasis. To explore target potential of PAUF forthe treatment of pancreatic cancer, we generated humanmonoclonal antibodies (mAbs) against PAUF. The mAbsexerted inhibition of proliferation, migration and invasionof human pancreatic cancer cells in vitro. For in vivoefficacy test, a monoclonal antibody was selected based onanti-migratory activity in pancreatic cancer cells in vitroand specific antigen-binding affinity judged by BiaCoreand ELISA. Treatment of pancreatic cancer cell-xenografted mice with the PAUF-specific mAb exhibitedsignificant reduction in tumor size. Our results suggest thatthe mAb specific to PAUF is a potential therapeutic agentfor the treatment of human pancreatic cancer. (The 62th

Annual Meeting of the Korean Association of BiologicalSciences : Seoul, 2008)

Keywords : mAbs, monoclonal antibody, pancreatic cancer,PAUF

A-84CTHRC1 promotes adhesion and motility ofpancreatic cancer cells

Eun Hye Park, Yangsoon Lee, and Sang Seok Koh


Genome-wide expression analysis revealed that collagentriple helix repeat containing-1 (CTHRC1), a secretedprotein present during embryonic development, wasexpressed in many human cancers including pancreaticadenocarcinoma. We explored the biological functions ofCTHRC1 in pancreatic cancer cell lines. Pancreatic cancercells treated with recombinant CTHRC1 (rCTHRC1)exhibited increased cell spreading, adhesion and migrationin a dose-dependent manner without changes in cellproliferation. Similar results were obtained with stasblecell lines over-expressing CTHRC1. Furthermore, knock-down of CTHRC1 expression by lentiviral-shRNA reducedspreading, adhesion and migration of pancreatic cancercells in vitro. Over-expression of CTHRC1 in pancreaticcancer cells promoted formation of focal adhesion andlamellipodia and reorganization of actin cytoskeleton. Keymigration factors, such as FAK and paxillin, were activatedby CTHRC1 as judged by Western analysis. Cells over-expressing CTHRC1 increased primary tumor progressionand local invasion in xenograft mice. (The 9th InternationalCongress on Cell Biology : Seoul, 2008)

Keywords : adhesion, migration, pancreatic cancer

Members

DirectorHyo Jeong Hong ([email protected])

Principal Researcher Young Woo Park ([email protected])Sang Seok Koh ([email protected])Jin San Yoo ([email protected])

Senior Researcher Se Mi Kim ([email protected])Jeong-Ki Min ([email protected])

Post-DocIn Soo Park ([email protected])Mi-Jin Shon ([email protected])Weon Sup Lee ([email protected])Sung Woo Kim ([email protected])So Young Choi ([email protected])

Researcher Eung Seuk Lee ([email protected])Jung Whoi Lee ([email protected])Jin Hong Kim ([email protected])Moon Sik Jeong ([email protected])Hyun Ho Yoon ([email protected])Joo Eun Lee ([email protected])Tae Woo park ([email protected])Se-Joing Yim ([email protected])Ji Hyun Moon ([email protected])Seul Ki Cho ([email protected])Taeh Young kwon ([email protected])Yang Soon Lee ([email protected])Su Jin Kim ([email protected])Eun Hye Park ([email protected])Soo Jeong Park ([email protected])Kyung Sook Yoo ([email protected])Hye Jin Min ([email protected])Sang Ryeol Shim ([email protected])Mi-Ra Lee ([email protected])Hee Young Kang ([email protected])Young Soon Jang ([email protected])Joon Goo Jung ([email protected])Ji Hyun Park ([email protected])Hye Won Lee ([email protected])Jung Yu ([email protected])Eun Jung Song ([email protected])Woo Chaang Kim ([email protected])Je Won Jeon ([email protected])


C-2Sophora flavescens attenuates atherosclerosisand hyperlipidemia in LDL receptor-deficientmice

Jong-Min Han1, Sojin An1, Min-Jung Kim1, Yue-Yan Jin1,Seung-Hwa Baek1, Ji-Seon Park1, Woo Song Lee1, Ki-Hoon Park2, and Tae-Sook Jeong1

1Metabolic Syndrome Research Center, KRIBB2Division of Applied Life Science, Gyeongsang NationalUniversity

Oxidative stress has attracted intense research interestrecently as the cause of cardiovascular events as well as avariety of vascular diseases. Overproduction of reactiveoxygen species (ROS) is considered a major mechanisminvolved in the pathogenesis of endothelial dysfunction,and might serve as a common pathogenic mechanism inconditions such as atherosclerosis, hypercholesterolemia,and hypertension. We have reported the isolation of 14compounds from Sophora flavescens, which include fouractive compounds, sophoraflavanone G, kurarinone,kuraridin, and norkurarinol, on copper-mediated LDLoxidation and lipopolysaccharide (LPS)-induced nuclearfactor- B (NF- B) dependent inflammatory mediators.Therefore, we have interest in developing the potentialtherapeutic value of ROS-antioxidants as inhibitors of NF-κB. In the present study, we used a Western diet-fed LDLreceptor deficient (LDLR-/-) mice to investigate whetherthe ethylacetate soluble fraction of S. flavescens (ESF)modulates early-stage atherosclerosis. The mice were fedwith a chow diet alone (ND group), a Western dietcontaining 0.15% cholesterol alone (control group) or adiet supplemented with 0.5% ESF (wt/wt diet) for 12weeks. Surprisingly, ESF with a Western diet significantlylowered plasma LDL cholesterol, total cholesterol, andtriglyceride levels as compared to the control group. Thehistopathological findings confirmed that the ESFsuppressed the progression of the fatty changes in the liverand some atherosclerotic lesions in the aorta. Thesupplementation with the ESF significantly reduced thepresence of atherosclerotic plaque by up to 95% in theaortic sinus (oil red O-stained lesions was 389.5±118.6µm2x103 in control group versus 63.3±49.9 µm2x103 in theESF-supplemented group) and in the ascending aorta.Moreover, the mRNA and protein levels such as, VCAM-1, ICAM-1, TNF- , IL-1β, and MCP-1 were suppressed inaorta. In conclusion, our results suggestthat supplementationwith ESF is a promising product for treatment ofhyperlipidemia and atherosclerosis by inhibiting NF- Band provides additional rationale for application of anti-inflammatory therapeutic approaches for prevention ofcardiovascular disease and atherosclerosis in human.(Arteriosclerosis, Thrombosis and Vascular BiologyAnnual Conference : Atlanta (USA), 2008)

Keywords : atherosclerosis, hyperlipidemia, nuclearfactor- B , Sophora flavescens

C-3Antioxidant property of eupatilin attenuatesatherosclerosis and improves adipokine profilesin LDL receptor-deficient mice

Jong-Min Han, Sojin An, Ji-Seon Park, Seung-Hwa Baek,Yue-Yan Jin, Moon-Hee Cho, and Tae-Sook Jeong

Metabolic Syndrome Research Center, KRIBB

Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone), apharmacologically active ingredient isolated fromArtemisia princes Pamp., has been reported to haveantioxidative and anti-inflammatory activities and toinhibit the activation of NF- B. In the current study, weinvestigated anti-atherosclerotic effects of eupatilin(0.02%, wt/wt diet) in LDLr-/- mice fed with a Western dietcontaining 0.15% cholesterol for 12 weeks. Eupatilinmarkedly reduced atherogenic-lesion formation as well asplasma lipid peroxidation (20.6 ± 1.5 MDA nmol/mlversus 23.0 ± 1.3 MDA nmol/ml, p<0.01) showing itsability as an antioxidant. Gene expression analysisdemonstrated that eupatilin restrained the inflammatory statenot only in aorta by decreasing levels of chemoattractants(ICAM-1, VCAM-1, MCP-1) and cytokines (IL-1β, IL-2)but also in white adipose tissues by improving adipokineprofiles, such as increased level of adiponectin anddecreased level of TNF- , IL-6, PAI-1, resistin. Adiposetissue is a major source of inflammatory and thromboticcytokines, called adipokines, which have very closerelationship with metabolic syndrome. Antioxidant canmodulate the expression of adipokine by inhibiting NF- Bactivation. In conclusion, the antioxidant properties ofeupatilin prevent the development of early stage ofatherosclerosis and lower metabolic risk by modulation ofadipokine expression. (Arteriosclerosis, Thrombosis andVascular Biology Annual Conference : Atlanta(USA),2008)

Keywords : adipokine, anti-atherosclerosis, anti-obesity,chemoattractants, cytokines, Eupatilin

C-4The ethanolic extracts of Rhodiola rosea preventatherosclerosis and obesity in LDLr-/- mice viastimulation of lipid decomposition pathway

Sojin An, Ji-Seon Park, Jong-Min Han, Yue-Jan Jin, Seung-Hwa Baek, and Tae-Sook Jeong

Metabolic Syndrome Research Center, KRIBB

Rhodiola rosea has been used for a very long time inRussian and Chinese folk medicine. There have beenseveral reports about biological activities of Rhodiolaspecies such as adaptogenic, antihypoxia, antifatigue,


Metabolic Syndrome Research Center

antioxidant, antiarrhythmic, cardioprotection, anticancer.In the current study, we investigated anti-atheroscleroticand anti-obesity effects of the ethanolic extracts ofRhodiola rosea (ERS) in LDLr-/- mice fed with a Westerndiet containing 0.15% cholesterol. Compared with thecontrol group, Supplement of ERS (1%, wt/wt diet) causeda 38.2% reduction (P<0.01) in atherosclerotic lesionformation, along with reduction of inflammation-relatedgene expression, such as ICAM-1, VCAM-1, MCP-1,MSRA, iNOS, MMP-9, TNF-α, IL-1β and IL-2, andincrement of ABCA1 gene expression. Concomitantly,ERS remarkably reduced obesity and the size of adipocytethat go beyond simple result of weight loss, possiblycaused by stimulated lipid decomposition pathway. Theanalyses of gene expression suggest that ERS stimulatelipid decomposition and excretion process not only inwhite adipose tissues but also in liver. As a result, therewas a noticeable decline of hepatic lipid accumulation andthe concentration of plasma triglyceride. Furthermore, theresults showed that ERS increased the expression level ofthyroid hormone in brain, which increases energyexpenditure systematically. These findings indicate thatthe extracts from Rhodiola rosea might be useful materialsfor prevention of obesity and atherosclerosis. (Arteriosclerosis,Thrombosis and Vascular Biology Annual Conference: Atalanta (USA), 2008)

Keywords : anti-atnerosclerosis, anti-obesity, stimulationof lipid decomposition, Rhodiola rosea

C-5Ethanolic extracts of Brassica campestris spprapa roots prevent high-fat diet-induced obesityvia beta3-adrenergic regulation of white adipocytelipolytic activity

Jang-Il Han1, Sojin An1, Min-Jung Kim1, Ji-Seon Park1,2,Jong-Min Han1, Nam-In Baek3, Hae-Gon Chung4, Myung-Sook Choi5, Kyung-Tae Lee6, and Tae-Sook Jeong1

1Metabolic Syndrome Research Center, KRIBB2Department of Food Science & Nutrition, ChungnamNational University

3Plant Metabolism Research Center, Kyung Hee University4Gangwha Agricultural Technology Service Center5Department of Food Science & Nutrition, KyungpookNational University

6College of Pharmacy, Kyung Hee University

The influence of ethanolic extracts of Brassica campestrisspp rapa roots (EBR) on obesity was examined in ICRmice fed a high-fat diet (HFD) and in 3T3-L1 adipocytes.ICR mice were divided into four groups and fed a regulardiet (RD), a HFD, HFD supplemented with EBR (50mg/kg/day), or HFD supplemented with the fat absorption

inhibitor Xenical (10 mg/kg/day). Molecular mechanismof the anti-obese effect of EBR was investigated in 3T3-L1 adipocytes as well as in HFD-fed ICR mice. EBRinhibited lipid accumulation in vivo and 3T3-L1adipocytes. In the obese mouse model, both weight gainand epididymal fat accumulation were highly suppressedby the daily oral administration of 50 mg/kg EBR for 8weeks, while the overall amount of food intake was notaffected. EBR treatment induced the expression in whiteadipocytes of lipolysis-related genes including beta 3adrenergic receptor (β3-AR), hormone sensitive lipase(HSL), adipose triglyceride lipase (ATGL), and uncouplingprotein 2 (UCP-2). Furthermore, the activation of cAMP-dependent protein kinase (PKA), hormone-sensitive lipase(HSL), and extracellular-signal-regulated kinase (ERK)were induced in EBR treated 3T3-L1 cells. The lipolyticeffect of EBR involved β3-AR modulation, as inferredfrom the inhibition by the β3-AR antagonist propanolol.These results suggest that EBR may havepotential as asafe and effective anti-obese agent via the inhibition ofadipocyte lipid accumulation and the stimulation of β3-ARdependent lipolysis. (한국지질동맥경화학회 추계학술대회: 서울, 2008)

Keywords : Banti-obese effect, beta3-adrenegic receptor,rassica campestris spp rapa, lipolysis, whiteadipose tissue

Members Director

Young Ik Lee ([email protected])

Principal Researcher Sung Uk Kim ([email protected])Tae-Sook Jeong ([email protected])

Post-DocJong-Min Han ([email protected])Jang-Il Han ([email protected])Tae Hoon Kang ([email protected])

Researcher Young Eun Jeon ([email protected])Jin Young Kim ([email protected])Seon Su Kim ([email protected])Seung-Hwa Baek ([email protected])Sung Eun Kim ([email protected])Hua Li ([email protected])Won Sik Choi ([email protected])Ji-Seon Park ([email protected])


Division of Biointegration Research

Medical Genomics Research Center

Disease Model Research Center

Regenerative Medicine Research Center

Omics and Integration Research Center

Translational Research Center



D-18Functional and clinical evidence for NDRG2 asa candidate suppressor of liver cancer metastasis

Dong Chul Lee1, Yun Kyung Kang2, Woo Ho Kim3, InYoung Park1, Bo Hwa Sohn1, Ye Jin Jang1, Hyun AhmSohn1, Soo Jung Kim1, Kyung Chan Park1, Jong SeokLim4, Jong Young Choi5, and Young Il Yeom1

1Medical Genomics Research Center, KRIBB2Department of Pathology, Inje University Seoul PaikHospital

3Department of Pathology, Seoul National UniversityCollege of Medicine

4Department of Biological Sciences, Sookmyung Women'sUniversity

5Department of Internal Medicine, Kangnam St.Mary'sHospital

We searched for metastasis suppressor genes that arefrequently down-regulated in HCC while being negativelycorrelated with clinical parameters relevant to tumormetastasis, and identified NDRG2 as a good candidate.NDRG2 expression was found to be significantly reducedin HCC due to promoter hypermethylation. Immunohistochemicalanalysis of NDRG2 protein in 100 HCC patient tissuesindicated that NDRG2 expression loss is significantlycorrelated with aggressive tumor behaviors such as lateTNM stage, differentiation grade, portal vein thrombi,infiltrative growth pattern, nodal/distant metastasis andrecurrent tumor as well as shorter patient survival rates.Ectopically expressed NDRG2 suppressed invasion andmigration of a highly invasive cell line, SK-Hep-1, andexperimental tumor metastasis in vivo. In addition,NDRG2 could antagonize TGF-β1-mediated tumor cellinvasion by down-regulating the expression of MMP2 andlaminin 332 pathway components, with concomitantsuppression of Rho GTPase activity. These results suggestthat NDRG2 can inhibit ECM-based, Rho-driven tumorcell invasion and migration, and thereby play importantroles in suppressing tumor metastasis in HCC. (The 20thAnnual Conference of the Korean Society for Molecularand Cellular Biology & The 9th International Congresson Cell Biology : Seoul, 2008)

Keywords : HCC, metastasis, NDRG2, TGF- 1

D-19The genome-wide effect of calsequestrin 2 (Casq2)knockdown on mouse myoblast differentiation

Seong-Min Park1, Moonkyung Kang2, Sukjin Yang1,Yeon-Soo Kim2, Sunmin Ahn1, and Yeong Il Yeom1

1Medical Genomics Research Center, KRIBB,

2Indang Institute of Molecular Biology, Inje University

Calsequestirn2 (Casq2) encodes the major calcium bufferprotein of endoplasmic reticulum, and plays a role inintracellular calcium signaling by regulating the sensitivityof ryanodine receptors (Ryrs). Ryrs are known to beessential for muscle differentiation. We generated twokinds of mouse myoblast cell lines that stably expressshRNA of Casq2 or scrambled shRNA as a control. Wefound that induction of muscle differentiation resulted insignificant morphological and genetic difference betweenthe two cell types. To identify the genetic background ofthis difference, we performed DNA microarray analyseson both cells after the induction of muscle differentiationfor 0, 2, 4 and 8 days. We detected 463 genes whosemRNA levels were significantly altered by both muscledifferentiation and Casq2 knockdown. Clustering analysisbased on gene expression pattern of the 463 genes as wellas Gene Ontology and pathway analyses revealed severalsignaling pathways which appear potentially essential formuscle differentiation that are also influenced by Casq2knockdown. We provide in this presentation theexperimental evidence to support our findings inmolecular cell biological terms. (The 9th InternationalCongress on Cell Biology : Seoul, 2008)

Keywords : calsequestrin, Casq2, microarray, muscledifferentiation, Ryr

D-20Genome-wide analysis of DNA methylationpatterns in gastric cancer cells using genomictiling microarrays

Han-Chul Lee, Jong-Lyul Park, Mirang Kim, Seon-YoungKim, and Yong Sung Kim

Medical Genomics Research Center, KRIBB

Epigenetic modifications play an important role in avariety of human diseases including cancer. DNAmethylation, one of such epigenetic modifications, plays acrucial role in human carcinogenesis by silencing of tumorsuppressor genes. Thus DNA methylation profiling of awhole eukaryotic genome is essential to our understandingof its relevance for normal development and diseasesincluding cancer. Recently, the enrichment of methylatedDNA by MeDIP (methylated DNA immunoprecipitation)was introduced and its combining with the high-densityoligonucleotide array has enabled high-throughput,genome-wide analysis of DNA methylation patterns. Inthis work, we isolated methylated DNA by MeDIP fromfragmented genomic DNA of four different cell lines,SNU-005, SNU-484, SNU-601 and SNU-620. Themethylated DNA was amplified by WGA method (whole

Medical Genomics Research Center


genome amplification) and applied to Affymetrixpromoter array. The result shows that the four cell lineswere divided into two groups according to the pattern ofglobal methylation, as we expected. The methylation statusat selected loci was also confirmed by pyrosequencing.Therefore, our result suggests that genome-wide DNAmethylation profiling can be reliably conducted on thehigh-density oligonucleotide array platform. (An AACRSpecial Conference in Cancer Research : Boston (USA),2008)

Keywords : epigenetics, MeDIP, methylaton

D-21Aberrant promoter methylation profile ingastric carcinoma by pyrosequencing of samplepools

Han-Chul Lee, Jeong-Hwan Kim, Jong-Lyul Park, andYong Sung Kim

Medical Genomics ResearchCenter, KRIBB

The epigenetic abnormality plays an important role in avariety of human diseases, particularly cancer. DNAmethylation is one of the epigenetic modifications.Pyrosequencing technology is precise method for thequantification of the DNA methylation in specific targetregions. Genomic DNAs from two hundred four normaland gastric cancer tissue pairs were pooled then analyzedmethylation status of 151 cancer related genes byPyrosequencing. Genes that showed different methylationpattern in pooling method were selected then themethylation status were compared with random selectedindividually samples (n=48). 40 of 151 genes were notamplified by PCR and 57 of 111 genes were failed byPyrosequencing. Only 55 genes were passed analysis ofPyrosequencing. Six of 55 genes (ABCB1, EBF3, GAD1,MYOD1, NEUROD1 and TNFRSF25) were shown thatdifferent methylation pattern between normal and cancerDNA pools. The methylation statuses of these genes weresignificant increasing in individual gastric cancer DNAsamples (ABCB1;p<0.0001, EBF3;p=0.0034, GAD1;p<0.0001, MYOD1;p<0.0001, NEUROD1;p<0.0001 andTNFRSF25;p<0.0001). However, the difference ofmethylation according to the carcinogenesis stage was notdetected. The methylation status of these genes in gastriccancer has not been reported yet. These six genes may be apromising risk marker for gastric cancer. We need tofunctional study of these genes in gastric cancer fordevelopment diagnostic marker. Furthermore, this poolingmethod may be sensitive enough for use in associationstudies involving cancer diagnosis and cancer researchwhere a small difference in methylation status betweencases and controls is expected. (The 17th KOGO

Conference : Seoul, 2008)

Keywords : mehtylation, pooling method, pyrosequencing

D-22Copy number variations (CNVs) identified innormal Korean individuals

Tae-Wook Kang, Yeo-Jin Jeon, Hee-Jin Kim, Jeong-Hwan Kim, Jong-Lyul Park, Yong Sung Kim, and Seon-Young Kim

Medical Genomics Research Center, KRIBB

Copy number variations (CNVs) are deletions, insertions,duplications, and more complex variations ranging from 1kb to sub-microscopic sizes. Recent advances in arraytechnologies have enabled researchers to identify anumber of CNVs from normal individuals. However, theidentification of new CNVs has not yet reached saturation,and more CNVs from diverse populations remain to bediscovered. We identified 205 copy number variationregions (CNVRs) in 118 normal Korean individuals byanalyzing Affymetrix 250K Nsp whole-genome SNP data.76 of these CNVRs were novel and not present in theDatabase of Genomic Variants (DGV). To increase thespecificity of CNV detection, two algorithms, CNAT andGEMCA, were applied to the data set. Only intersectingregions from the two algorithms were identified as CNVs.Most CNVRs identified in the Korean population wererare (<1%), occurring just once among the 118individuals. When CNVs from the Korean populationwere compared with CNVs from the three HapMap ethnicgroups, African, European, and Asian; our Koreanpopulation showed the highest degree of overlap with theAsian population, as expected. However, the overlap wasless than 22%, implying that more CNVs remain to bediscovered from the Asian population as well as fromother populations. Genes in the novel CNVRs from theKorean population were enriched for genes involved insensory perception and multicellular processes. CNVs arerecently-recognized structural variations amongindividuals, and more CNVs need to be identified fromdiverse populations. Until now, CNVs from Asianpopulations have been studied less than those fromEuropean or American populations. In this regard, ourstudy of CNVs from the Korean population will contributeto the full cataloguing of structural variation amongdiverse human populations. (The 17th KOGO Conference :Seoul, 2008)

Keywords : CNV, copy number variation, database ofgenomic variants, DGV, SNP array


D-23LRRC3B, encoding a leucine-rich repeat-containingprotein, is a putative tumor suppressor gene ingastric cancer

Mirang Kim1, Hee Jin Kim1, Jeong-Hwan Kim1, Hay-RanJang1, Hwan-Mook Kim1, Seung-Moo Noh2, Kyu-SangSong2, Hyang-Sook Yoo1, and Yong Sung Kim1

1Medical Genomics Research Center, KRIBB2Chungnam National University

LRRC3B (Leucine-rich repeat-containing 3B) is anevolutionarily highly conserved leucine-rich repeat-containingprotein, but its biological significance is unknown. Usingrestriction landmark genomic scanning (RLGS) andpyrosequencing, we found that the promoter region ofLRRC3B was aberrantly methylated in gastric cancer.Gastric cancer cell lines displayed epigenetic silencing ofLRRC3B, but treatment with the DNA methylationinhibitor 5-aza-2'-deoxycytidine and/or the histonedeacetylase inhibitor trichostatin A increased LRRC3Bexpression in gastric cancer cell lines. Real-time RT-PCRanalysis of 96 paired primary gastric tumors and normaladjacent tissues showed that LRRC3B expression wasreduced in 88.5% of gastric tumors as compared to normaladjacent tissues. Pyrosequencing analysis of the promoterregion revealed that LRRC3B was significantly hypermethylatedin gastric tumors. Stable transfection of LRRC3B in SNU-601 cells, a gastric cancer cell line, inhibited anchorage-dependent and anchorage-independent colony formation,and LRRC3B expression suppressed tumorigenesis in nudemice. Microarray analysis of LRRC3B-expressing xenografttumors showed induction of immune response-relatedgenes and interferon signaling genes. Hematoxylin andeosin-stained sections of LRRC3B-expressing xenografttumors showed lymphocyte infiltration in the region. Wesuggest that LRRC3B is a putative tumor suppressor genethat is silenced in gastric cancers by epigenetic mechanismsand that LRRC3B silencing in cancer may play an importantrole in tumor escape from immune surveillance. (An AACRSpecial Conference in Cancer Research : Boston (USA), 2008)

Keywords : LRRC3B, methylation, RLGS, tumor suppressorgene

D-24Stroke web portal based on Korean orientalmedicine

Hoon Jin1, Hyun-Ho Kyung1, Sang-Uk Han1, Young-UkKim1, Sun-Mi Choi2, Ok-Sun Bang2, and Young-Joo Kim1

1Medical Genomics Research Center, KRIBB2Oriental Medicine Information Center, KIOM

According to the Korea National Statistical Office(http://www.nso.go.kr/), deaths by cerebrovascular diseasewere 30,036 (61.4 persons per 100,000 population), thesecond highest following cancer in 2006. Moreoverdiagnosis by experts was shown 13.87% while the strokeprevalence rate was 16.14%, which indicates that peoplewho are suffering from the disease may not be treatedsufficiently from medical services (3rd People HealthQuestionnaire, KNIH, 2006). As for a complementary andalternative medicine, Korean oriental medicine, such asmedical herbs, medical soup (stuff), acupuncture andmoxa treatment has been vastly utilized along with Qitherapy, aroma therapy and chiropractic. We developedthe Stroke Web Portal in order to provide the informationof self-guide, self-diagnosis, self-prevention, self-treatment and/or self-rehabilitation to those peopleincluding pre-patients, patients and their family andMedical Practicing Physicians. We collected informationfrom off-line and on-line sources including a series of folktraditional remedies, Web sites and professional journalsand textbooks. Then we extracted useful data and re-organize it. On-line sources included Korean StrokeSociety website, Korea Institute of Oriental Medicinewebsite and medcity.com. The Stroke Web Portal willprovide valuable information not only to people who arepre-patients or rehabilitation patients but also the expertswho are trying to provide the best medical services to theirpatients. (The 17th KOGO Conference : Seoul, 2008)

Keywords : oriental medicine, portal, stroke, Web site

D-25Genome deletion project in S. pombe

Dong-Uk Kim1, Jacquline Hayles2, Miyoung Nam1, SangjoHan3, Haemi Lee1, Misun Won1, Kyung-Sook Chung1,Young-Joo Jang1, Minho Lee1, Shin-Jung Choi1, Han-OhPark4 , Dongsup Kim3, Paul Nurse5, and Kwang-Lae Hoe1

1Medical Genomics Research Center, KRIBB2Cancer Research London, UK 3Department of Bio and Brain Engeering, KAIST4Bioneer Corp.5Rockefeller University, USA

The fission yeast has served as an excellent modelorganism for mechanism studies of cell cycle control,mitosis and meiosis, DNA repair and recombination, thecheckpoint controls and genome stability. Therefore,systematic generation of deletion mutants by targetedmutagenesis would accelerate the use of S. pombe forfunctional and comparative studies of eukaryotic cellprocesses. We report here that more than 96% of totalgenes of S. pombe have been deleted by using PCR-generated deletion cassettes, which were designed to


facilitate the later parallel analysis. Lowering the dosageof a single gene from two copies to one copy in diploidresults in HAPLOINSUCFFICIENCY, that is sensitized toany conditions or drug that acts on the product of thisgene. With the heterozygous deletion pool, we have setupa parallel assay system, which would be useful for agrowth fitness assay under a variety of conditions. (The3rd North American Regional Fission Yeast Meeting :Los Angeles (USA), 2008)

Keywords : deletion, essentiality, yeast

D-26Fission yeast-based screening to identify putativeHDAC inhibitors using a telomeric reporterstrain

Jiwon Ahn1, Chung-Hae Choi1, Nam Hui Yim1, Chang-MoKang3, Chun-Ho Kim3, Kyeong Lee2, Kyung-Bin Song4,Misun Won1,*, and Kyung-Sook Chung1,*

1Medical Genomics Research Center, KRIBB2Molecular Cancer Research Center, KRIBB3Laboratory of Cytogenetics and Tissue Regeneration, KIRAMS4Agriculture and Life Science, Chungnam National University

Transcriptional silencing is regulated by promoter methylationand histone modifications, such as methylation andacetylation. As a a model system to search for modifiers oftranscriptional silencing, we constructed a Schizosaccaromycespombereporter strain, KCT120a, to study transcriptionalsilencing by inserting the ura4+ gene into a telomere, oneof the heterochromatin regions. The strain KCT120a wasused to investigate compounds that are involved in themodification of transcriptional silencing. Among them,two compounds, reproducibly positive by dose-dependentassays in a screen system, inhibited the activity of histonedeacetylases and subsequently induced the acetylation ofhistone H3 and apoptotic cell death in HeLa cells. Theexpressions of gelsolin and p21waf1/cip1 were also increasedby the two compounds in a manner consistent with HDACinhibitors such as TSA. Therefore, they might also bepotent inhibitors of HDACs, making them possible anti-cancer drugs. This result suggests that a yeast cell-basedassay system for transcriptional silencing is very useful foridentifying histone deacetylase inhibitors and other agentsinvolved in the modification of chromatin remodeling.(Under preparation)

Keywords: HDAC inhibitor, heterochromatin,Schizosaccharomyces pombe, telomere, yeast-drug screen

Members

DirectorYoung Il Yeom ([email protected])

Principal Researcher Dong Soo Im ([email protected])Yong Sung Kim ([email protected])Nam Soon Kim ([email protected])Young-Joo Kim ([email protected])Kwang Lae Hoe ([email protected])Mi Sun Won ([email protected])Kyung-Sook Chung ([email protected])

Senior Researcher Soo Jung Kim ([email protected])Kyung Chan Park ([email protected])Seon-Young Kim ([email protected])Dong Wook Kim ([email protected])Cho-Rok Jung ([email protected])

Post-DocDong Chul Lee ([email protected])In Young Park ([email protected])Ye Jin Jang ([email protected])Mi Rang Kim ([email protected])Han-Chul Lee ([email protected])In Sung Song ([email protected])Seung Beom Kim ([email protected])Hoon Jin ([email protected])Eun Joo Noh ([email protected])Sun-Jung Cho ([email protected])

Researcher Bo Hwa Sohn ([email protected])Seok Jin Yang ([email protected])Hyun Ahm Sohn ([email protected])Seong-Min Park ([email protected])Jeong Joo Lee ([email protected])Tae-Woo Kim ([email protected])Jeong-Hwan Kim ([email protected])Tae-Wook Kang ([email protected])Hay-Ran Jang ([email protected])Hee Jin Kim ([email protected])Yeo-Jin Jeon ([email protected])Sung-Joon Park ([email protected])Jong-Lyul Park ([email protected])Kee Ok Haam ([email protected])So Young Jeong ([email protected])Nang Soo Oh ([email protected])Ga Hee Ha ([email protected])Young Kyu Park ([email protected])Mi Hyun Ji ([email protected])Young Uk Kim ([email protected])Mi Young Nam ([email protected])A Reum Lee ([email protected])Hye Mi Lee ([email protected])Dong Myung Kim ([email protected])Shin-Jung Choi ([email protected])Yu Jin Jung ([email protected])


Jung-Eun Kang ([email protected])Ji Won Ahn ([email protected])Chung-Hae Choi ([email protected])Jung-Hwa Lim ([email protected])Yoon-Jung Choi ([email protected])

B-26Activation of PI2-K and p38 MAPK signalpathways is required for lipopolysaccharide-induced microglial phagocytosis

SU Kim1, HN Sun1, SK Kim2, JM Kim3, DS Lee4, and DYYu1

1Disease Model Research Center, KRIBB2College of Veterinary Medicine, Chungnam National University3Department of Pathology, Chungnam National University4College of Natural Sciences, Kyungpook National University

The importance of microglial reactive oxygen species(ROS) signaling in neuroinflammatory processes has beenwell demonstrated; however, relatively little is known regardingthe related mechanisms underlying these processes. Here,we show that ROS-dependent signal pathways that governmicroglial phagocytosis are highly dependent uponNADPH oxidase (Nox) activation. Specifically, phagocytosiswas greatly reduced by both antioxidant and Nox inhibitortreatments in lipopolysaccharide (LPS)-stimulated BV-2microglia. Additionally, there was a marked reduction inintracellular ROS content. These results suggest that Noxis the main ROS source for LPS-induced microglialphagocytosis. More decisive evidence for the involvementof ROS in phagocytosis was obtained from an examinationof PI3-K and p38 MAPK signal pathway activation underreduced ROS levels. These two kinases were activated byLPS treatment and inhibited by ROS neutralization andNox inhibition. We conclude that microglial phagocytosisrequires ROS-dependent PI3-K and p38 MAPK activationand that Nox-derived ROS functions as an upstream regulatorof both PI3-K and p38 MAPK. (The 9th International Congresson Cell Biology : Seoul, 2008)

Keywords : microglial phagocytosis, nicotineamideadenosine dinucleotide phosphate oxidase,phosphatidylinositol 3-kinase, reactive oxygenspecies

B-27Antihypertensive effect of MB12066 compoundassociated with e modulation in spontaneouslyhypertensive rats

Yong-Hoon Kim, Yong-Seong Kim, Keum-Jin Yang, andChul-Ho Lee

Disease Model Research Center, KRIBB

We have assessed anti-hypertensive effect of MB12066, anatural single compound derived from plant in spontaneouslyhypertensive rats (SHR). Experimental animals wereassigned to 3 groups: group 1 was supplemented with a

Disease Model Research Center


regular chow diet, group 2 was MB12066 in diet, group 3was the same MB12066 diet for the first 4 weeks and thenfollowed with nitro-L-arginine methyl ester (L-NAME)drinking every other weeks for next 4 weeks. MB12066supplementation lowered systolic BP (SBP) levels of -lapgroup and L-NAME group by 3 weeks post treatment.However BP lowering effect of L-NAME group wasblocked and rebounded to beyond SBP level of controlgroup by additional a week treatment of L-NAME.Meantime, in order to investigate that anti-hypertensionactivity of MB12066 is related with the inhibition ofactivity of angiotensin converting enzyme (ACE) activity,lung ACE activity was determined with enzymatic assay.No difference in MB12066 and control SHR. By real-timePCR, mRNA expressions of angiotensin II receptor type1a and 1b in thoracic aorta were not significantly differentbetween control and MB12066-treated groups. Althoughthe eNOS expression levels were similar, plasma NO levelwas slightly but elevated in MB12066-treated SHR,compared to control group. From these findings, it issuggested that anti-hypertensive action of MB12066 couldbe conferred by NO-mediated vascular relaxation througheNOS activity modulation rather than eNOS expression orinhibition activity of rennin-angiotnesin system in SHR.We speculate that MB12066, as natural single compound,is beneficial for control of hypertension. (Arteriosclerosis,Thrombosis and Vascular Biology, Annual Conference: Atlanta (USA), 2008)

Keywords : endothelial nitric oxide synthase, hypertension,MB12066, nitric oxide

B-28Acceleration of K-rasG12D-driven tumor progressionby deleted mutation of Peroxiredoxin I in mouseembryonic fibroblasts

BK Lee1, SU Kim1, YH Park1, HS Kim1, SW Kang2, ZWRyoo3, and DY Yu1

1Disease Model Research Center, KRIBB 2Division of Molecular Life Sciences, Ewha Womans University3Department of Genetic Engineering, Kyungpook NationalUniversity

Peroxiredoxin (Prx) I, a recently-characterized antioxidant,is frequentlyreported to be increased in a variety of neoplasticregions however, little is known regarding the molecularmechanisms governed by the augmented Prx I levels.Thus, the current study was carried out to define the rolesof Prx I in oncogene-driven tumor progression. We firstlyestablished 3T3-like cell lines from wild type and Prx I-deficient mouse embryonic fibroblasts (MEFs) by passingfifty times and transfected with K-rasG12D overexpression

vector driven by chicken -actin promoter. K-rasG12D

overexpression resulted in induction of anchorage-independent growth and elevation of ROS content, whichwere more prominent in Prx I-deficient 3T3-like cells thanwild types. However, either Prx I overexpression or NACaddition resulted in alleviation of ROS generation andanchorage-independent colony formation in K-rasG12D/Prx I-/-

cells. Taken together these results suggest that Prx Ifunctions as a negative regulator of K-rasG12D-driventumorigenesis. (The 9th International Congress on CellBiology : Seoul, 2008)

Keywords : K-ras, peroxiredoxin I, ROS, tumor progression

Members

DirectorDae-Yeul Yu ([email protected])

Principal Researcher Byung Hwa Hyun ([email protected]) Chul-Ho Lee ([email protected])

Post-DocSun-Uk Kim ([email protected])Ying-Hao Han ([email protected])Keum-Jin Yang ([email protected])

Researcher Sang-Mi Cho ([email protected])Hye-Jun Shin ([email protected])Mi-Ra Jung ([email protected])Young-Ho Park ([email protected])Hye-Lin Ha ([email protected])Bo-Kyoung Lee ([email protected])Tae-Ho Kwon ([email protected])Dong-Hee Choi ([email protected])Min-Suk Choi ([email protected])Yong-Hoon Kim ([email protected])Ji-Sun Moon ([email protected])Jung-Ran Noh ([email protected])Gil-Tae Gang ([email protected])Keong-Suk Hwang ([email protected])Hyo-Jung Kim ([email protected])


C-12Expression patterns of protein kinase Asubunits during meiotic maturation periods inporcine oocytes

Ji-Su Kim, Yee-Sook Cho, Bong-Seok Song, Xue-LongJin, Kyung-Kwang Lee, and Deog-Bon Koo

Regenerative Medicine Research Center, KRIBB

Many extracellular ligands activate adenylyl cyclase andthen altering the intracellular concentrations of the secondmessenger, cAMP. A rise in cAMP levels affects cellsmainly by stimulating protein kinase A (PKA). In thisstudy, we examined the role of PKA in porcine embryo duringpreimplantation stages. Porcine oocytes were matured inthe NCSU-23 medium supplemented with 10% (v/v) porcinefollicular fluid, 10 ng/ml EGF, 25 µM -mercaptoethanol,0.57 mM cysteine, 10 IU/ml PMSG, 10 IU/ml hCG, 1 mMdbcAMP for 22 h and further cultured without cAMP for22 h. Then, porcine oocytes were cultured additionally inhormone free medium. After in vitro fertilization, porcineeggs were cultured in NCSU23 medium with 4 % BSA at39˚C, 5 % CO2 in air for 6d. All experiments were repeatedmore than three times. All data were analyzed by usingDuncan test of one way -ANOVA by Statistical AnalysisSystem (SAS). We found that typeI, typeII and typeCregulatory subunits (RI,RIIandC) of PKA wereexpressed in the germinal vesicle (GV) and metaphase II(MII) stages of porcine oocytes, and their expression weredynamically changed during meiosis. The expressionlevels of PKA regulatory subunits type I and type II wereevaluated by Western blot analysis by using specificantibodies such as anti-PKAc,anti-PKARI , anti-PKARII , andanti-PKARII in porcine GV and MII oocytes (n=25). Foranalyzing the effect of PKA on MPF or MAPK duringmeiosis, the activity of MPF or MAPK were evaluated byWestern blot with anti-cdc2 and anti-ERK1/2. As a result,the expressions of type I and type II regulatory subunits(RI and RII) of PKA were decreased during maturation ofoocytes, whereas the activities of MPF or MAPK wereincreased. In conclusion, our results indicate that thedynamics of PKA subunits are important during oocytematuration, which it also implicates the changes of cAMPconcentration during meiosis in porcine oocytes. (The 34thProceedings of the Annual Conference of the InternationalEmbryo Transfer Society : Denver (USA), 2008)

Keywords : meiotic maturation, oocyte, pig, protein kinaseA subunit

C-13Porcine XBP-1 is a transcription factor involvedin UPR response

Kyu-Sun Lee, Jin-Yu Zhang, Ji-Su Kim, Kyung-KwangLee, Kweon Yu, and Deog-Bon Koo*


The unfolded protein response (UPR) is an evolutionaryconserved adaptive reaction for increasing cell survivalunder endoplasmic reticulum (ER) stress. The occurrenceof chronic ER stress has been extensively described in awide variety of diseases linked to protein misfolding andaggregation. However, the genes involved in the UPRpathway of porcine have not been reported, which takepart in the response to a challenge with ER stress. Transcriptionfactor X-box-binding protein-1 (XBP-1), a key componentof the ER stress response, is required for development ofspecific lineage of cells and maintenance of cellularfunctions. In this study, we describe the cloning strategyand the functional characterization of porcine Xbp-1(pXbp-1). We analyzed the molecular characters of pXbp-1 to establish its role in response to ER stress using porcineembryonic fibroblast (PEF) cells. DTT and tunicamycin,well-known ER stressors, lead to activation of UPRmarker, IRE-1 and BiP in PEF cells. The pXbp-1 mRNAundergoes IRE-1-mediated unconventional splicing in theresponse to ER stress and produces two protein isoforms,active pXBP-1 and inactive pXBP-1. In normal condition,pXBP-1 proteins predominantly localize in cytoplasm,whereas pXBP-1 proteins translocate into nuclei by ERstress. These results demonstrate that pXbp-1 could be useas a specific UPR marker in porcine system and open newopportunities for examining the UPR in vitro developmentof porcine embryos. (The 9th International Congress onCell Biology : Seoul, 2008)

Keywords : endoplasmic reticulum stress, porcine, unfoldedprotein response, xbp-1

C-14Characterizations of seven Drosophila insulin-like peptide genes

Ae-Kyeong Kim, Kyu-Sun Lee, and Kweon Yu*


Insulin/insulin-like growth factor signaling (IIS) is anevolutionarily conserved neuroendocirne signal transductionpathway which regulates various physiological processes.IIS affects growth, development, energy homeostasis,stress resistance, reproduction, and lifespan in animals.Drosophila insulin signaling studies provided valuableinsights to understand mechanisms of IIS in other biologicalsystems. In Drosophila, seven insulin-like peptides (Dilps)genes were identified in the fly genome. Although overallfunctions of Dilps were investigated, roles of each Dilp are

Regenerative Medicine Research Center


not well understood. Here, we examined tissue- and stage-specific expression of seven Dilps and analyzed theirfunctions. Dilp1, 2, 3, 5 and 7 transcripts were predominantlyexpressed in CNS while Dilp3 and Dilp6 were expressednot only in CNS but also in gut and fat body, respectively.Expression level of Dilp4 was very low. Over-expressionof seven Dilps in eyes showed different growth phenotypes.These results indicate that Dilp genes may have partlyoverlapped but different functions in various physiologicalprocesses. (The 9th International Congress on Cell Biology: Seoul, 2008)

Keywords : Drosophila, Drosophila insulin-like peptide,growth, insulin/IGF signaling

C-15Isolation of differentially expressed genes inrosette-enriched neuroectodermal spheres derivedfrom human embryonic stem cells

Hyo-Won Han1, Janghwan Kim1, Jung-Il Chae1, Sun-MiWoo1, Mi-Young Son1, Seong-Ju Moon2, YeeSook Cho1,Yong-Kook Kang1, and Deog-Bon Koo1

1Regenerative Medicine Research Center, KRIBB2Department of Animal Science, Chonnam National University

Multipotent neural stem cells (NSCs) isolated from embryonicand adult CNS can produce neural cells such as neurons,astrocytes and oligodendrocytes. Therapeutic potential ofNSCs in neurological disorders has been largely demonstrated,however, the human CNS tissue for isolating NSCs islimited and in vitro expansion of highly potent NSCs isgenerally restricted. Recent studies showed that humanembryonic stem cells (hESCs) are the promising cellsource of self-renewing, multipotent NSCs. HESC-derivedNSCs form unique structure called neural rosette, whichcan not be found in neurospheres, cluster of undifferentiatedNSCs derived from embryonic or adult brain. HESC-derived neural rosettes resemble the developing neuraltube in early embryonic development. During hESCcultivation, we isolated hES cell line acquired an additionalchromosome 12 (hESC/Chr12) which has no significantdifference in terms of morphology and hESC-markerexpression. In this study, we questioned whether theneural differentiation, especially the formation of rosette-enriched neuroectodermal spheres (NESs), is affected byacquisition of extra chromosomes in hESCs using 2independently isolated hES cell lines, CHA-hES3 and H9.We observed that hESC/Chr12-derived NESs displayedlarge, clear rosette structure compared to NESs fromnormal hESCs. During NESs cultivation, rosette formationwas gradually reduced in normal hESC-derived NESs,however, rosette structure was well maintained in

hESC/Chr12-derived NESs. To determine the molecularbasis for these observations related to neural rosettes, weused DNA microarray to analyze gene expression patternsof hESC/Chr12-derived NESs compared with NESs fromnormal hESCs. We identified 1047 genes which were up-regulated in hESC/Chr12-derived NESs compared with genesfrom normal hESC-derived NESs by Kyoto Encyclopediaof Genes and Genomes (KEGG) pathway analysis. Amongthose, 22 neuroactive ligand-receptor interaction, 16 Focaladhesions, 8 ECM-receptor interaction-related genes had asignificant fold-change. Specifically, 7 genes includingLGR5, WNT5B, FOXN4, PPFIA2, BICD1, FBXL14, and oneunknown were located in chromosome 12 and differentiallyup-regulated during EB to NES differentiation. Furthercharacterization of these genes may provide important cluesto the molecular mechanisms involved in the regulation ofNSC expansion and maintenance, and embryonic neuraldevelopment. (6th Annual Meeting of the InternationalSociety for Stem Cell Research : Philadelphia (USA), 2008)

Keywords : DNA microarray, human embryonic stemcells, neuroectodermal spheres, NSC, rosette

Members

DirectorDeog-Bon Koo ([email protected])

Principal Researcher Kyung-Kwang Lee ([email protected])Kweon Yu ([email protected])Yong-Kook Kang ([email protected])Yee-Sook Cho ([email protected])

Senior Researcher Janghwan Kim ([email protected])Jeong Woong Lee ([email protected])Kyu-Sun Lee ([email protected])Jung Sun Park ([email protected])Mi-Young Son ([email protected])Sun-Mi Woo ([email protected])

Post-DocJung-Il Chae ([email protected])Seung Hyun Hong ([email protected])

Researcher Ji-Su Kim ([email protected])Bong-Seok Song ([email protected])Jin-Yu Zhang ([email protected])Young Sun Jeong ([email protected])Sun Hwa Cho ([email protected])Ae-Kyeong Kim ([email protected])Bong Su Kim ([email protected]) Hyo-Won Han Min Jeong Kim ([email protected])Dong Min Jang ([email protected])Mira Chang ([email protected])Seungeun Yeo ([email protected])


A-65Reconstruction of sulfur metabolism pathwayin Hansenula polymorpha based on transcriptomeand metabolite analysis

Min Jeong Sohn1,2,4, Vera M. Ubiyvovk3, Doo-Byoung Oh1,Ohsuk Kwon1, Sang Yup Lee2, Anderi A Sibirny3, andHyun Ah Kang1,4,*

1Omics and Integration Research Center, KRIBB2Korea Advanced Institute of Science and Technology (KAIST)3Institute of Cell Biology, Ukraine4Department of Life Science, Chung-Ang University

Biosynthesis of sulfur-containing amino acids and glutathione(GSH) has been studied extensively at the genetic, enzymaticand regulatory levels in various microorganisms becauseof interest in the gene regulation, the complexity of thepathway and the essential roles of sulfur compounds forseveral important metabolisms. The construction of sulfurpathway in the thermotolerant methylotrophic yeastHansenula polymorpha was made by the combined analysisof amino acid sequence alignment and transcriptomeprofile data. These analyses led to the proposal that thesulfur accumulation pathway of H. polymorpha is rathersimilar to that of the fission yeast Schizosaccharomycespombe. The microarray data obtained under the conditionof cadmium exposure and sulfur deficiency stronglyindicate that the main pathway from sulfide to methioninein H. polymorpha is through homocysteine mediated byMET6 protein and through cysteine and cystathionine, thedirect transsulfuration reaction, which are mediated by thereaction of CYSD and STR2 proteins. This is good agreementwith the observation that the growth of H. polymorphamet4 mutant (hpmet4) was dependent on the supplementationof GSH or cysteine, contrary to the methionine auxotrophicphenotype of Saccharomyces cerevisiae met4 mutant.Furthermore, the GSH synthesis analysis using 35S-methionineand 35S-cysteine labeling of the wild type, hpmet4, and H.polymorpha gsh2 mutant strains strongly supports the notionthat H. polymorpha might lack the reverse transsulfurationpathway and thus can not use methionine as the sole sulfursource, differently from S. cerevisiae. Overall, our datashowed that the sulfur metabolism pathway and its regulationin H. polymorpha retains a similar design but evolves witha significant diversity in the nature of the components andthe regulation mechanism, distinctive among yeast species.(International Congress on Yeasts : Kyiv (Ukraine), 2008)

Keywords : Hansenula polymorpha, methylotrophic yeast,sulfur metabolism, transcriptome

A-66Analysis of the unfolded protein response

pathway in the methylotrophic yeast Hansenulapolymorpha

Hye-Yun Moon1,2,3, Seon Ah Cheon1, Doo-Byoung Oh2,Ohsuk Kwon2, Jeong-Yoon Kim3, MO Agaphonov4, andHyun Ah Kang1,*

1Department of Life Science, Chung-Ang University2Omics and Integration Research Center, KRIBB3Department of Microbiology, Chungnam National University4Institute of Experimental Cardiology, Cardiology ResearchCenter, Russia

Accumulation of misfolded proteins in the ER (endoplasmicreticulum) activates an intracellular signaling pathway,UPR (unfolded protein response), which brings aboutmultiple cellular protective events for proper protein foldingand misfolded protein degradation. In the Saccharomycescerevisiae, the transmembrane protein Ire1p signals ERstress by removing an intron of the precursor HAC1mRNA. The spliced HAC1 mRNA is then translated to anactive transcription factor, which up-regulates UPR genes.We have investigated the global changes of geneexpression induced by UPR in the methylotrophic yeastHansenula polymorpha, a promising host for theproduction of recombinant proteins to identify the UPRtarget genes under the control of HAC1 and IRE1 in H.polymorpha. (International Congress on Yeasts : Kyiv(Ukraine), 2008)

Keywords : Hansenula polymorpha, methylotrophic yeast,transcriptome, unfolded protein response,UPR

A-67Functional characterization of Yarrowialipolitica homologues to Saccaromyces cerevisiaeMNN4p and MNN6p.

Jeong-Nam Park1, Yunkyoung Song2, Seon Ah Cheon1,Ohsuk Kwon1, Doo-Byoung Oh1, Jeong-Yoon Kim2, andHyun Ah Kang1,3*

1Omics and Integration Research Center, KRIBB2School of Bioscience and Biotechnology, ChungnamNational University

3Department of Life Science, Chung-Ang University

The non-conventional yeast Yarrowia lipolytica has beenutilized in many industrial areas, and recently developedas a promising host system for production of recombinantproteins for its high secretion capacity. Our previouslystudy showed that N-linked glycans of Y. lipolytica werecomposed of major neutral sugars and minor acidic sugars.

Omics and Integration Research Center


The neutral sugars of N-linked glycans are high-mannoseoligosaccharides, mainly Man7-12GlcNAc2, whereas theacidic sugars of N-linked glycans are consisted ofmonomannosylphophorylated sugars. In the conventionalyeast Saccharomyces cerevisiae, both Mnn4p and Mnn6pwere reported to participate in mannosylphosphorylationof N-linked or O-linked glycans on secretory proteins. Byhomology search of Y. lipolytica genome data base, one Y.lipolytica homologue (YlMpo1 ) to ScMnn4p and four Y.lipolytica homologues (YlMnn6a, 6b, 6c, and 6d) toScMnn6p were found. Deletion mutant strains (Ylmpo1 ,Ylmnn6a , Ylmnn6b , Ylmnn6c , and Ylmnn6d ) of eachY. lipolytica homologue were constructed and analyzed for thestructure of N-linked glycans from cell wall mannoproteins(CWP) by using HPLC and MALDI-TOF. Only theYlmpo1 mutant showed the apparent disappearance ofacidic sugar moieties in N-linked glycans derived from thesecretory recombinant EGI protein and the CWPs.Furthermore, alcian blue staining also showed a significantdecrease of blue color intensity in the Ylmpo1 strain,supporting the loss of cell wall mannosylphosphate by theinactivation of YlMPO1. These results strongly supportedthat YlMpo1 plays an essential role in mannosylphosphorylationof the N-linked glycan moiety on secretory or cell wallglycoproteins in Y. lipolytica. (제6회 당전이 효소 국제심포지엄 (GlycoT) : Atlanta(USA), 2008 )

Keywords : glycoprotein, mannosylphosphorylation, N-linked glycan, Yarrowia lipolytica

A-68Global gene expression profiles of the capnophilicrumen bacterium Mannheimia succiniciproducensgrown under anaerobic conditions

Jong Moon Shin1, Wonseok Jung1, Youngryul Jung1, Doo-Byoung Oh1, Hyun Ah Kang1, Sang Yup Lee2, and OhsukKwon1

1Omics and Integration Research Center, KRIBB2Department of Chemical and Biomolecular Engineering,KAIST

Mannheimia succiniciproducensis a capnophilic Gram-negative bacterium isolated from bovine rumen and able toproduce a large amount of succinic acid under anaerobicgrowth conditions. In this study, we investigated thegenome-wide response of M. succiniciproducens tooxygen availability by using a whole genome DNAmicroarray. When the transcriptome of anaerobicallycultured cells was compared to that of aerobically culturedcells, it was found that 15% of the genes encoded in thegenome were up-regulated and 13% of the genes weredown-regulated by at least two-fold during anaerobicgrowth. About 41% of the up-regulated and 23% of the

down-regulated genes encoded proteins of hypothetical orunknown functions. The majority of up-regulated genescan be functionally grouped into those responsible foramino acid transport and metabolism (8%), energyproduction and conversion(7%), and carbohydratetransport and metabolism (6%). In contrast, the majority ofdown-regulated genes can be categorized into thoseresponsible for translation (22%), energy production andconversion (10%), and amino acid transport andmetabolism (7%). When the genome sequence of M.succiniciproducens was searched for the putative targetgenes for the ArcA response regulator and FNR transcriptionalregulator by using the positional weight matrices, about160 and 70 genes were identified as the putative targets forArcA and FNR, respectively. Interestingly, one third ofthese putative targets were differentially expressed duringanaerobic growth. Our transcriptome profiling and insilico prediction data strongly suggest that the ArcB/Atwo-component signal transduction system and FNRwhich are highly conserved among various bacterial speciesalso play pivotal roles in redox responsive transcriptionalregulation in M. succiniciproducens. (American SocietyFor Microbiology, 108TH General Meeting : Boston (USA),2008)

Keywords : anaerobiosis, global gene expression,Mannheimia succiniciproducens, transcriptomeprofiling

A-69Genome-wide transcriptional responses ofdifferent Escherichia coli strains to recombinantprotein overproduction

Min Jee Kim, Su Jeong Park, Doo-Byoung Oh, Hyun AhKang, and Ohsuk Kwon

Omics and Integration Research Center, KRIBB

The Gram-negative bacterium Escherichia coli is one ofthe most widely used hosts for heterologous proteinexpression and perhaps the best characterized organism forgenetic and molecular biology studies. In this study, weinvestigated the global transcriptome profiles of fourcommonly used E. coli strains, JM10 (DE3), BL21(DE3),C41(DE3), and C43(DE3), in response to overproductionof heterologous cytosolic or secretory proteins. When asecretory protein was overexpressed in JM109(DE3) orBL21(DE3), between 120 and 150 genes in each strainwere either up- or down-regulated by at least two-fold.However, in cases of cytosolic protein overexpression, thenumbers of up- or down-regulated genes were between170 and 200. Interestingly, the numbers of differentlyexpressed genes in both C41(DE3) and C43(DE3) strainswere less than half compared to those observed in


JM109(DE3) or BL21(DE3). Even though more than 20%of the differently expressed genes in each case were foundto encode proteins of hypothetical or unknown functions,the majority of up-regulated genes upon recombinantprotein overexpression were functionally grouped intothose responsible for translation and post-translationalmodification, and cell processes. In contrast, the majorityof down-regulated genes were functionally categorizedinto transport and binding proteins, and amino acidbiosynthesis and metabolism. It is noteworthy that variousstress responsive genes, such as heat shock and phageshock were commonly induced irregardless of the type ofoverexpressed proteins. However, the secretory proteinoverproduction specifically induced genes involved in theenvelop stress response. Since our transcriptome profilingdata also include various target or strain specific genes, itwill provide useful information to understand and todevelop E. coli as an efficient cell factory for recombinantprotein production. (American Society For Microbiology,108th General Meeting : Boston (USA), 2008)

Keywords : Escherichia coli, global gene expression,recombinant protein overproduction, transcriptomeprofiling

A-70

Characterization of the YgiXY two-componentsignal transduction system of the capnophilicrumen bacterium Mannheimia succiniciproducens

Eun-Gyeong Lee1, Wonseok Jung1, Jong Moon Shin1, Doo-Byoung Oh1, Hyun Ah Kang1, Sang Yup Lee2, and OhsukKwon1

1Omics and Integration Research Center, KRIBB2Department of Chemical and Biomolecular Engineering,KAIST

Two-component signal transduction systems, whichconsist of a membrane bound sensor kinase and a responseregulator, are highly conserved in nature and mediateadaptive responses to a variety of environmental changes.In this study, we identified and characterized the YgiXYtwo-component signal transduction system of thecapnophilic Gram-negative bacterium Mannheimiasucciniciproducens isolated from bovine rumen. Thisbacterium gains increasing attentions for its ability toproduce a large quantity of succinic acid under anaerobicgrowth conditions. According to its recently determinedfull genome sequence, five putative two-component signaltransduction systems exist in M. succiniciproducens. TheygiX and ygiY genes encode, respectively, proteins havingabout 70% and 40% of amino acid sequence homologywith QseB response regulator and QseC sensor kinase of

the Qse two-component system involved in quorum sensingin other bacteria. An error in the previously reportedsequence of ygiY gene has been found, thus increasing itsopen reading frame. The purified N-terminally truncatedYgiY which was deleted for its transmembrane domainwas able to autophosphorylate and transphosphorylateYgiX, demonstrating that these two proteins are a functionalsensor kinase and a response regulator, respectively. In anattempt to identify the target operons regulated by Ygisystem, we investigated the genome-wide transcriptomeprofiles of M. succiniciproducens in response tooverexpression of YgiX response regulator by using a wholegenome DNA microarray. The up- or down-regulatedgenes by at least two-fold upon overexpression of YgiXwere chosen as the putative targets and further analyzed byelectrophoretic mobility shift assay. (American SocietyFor Microbiology, 108th General Meeting : Boston(USA), 2008)

Keywords : global gene expression, Mannheimiasucciniciproducens, transcriptome profiling,two-component signal transduction system

A-71Tomato chromosome 2: BAC assemble andgene annotation

Jungeun Kim1,2, Sung-Hwan Jo1,2, Bo-Ra Kang1, Bong-Woo Lee1, Doil Choi3, and Cheol-Goo Hur1

1Omics and Integration Research Center, KRIBB2University of Science and Technology3Department of Plant Sciences, Seoul National University

As a member of International tomato genome project, weassigned 26 Mb of euchromatin in the long arm of chromosome2. From 45 contigs, 3,156 pseudo genes are predictedusing FGeneSH which is trained with tomato mRNA,EST, proteins. 1,285 consensus and 1,858 singletons aremapped using sim4 with criteria above 90% identity andabove 90% coverage. 71 tRNAs are predicted using tRNA-SE. On average, a gene occurs every 1.95 kb, is about 3 kb inlength (from start to stop codon) and contains 5.3 exons.The predicted proteins are annotated by similarity searchagainst Arabidopsis and Uniprot proteins. 2,228 (70.59 %)and 2,287 (72.47 %) of proteins are similar to those ofArabidopsis and Uniprot, respectively. Classification byfunctional analysis comparing to Arabidopsis proteinshows that most major proteins (6.24 %) are involved inmetabolism function and 5.99 % proteins are involved incellular process. The physical map based on seed BACcoordinates related to genetic map and related coordinateof extended BACs. On the Website, we represent BACcontigs, gene structure, annotation results and otherbioinformatic results (http://sol.kribb.re.kr/tomatogenome/


index.php). We also analyzed and represented other chromosome'sequences downloaded from SGN homepage. Our websiteis useful to analyze the sequencing status and to understandfeature of each chromosome. (SOL 2008 SequencingMeeting : Cologne (Germany), 2008 )

Keywords : BAC assemble, gene annotation, gene prediction

Members

DirectorCheol-Goo Hur ([email protected])

Principal Researcher O-Suk Kwon ([email protected])Sang Ki Rhee ([email protected])Myung Kyu Lee ([email protected])Ki-Sun Kwon ([email protected])Sung Sup Park ([email protected])In Sun Chu ([email protected])

Senior Researcher Doo Byeong Oh ([email protected])

Post-DocHyun-Seo Jang ([email protected])Tae Woo Ryu ([email protected])Surisa Suwannarangsee ([email protected])Jeong-Nam Park ([email protected])Sung-Kyu Ju ([email protected])Chae Young Hwang ([email protected])Seung-Min Lee ([email protected])So Hee Dho ([email protected])Keun Woo Lee ([email protected])

Researcher Jun-Kyoung Choe ([email protected])Hyun-Jin Kim ([email protected])Jeong Su Oh ([email protected])Seung-Won Lee ([email protected])Won Young Jung ([email protected])Hyo Jung Paik ([email protected])Ji-Man Hong ([email protected])Bong-Woo Lee ([email protected])Jae Pil Choi ([email protected])Bo-Ra Kang ([email protected])Mi Jeong Byun ([email protected])Mi-Ryoung Kim ([email protected])Jung Eun Kim ([email protected])Sung-Hwan Jo ([email protected])Sang-Mi Kim ([email protected])Min Jee Kim ([email protected])Min Jung Sohn ([email protected])Jong Moon Shin ([email protected])Yun Mi Lee ([email protected])Jung Mi Lee ([email protected])Yu Jin Kim ([email protected])Gyeong Jin Lee ([email protected])Eun Gyeong Lee ([email protected])Mun Kyeong Min ([email protected])Eun Mi Jung ([email protected])Hye-Yun Moon ([email protected])Eun-Ah Cho ([email protected])Hyun Kung Song ([email protected])Young Lang Lee ([email protected])Jung-I Choi ([email protected])Hye Lim Cho ([email protected])Jong Seong Ha ([email protected])


A-72Comparative proteomic analysis of peripheralblood mononuclear cells from atopic dermatitispatients and healthy donors

Won Kon Kim1, Hyun Ju Cho1, Su In Ryu1, Hyang-RanHwang1, Do-Hyung Kim1, Hye Young Ryu2, Jin WoongChung3, Byoung Chul Park1, Kwang-Hee Bae1, and SangChul Lee1

1Translational Research Center, KRIBB2Department of Pediatics, SUN General Hospital3Stem Cell Research Center, KRIBB

Atopic dermatitis (AD) is a chronic inflammatory skindisease that induces change in various inflammatory cells.The prevalence rate of AD is as high as 18% and issteadily rising, especially in developed countries. However,the pathophysiology of AD is poorly understood. Toidentify the genes involved in AD pathogenesis, weconducted the comparative proteomic analysis of proteinexpression in peripheral blood mononuclear cells isolatedfrom AD patients and healthy donors. The significantquantitative changes were shown in fourteen proteinsincluding vinculin, RAB11B, and FLNA protein. Amongthem, -SNAP and FLNA (Filamin A) prominently decreasedin AD patients compared to controls, which is furtherconfirmed by RT-PCR or western blot analysis. Thesecomparative proteome data may provide a valuable cluefor the understanding of AD pathogenesis, and severaldifferentially regulated proteins can be used as biomarkersfor diagnosis and as target proteins for new drug development.(KHUPO 8th Annual International Proteomics Conference: Seoul, 2008)

Keywords : atopic dermatitis, biromarkers, peripheralblood mononuclear cells, proteomics

A-73Identification of proteins involved in hepatocellularcarcinogenesis using HBx transgenic mouse

Sun Young Kim1,2, Phil Young Lee1, Sang Won Kang2,Dae Yeol Yu1, Kwang Hee Bae1,*, and Sang Chul Lee1,*

1Translational Research Center, KRIBB2Department of Life Sciences and Center for Cell Signalingand Drug Discovery, Ewha Womans University

Chronic infection of Hepatitis B virus (HBV) is one of themajor causes of hepatocellular carcinoma (HCC) in theworld. Hepatitis B virus X protein (HBx) is suspected tobe involved in hepatocarcinogenesis, although its oncogenicrole still remains controversial. HBx is a multifunctionalregulator that modulates transcription, signal transduction,

cell cycle processes, protein degradation pathway andapoptosis. Recently, the Disease Model Research Centerhas generated transgenic mice expressing HBx (HBxHb).It has been characterized as a model for steatosis andhepatic cancer. Aim of our study is to profile the keyproteins expressed in HBx transgenic mouse liver. Theliver tissues were processed for protein extraction andwere analyzed by 2-dimensional gel electrophoresis. Spotsshowing quantitative changes in the HBx transgenicmouse liver were identified by Autospec-Ultima OA TOF(Micromass) or Synapt High definition mass spectrometer(Waters), and validated by real-time PCR and westernblot. Our analysis revealed that 25 proteins are differentiallyexpressed in HBx transgenic mouse during dysplasia, and27 proteins were identified during hepatocellular adenoma(HCA). Through this approach, a more detailed understandingabout target discovery for liver cancer will be possible. Weare further probing the probability of these proteins to beused as potential cancer biomarkers. (The 8th AnnualInternational Proteomics Conference : Jeju, 2008)

Keywords : Dysplasia, HBx, hepatocellular adenoma,hepatocellular carcinoma, proteomics

A-74

Role of autophagy in oxidative stress-inducedcytotoxicity in HT22 cells

Hansoo Kim1,2, Jinsun Choi1, Joohyun Ryu1, Sung GooPark1, Kwang Hee Bae1, Sunghyun Kang1, SeongmanKang2, Byung Chul Park1, and Do Hee Lee1,*

1Translational Research Center, KRIBB2School of Life Science and Biotechnology, Korea University

Glutamate-induced oxidative stress leads to neuronal celldeath, which is often implicated in several neurodegenerativediseases. During the cell death, HT22 cells exhibit both theapoptotic and non-apoptotic features. Recently, evidenceis accumulating that macroautophagy, an intracellular bulkdegradation process, plays a role in the oxidative stressinduced cell death process. These findings led us tospeculate that the autophagic pathway also be involved inthe glutamate-induced HT22 cell death. In the presentstudy, we investigated the role of autophagy during HT22cell death. First, we observed an activation of LC3, anautophagic effector protein, during HT22 cell death. Inaddition, 3-MA, an autophagy inhibitor, effectively suppressedHT22 cell death. Moreover, activation of ERK1/2 wasreduced by 3-MA treatment. Previously we reported thatseveral molecular chaperones including hsp60 underwentsubstantial changes during HT22 cell death. Interestingly,siRNA ablation of hsp60 affected LC3 activation as wellas cell viability. Taken together, our results suggest that

Translational Research Center


glutamate-induced HT22 cell death involves autophagicpathway and hsp60 likely plays a role in this process. (The9th International Congress on Cell Biology & The 20th

Annual Conference of the Korean Society for Molecularand Cellular Biology : Seoul, 2008)

Keywords : autophagy, Hsp60, HT22, oxidative stress

A-75A proteome profiling of differentiating humanembryonic neural stem cells

Hansoo Kim1,2, Joohyun Ryu1, Sung Goo Park1, KwangHee Bea1, Sunghyun Kang1, Seongman Kang2, Ki HwanLee3, Yong Sung Lee3, Byung Chul Park1, and Do HeeLee1,*

1Translational Research Center, KRIBB2School of Life Science and Biotechnology, Korea University, 3Departmen tof Biochemistry, College of Medicine,Hanyang University

Human embryonic stem cells (hESCs) have the ability torenew themselves and differentiate into multiple cell types.To study the changes in proteome pattern associated withneural differentiation and identify individual up- or down-regulated proteins which may function in this process, weprofiled proteome derived from differentiating human F6embryonic neural stem cell line. To this end, we collecteddifferentiating F6 cells at different time points (3 daysinterval, 0-9 day) and separated soluble proteins (200-350g) by 2-DE (pI4-7) and then identified proteins showingdifferential expression pattern by MALDI-TOF MS. Usingthe Phoretix¢‚ 2D gel analysis software, the images wereanalyzed and compared. During the course of neuraldifferentiation, we observed no substantial change inoverall proteome pattern and as a re sult were able toidentify ~30 individual proteins only. Interestingly, mostof the proteins showing differential expression patternwere down-regulated ones. We also compared our proteomeresults with the previous microarray data obtained withdifferentiating mouse neural ESCs and found certaindiscrepancy in data. For example, peroxiredoxin 6 andannexin A5 which were found to be up-regulated in themicroarray analysis, were actually down-regulated in theprotein level. In addition, enolase 1 and Hsp60 did notshow any significant change in the protein level, whilethey were clearly down-regulated in mRNA level. Bycontrast, PA28 alpha subunit for proteasome activator andtriosephosphate isomerase were donw-regulated both inmRNA and protein levels. We also compared our resultswith the previous reports on the proteome changesassociated with differentiating neural ESCs and foundsome similarity. The neural differentiation process, or atleast the proteome changes associated with this process,

for each hESC might differ substantially from other neuralcell lines and more rigorous studies may be necessary tofurther elaborate the molecular event occurred during thedifferentiation of each hESCs. (KHUPO 8th AnnualInternational Proteomics Conference : Jeju, 2008)

Keywords : 2DE, embryonic stem cell, stem cell

A-76Comparative proteome analysis of 3T3-L1 celldifferentiation into adipocytes

Ji Young Choi1,2, Kwang Hee, Bae1, Do Hee Lee1, SungGoo Park1, Byoung Chul Park1, Jong Kil Lee2, and SungHyun Kang1,*

1Translational Research Center, KRIBB2College of Pharmacy, Chungbuk National University

Adipocytes are central players in energy metabolism andthe obesity epidemic. 3T3-L1 is a derived from and widelyused in the studies on adipogenesis. To identified adipogenesisrelated-proteins, we differentiated 3T3-L1 cells into adipocytesand harvested cells at different time points (2days interval,0-14day). Using two-dimensional gel electrophoresis,high-throughput image analysis, and candidate pickingcombined with liquid chromatography mass spectrometry(LC-MS/MS), proteins which showed different levels ofexpression were identified. Our studies may provide usefulclues to understanding the molecular mechanism ofAdipogenesis. (The 9th International Congress on CellBiology & The 20th Annual Conference of the KoreanSociety for Molecular and Cellular Biology : Seoul,2008)

Keywords : 3T3-L1, adipocyte, LC-MS/MS

A-77

Role of Hsp70 Co-chaperones in the degradationof neurodegenerative proteins

Jinsun Choi, Hansoo Kim, Sung Goo Park, Kwang Hee Bae,Sung hyun Kang, Byung Chul Park, and Do Hee Lee1,*

Translational Research Center, KRIBB

Recently, the autophagy as well as the ubiquitin-proteasomesystem has been shown to play an important role in thedegradation of misfolded proteins implicated inneurodegenerative diseases. We previously demonstratedthat co-chaperone CHIP (Carboxyl terminus of Hsc70-Interacting Protein) is associated with mutant SOD1


proteins and promotes their degradation via catalyzing theubiquitylation of mutant SOD1. In this study, we provideevidence that HspBP1, another co-chaperone of Hsp70,increases the degradation of misfolded proteins linked toneurodegenerative diseases including mutant SOD1apparently by activating autophagic process. HspBP1over-expression stimulates the formation LC3 , a keymolecule involved in macroautophagy, whereas CHIPdoes not. By contrast, reducing the cellular level ofHspBP1 by RNAi causes inhibition of LC3 formation aswell as the SOD1 degradation. Interestingly, knock-downof CHIP leads to the activation of authophagy, suggestingthat macroautophagy is induced due to the proteasomalimpairment caused by the defect of CHIP function. Basedon these results, we conclude that CHIP is the Hsp70 co-chaperone promoting the ubiqutin-proteasome system forthe degradation of mutant SOD1 while HspBP1 is the co-chaperone to facilitate the macroautophagy system for thedegradation of mutant SOD1. (The 9th InternationalCongress on Cell Biology & The 20th Annual Conferenceof the Korean Society for Molecular and Cellular Biology: Seoul, 2008)

Keywords : CHIP, HspBP1, macroautophagy, SOD1,ubiquin-proteasome

Members

Director Byung Chul Park ([email protected])

Principal Researcher Sang Chul Lee ([email protected])Seong Goo Park ([email protected])Seung Jun Kim ([email protected])Gwang Hui Bae ([email protected])Seung Uk Ji ([email protected])

Senior Researcher Seong Hyeon Gang ([email protected])Ui Jeon U ([email protected])Jae Ran Lee ([email protected])

Post-Doc Jae Eun Park (jepack@hanmail,net)Mi Jang ([email protected])

Researcher Jin Sun Chai ([email protected])Sun Young Lee ([email protected])Pil Young Lee ([email protected])Young Ju Jeon ([email protected])Ju Hyeon Yu ([email protected])Ji Young Choe ([email protected])Han Su Kim ([email protected])Ji Young Kim ([email protected])Ha Na Lee ([email protected])Young Ji Kang ([email protected])Min Beon Heo ([email protected])Ki Beon Heo ([email protected])Min Young Bang ([email protected])Se Mi Yun ([email protected])So Hee Im ([email protected])Hye Yun Jeong ([email protected])Sun Young Kim ([email protected])Won Kok Kim ([email protected])Suk Kyung Jeong ([email protected])Hyeong Nam Song ([email protected])Arti ([email protected])Tae Yang Jeong ([email protected])Eun Ha Park ([email protected])A Reum Park ([email protected])Eun Bi Cho ([email protected])Eun Young Won ([email protected])Ji Hyang Ha ([email protected])Eun Young Kim ([email protected])

Division of Molecular Therapeutics

Molecular Cancer Research Center

Natural Medicines Research Center

Functional Metabolite Research Center



D-15Characterization of tailoring genes involved inthe modification of geldanamycin polyketide inStreptomyces hygroscopicus JCM4427

Shin, Jin Chul1,2, Zhu Na1,3,4, DongHo Lee5, WonCheolKim1,2, Kyeong Lee1, Yue-Mao Shen3, Sang-Gi Paik2,Young-Soo Hong1,*, and Jung Joon Lee1,*

1Molecular Cancer Research Center, KRIBB2Chungnam National University3State Key Laboratory of Phytochemistry and Plant Resourcesin West China, China

4Graduate University of Chinese Academy of Sciences, China5Division of Biotechnology, Korea University

Geldanamycin and its analogs are important anti-canceragents that inhibit of the newly targeted, heat shock protein(Hsp) 90, which is a chaperone protein in eukaryotic cells.To resolve which geldanamycin biosynthetic genes areresponsible for particular post-polyketide synthase (PKS)processing steps and in which order the reactions occur,we individually inactivated candidate genes in Streptomyceshygroscopicus subsp. duamyceticus JCM4427, andisolated and elucidated the structures of intermediatesfrom each mutant. The results obtained that gel7 governsat least one of the benzoquinone ring oxidation steps. Inaddition, gel16 was founded to be involved in double-bondformation between C-4 and C-5 of 4,5-dihydrogeldanamycin,which confirmed our previous findings that this doublebond is reduced during the post-PKS modification of thepolyketide assembly. In addition, progeldanamycin, whichdoes not possess a double bond at C-4/5, was purified fromgel7 and 8 double gene inactivated mutant. (InternationalSymposium & Annual Meeting of The Korean Societyfor Microbiology and Biotechnology : Seoul, 2008)

Keywords : anticancer agents, biosynthesis, geldanamycin,polyketide, Streptomyces

D-16Cryptotanshinone inhibits constitutive STAT3function through blocking the dimerization inDU145 prostate cancer cells

Dae-Seop Shin1,2, Hyae-Nan Kim1, Ki Deok Shin1, YoungJu Yoon1,2, Seung-Jun Kim1, Dong Cho Han1, and Byoung-Mog Kwon1,2

1Molecular Cancer Research Center, KRIBB2Biomolecular Science, UST

We screened for natural compounds which inhibit the activityof STAT3 using a dual-luciferase assay. Cryptotanshinonewas identified as a potent STAT3 inhibitor. Cryptotanshinone

rapidly inhibited STAT3 Tyr705 phosphorylation in DU145prostate cancer cells and the growth of cells through 96 hof treatment. Inhibition of STAT3 Tyr705 phosphorylationin DU145 cells decreased the expression of STAT3downstream target proteins such as cyclin D1, survivin, andBcl-xL. To investigate the cryptotanshinone inhibitorymechanism in DU145 cells, we analyzed upstreamproteins. Although phosphorylation of JAK2 was inhibitedby 7 mol/L cryptotanshinone at 24 h, inhibition of STAT3Tyr705 phosphorylation occurred within 30 min and theactivity of the other proteins was not affected. Theseresults suggestthat inhibition of STAT3 phosphorylationoccurs by an independent mechanism, with suppression ofJAK2 phosphorylation a secondary effect of cryptotanshinonetreatment. Continuing experiments revealed the possibilitythat cryptotanshinone might directly bind to STAT3molecules. Cryptotanshinone was co-localized withSTAT3 molecules in the cytoplasm and inhibited theformation of STAT3 dimers. Computational modelingshowed that cryptotanshinone could bind to the SH2domain of STAT3. These results suggest that cryptotanshinoneis a potent anticancer agent targeting the activation STAT3protein. It is the first report that cryptotanshinone has antitumoractivity through the inhibition of STAT3. (AACR-NCI-EORTC International Conference : San Francisco(USA),2007)

Keywords : cryptotanshinone, JAK2, prostate cancer,STAT3

D-17YJ-1 induces apoptosis through inhibition ofHSF1 in human cancer cells

Young Ju Yoon, Ki-Deok Shin, Dae-Seop Shin, Young-Lim Kang, Byoung-Mog Kwon, and Dong Cho Han

Molecular Cancer Research Center, KRIBB

Heat shock proteins (HSPs) are highly expressed in a widerange of tumors and are correlated with a poor prognosisand resistance to therapy. Heat shock factor 1 (HSF1) isthe master switch of HSP expression in eukaryotes. Knockdownof HSF1 expression using siRNA decreased proliferationof cancer cells. Targeting HSF1 using small molecules istherefore an attractive for cancer therapy. In order toidentify HSF1 inhibitors, we carried out chemical libraryscreening using cell-based reporter assay. From the screening,we identified YJ-1. YJ-1 inhibited HSF1-dependent expressionof HSP70 and HSP27 in a concentration-dependent manner,causing cell cycle arrest and apoptosis. In addition, whenYJ-1 was administered intraperitoneally into nude mice at50 mg/kg, it inhibited 47% of tumor growth without bodyweight change. However, when adriamycin (currently usedanticancer drug) was administered at 2 mg/kg, it inhibited

Molecular Cancer Research Center


32% of tumor growth with body weight loss, suggestingcytotoxicity of adriamycin. Collectively, these data indicatethat YJ-1 induces apoptosis through inhibition of HSF1and can be a good lead compound for anti-cancer drug.(Under preparation)

Keywords : anticancer activity, apoptosis, hear shockprotein 27, heat shock protein 70, heat shocktranscription factor 1, HSF1, Hsp27, hsp70

Members

DirectorByoung-Mog Kwon ([email protected])

Principal Researcher Jung Joon Lee ([email protected])Kyou-Hoon Han ([email protected])Kwang-Hee Son([email protected])Young-Soo Hong ([email protected])Dong Cho Han ([email protected])

Senior Researcher Kyeong Lee ([email protected])

Post-DocDae-Seop Shin ([email protected])Ki-Deok Shin ([email protected])Won Cheol Kim ([email protected])Xue Jun Jin ([email protected])

Researcher Young Ju Yoon ([email protected])Young Ran Ha ([email protected])Jina Kim ([email protected])Hye-Nan Kim ([email protected])Yeong-Rim Kang ([email protected])Young-Min Han ([email protected])Hong Mei Li ([email protected])Eun Jin Lee ([email protected])So Young Lee ([email protected])Lan Hee Lee ([email protected])Do-Hyoung Kim ([email protected])Si-Hyung Lee ([email protected])Hong Ri Jin ([email protected])Cheng Zhu Wu ([email protected])Chol Ok SikSu Heun Roh ([email protected])Yan Xia ([email protected])Yinglan Jin ([email protected]) Jin Ju Kim ([email protected])

C-16Diacylglycerol acyltransferase inhibitory effectby sesquiterpenoids isolated from the flowerbuds of Tussilago farfara

Jee Hee Seo1, Mi Young Yoo1, Hye Ran Park2, Il SoonKim1, Nam Ye Kim1, Long Cui1, Chang Soo Lee2, Chul-Ho Lee1, and Hyun Sun Lee1

1Natural Medicine Research Center, KRIBB2Department of Applied Biochemistry, Konkuk University

Inhibition of acyl CoA: diacylglycerol acyltransferase(DGAT), which is a key enzyme in triglyceride synthesisin eukaryotic organisms, has been proposed as one of thedrug targets for treating obesity, type II diabetes mellitusand metabolic syndrome. Bioassay-guided fractionation ofEtOH extract of the flower buds of Tussilago farfara, usingan in vitro DGAT enzyme assay, resulted in the isolation offour known sesquiterpenoids. DGAT1 inhibitory activitywas studied by in vitro DGAT assay using rat livermicrosomes and HepG2 cell microsomes. They showedDGAT1 inhibition with IC50 values of 99.2(1), 18.8(2),47.0(3), 211.1(4) µM (for rat liver microsomes) and >500 M (1),49.1(2), 160.7(3), 294.4(4) µM (for HepG2 cell microsomes)respectively. Among them, tussilagone (2) showed mostpotent inhibition against microsomal DGAT1 derivedfrom rat liver and human hepatocellular carcinoma,HepG2 cells and also significantly inhibited triglyceridesynthesis by suppressing incorporation of [14C]acetateor[14C]glycerol into triglycerides in HepG2 cells. Thesefindings suggest that tussilagone is a potential leadcompound in the treatment of obesity and type 2 diabetes.(The 4th KSP-CCTCNM Joint Symposium on Pharmacognosy: Gangneung, 2008)

Keywords : diacylglycerol acyltansferase, obesity,sesquiterpenoid, triglyceride synthesis,Tussilago farfara, typeII diabetes mellitus

C-17Synthesis and biological evaluation ofbenzoimidazole derivatives as inhibitors of DGAT

Jee Hee Seo1, Sun Jung Park2, Hail Gu2, Hwa Young Jung2,Nam Ye Kim1, Il Soon Kim1, Youngseok Choi2, KyeongLee1, and Hyun Sun Lee1

1Natural Medicine Research Center, KRIBB2Laboratory of Molecular Therapeutics, Korea University

Diacylglycerol acyltransferase (DGAT) is the key enzymewhich catalyzes the final reaction in the synthesis of triglyceride(TG) facilitating the joining of a diacylglycerol with afatty acyl CoA. The accumulation of TG in adipose tissue

Natural Medicine Research Center

is known to result in increased risks of obesity and type 2diabetes. Recently, it was reported that DGAT-1 deficientmice allowed the expenditure of more energy and wereresistant to diet-induced obesity (DIO), making it apotential therapeutic target for these diseases. Our effortsto discover DGAT inhibitors led to novel benzimidazoleanalogues, which showed in vitro DGAT inhibitory activity.In addition, the benzimidazole analogues suppressed TGformation in HepG2 cells and inhibited increment of bodyweight of high fat diet induced mice. Detailed synthesisand biological evaluation of these derivatives will be presented.(The 102nd Fall Meeting of the Korean Chemical Society :Jeju, 2008)

Keywords : benzoimidazole derivatives, diacylglycerolacyltansferase, obesity, triglyceride synthesis

C-18ACAT inhibitors from the fruits of Piper longumand Piper nigrum

Young-Kook Kim1, Mun-Chual Rho1, Seung Woong Lee1,Soon-Yong Choi2, and Yongseok Choi3

1Natural Medicine Research Center, KRIBB2Department of Biotechnology, Hannam University3School of Bioscience and Biotechnology, Korea University

ACAT is an intracellular enzyme that catalyzes theformation of cholesteryl esters from free cholesterol and long-chain fatty acyl coenzyme A. This enzyme plays pivotalroles in intestinal absorption of cholesterol, hepatic productionof lipoproteins and accumulation of cholesterol ester withinmacrophages and smooth muscle cells of the atheroma. Bioactivity-guided fractionation of chloroform extracts ofPiper species (Piper longum and Piper nigrum) led thatcatalyzes the formation of cholesteryl esters from cholesteroland to the isolation of eleven alkamide compounds (1-11).ACAT inhibitory activities of alkamides were positivelyinfluenced by the presence of isobutyl moiety and numberof conjugated double bond. 1 and 6 showed dose dependentinhibition of cholesteryl oleate formation in cells, the IC50

values were observed at 1.87 (1) and 0.7 (6), <2.6 (1) (inHepG2) and 5.1 uM (6) (in CaCo2) respectively. Also,they (1 and 6) showed inhibitory effects of the secretion ofapolipoprotein B in HepG2 cells. 1 showed no inhibition effecton the foam cell formation and cholesterol esterification inTHP-1-derived macrophages. Therefore, compounds 1 and6 were thought to be more effect against ACAT2 thanACAT1. (XXth International Symposium for MedicinalChemistry : Vienna (Austria), 2008)

Keywords : ACAT 2 inhibitors, alkamides, Piper longum,Piper nigrum

C-19Tiarellic acid and randianin induce apoptosisin clone 15 HL-60 cells via caspase-3- andmitochondria-mediated pathway

Ok-Kyoung Kwon1, Kyung-Seop Ahn1, Jaewha Kim2,Guanghai Shen3, Sei-Ryang Oh1, and Hyeong-Kyu Lee1

1Natural Medicine Research Center, KRIBB2Stem Cell Research Center, KRIBB3College of Pharmacy, Yanbian University, China

Triterpenoid saponins including tiarellic acid and randianinwere isolated from Tiarella polyphylla in our previouswork. In the present study, both tiarellic acid and randianinwere found to induce apoptosis of clone 15 HL-60 humanpromyelocytic leukemic cells which have eosinophiliccharacter. Both tiarellic acid and randianin exhibited thecaspase 3 activation, induction of DNA fragmentation, andelevation of annexin V stained cells showing cytoplasmicmembrane flipping typically detected in early apoptoticcells. Following experiment showed that Bcl-2 was down-regulated and Bax expression and cytochrome C release waspromoted by both compounds which mean these compoundsseem to be involved in mitochondrial pathway. Dot blotanalysis using an apoptosis expression array showed thatTRAF-1, TRAF-2 and IL-4R were up-regulated by tiarellicacid, but no significantly modulated genes were found inthe randianin- treated cells. These results suggest that bothtiarellic acid and randianin is able to induce apoptosis ofclone 15 HL-60 cells via caspase-3- and mitochondria-mediatedpathway, even though some apoptosis-related genes aremodulated differently by two compounds. (Spring InternationalConvention of the Pharmaceutical Society of Korea :Jeju, 2008)

Keywords : apoptosis, caspase-3 activity, DNA fragmentation,tiarellic acid, triterpenoid saponin

C-20ERK1/2 signaling pathway is involved inverproside-mediated anti-inflammation in amouse model of allergic asthma

Meeyoung Lee, Kyungseop Ahn, Soyoung Kim, OkkyoungKwon, Huiseong Kim, Seiryang Oh, and Hyeong-Kyu Lee

Natural Medicine Research Center, KRIBB

We reported previously the suppressive effects of verprosideisolated from Pseudolysimachion longifolium on asthmaticparameters such as immunoglobulin E (IgE) level, Th2-typecytokine release, eosinophilia, airway hyperresponsivenessand mucus hypersecretion in an OVA-sensitized/challengedmouse model. To study this action mechanism, we focused



on verproside-mediated modulation of the extracellularregulated kinase 1 and 2 (ERK1/2) pathway in OVA-sensitized/challenged mouse model. ERK1/2 phosphorylation wasblocked by verproside (30 mg/kg, oral administration) inlung tissues from OVA-sensitized/challenged mice. Inaddition, U0126, a specific inhibitor of the MAPK/ERK1/2signaling, significantly inhibited OVA-induced increasesof total cell counts, eosinophil counts, IL-4, IL-5,IL-13and airway mucus secretion levels in bronchoalveolarlavage fluid. Furthermore, Immunohistological study usinganti-pERK1/2 Ab revealed that verproside down-regulatedthe expression and activity of ERK1/2 in lung tissue. Resultsof the present study indicate that the ERK1/2 pathway isinvolved in verproside-mediated anti-inflammation effectin a mouse model of allergic asthma. (Spring InternationalConvention of the Pharmaceutical Society of Korea :Seoul, 2008)

Keywords : asthma, ERK1/2, IgE, verproside

C-21Nicotine attenuates iNOS expression andcontributes to neuroprotection in a compressivemodel of spinal cord injury

Mee-Young Lee1, Lei Chen2, Bernhard Hennig3, Hyeong-Kyu Lee1, and Michal Toborek2

1Natural Medicine Research Center, KRIBB2Department of Neurosurgery, University of Kentucky,USA

3College of Agriculture, University of Kentucky, USA

Primary impact to the spinal cord results in stimulation ofsecondary processes that potentiate the initial trauma. In thepresent study, we hypothesize that the altered expressionof nitric oxide synthase (NOS) may contribute to theseeffects. Recent evidence indicates that nicotine can exertpotent antioxidant and neuroprotective effects in spinalcord injury (SCI). Therefore, the aim of the present studywas to evaluate whether the administration of nicotine caninfluence expression of inducible NOS (iNOS) and/orneuronal NOS (nNOS) in injured spinal cords. Adult maleLong-Evans rats were subjected to a moderate contusionmodel of SCI and received a single i.p. injection of eithersaline or nicotine (0.35, 3.5 or 7 mg/kg) 2 h after trauma. SCIdramatically increased iNOS (but not nNOS) mRNA andprotein levels in microglial cells in the thoracic and lumbarregions of spinal cords. iNOS overexpression resulted inincreased nitrotyrosine formation, decreased number ofNeuN (neuronal nuclei) immunoreactive cells,and upregulationof inflammatory genes. Most importantly, these effectswere markedly attenuated by nicotine acting via a receptor-mediated mechanism. These data may have significant

therapeutic implications for the targeting of nicotine receptorsin the treatment of compressive spinal cord trauma. (SpringInternational Convention of the Pharmaceutical Societyof Korea : Jeju, 2008)

Keywords : inflammatory responses, neuronal nicotinicreceptors, nicotine, nitric oxide synthase, spinalcord injury

C-22Anti-influenzavirus acitivity of quercetin 7-rhamnoside isolated from Korean medicinalplants

Hwa-Jung Choi1, Jae-Hyoung Song2, Young-Joon Ahn3,Seung-Hwa Baek2, and Dur-Han Kwon1

1Natural Medicines Research Center, KRIBB2Department of Herbal Resources, WonKwang University3School of Agricultural Biotechnology, Seoul NationalUniversity

Influenza viruses cause severe respiratory disease in humansand animals with high morbidity and mortality rates. Now,frequently occurrence of genetically variant of influenzaviruses is hard to treat with current vaccine or antiviral drug.In this study we have isolated quercetin 7-rhamnoside (Q7R)isolated from Houttuynia cordata Thunb. (Saururaceae),this compound inhibited replication of influenza A and Bvirus in cell-based assay. Furthermore, Q7R oral administrationof mice infected with influenza A virus (A/WS/33) protectedthe animals against clinical disease symptoms. No toxic effectsof Q7R were observed in cell-based assay or oral administrationof mice. Q7R may play very important role for the treatmentof influenza virus infections. (Chemistry and the BiosphereConference : Dunedin (NewZealand), 2008)

Keywords : antiviral agent Houttuynia cordata, coronaviruses,PEDV

Members

DirectorHyeong-Kyu Lee ([email protected])

Principal Researcher Hyun Sun Lee ([email protected])Young-Kook Kim ([email protected])Kyung Seop Ahn ([email protected])Sei-Ryang Oh ([email protected])Sang Ku Lee ([email protected])

Senior Researcher Dur-Han Kweon ([email protected])

Joong Ku Lee ([email protected])

Post-DocCai Xing Fu ([email protected])Tran Man Hung ([email protected])Bo Young Park ([email protected])Mee-Young Lee ([email protected])Hwa Jung Choi ([email protected])

Researcher Jung Hee Kim ([email protected])Doo Young Kim ([email protected])Soo Hyun Kim ([email protected])Sun Ja Choi ([email protected])Piseth Khiev ([email protected])Hye Sun Lim ([email protected])Quan QuiHua ([email protected])Kim Jin Young ([email protected])Eun Ah Kim ([email protected])Ok Kyoung Kwon ([email protected])Jee Eeun Yook ([email protected])Hui Seong Kim ([email protected])Jee Hee Seo ([email protected])Il Soon Kim ([email protected])Nam Ye Kim ([email protected])Min Seok Kim ([email protected])Gi Woong Kang ([email protected])Jung Ho Choi ([email protected])Ji Na Choi ([email protected])Seung Yoon Lee ([email protected])Je Hyeong Song ([email protected])Geon Rae Kim ([email protected])Jin Ki Kim ([email protected])Sang-Hong Kim ([email protected])Jae Hyeong Song ([email protected])

A-5New type of FabI-directed antibacterial agentfrom microbial metabolites

Chang Ji Zheng, Mi-Jin Sohn, and Won-Gon Kim

Functional Metabolite Research Center, KRIBB

Bacterial enoyl-acyl carrier protein (ACP) reductase (FabI)catalyzes the final and rate-limiting step of the chainelongation process of bacterial fatty acid synthesis and hasbeen validated as an attractive target for the developmentof new antibacterial drugs. New FabI inhibitors have beenscreened from natural resources for discovery of new anti-bacterial lead compound. Two active compounds namedcompound 251A and 251B were isolated from thefermentation broth of an actinomycetal strain throughbioassay-guided fractionation using ethyl acetate extraction,various chromatographic methods and C18 HPLC.Chemical structure of the purified compounds 251A and251B were elucidated to be methylated unsaturated fattyacids by MS and NMR spectral data. Compound 251Awas isolated as a natural compound for the first time inthis study. Compounds 251A and 251B inhibited S. aureusFabI with IC50 (uM) of 21 and 25, respectively. Theinhibition mechanism of compound 251B on FabI wascompetitive with both the substrate and NADPH. Theyalso prevented the growth of S. aureus with MIC (µg/ml) of32. Using biochemical and genetic approaches such asintracellular fatty acid biosynthesis and overexpressionresistance assay, it has been confirmed that the mode ofantibacterial action of compounds 251A and 251B is viainhibition of FabI. Non-methylated unsaturated fatty acids,however, turned out not to target FabI in vivo, indicatingthat the methyl groups in compounds 251A and 251B areimportant for the in vivo FabI-inhibition. This work wassupported by the 21C Frontier Microbial Genomics andApplication Center Program. (KMB InternationalSymphosium : Seoul, 2008)

Keywords : ACP, enoyl-acyl carrier protein, FabI, inhibitors,reductase, Staphylococcus aureus

A-6New anti-MRSA agent from Penicillium sp.FR0011

Chang Ji Zheng, Hyung-Eun Yu, and Won-Gon Kim


Methicillin-resistant Staphylococcus aureus(MRSA) isknown as a major nosocomial pathogen that has alsodeveloped resistance to many other antibiotics. Moreover,MRSA has been reported to acquire resistance to the

Functional Metabolite Research Center



antibiotic of last resort, vancomycin. It has been suggestedthat S. aureus will acquire full resistance to vancomycin inthe near future. Therefore, it is increasingly important andnecessary to find new classes of antibacterial. In the courseof screening new antibacterial against MRSA, we foundtwo potent substances, compounds 11A and 11B, from thebroth cultures of Penicillium sp. FR0011. Compounds11A and 11B were isolated from the fermentation broththrough bioassay-guided fractionation using ethyl acetateextraction, silica gel chromatography and C18 HPLC.Compound 11B was determined to be new tetracycline-type by NMR and MS spectral data. They exhibited potentantibacterial activity against S. aureus including MRSAand quinolone-resistant S. aureus (QRSA) with MIC (µg/ml)of 0.5, which is similar with that of vancomycin. Thiswork was supported by the 21C Frontier Microbial Genomicsand Application Center Program. (KMB InternationalSymphosium : Seoul, 2008)

Keywords : MRSA, new antibacterial, Penicillium,tetracycline

A-7Cordyformamide and chrysophanol, phenolicantioxidative metabolites from the fungusEupenicillium shearii F80695

Hahk-Soo Kang, Soon-Hye Park, Kyoung-Rok Kim, andJong-Pyung Kim


We reported new potent antioxidants, melanocins A, B,and C from the fungal metabolite of Eupenicillium sheariiiF80695. Among them, the isocyanide compound, melanocinA, exhibited tyrosinase inhibitory activity, melaninsynthesis inhibitory activity and antiwrinkle effect. Thus,we have further investigated new bioactive compounds forcosmeceutical materials from the EtOAc extract of thefungus, and isolated two phenolic antioxidants. Thecompounds were purified by chromatographic methodsincluding centrifugal silica TLC and Sephadex LH-20 ccand HPLC. The chemical structures of the compoundswere determined by MS and NMR, and were identified ascordyformamide and chrysophanol. Cordyformamide andchrysophanol showed strong free radical scavengingactivity against ABTS+, DPPH, and hydroxyl radicals. (한국응용생명화학회춘계학술대회 : 서울, 2008)

Keywords : antioxidant, chrysophanol, cordyformamide,Eupenicillium shearii

A-8New lactone compounds from the BasidiomyceteStereum ostrea

Hahk-Soo Kang1, Eun-Mi Jun1, Soon-Hye Park1, Kyoung-Rok Kim1, Tae-Soo Lee1, and Jong-Pyung Kim1

1Functional Metabolite Research Center, KRIBB2University of Incheon

Mushrooms have been known as a potential source ofvarious secondary metabolites with diverse chemicalstructures and biological activities. We have collectedKorean wild mushroom strains, and have constructed theirmetabolite library. During the screening of natural antiobesityagents from the library, we found that Stereum ostreashowed significant inhibitory activity against humanpancreatic lipase. Purification of the active constituentsthrough chromatographic methods, including siligica gelcc, Sephadex LH-20 cc and C18 HPLC, led four newunique lactone compounds. The structures of the compoundswere determined by NMR and MS spectroscopic analyses.(한국응용생명화학회춘계학술대회 : 서울, 2008)

Keywords : antiobesity, lipase, loactone, mushroom,Stereum ostrea

A-9Chemical and biological properties of the pinebark extract of Pinus densiflora Siebold etZuccarini

Hahk-Soo Kang1, Kyoung-Rok Kim1, Ohan Taek Han2,and Jong-Pyung Kim1

1Functional Metabolite Research Center, KRIBB 2Nutrapharm Co. Ltd.

The procyandin-rich French maritime pine (Pinus martima)bark extract Pycnogenol is a mixture of flavonoids. It ispromoted as a polyphenol-rich food supplement and isbelieved to have strong antixoidant activity and variousbeneficial health effects. To develop Pycnogenol-likeantioxidant supplement form Korean pine bark, Pinusdensiflora was selected due to its comparable antioxidantactivity to Pycnogenol. Polyphenol content, catechinprofile, and antioxidant properties of PinExol, the barkextract of Pinus densiflora, is the equal of Pycnogenol.(한국응용생명화학회춘계학술대회 : 서울, 2008)

Keywords : antioxidant, pine bark extract, Pinus densiflora

A-10Endoplasmic reticulum stress induction byhonokiol isolated from Magnolia obovata

Sun-Ok Kim, Hiroshi Kamiyama, Long He, HiroyukiOsada, Bo Yeon Kim, and Jong-Seog Ahn


Endoplasmic reticulum stress (ER stress) is a key mediatorfor various types of diseases, including cancer, obesity,diabetes and immune system. Thus, development of ERstress regulator would be a promising drug for the therapyof those diseases. Recently, honokiol having a stronginduction of endoplasmic reticulum stress was isolatedfrom a plant Magnolia obovata. Honokiol induced theXBP-1 mRNA splicing and accordingly upregulated theexpression of Grp78/Bip chaperone in HeLa and PC3cells. Moreover, the data shows that honokiol induced theactivation of several ER stress-relevant regulator, includingCHOP/GADD153, JNK and c-Jun. Also, JNK and caspaseinhibition does not affect XBP-1 splicing by honokiol anddecreases honokiol-induced cell death. In this study, themechanism of honokiol compound for induction of ERstress and cancer cell death is presented. (The 4th Korea-Japan Joint Symposium, Chemical Biology for BioactiveMolecules : Nikko (Japan), 2008)

Keywords : ER-stress, GRP78, honokio

A-11Repression of E-cadherin by oncogenic K-rasinvolving DNA methyltransferase activity

Osong Kwon, Sook Jung Jeong, Sun Ok Kim, Long He,Eun Young Cha, Jong Seog Ahn, and Bo Yeon Kim


E-cadherin, transmembrane glycoprotein, is essential fororganogenesis in epithelial tissues and plays an importantrole for Ca2+-dependent intracellular adhesion in normalepithelium. E-cadherin expression in human cancers isfrequently reduced via methylation of E-cadherin promoter.Here, we report that oncogenic Ki-ras repress E-cadherinexpression in Ki-Ras-transformed human prostateepithelial cells. The loss of E-cadherin expression resultsfrom hypermethylation of CpG-rich promoter region of E-cadherin, as revealed by methylation-specific PCR,bisulfite sequencing, real-time reverse transcription PCR,ChIP, and Western-blotting data. The DNA methyltransferaseinhibitor, 5'-Aza-2'dC, restored E-cadherin promoteractivity and protein expression in Ki-Ras-expressing cells,

which in turn blocked collagen invasion ability.Furthermore, hypermethylation of CpG-rich promoterregion of E-cadherin is induced with no change of DNAmethyltransferase 1 between normal and Ki-Ras-expressingcells. These observations suggest that Ki-ras inducehypermethylation of E-cadherin promoter and repress E-cadherin gene expression. Moreover, Ki-ras-mediatedepigenic silencing might occur through indirect DNAmethyltransferase activity. (The 4th Korea-Japan JointSymposium, Chemical Biology for Bioactive Molecules: Nikko (Japan), 2008)

Keywords : DNA methylation, E-cadherin, Ki-ras

A-12DNA methylation and moderately decreasedCBR1 gene expression in K-ras overexpressedprostate cancer cell lines

Eun Young Cha, Sook Jung Jeong, Osong Kwon, JongSeog Ahn, and Bo Yeon Kim


We performed microarray and real-time PCR analysis with267B1 human prostate epithelial cells and viral Kirsten-rastransformed cells (267B1/Ki-ras), and found that Ki-rasoverexpression moderately represses carbonyl reductase(CBR1) expression at the transcription level. Carbonylreductase (NADPH:secondary-alcohol oxidoreductase)belongs to a group of NADPH-dependent cytosolicenzymes called aldo-keto reductases. Human CBR1 (hCBR1)is known as prostaglandin 9-keto reductase and 15-hydroxy dehydrogenase, and regulates the metastasis ofcancer cells through the regulation of prostaglandinmetabolism. Validation of mRNA level and protein levelconfirmed down-regulation of CBR1 gene in 267B1/Ki-ras cells and in cells transiently transfected with Ki-ras,however, CBR1 was restored by DNA methyltransferaseinhibitor 5'-Aza-2'dC. To determine which region containsmethylation CpG site, we used bisulfite sequencing of thepromoter upstream region and exon-1 of CBR1 gene.Interestingly, CBR1 gene was not hypermethylated in thepromoter region but in exon-1 in 267B1/Ki-ras cells anddemethylation occurred upon 5'-Aza-2'dC treatment.These observations suggest that repression of CBR1expression by exon-1 hypermethylation could be relatedwith Ki-ras induced cell transformation. (제20회 한국분자세포생물학회학술대회 : 서울, 2008)

Keywords : carbonyl reductase, DNA methylation, Ki-ras,prostate cancer



A-13Isolation and identification of Micromonosporasp. showing nematocidal activity against Pinewood nematode

Dong-Jin Park, Jae-Chan Lee, Pan Kyung Kim , Yong HaChang, and Chang-Jin Kim


For the isolation of Actinomycetes showing nematocidalactivity against Pine wood nematode, Bursaphelenchusxylophilus, about 2000 culture broth of Actinomyceteswere tested and their activity were compared with that ofStreptomyces avermitilis resulting a selected strainAW050027. The cultural, morphological and physiologicalanalysis was performed for the identification of a selectedstrain. Phylogenetic analyses based on 16S rDNA genesequences showed that the selected strain AW050027belonged to the genus Micromonospora and M. coriariaeNAR01T was the closest neighbors, sharing 98.9% 16SrDNA gene sequence similarity. (한국식물병리학회춘계학술대회 : 진주, 2008)

Keywords : Bursaphelenchus xylophilus, nematocide,pine wood nematode

A-14Diversity of culturable prokaryotes in BigeumIsland, established by phylogenetic approach

Yoon-Jung Ju, Jae-Chan Lee, Syed G Dastager, Dong-JinPark, and Chang-Jin Kim


A study was made on diversity of culturable prokaryotesacross Bigeum Island, in South Korea, and also attempts toinfer them from phylogenetic data for possible presence ofindicative phenotypes which might contribute to a functionalmicrobial community. To address the richness in Bigeum'smicrobiota, we cultivated bacteria from different sites inBigeum Island, each site hosted a distinct prokaryoticcommunity revealed by 16S rRNA gene sequencing. Twentyseven bacterial isolates with different morphotypes wereisolated on R2A medium and were subjected to polyphasicanalysis including morphological, physiological andbiochemical characterization. The phylogenetic tree basedon 16S rRNA gene sequences placed the 27 isolates into 9different major bacterial groups, each group comprised ofpure cultures that shared ≤ 97% sequence identity. Themost abundant and diverse isolates were members ofGram-positive bacteria, particularly the Nocardioides

(59.0%) as a dominant group in Bigeum Island culturablepopulations. Results of the Jukes-Cantor evolutionarydistance matrix suggested that the vast majority of theisolates were different strains of known species like,Nocardioides, Marmoricola,, Rubellimicrobium, Phycicoccus,Agrococcus, Frigoribacterium, Leifsonia, Cryobacteriumand Microbacterium. Novel sequences that affiliate with aBigeum habitats provide unique niches for the evolution ofnovel communities and microorganisms. (InternationalSymposium & Annual Meeting of the Korean Society forMicrobiology and Biotechnology : Seoul, 2008)

Keywords : diversity, habitat, microbial community, population

A-15Clitocybin A, a new antioxidative compoundfrom the culture broth of Clitocybe aurantiaca

Young-hee Kim, Soo-Jin Choo, In-ja Ryoo, Guang-HuaXu, and Ick-Dong Yoo*

1National Cosmeceuticals Research Center, KRIBB

Clitocybin A , a new antioxidative compound was isolatedfrom the culture broth of Clitocybe aurantiaca. Thiscompound was purified by solvent extraction, silica gelchromatography, Sephadex LH-20 column chromatography,and finally by preparative HPLC. Its structure wasdetermined as 5, 7-dihydroxy-2--hydroxy phenyl-isoindol-1-one on the basis of the UV, NMR, and MS spectroscopicanalysis. The antioxidant activity of clitocybin A wasevaluated by measuring free radical scavenging effectsusing three different assays, the superoxide radical anionscavenging activity by the xanthine/xanthine oxidasemethod, ABTS radical scavenging activity by using ABTSradical cation decolorization assay, and DPPH radicalscavenging assay. Clitocybin Aexhibited moderate superoxideradicalscavenging activity with IC50 value of 10.3 M(Catechin 10.4M), and displayed significant ABTS radicalscavenging activity with IC50 value of 6.4 M (Catechin4.8 M), respectively, while exhibited no DPPH radicalscavenging activity. The comet assay showed that theexposure of the cells to H2O2 increased their tail length to61.11, whereas their treatment with 1 resulted in adecrease of their tail length to 35.39, as shown in Table 4.This data suggests that 1 had a protective effect against cellularDNA damage induced by oxidative stress. (InternationalSymposium & Annual Meeting of the Korean Societyfor Microbiology and Biotechnology : Seoul, 2008)

Keywords : antioxidant activity, Clitocybe aurantiaca,Clitocybin A, comet assay

A-16Etoposide-resistant HT-29 human colon carcinomacells during glucose deprivation are sensitive toPiericidin A, a GRP78 down-regulator

In-Ja Ryoo1, Soo-Jin Choo1, Suns-Min Cho2, Young-HeeKim1, Guang- Hua Xu1, and Ick-Dong Yoo1

1Functional Metabolite Research Center, KRIBB2GenExel-Sein Bio Model system Park (E-18), KAIST

Downregulating GRP78 expression may be a novel strategyanticancer drug development. Based on thatpremise, weestablished a screening program for anticancer agents thatexhibit preferential cytotoxic activity for etoposide-resistantcancer cells under glucose-deprived conditions. Werecently isolated an active compound, AR-054, from theculture broth of Streptomyces sp., which prevents stress-inducedetoposide resistance in vitro. AR-054 was identified aspiericidin A, a prototypical compound, by ESI-MS analysisand various NMR spectroscopic methods. Here, weshowed that piericidin A suppressed the accumulation ofGRP78 protein and was also highly toxic to etoposide-resistantHT-29 cells, with IC50 values for colony formation of 6.4nM and 7.7 nM under 2-deoxyglucose supplemented andglucose-deprived conditions, respectively. Interestingly,piericidin A had no effect under normal growth conditions.Therefore, we suggest that prevents up-regulation of GRP78 andexhibits a selective cytotoxicity in glucose-deprived HT-29 cells that are resistant to etoposide. (InternationalSymposium & Annual Meeting of the Korean Society forMicrobiology and Biotechnology : Seoul, 2008)

Keywords : etoposide resistance, GRP78, HT-29 cells,Hypoglycemia, Piericidin A

Members

Director Jong-Seog Ahn ([email protected])

Principal Researcher Jong-Pyung Kim ([email protected])Chang-Jin Kim ([email protected])Ick-Dong Yoo ([email protected])Won-Gon Kim ([email protected])Bo Yeon Kim ([email protected])

Senior Researcher Dong-Jin Park ([email protected])In-ja Ryoo ([email protected])

Post-DocJae-Chan Lee ([email protected])Guang-Hua XU ([email protected])Chang-Ji Zheng ([email protected])Sun-Og Lee ([email protected])Sook Jung Jeong ([email protected])Myung Sun Lee ([email protected])Jin Hee Kim Eun Young Bae ([email protected])

Researcher Hahk-Soo Kang ([email protected])Kyung-Rok Kim Yoon-Jung Ju ([email protected])Yong Ha Chang ([email protected])Pan Kyung Kim ([email protected])Young-Hee Kim ([email protected])Soo-jin Choo ([email protected])Sung-Min Cho ([email protected])Yi Fang ([email protected])Osong Kwon ([email protected])Eun Young Cha ([email protected])Long He ([email protected])


Division of Biomaterials Science

Plant Genome Research Center

Systems Microbiology Research Center

Environmental Biotechnology Research Center



C-6Repression of BiP, an ER-resident protein, preventsR-gene-mediated hypersensitive response (HR)but enhances non-host pathogen-inducednecrosis in N. benthamiana.

Jeong Hee Lee, Sora Choi, Sanghun Lee, and Jeong Mee Park*

Plant Genome Research Center, KRIBB

HRT, encoded a classic leucine zipper/nucleotide bindingsite/leucine rich repeat containing protein, is required forHR development and resistance after turnip crinkle virus(TCV) infection. In tobacco leaves, transient co-expression ofthe HRT and its elicitor, the TCV coat protein (CP),results in rapid cell death. To isolate genes involved inHRT/CP-mediated cell death in Nicotiana benthamiana,we analyzed pepper cDNA microarray. Based on virus-induced gene silencing analysis, we identified 10genesthat were responsive to the HRT/CP-mediated celldeath in N. benthamiana. Silencing of one of genes,encoding a luminal binding protein 5 precursor (BiP 5-ERresident heat shock protein 70), showed the inhibition ofplant growth and development. Moreover, VIGS of BiP5resulted in the abolishment of R-gene mediated HRsignaling including HRT and Rx which confer resistance toturnip crinkle virus (TCV), potato virus X (PVX), respectively,and cell death by transient expression of Bax whichencoded a protein that promotes apoptosis in animalsystem. However, cell death induced by non-host pathogen,Pseudomonas syringae pv. syringae strain 61, was morerapidly progressed in BiP5-silenced plants. These resultssuggestthat BiP5 might play an opposite role in cell deathbetween host pathogen and non-host pathogen. Inmammals, BiP is known as negative regulator of signaltransducer of unfolded protein response (UPR) followingER stress. To investigate the involvement of the UPRpathway during plant-pathogen interactions, we are testingwhether a typical UPR, triggered by treatment withtunicamycin, will affect the HRT/CP-induced cell death.(The 9th International Congress on Cell Biology :Seoul, 2008)

Keywords : BiP, HRT, hypersensitive response, UPR

C-7In silico screening and promoter analysis ofpathogen-induced genes in pepper

Yeon Lee1, Young-Im Ha1, Sora Choi1, Cheol-Goo Hur2,and Jeong Mee Park1,*

1Plant Genome Research Center, KRIBB2Omics and Integration Research Center, KRIBB,

We report here in silico screening of non-host pathogen-

induced genes (PIGs) in pepper and its promoter analysis.In an attempt to identify specific pathogen-induced genesin pepper, we have used an in silico subtraction methodology.A total of 115,598 EST sequences obtained from 26pepper tissue/organ categories were used as the initialdataset. These techniques led to the identification of 16pathogen-specific genes. Five PIG genes were preferentiallyincreased in pepper plants in response to non-hostpathogen inoculation and also during the host resistanceresponse. Although, their expressions were differentiallymodulated by various defense-related signals, such assalicylic acid (SA), methyl jasmonate (MeJA), andethylene. To increase our comprehension of PIGsexpression, we isolated putative promoter regions for allPIGs by genome walker technique. Putative promoters ofPIGs fused to GUS were transiently assayed in N.benthamiana leaves by using an Agrobacterium-mediatedtransient expression assay. Although differences in timeand levels were observed among the promoters of PIGs,GUS activities were significantly increased followinginfection of non-host pathogen, Pseudomanas syringae pv.syringae 61. These results indicated that all PIGspromoters are sufficient for pathogen specific expression.Further studies combined computational analysis are inprogress to isolate individual cis elements within thepromoters as well as their combinatorial effects in PIGsexpression. (Symposium on Genetic Study and PlantBreeding for Biotic and Abiotic Stress Tolerance :Daegu, 2008)

Keywords : Agrobacterium-mediated transient expressionassay, GUS activity, non-host pathogen, PIGpromoter

C-8Silencing of an NbLytB gene involved in theplastid nonmevalonate pathway compromisedhypersensitive response

Sanghun Lee1,2, Jeong Hee Lee1, Byeongjin Cha2, andJeong Mee Park1.

1Plant Genome Research Center, KRIBB2Department of Plant Medicine, Chungbuk NationalUniversity

Using pepper cDNA microarray analysis, we identified 44pepper genes that were responsive to the HRT/CP- or Bax-mediated cell death in N. benthamiana. To investigatefunctional contributions of selected genes in plant celldeath, virus-induced gene silencing (VIGS)method wasused. As results of VIGS, one of genes, NbLytB, which isknown that involved in the last step of the nonmevalonatepathway was isolated. VIGS of NbLytB resulted in growtharrest and an albino phenotype. In addition, the silenced

Plant Genome Research Center


plants suppressed HRT/CP- and Bax-mediated cell death.To determine whether suppression of cell death in NbLytBdepleted plants correlate with production of reactiveoxygen species, nitro blue tetrazolium (NBT) anddiaminobenzidine (DAB) assays were performed. NbLytB-silenced plant displayed significantly reduced levels ofhydrogen peroxide and also ROS. Furthermore, NbLytB-silenced plants showed reduced expression of PR1 geneduring hypersensitive response, while PR5 transcriptswere not changed. These results suggest that theNbLytBgene may involved in plant cell death and itsinvolvement is correlated with the formation of reactiveoxygen species and induction of PR genes. (2008 CropFunctional Genomics Workshop : Chungmu, 2008)

Keywords : LytB, ROS, VIGS

C-9Increased salinity tolerance by carbon metaboliteproduction in chloroplast transgenic Nicotianatabacum

Hyun Tae Kim1, Sung Ran Min1, Hwa Jee Chung1,2,Junghe Hur1,2, Joo Young Park1,2, and Jang Ryol Liu1

1Plant Genome Research Center, KRIBB2GenDocs

Many crops are classified as C3 plants as the first productof atmospheric CO2 fixation is the 3-carbon compound, 3-phosphoglycerate (3-PGA) that is produced in the Calvincycle by Rubisco in the chloroplast stroma, at the expenseof ATP and NADPH produced from the light-dependentreaction. Researchers have attempted to improve thephotosynthesis and crop yield by touching Calvin cyclegenes. A number of key enzymes involved in the Calvincycle including phosphoribulokinase (PRK), Rubisco,glyceraldehydes-3-phosphate dehydrogenase (GAPDH),chloroplastic fructose-1,6-bisphosphatase (FBPase), andsedoheptulose1,7-bisphosphatase (SBPase) have beenidentified, and their roles in the regulation of the Calvincycle and carbohydrate metabolism have been analyzed byproducing transgenic plants. Also, plastid transformationhas become one of the powerful tolls of plantbiotechnology because of several advantages over anuclear transformation such as a high level of the desiredgene expression and an avoidance of environmentaldissemination of pollen-mediated foreign gene. Plants aresubjected to several harsh environmental stresses thatadversely affect growth, metabolism, and yield. Drought,salinity, low and high temperatures, flood, pollutants, andradiation are the important stress factors limiting theproductivity of crops. Several biotic (insects, bacteria,fungi, and viruses) and abiotic (light, temperature, wateravailability, nutrients, and soil structure) factors affect the

growth in higher plants. Among these, drought and salinityare a major abiotic factor that limits agricultural cropproduction. As the results of the project, chloroplast-transformed plants overexpressing FBP/SBPase exhibitedincreased carbon metabolite and enzyme activity forCalvin cycle compared to control plants. Transgenic plantsled to significant improvement in salt and drought stress.Also, transgenic plants have the higher concentrations ofglucose, fructose, sucrose and starch than control plant inresponse to abiotic stresses. Therefore, high carbonmetabolites play a part in the Osmoprotectant. (2008Crop Functional Genomics Workshop : Chungmu,2008)

Keywords : chloroplast-transgenic, FBP/SBPase,photosynthesis, salinity tolerance

C-10Expression of RNAi suppressor codon-optimizedfor Chlamydomoans reinhardtii

Yaw Joo Kim, Chun ji Yin, Seo Hee Jung, Seyed JavadDavarpanah Shahri, Sung Ran Min, Jang Ryol Liu, andWon Joong Jeong*

Plant Genome Research Center, KRIBB

Codon-optimized RNAi suppressor (CrCMV2b) gene,synthesized for expression in Chlamydomonas reinhardtiiwith high GC content in codong region, was active forsuppressing gene silencing in Nicotiana benthamiana andC. reinhardtii. Strong suppressor activity of CrCMV2bagainst gene silencing machinery was confirmed withtransient expression in N. benthamiana usingagroinfiltration experiment. The accumulation of miRNAs,siRNAs, and their putative target mRNA were altered inC. reinhardtii transformed with CrCMV2b gene. Theseresults suggest that codon optimized 2b protein interfereswith RNAi pathway in C. reinhardtii as in higher plants.(한국육종학회정기학술발표회 : 대구, 2008)

Keywords : Chlamydomoans reinhardtii, gene silencing,RNAi suppressor

C-11

Idenification and characterization of the PAMPsand effectors of Burkholderia glumae, thecausative agent of bacterial grain rot of rice.

Serry Koh1, Ran Hee Yoo2, Moon Nam3, Jung Kyu Kim4,Ingyu Hwang5, and Jae Sun Moon1

1Plant Genome Research Center, KRIBB


2Department of Functional Genomics, UST3Department of Applied Biology, CNU4Department of Crop Science, CNU5Department of Agricultural Biotechnology, SNU

Burkholderia glumae, the causative agent of bacterialgrain rot of rice, genome sequence has been completedrecently. With newly available genome information, thepathogen-associated molecular patterns (PAMPs) oreffectors of B. glumae were screened systematically in thisstudy. PAMPs and effectors, mostly secreted proteins viaeither Type II (T2SS) or Type III protein secretion systems(T3SS), have been reported to play important roles inbacterial pathogenecity and triggering innate immunity ofeukaryotic host cells. Over 200 genes with PIP (PlantInduced Promoter) box (TTCG-n16-TTCG), a cis-actingregulatory element, were identified in B. glumae genomedatabase, and the proteins of these PIP box containinggenes were investigated to characterize their possible rolein pathogenecity. B. glumae genomic library with a cya(adenylate cyclase) reporter gene was also constructed andscreened in vivo, for the identification of effectorstranslocated by T3SS. As PAMP candidates, total of 34extracellular accumulated proteins were identified by 2-DE and ESI-MS/MS, in plant apoplast-mimickingconditions, and 16 were secreted via a T2SS (Kang YS, etal., 2007). In order to elucidate the molecular mechanismof B. glumae pathogenecity in rice, PAMPs/effectorsinteracting proteins of rice have been searched using yeasttwo hybrid assay, as well. (XII Internatioanl Congress ofBacteriology and Applied Microbiology : Istanbul(Turkey), 2008)

Keywords : Burkholderia glumae, effectors, PAMPs,pathogen-associated molecular patterns, TypeIII protein secretion systems

Members

Director Suk Yoon Kwon ([email protected])

Principal Researcher Jang Ryol Liu ([email protected])Jeong Mee Park ([email protected])Jae Heung Jeon ([email protected])

Senior Researcher Won Joong Jeong ([email protected])Sung Ran Min ([email protected])Jae Sun Moon ([email protected])Hyun Soon Kim ([email protected]) Hye Sun Cho ([email protected])

Post-DocJong Hyun Kim ([email protected])Jeong Hee Lee ([email protected])Serry Koh ([email protected])

Researcher Ryong Nam Kim ([email protected])Hyun Tae Kim ([email protected])Myung Suk Ahn ([email protected])Myung Jin Oh ([email protected])Yaw Joo Kim ([email protected])Chon Ji Yin ([email protected])Mina Kim ([email protected])Mi Kyung Yoon ([email protected])Yeon Lee ([email protected])Sang Hun Lee ([email protected])Ran Hee Yoo ([email protected])


A-43Spectrum of proton irradiation-induced mutationsin the tonB gene of Escherichia coli

Jihee Han and Haeyoung Jeong

Systems Microbiology Research Center, KRIBB

Ionizing radiation induces damages in DNA such as abasicsites, oxidized bases and strand breaks depending on thetypes and LET of radiation. The most important biologicalimplication of such lesions is the cell-killing effect, butcellular responses to repair them and to restore normalphysiological status often result in mutations that can bedirectly applicable to the improvement of biologicalresources. To investigate the specificity of mutationscaused by the proton irradiation, nucleotide sequences ofthe endogenous tonB gene of phage T1-resistantEscherichia coli BL21 mutants induced by 45-MeV protonbeam (KIRAMS MC-50 Cyclotron) were determined. Adose of 125 Gy induced a mutant frequency of 9.03 x 10-7per survivor (56.3% survival), which was a 2.1-foldincrease from the background level. In contrast to insertionsequence transposition and G:C®A:T transition that areknown to dominate in the spontaneous mutations occurringin the tonB gene, a rather broad types of mutations (ThreeG:C®A:T transitions, two G:C®T:A transversions, andnine -1 bp frameshifts) were identified from 149 proton-induced T1-resistant colonies. The mutational spectrumwas similar with that of X-ray-induced mutations in thetonB gene, but was different from a previous study onproton beam-induced mutation in the crp gene cloned in aplasmid, followed by transformation and sequence analysis.Our result, the first report describing nucleotide sequenceanalysis of proton beam-induced mutations in an endogenousgene, suggests that the mutational nucleotide changepatterns induced by protons can be different according tothe target gene and the assay system. We will also presentpreliminary results of a combined approach of protonirradiation and adaptive evolution for the enhancement ofbutanol tolerance in E. coli that can be utilized for biofuelproduction. (The 12th International Workshop onAccelerator & Bean Utilization : Gyeongju, 2008)

Keywords : bacteriophage T1, mutation, proton beam, tonB

A-44Genome sequence of the probiotic bacteriumBifidobacterium bifidum BGN4

Dong Su Yu1, Haeyoung Jeong1, Myeong-Soo Park2, GeunEog Ji3, Tae Kwang Oh1,4, and Jihyun F. Kim1

1Systems Microbiology Research Center, KRIBB 2Anyang Technical College

3BIFIDO Co.421C Frontier Microbial Genomics and Applications Center

Probiotics are live microorganisms which when administeredin adequate amounts confer a health benefit to the host.They are often included in dairy products which have beenhistorically consumed by humans. Bifidobacteriumbifidum, a probiotic bacterium that resides in the humangut, is known to exhibit a prominent adhesive capacity forthe intestinal epithelial cell and high immunomodulatoryactivities as well. We determined the whole genomesequence of B. bifidum BGN4 producing chiro-inositolthat has a growth inhibitory effect against various humancolon cancer cell lines. 27 contigs produced from 20X-coverage of 454 pyrosequencing (Roche GS FLX) werejoined by primer-walk reads on combinatorial PCRproducts. The assembled genome consists of a singlecircular chromosome of 2,218,454 bp with 62.65% G+C.Functional annotation of 1,924 protein-coding genes,predicted by Critica and Glimmer, was performed throughdatabases searches, and putative functions were assignedto 85.9% of them. Comparative genomic analysis withother sequenced Bifidobacterium species (B. longumNCC2705, B. adolescentis ATCC 15703 and B. longumDJO10A) revealed that B. bifidum BGN4 has 1,175common genes with an average percent identity of 75.8%.(International Meeting of the Federation of KoreaaMicrobiological Societies : Seoul, 2008)

Keywords : Bifidobacteria, Bifidobacterium bifidumBGN4, genome sequencing, probiotic

A-45Identification of a new gene cluster for type IIsecretion in Escherichia coli BL21(DE3)

Ji Hoon Shim, Sung Ho Yoon, and Jihyun F. Kim

System Microbiology Research Center, KRIBB

Secretory production of recombinant proteins has severaladvantages overproduction as inclusion bodies. Generally,targeting protein toperiplasmic space or the culturemedium facilitates downstream processing much easier ata reduced process cost, especiallythe host strainsecretes itsown proteins. Laboratory E. coli strains normally does notsecrete extracellular proteins because genes encoding thetype II secretion (T2S) pathway are silenced by H-NS.From the analysis of two E. coliB strains, REL606 andBL21(DE3), we found that the B genomes have an additionalgene cluster for T2S as compared to the laboratory strainof K-12 MG1655 or W3110. Phylogenetic analysis showedthat clear distinctions could be made between the homologsof the T2S system which is conserved in B and K-12 andthose of the additional T2S system in B strains. To find

Systems Microbiology Research Center


out the role of the new T2S, transcriptional activities ofgene clusters of T2S in BL21(DE3) and MG1655 wereanalyzed by semi-quantitative RT-PCR analysis. (InternationalMeeting of the Federation of Koreaa MicrobiologicalSocieties : Seoul, 2008)

Keywords : Escherichia coli, RT-PCR, type II secretion

A-46Genome sequence analysis of the marine microbeDonghaeana dokdonensis DSW-6

Soon-Kyeong Kwon1,2, Haeyoung Jeong1, Jung-HoonYoon1, Tae-Kwang Oh1,3, and Jihyun F. Kim1,2

1Systems Microbiology Research Center, KRIBB 2Functional Genomics Field, School of Science, UST321C Frontier Microbial Genomics and ApplicationsCenter, KRIBB

A warm branch of the Kuroshio Current and the coldNorth Korea Current, an extension of the Liman Current,meeting to get mixed and merged, the East Sea of Koreaaround Ulleungdo and Dokdo incubates a wealth of marinelives of both macroscopic and microscopic levels. Isolatedfrom the sea water sampled in Dokdo, Donghaeanadokdonensis DSW-6 represents a novel genus and speciesbelonging to the Cytophaga-Flavobacterium-Bacteroidesgroup that also includes Dokdonia donghaensis. Onestandard run of GS FLX produced 55 effective contigs(3.8 Mb of total contig length), from which genesencoding non-ribosomal peptide synthetases or polyketidesynthases were identified frequently in some short, high-depth contigs or at the boundaries of some contigs. Thesegenes are presumed to be involved in the biosynthesis ofsecondary metabolites in D. dokdonensis. To aid scaffoldingthe contigs and to resolve possible misassemblies, endsequencing of fosmid library has been performed. Functionalassignment of the genome was accomplished by searchingtranslated CDSs against public protein databases. Here, wepresent the results of D. dokdonensis genome sequenceanalysis. This genome information provides insights into abetter understanding on the evolution of the flavobacteriagroup, whose members are associated with a diversity oflifestyles and habitats. (Genomes 2008 : Paris(France), 2008)

Keywords : Donghaeana dokdonensis, flavobacteria,genome sequence

A-47Multidimensional comparative omics analysisof Escherichia coli B and K-12

Jihyun F. Kim1, Sung Ho Yoon1, Mee-Jung Han2, HaeyoungJeong1, Choong Hoon Lee3, Xiao-Xia Xia2, Ji-hoon Shim1,Sang Yup Lee2,4, and Tae Kwang Oh5

1Systems Microbiology Research Center, KRIBB2Center for Systems and Synthetic Biotechnology, andInstitute for the BioCentury, KAIST

3Department of Biological Sciences, KAIST4Department of Bio and Brain Engineering, and BioinformaticsResearch Center, KAIST

521C Frontier Microbial Genomics & Applications Center,KRIBB

Attaining a system-level understanding of an organismrequires integration of multifaceted holistic data. Here, wepresent a comprehensive analysis of genomes, transcriptomes,proteomes, and phenomes of closely related Escherichiacoli B and K-12. This 'multi-omics' comparative approachdemonstrates that B strains REL606 and BL21(DE3) arewell-suited for production of recombinant proteins througha greater capacity for amino acid biosynthesis, fewerproteases, and lack of flagella. They also have an additionaltype II secretion system and different cell wall and outermembrane compositions, which should be more feasible forsecretion of protein products. In contrast, MG1655 andW3110 show higher expression of heat shock genes and aremore tolerant to certain stress conditions. This comparativeand integrative systems approach thus provides a high-resolution system-wide view and reveals significantinsights into why the two groups manifest a number ofdistinct phenotypes. (Genomes 2008 : Paris (France), 2008)

Keywords : cell factory, comparative genomics, metabolicnetwork, microarray, systems biology

A-48Enzyme platform based on yeast TFP technologyfor the production of cellulosic bioethanol

Jong-Ok Jang1, Kwang-Mook Lim1, Jung-Hoon Bae1, Gi-Seok Kwon2, Jeong-Jun Yoon3, and Jung-Hoon Sohn1

1Systems Microbiology Research Center, KRIBB2School of Bioresource Science, Andong National University3New & Renewable Energy Team, KITECH

Sustainable energy sources are required to solve the crisesof the fossil energy and the global warming. Bioethanolfrom biomass is a promising alternative energy especiallyfor transportation. Up to date, most of bioethanol is producedfrom starch biomass, but it causes the feedstock priceincrease. To confront the rising demand of bioethanol,cellulosic biomass should be utilized as substrate forbioethanol. However, the recalcitrance of cellulosic biomassto digestion remains a significant obstacle for the production


of bioethanol. Cellulosic biomass needs 40 to 100 fold moreenzymes for saccharification compared to starch biomass.Thus, the major bottleneck for cellulosic bioethanol is thecost of cellulase enzymes. In this study, 4 cellulases, CEL1and CEL2 from Polyporus arcularius, EGL2 and BGL1 ofTrichoderma reesei were cost-effectively produced up toseveral grams/liter using yeast TFP(translational fusionpartner) technology. Optimal TFPs inducing hyper-secretion of each cellulase were selected from the pre-made TFP libraries. All cellulases showed proper activitieseven under yeast glycosylation pattern. Reconstitutedcellulase cocktail using 4 recombinant cellulases secretedfrom yeast could hydrolyze the insoluble filter papereffectively. (International Symposium & AnnualMeeting of the Korean Society for Microbiology andBiotechnology : Seoul, 2008)

Keywords : bioethanol, cellulase, recombinant yeast, secretion

A-49Development of FRET-based biosensors forrapid and quantitative analysis of sugars inbiological samples

Jae-Seok Ha1,3, Jae Jun Song2, Chul Soo Shin3, and Seung-Goo Lee1,*

1Systems Microbial Research Center, KRIBB2Molecular Bioprocess Research Center, KRIBB3Department of Biotechnology, Yonsei University

Genetically encoded biosensors were developed for thedetection of sugars in vitro and in vivo, which were constructedby fusion of bacterial periplasmic-binding proteins (PBPs)between enhanced cyan fluorescent protein (ECFP) andenhanced yellow fluorescent protein (EYFP). The PBPsundergo a large, ligand-mediated conformational change,thus transform their hinge-bend movements into fluorescenceresonance energy transfer (FRET) changes between twoGFP variants. Among various PBPs, five sugar-bindingproteins were introduced as recognition element of biosensors,and thus we achieved 5 sugar sensors for detection of theallose, arabinose, glucose, maltose, and ribose. To verifythe usability, these biosensors were applied to analysis ofvarious sugars in the broth during Escherichia coli cultivation. Asa result, the sugar concentrations calculated from the sugarsensors were comparable with results obtained by HPLC,and both sigmoid curves were identical each other. Inaddition, when FRET-based maltose sensors were employed asan in vivosensor, highly responsive FRET images weregenerated with application to the real-time analysis ofmaltose uptake in living Saccharomyces cerevisiae. Moreover,these FRET-based biosensors could be used as an in vivosensor for detection of the maltose in the cytoplasm of

living HeLa cells. (International Symposium & AnnualMeeting of the Korean Society for Microbiology andBiotechnology : Seoul, 2008)

Keywords : FRET, GFP, live molecular imaging, proteinengineering, protein-only biosensors

A-50Rapid cloning and expression of target genesby homologous recombination system, pRMT-iTGR

Jeong-Min Lee1, Eugene Rha1, Jae-Seok Ha1, Yu JungKim1, Jae Jun Song2, Minsu Ko3, and Seung-Goo Lee1,*

1Systems Microbiology Research Center, KRIBB2Molecular Bioprocess Research Center, KRIBB3Solgent Co.

This study presents a method for cloning and expressing atarget gene usinghomologous recombination in Escherichiacoli without the need for complicated gene manipulationsteps, such as restriction digestion and ligation between thevector and the insert. More particularly, wherein a host celltransformed with a recipient vector (pRMT) and a plasmidcontaining a recombinase system is introduced witha linearDNA fragment (iTGR) that contains the target gene and asequence with a homology to the recipient vector. Sincethe technique is completed by preparation of the iTGR usingPCR amplification of thetarget genes and transformation in E.coli, the gene cloning and expression can be performedwithout the need for a high degree of skill and expensiveenzyme costs. The working principle of pRMT-iTGR wasverified based on the cloning and simultaneous expressionof complicated target genes, including a single ORF (EGFP,DsRed, GFPuv), fusion protein (MBP-YFP), multi-gene(crtEBIY), and multi-transcripts (crtW, crtZ, crtEBIY). Thetargets were amplified by a PCR to produce iTGR fragments,and then introduced to the pRMT host by electroporation. Theresulting expression products were detected by the color ofthe metabolic products or a fluorescence change in thecolonies on agar plates. (International Symposium &Annual Meeting of the Korean Society for Microbiologyand Biotechnology : Seoul, 2008)

Keywords : genome engineering, homologous recombination,lambda red recombinase, recombineering,synthetic biology

A-51Catalytic protein particles based on cellulose-


binding domain fusion

Su-Lim Choi1,3, Sun-Hwa Kim1, Jae Jun Song2, Young HaRhee3, and Seung-Goo Lee1,*

1Systems Microbiology Research Center, KRIBB2Molecular Bioprocess Research Center, KRIBB3Department of Microbiology, Chungnam National University

The Cellulomonas fimi CBD consisting of solely ß-strandstends to accelerate insoluble aggregates in Escherichiacoli, such as ß-amyloid proteins. Interestingly, when greenor red fluorescent proteins were fused with the C. fimiCBD on the C-terminal they formed aggregated yet highlyfluorescent particles in E. coli cells. Moreover, when thecatalytic proteins, GusA or BglA, were fused with the C.fimi CBD, both proteins formed insoluble particles in E.coli, exhibiting a 7~2.5-fold higher activity than in thesoluble fractions. The catalytic activity of the proteinparticles was increased when broken by sonification ortreatment with anti-aggregation chemicals, suggesting thatthe enzyme trapped inside the particles is catalyticallyactive. When separated from the E. coli cellsand treatedwith glutaraldehyde as a cross-linker, the active proteinparticles could be recovered in a high yield and repeatedlyused as a biocatalyst for the hydrolysis of a ß-glycosidesubstrate, thereby demonstrating that the CBD of C. fimimay be useful for the generation of catalytic proteinparticles in E. coli. (International Symposium & AnnualMeeting of the Korean Society for Microbiology andBiotechnology : Seoul, 2008)

Keywords : ß-glucuronidase, ß-glycosidase, cellulose bindingdomain, DsRed, EGFP, protein engineering

A-52Protein engineering supported by homologymodeling and docking simulation

Su-Jin Kim, Su-Lim Choi, Eugene Rha, Jae-Seok Ha, Jae-Gu Pan, and Seung-Goo Lee*


Predicting the 3-dimensional structures of proteins andsimulating molecular docking are now popular in manyfields of biotechnology, including enzyme engineering.Meanwhile, directed evolution technologies have had asignificant impact on enzyme engineering, although therepetitive screening of a gene library creates problems dueto the limited throughput of the screening stage relative tothe vast genetic diversity. Thus, various studies havesuggested that structure prediction may be an effectivemethod for improving directed evolution technologies ifadequate structural homologues are available. Docking

simulation can also be applied to optimize the binding siteand improve substrate specificity. Therefore, to build aworking example, this study employed homology-basedmodeling and a docking simulation for several catalyticenzymes and microbial binding proteins based onDiscovery Studio 2.0. The same principles were alsoapplied to a comprehensive estimation of the active siteand an evaluation of the binding free energy of smallmolecule-protein interactions. (International Symposium& Annual Meeting of the Korean Society forMicrobiology and Biotechnology : Seoul, 2008)

Keywords : directed evolution, homology modeling, moleculardocking, protein engineering

A-53Novel cold-adapted alkaline lipase from intertidalflat metagenome and proposal of a new familyof bacterial lipase

Eun-Young Kim, Ki-Hoon Oh, Mi-Hwa Lee, Chul-HyungKang, Tae-Kwang Oh, and Jung-Hoon Yoon*


A lipase (LipEH166) was selected from a Koreanintertidal flat sediment metagenome via activity screening.This putative lipase gene was 1,143 bp encodingapproximately 382 amino acids, 42 kDa. LipEH166effectively hydrolyzed -nitrophenyl esters, whose carbonchain was longer than C8, triacylglycerol, and natural oils.This enzyme displayed the apparent optimum activityaround 30 °C and retained approximately 50 % activitywithin 5 to 20 °C. The thermal unfolding curve which wasobtained by the change of ellipticity at 222 nm showedthat LipEH166 started to be unfolded around 25 °C andfully denatured at 55 °C. It exhibited significant activityand stability under alkaline conditions. Thesecharacteristics show that LipEH166 is a cold-adaptedalkaline lipase. Most metal ions had little effect on theenzymatic activity. Copper and zinc ions, however,showed an inhibitory effect. Detergents severely inhibitedactivity of the enzyme. In addition, LipEH166 and relateduncharacterized secreted proteins appear to be included ina novel lipolytic enzyme family as they have no significantamino acids similarity to any known lipases or esterases.Remarkably, stability and activity of LipEH166 overbroad alkaline conditions and low temperatures represent aunique feature that does not appear to be related to anenvironment derived from it. (IUMS 2008-Meetings ofthe Three Division of the International Union ofMicrobiological Societies : Istanbul (Turkey), 2008)

Keywords : bacterial lipase, LipEH166, metagenome


A-54Mycotoxins, a group of fungal secondarymetabolites, can induce motility of bacterial cells

Soo-Young Park1, Rumi Kim2, Choong-Min Ryu1, Soo-KeunChoi1, Choong-Hwan Lee3, and Seung-Hwan Park1

1Systems Microbiology Research Center, KRIBB 2Institute of Hadong Green Tea3Division of Bioscience and Biotechnology, Konkuk University

Paenibacillus polymyxa, a Gram-positive low-G+C spore-forming bacterium, is a soil bacterium belongs to the groupof plant growth promoting rhizobacteria and exhibitsantagonizing ability against bacterial and/or fungal plantpathogens. We observed that the swarming motility of P.polymyxa strain E681, isolated from the rhizosphere ofwinter barley, was greatly increased by adjacent growth ofa fungal strain, Penicillium citrinum KCTC6549. From aculture supernatant of the fungal strain which showed thesame effect an active compound was purified and identifiedas citrinin by LC/MS analysis. On TSA plates containingcitrinin the P. polymyxa E681 strain showed remarkablyincreased swarming motility. Microscopic observation ofthe P. polymyxa cells revealed that the expression of flagellawas increased by citrinin dose dependently, and thisphenomenon was supported by RT-PCR data which showingincreased transcription level of some genes related toexpression of flagella of P. polymyxa E681 in the presenceof citrinin. The citrinin effect on enhancing bacterial swarmmotility was also observed in P. polymyxa ATCC842, atype strain of Paenibacillus species, Bacillus subtilis C9and Pseudomonas fluorescence pf5. Interestingly, othermycotoxins including fusaric acid, ochratoxin A and patulinalso were capable of inducing swarm motility of P. polymyxaE681. The results presented here will provide an insightfor understanding ecological role of fungal mycotoxinsbesides their well-known toxicity to man and/or animalssuch as nephrotoxins. That is, we found that mycotoxins,has a newly identified function of inducing bacterial motility.(The 48th MSK Annual Meeting and 2008 InternationalSymposium on Microbiology : Daejeon, 2008)

Keywords : citrinin, mycotoxin, Paenibacillus polymyxa,swarm motility

A-55Functional analysis of the fusaricidin biosyntheticgene of Paenibacillus polymyxa E681 and apossible role of fusaricidin in biofilm formation

Soo-Young Park1, Soo-Keun Choi1, Jihyun F Kim1,Choong-Hwan Lee2, and Seung-Hwan Park1

1Systems Microbiology Research Center, KRIBB

2Division of Bioscience and Biotechnology, KonkukUniversity

Fusaricidin, a peptide antibiotic consisting of six aminoacids, has been identified as a potential antifungal agentfrom Paenibacillus polymyxa. Recently, we reported thecomplete sequence of the fusaricidin synthetase gene (fusA)identified from the genome sequence of a rhizobacterium,P. polymyxa E681. The gene encodes a polypeptide consistingof six modules in a single open-reading frame. Theinactivation of fusA led to the complete loss of antifungalactivity against Fusarium oxysporum. LC/MS analysisconfirmed the incapability of fusaricidin production in thefusA mutant strain, thus demonstrating that fusA isinvolved in fusaricidin biosynthesis. On the other hand wehave constructed several a recombinant E. coli strains strainharboring fusA and sfp (encoding phosphopantetheinyltransferase of Bacillus subtilis), and the recombinantstrains showed clear growth-inhibiting activities against F.oxysporum. This result means that the fusA gene wasexpressed successfully in E. coli. Interestingly, we observedthat major part of fusaricidin was extracted in cell pelletsnot in culture supernatant and the fusA mutant of E681strain formed a different shape of colony on solid mediumand also showed significantly reduced level of biofilmwhen compared with parent strain. Our findings suggest thatthe peptide antibiotic fusaricidin might be linked tobacterial cell wall or membrane and involved in thedevelopment expression of biofilm matrix. (InternationalMeeting of the Federation of Korean MicrobiologicalSocieties : Seoul, 2008)

Keywords : biofilm, fusA, fusaricidin, Paenibacilluspolymyxa

A-56Airborne induction and priming of plant defensesagainst a bacterial pathogen

Hwe-Su Yi1,2, Martin Heil3, Daniel Ballhorn4, and Choong-Min Ryu1

1Systems Microbiology Research Center, KRIBB2School of Life Science, Kyungpook National University3Departamento de Ingenieria Genetica, CINVESTAVIrapuato, Mexico

4Department of General Botany-Plant Ecology, Universityof Duisburg-Essen, Germany

Herbivore-induced plant volatiles affect the systemicresponse of plants to local damage and hence represent akind of plant hormone. These signals are transportedoutside the plants and can, therewith, also lead to 'plant-plant communication', a defense induction in yet undamagedplants that grow close to damaged neighbors. We observed


a similar phenomenon in the context of disease resistance.Lima bean plants became more resistant against a bacterialpathogen, Pseudomonas syringae pv. syringae, when theywere located close to conspecific neighbors in whichsystemic acquired resistance (SAR) to pathogens had beenchemically induced with benzothiadiazole (BTH) orbiologically by infection with avirulent P. syringae. Challengeinoculation after exposure to induced and non-inducedplants revealed that the air coming from induced plantsmainly primed resistance, since expression of pathogenesis-related protein 2 (PR-2) was significantly stronger inexposed than in non-exposed individuals when the plantswere subsequently challenged by P. syringae. Amongothers, the plant-derived volatile, nonanal, was present inthe headspace of BTH-treated plants and significantlyenhanced PR-2 expression in the exposed plants. Negativeeffects on growth of BTH-treated plants, which usuallyoccur as a consequence of high costs of direct resistanceinduction, were not observed in VOC-exposed plants.Volatile mediated priming appears to be a highly attractivemeans for the tailoring of SAR against plant pathogens.(KSPP International Symposium - New Approaches toPlant Disease Management : Muju, 2008)

Keywords : PGPR, PR, SA, SAR, VOC

A-57Insight into plant defense signalings against Ralstoniasolanacearum in Nicotiana benthamiana

Hyun-Kyung Kim1 and Choong-Min Ryu1,2

1Systems Microbiology Research Center, KRIBB2Field of Functional Genomics, School of Science, UST

Ralstonia solanacearum is a Gram negative and rod-shaped bacterium that cause bacterial wilt in over 200plant species including solanaceous family such as tomato,tobacco and pepper. R. solanacearum is a typical soil-borne pathogenic bacterium that infects root hairs andmultiplies in xylem vesselswhere produces excessiveexopolysaccharides resulting in blocking water transportationfrom root to foliar parts and finally inducing wiltsymptoms. Here we utilized R. solanacearum strainsGMI1000 and 1931 and Nicotiana benthamiana as amodel system to understand plant defense signalingmechanisms against bacterial wilt disease. Up to now, N.benthamiana has known as a nonhost plant to R.solanacearumdue to elicitation of hytpersensitive responsewhen infiltrated on the leaf. However we selected virulentstrain 1931 which cause severe wilt symptom 7 days afterdrench-inoculations while avirulent strain GMI1000 didnot show any symptoms. To dissectdefense signaling,virus-induced gene silencing was employed with suppressionof plant defense signaling related genes that include

salicylic acid, jasmonic acid, ethylene and protein-degrading dependent pathway genes. Through massivescreening, we defined many defense-related genesincluding jasmonic acid signaling genes (e.g. NbCOI1)that previously known as involvement on resistance againstinsect and necrotizing pathogens. Our result indicates thatplant defenses against a bacterial wilt pathogen can befollowed a unique signaling pathway. (KSPP InternationalSymposium - New Approaches to Plant DiseaseManagement : Muju, 2008)

Keywords : jasmonic acid, NbCOI1, Nicotiana benthamiana,Ralstonia solanacearum strains GMI1000 and 1931

A-58Plant innate immunity by bacterial geneticmaterials

Boyoung Lee, Soohyun Lee, Jung Wook Yang, and Choong-Min Ryu


Plant need to protect themselves against multiple pathogenson the infected site as well as at the distal part. Recentreport presented that plant percept general cue frommicrobes referred to as pathogen associated molecularpatterns (PAMPs). The plant innate immunity elicited byrecognition of PAMPs such as fllagellin and elongationfactor and structural materials has intensively studied.Here, we provide a evidence that bacterial genetic materialcan be a PAMPs to increase plant defense responsesagainst bacterial pathogen. Infiltration of RNA fromPseudomonas syringae pv. tomato DC3000 resulted inenhancing resistant to subsequent infection by P. syringaepv. tomato DC3000 compared to water control onArabidopsis thaliana. 50K Arabidopsis microarray wasemployed to analyze RNA transcripts of Arabidopsisplants following treatment of bacterial RNA frompathogenic bacteria P. syringae pv. tomato DC3000. Thetranscriptional expression of plant defense related genesand its transcription factor was up-regulated. Our resultsindicate that bacterial genetic material can be a potentialPAMP. (9th International Congress of Plant Pathology: Torino(Italy), 2008)

Keywords : Arabidopsis thaliana, bacterial RNA, ISR,MAMPs, PAMPs, plant immunity

A-59

Role of priming resistance genes on bacilli-elicited induced systemic resistance


Jung Wook Yang1,2 and Choong-Min Ryu1

1Systems Microbiology Research Center, KRIBB2Department of Agriculture and Life Science, ChungNamNational University

Within an ecosystem, many organisms survive in a constantrelationship with their environment and among the organismswhich can be positive or negative depending on the type ofneed required by each one of them. Many root-colonizingbacterial species including Bacillus spp. (bacilli) havebeen utilized as microbial inoculants to decrease plantdiseases and to increase plant productivity. We attemptedto screen endospore-forming bacilli strains that is capableto elicit induced systemic resistance (ISR) against bacterialspot disease caused by Xanthomonas axonopodis pv.vesicatoria on pepper. We focused on define plant defensepriming genes that make plants rapidly and stronglydefense responses against X. axonopodis pv. vesicatoria.Two week-old pepper seedlings were drench-inoculatedwith bacterial suspension of 741 bacilli strains that wereisolated from diverse soils and plant samples in SouthKorea. ISR was assessed by disease severity (0 5 diseasescale) at seven days after pathogen challenge. After seriesof screening in the greenhouse, Bacillus subtilis strainBS101 and B. megaterium strain BS107 were selected forfurther experiments. In addition to elicitation of ISR, thetwo strains BS101 and BS107 enhanced fresh weight asmuch as 60 and 44 % compared to control treatmentrespectively. To understand priming status during elicitationof ISR by strain BS 107, we employed pepper oligonucleotidemicroarrays to detect differentially expressed genes onbacterial treated pepper plant leaves after pathogen challenge.The selected putative priming genes were validated byquantitative real-time PCR. Our results provide new insightinto bacterial role on elicitation of ISR in pepper plants.(KSPP International Symposium - New Approaches toPlant Disease Management : Muju, 2008)

Keywords : induced systemic resistance, ISR, priming,Xanthomonas axonopodis pv. vesicatoria

A-60Improved thermostability and acetic acid toleranceof Escherichia coli by directed evolution ofMetA

Elena Mordukhova1, Hee-Soon Lee2, and Jae-Gu Pan1

1Systems Microbiology Research Center, KRIBB2Korea Ocean Research & Development Institute

In Escherichia coli, growth Is limited at elevated temperaturesmainly because of the instability of a single enzyme,homoserine o-succinyltransferase(MetA), the first enzymein the methionine biosynthesis pathway. The metA gene

from the thermophile Geobacillus kaustophilus cloned intothe E.coli chromosome was found to enhance growth ofthe host strain at elevated temperature(440C), thusconfirming the limited growth of E.coli due to MetAinstability. In order to improve E.coli growth at highertemperatures, we used random mutagenesis to obtain athermostable MetA protein. Sequencing of the thermotolerantmutant showed five amino acid substitutions: Ser61Thr,Glu213Val, Ile229Thr, Asn267Asp and Asn271Lys. AnE.coli strain with the mutated metA chromosomally insertedshowed accelerated growth in the temperature range 34-440C. We used the site-directed metA mutants to identifytwo amino acid residues responsible for both heat and acidsensitivity of MetA. Replacement of asparagine-267 withaspartic acid and isoleucine-229 with threonine stabilizedthe protein. The thermostable MetA showed less aggregationin vivo at higher temperature as well as with acetic acidtreatment. The data presented here are the first to showimproved E.coli growth under stress conditions solely dueto the MetA stabilization. (Metabolic Engineering 9 : PuertoVallarta (Mexico), 2008)

Keywords : E. coli, growthrate, MetA, metabolicengineering,thermotoelrance

A-61Autoinduction expression system in Escherichia coli

Eui-Joong Kim2, Soo-An Shin1, Kook-Ki Hong1, and Jae-Gu Pan1

1Systems Microbiology Research Center, KRIBB2GenoFocus

Acetyl-CoA synthetase(Acs) is responsible for theconsumption of acetate accumulated during growth phase.Repressed in the presence of glucose and highly expressedat the stationary phase and additionally induced by aceticacid, it suits many aspects of the ideal expression systemin synthetic biology applications. pACE plasmid vectorsderived from a pUC containing acs promoter wasconstructed and tested for the expression of LacZ andGFPuv as reporter proteins. Expression of the reporterproteins was tightly controlled with the carbon source:When glucose was used as a sole carbon source, theexpression of LacZ was completely repressed duringexponential growth phase and induced only at thestationary phase. When glycerol or organic acids wereused, the LacZ was constitutively expressed. The uniformexpressionof GFPuv in each cell at the stationary phase wasconfirmed by flow cytometry analysis. This acs-basedexpression would provide a novel autoinduction system, avarious expression systems available currently in E.coli.(Synthetic Biology4.0 : HongKong, 2008)

Keywords : Acs, autoinduction, E. coli, expression system


A-62Crystal structure of fully oxidized human thioredoxin1containing disulfide between Cys62 and Cys69

Jungwon Hwang1,2, Jie-Oh Lee2, Tae-Kwang Oh1, and MyungHee Kim1

1Systems Microbiology Research Center, KRIBB2Department of Chemistry, KAIST

Human thioredoxin1 (hTRX1) is a small 12-kD oxidoreductaseenzyme consisting of 105 amino acids and containing adithiol/disulfide active site with multiple cellular functions.This enzyme has activity as a cellular reductase by a dithiol-disulfide exchange reaction using two cysteine residues(Cys32 and Cys35) in the conserved active site sequence.Apart from the two cysteines, there are three additionalconserved cysteines, Cys62, Cys69, and Cys73 in themammalian TRX, which have not been known to theirbiological functions. Although it has been identified thatthe Cys73 residue is involved in dimerization of hTRX viaan intermolecular disulfide bond formation between Cys73of each monomer in the oxidized state, biological functionof the Cys62 and Cys69 residues in the non-active remain tobe fully elucidated. In the previous paper, researchersproposed that the formation of a disulfide bond betweenCys62 and Cys69 could give a way to transiently inhibithTRX activity for redox signaling or oxidative stress.Furthermore, they proposed a model structure of the non-active site disulfide in the hTRX. Here, we present the high-resolution crystal structure of fully oxidized hTRX1, whichshows an intramolecular disulfide bond between Cys62and Cys69. The disulfide bond formation disengages a helixproximal to the active site and results in a conformationalchange of the hTRX enzyme, providing a structural basisfor understanding the regulation mechanism of redoxsignaling or oxidative stress. (XXI Congress and GeneralAssembly of International Union of Crystallography :Osaka (Japan), 2008)

Keywords : crystal structure, hTRX1, Human thioredoxin1,oxidoreductase enzyme

A-63Genomic basis of the two E. coli workhorsestrains for their improved capacity to synthesizemembrane proteins at high levels

Soon-Kyeong Kwon1,2, John E. Walker3, and Jihyun F.Kim1,2

1Systems Microbiology Research Center, KRIBB 2Functional Genomics Field, School of Science, UST3Dunn Human Nutrition Unit, Medical Research Council,Cambridge, United Kingdom

Membrane proteins constitute about 30% of the total proteinsin a living organism, and play important roles in cellmetabolism. They are the causes of many metabolic diseasesand therefore be regarded as major drug targets. However,though over-expression of membrane proteins is pivotalfor their studies through biochemical, structural, andfunctional approaches not to mention their applications andcommercial developments, it often is considered a majorbottleneck due to toxicity and other problems. Molecularstructures of the membrane proteins have been verified forless than 1% of them in spite of their impact. To overcomethe hurdle, E. coli strains C41(DE3) and C43(DE3) thatare superior in toxic-protein expression including membraneproteins were selected from spontaneous mutants of thecommon protein expression host BL21(DE3). To understandthe genetic and biochemical basis of this unique feature,the genome sequences of C41(DE3) and C43(DE3) weredetermined using GS FLX to result in 43-55 Mb each ofhigh-quality reads that covers each genome about ten totwelve times. Comparative analysis revealed that there arefive SNPs in C41(DE3) and six in C43(DE3) as comparedto BL21(DE3). Interestingly and perplexingly, only one ofthem overlaps between the two strains. Also, there are twoIS-mediated deletions that have been observed - one in bothC41(DE3) and C43(DE3) and the other only in C43(DE3).In order to figure out which of these changes areresponsible for the traits of C41(DE3) and C43(DE3),individual mutations are being validated in BL21(DE3).Recombination cassettes have been designed to mediate targetedmutation in the chromosome of BL21(DE3), and expressionlevels of a membrane protein are being measured in everyestablished BL21(DE3) mutant strain. (Synthetic Biology4.0 : Hong Kong, 2008)

Keywords : E. coli, membrane proteins, BL21(DE3)

A-64Characterization and molecular modeling of athermophilic and alkaliphilic galactokinasefrom Thermus caldophilus GK24

Dooil Kim, BoHyun Park, and Dae-Sil Lee


The gene encoding for a galactokinase (TcaGalK)from anextremely thermophilic Thermus caldophilus GK24 wascloned and the biochemical characteristics of the resultingrecombinant protein as well as its substrate specificitywere examined. The Tca GalK was over expressed from apTrc99A-based plasmid under the control of the trcpromoterin E.coli. There combinant purified enzyme wasoptimally active in the range of 75-90˚C, showing the highthermostability, particularly in the presence of ATP. Andthe Tca GalK enzyme had unique optimum pH range of


9.0 and 10.0 at 70˚C. The Tca GalK phosphorylated notonly D-galactose to D-galactose-1-phosphate in thepresence of ATP-Mg2+, but also did2-deoxy-D-galactose,D-galactosamine as well. Moreover, the enzyme was ableto efficiently utilize TTP, GTP, UTP, or CTP in the placeof ATP as phosphate donor. In order to understand the sugarspecificity at a molecular level, the enzyme-sugar bindingmodel shave constructed. It has been shown that the sugarspecificity is probably dependent on the interaction energyoccurred by the positional proximity of sugars bound inthe active site of the enzyme. (Spring Symposium of theKorean Society for Glycoscience : Seoul, 2008)

Keywords : alkaliphilic galactokinase, GK24, Tca GalK,Termus caldophilus

Members

Director : Jae Gu Pan ([email protected])

Principal Researcher Dae Sil Lee ([email protected])Eui Sung Choi ([email protected])Seung Hwan Park ([email protected])Jung Hoon Sohn ([email protected])Seung Goo Lee ([email protected])Jihyn F. Kim ([email protected])Jung Hoon Yoon ([email protected])Choong Min Ryu ([email protected])Myung Hee Kim ([email protected])

Senior Researcher Soo Keun Choi ([email protected])Hae Young Jeong ([email protected])Sung Ho Yoon ([email protected])

Post-DocJung Hoon Bae ([email protected])Jung Don Bae ([email protected])Eun Ah Cho ([email protected])Jae Seok Ha ([email protected])Doo Il Kim ([email protected])Soo Young Park ([email protected])Won Chan Choi ([email protected])Ju Yeon Song ([email protected])Elena ([email protected])Eu Gene Rha ([email protected])Chang Soo Lee ([email protected])Ki Hoon Oh ([email protected])Ji Hee Han ([email protected])

Researcher Dong Su Yu ([email protected])Ji Hoon Shim ([email protected])Soon Kyeong Kwon ([email protected])Ki Jin Yu ([email protected])Jeong Min Lee ([email protected])Su Lim Choi ([email protected])Su Jin Kim ([email protected])Hwe Su Yi ([email protected])Hyun Kyung Kim ([email protected])Bo Young Lee ([email protected])Jung Wook Yang ([email protected])Jung Won Hwang ([email protected])


B-13Transgenic crops with enhanced tolerance tomultiple environmental stresses for sustainableagriculture

Sang-Soo Kwak, Raza Ahmad, Li Tang, Soon Lim, Yun-Hee Kim, Myung-Duck Kim, and Haeng-Soon Lee

Environmental Biotechnology Research Center, KRIBB

The dramatic increase in population accompanied by rapidindustrialization in developing countries has causedimbalances in the supply of food and energy. To cope withthese global crises over food and energy supplies as well asenvironmental problems, it is urgently required to developnew crop varieties to be grown in marginal lands includingdesertification lands by molecular breeding. In particular,cash crops to increase the income of local farmers inmarginal areas should be developed. Oxidative stressderived from reactive oxygen species (ROS) is one of themajor factors causing injury to plants exposed toenvironmental stress. In order to develop varioustransgenic plants with an enhanced tolerance to multipleenvironmental stresses, we are focusing on themanipulation of antioxidant genes in plant cells. Recentlywe developed several transgenic crops such assweetpotato, potato, tall fescue expressing genes of bothCu/Zn superoxide dismutase (CuZnSOD) and ascorbateperoxidase (APX) in the chloroplasts under the control ofan oxidative stress-inducible peroxidase (SWPA2) promoter(Kim et al. 2003)(referred to SSA plants). Transgenic cropsexpressing nucleoside diphosphate kinase 2(NDPK2)(Moon et al. 2003) gene in cytosols underSWPA2 promoter (SN plants) were also developed. Inaddition, transgenic potato plants with the ability tosynthesize glycinebetaine in chloroplasts under the SWPA2promoter (SC plants) were developed via the introductionof the bacterial choline oxidase (codA) gene. In the presentation,characterization of transgenic plants such as SSA plantsand SN plants will be introduced in term of enhancedtolerance to multiple environmental stresses in detail. (16th

Congress of the Federation of European Societies of PlantBiology (FEPBS) & 8th International Peroxidase Symposium: Tampere (Finland), 2008)

Keywords : ascorbate peroxidase, choline oxidase, nucleosidediphosphate kinase 2, oxidative stress, peroxidasepromoter, potato, superoxide dismutase,sweetpotato

B-14Expression of sweetpotato IbMYB1 transcriptionfactor gene induced anthocyanin accumulationin plants

Cha Young Kim1, Sang-Yeop Seong1,2, Young Ock Ahn1,Kwan Sam Choi2, Joon-Seol Lee3, Hang-Soon Lee1, andSang-Soo Kwak1

1Environmental Biotechnology Research Center, KRIBB2Department of Applied Biology, Chungnam NationalUniversity

3Mokpo Experiment Station, National Institute of CropScience, RDA

MYB transcription MYB transcription factors play importantroles in transcriptional regulation of many secondarymetabolites including anthocyanins. We cloned the R2R3-MYB IbMYB1 cDNA from purple-fleshed sweetpotato(Ipomoea batatas L. cv. Sinjami). The transcript of IbMYB1was specifically expressed only in the purple-fleshedstorage roots, but not in other cultivars. To generate transgenicsweetpotato by use of intragenic vector system, we firstevaluated the utilization of the sweetpotato IbMYB1transcription factor as a visible selectable marker. TheIbMYB1 gene was expressed in plants under the control ofCaMV 35S promoter, sweetpotato sporamin promoter andSWPA2 promoter, respectively. Here, we show that theexpression of IbMYB1 induced anthocyanin accumulationin tobacco leavesin transient assays. In addition, stabletransgenic Arabidopsis plants produced highly pigmentedseedling plants and generated a deep purple color in seeds.These results suggest that this transcription factor can beapplicable to a useful selectable marker for developmentof transgenic sweetpotato with intragenic vector system, aswell as the production of anthocyanin in other plant species.(Joint Meeting of The Botanical Society of Korea and TheKorean Society for Plant Biotechnology : Busan, 2008)

Keywords : anthocyanin, intragenic vector, MYB, selectablemarker, sweetpotato

B-15Characterization of sweetpotato orange geneinvolved in carotenoid biosynthesis

Young Ock Ahn1, Sun Ha Kim1,2, Cha Young Kim1, Joon-Seol Lee3, Kwan Sam Choi2, Haeng-Soon Lee1, and Sang-Soo Kwak1

1Environmental Biotechnology Research Center, KRIBB2Department of Applied Biology, Chungnam NationalUniversity

3Mokpo Experiment Station, National Institute of CropScience, RDA

Sweetpotato (Ipomoea batats L.) is an important food andindustrial crop to produce starch and useful componentsincluding various antioxidants. Yellow-fleshed sweetpotatocultivars with high levels of carotenoids are very popular

Environmental Biotechnology Research Center


as the healthy food. We are trying to develop industrialsweetpotato with enhanced tolerance to environmentalstresses by manipulation of antioxidant genes for sustainableagriculture. In this study, orange gene (IbOr) involved incarotenoid biosynthesis was isolated from the leaves ofyellow-fleshed sweetpotato (cv. Sinwhangmi) andcharacterized. The deduced IbOrshowed a high homologywith various plant orange genes and has a well-knownfeature of Cys-rich zinc finger domain specific to DnaJ-likemolecular chaperones. IbOr was expressed predominantlyin leaves and fibrous roots. We investigated the expressionpatterns of genes related to carotenoid biosynthesis andIbOr gene in response to various hormone treatments.(Joint Meeting of The Botanical Society of Korea and TheKorean Society for Plant Biotechnology : Busan, 2008)

Keywords : antioxidant, carotenoid, orange, sweetpotato

B-16

Enhanced tolerance of transgenic potato plantsexpressing 2-Cys peroxiredoxin in chloroplastsagainst oxidative stress and high temperature

Myoung Duck Kim1, Yun-Hee Kim1, Bo-Young Jang1,Sang Yeol Lee2, Sang-Soo Kwak1, and Haeng-Soon Lee1

1Environmental Biotechnology Research Center, KRIBB 2Department of Biochemistry, Gyeongsang National University

Oxidative stress is one of major factors causing injury to plantsexposed to environmental stresses. Under the oxidativestress or heat shock conditions, 2-Cys peroxiredoxin (Prx)in eukaryotes rapidly switches its protein structure toprotect host cells against external stresses. Chloroplast isrich source of reactive oxygen species due to its highenergetic photosynthetic electron transport system and agenerous supply of oxygens. In this study, transgenicpotato (cv. Atlantic) by expressing 2-Cys Prx gene in thechloroplasts under the control of an oxidative stress-inducible SWPA2 promoter or enhanced CaMV 35Spromoter, respectively (referred to as SP and EP plants)were generated and characterized in terms of stress. Theintegration of foreign gene in transgenic plants was confirmedby PCR and Southern blot analysis. When 300 µM methylviologen was sprayed onto the whole plants, SP and EPplants showed less decrease of the photosynthetic activitycompared to non-transgenic plants. The further characterizationincluding high temperature tolerance in transgenic potatoplants will be introduced in detail. (Joint Meeting of TheBotanical Society of Korea and The Korean Society forPlant Biotechnology : Busan, 2008)

Keywords : 2-Cys peroxiredoxin, oxidative stress-inducibleSWPA2 promoter, potato

B-17Transgenic sweetpotato plants expressing a cold-inducible zinc finger protein, SCOF-1

Myoung Duck Kim1, Kyoung-Sil Yang1, Hyun-Sook Woo1,Jung-Mae Liu1, Suk-Yoon Kwon2, Sang-Soo Kwak1, andHaeng-Soon Lee1

1Environmental Biotechnology Research Center, KRIBB 2Plant Genomics Research Center, KRIBB

Sweetpotato [Ipomoea batatas (L.) Lam.] is one of themost important crops to produce food, feed and industrialmaterials. However, sweetpotato is sensitive to low temperaturedue to its origin from tropical and subtropical regions.SCOF-1 encodes a cold-inducible zinc finger protein insoybean, which functions as a positive regulator of cold-regulated gene expression (Kim et al. 2001). In this study,to develop the transgenic sweetpotato (cv. Yulmi) with enhancedtolerance to low temperature, we generated SCOF-1expressing transgenic sweetpotato under the control of anoxidative stress-inducible SWPA2 promoter (Kim et al.2003) by Agrobacterium-mediated transformation.Hygromycin-resistant embryogenic calli were selected onselection medium and converted into plantlets whichdeveloped into morphologically normal plants. Putativetransgenic plants were selected by PCR with hygromycinand SCOF-1 specific primers. The expression level of theSCOF-1 gene was confirmed by RT-PCR analysis in theleaves of transgenic sweetpotato plants. Further characterizationof transgenic plants is under study response to cold stress. (3rd

China-Japan-Korea Workshop on Sweetpotato : Beijing(China), 2008)

Keywords : Agrobacterium-mediated transformation, coldstress, SCOF-1, sweetpotato, SWPA2 promoter

B-18Transgenic sweetpotato plants overexpressingnucleoside diphosphate kinase 2 showed increasesof antioxidant enzymes activities and enhancedtolerance to multiple environmental stresses

Yun-Hee Kim1, Hyun-Sook Woo1, Soon Lim1, Suk-YoonKwon2, Haeng-Soon Lee1, Dae-Jin Yun3, and Sang-SooKwak1

1Environmental Biotechnology Research Center, KRIBB 2Plant Genomics Research Center, KRIBB 3Division of Applied Life Science, Gyeongsang NationalUniversity

Oxidative stress is one of the important factors causinginjury to plants exposed to environmental stress. Nucleosidediphosphate kinase 2 (NDPK2) is known to regulate the


expression of antioxidant genes via hydrogen peroxide (H2O2)-dependent signal pathway in plant under stress conditions.In this study, we developed transgenic sweetpotato plants(Ipomoea batatas L. cv. Yulmi) expressing ArabidopsisNDPK2 (AtNDPK2) gene under the control of an oxidativestress-inducible sweetpotato peroxidase (SWPA2) promoter(referred to as SN plants), and evaluated their tolerance tovarious environmental stress, including methyl viologen(MV)-mediated oxidative stress, cold, salt and drought stresses.When leaf discs of SN plants were subjected to 5 M MV,they showed less damage compare to non-transformed (NT)plants. Expression level of AtNDPK2 gene and NDPKactivity in SN plants following MV treatment wellreflected the plant phenotype. Interestingly, the activitiesof H2O2-scavenging enzymes such as peroxidase, ascorbateperoxidase and catalase were also increased in MV-treatedSN plants. In addition, SN plants showed enhancedtolerance to cold, high salinity and drought stresses byincrease of the H2O2 scavenging enzyme activities. Theseresults indicate that overexpression of AtNDPK2 under thecontrol of SWPA2 promoter in sweetpotato might efficientlyregulate the oxidative stress derived from various environmentalstresses. Therefore, SNplants would be a useful plant crop forcommercial cultivation under unfavorable growth conditions.(Joint Meeting of The Botanical Society of Korea and TheKorean Society for Plant Biotechnology : Busan, 2008)

Keywords : nucleoside diphosphate kinase 2, oxidativestress, peroxidase promoter, sweetpotato

B-19Differential response of thirteen sweetpotatoperoxidase genes under various abiotic stresses

Yun-Hee Kim, Haeng-Soon Lee, and Sang-Soo Kwak


Plants possess a large set of the class III peroxidase (PODE.C. 1.11.1.7). Recent genomic sequence analyses haverevealed that there are 73 POD genes in Arabidopsis and138 genes in rice. The roles of diverse POD isoenzymeshave been implicated in broad range of physiologicalprocesses, including the response to various abioticstresses. In our previous studies, 10 POD cDNAs wereisolated from cell cultures of sweetpotato (Ipomoeabatatas) (Mol Gen Genet 255:382-397, 1997; 261:941-947, 1999; Mol Gen Genome 269:542-552, 2003), and 3cDNAs were isolated from dehydrated-fibrous roots ofsweetpotato (Biochem Mol Biol Rep, in press) via thescreening of a cDNA library. In this study, to understandthe physiological function of each POD isoenzyme insweetpotato, their expressions were assessed to characterizefunctions of each POD in relation to environmentalstresses such as drought, salt, chemicals and air pollutions.

Among them, the expressions of 4 acidic PODs, such asswpa1, swpa2, swpa3 and swpa4, were highly induced byseveral abiotic stresses, suggesting that these POD genesare inducible by various stress conditions. Interestingly,our studies indicated that the responses of the 4 acidicPOD genes under various abiotic stresses are well-correlatedwith the phylogenetic tree of the 13 POD genes on thebasis of amino acid sequences. The results suggested thateach POD gene might be specifically involved in theevolutional adaptation to each environmental stress. (16th

Congress of the Federation of European Societies ofPlant Biology (FEPBS) & 8th International PeroxidaseSymposium : Tampere (Finland), 2008)

Keywords : environmental stress, peroxidase, sweetpotato

B-20Transgenic sweetpotato plants expressing spikeprotein of porcine epidemic diarrhea virus

Kyoung-Sil Yang1, Suk-Yoon Kwon2, Joon-Seol Lee3,Sang-Soo Kwak1, and Haeng-Soon Lee1

1Environmental Biotechnology Research Center, KRIBB2Plant Genomic Research Center, KRIBB3Mokpo Experiment Station, National Institute of CropScience, RDA

Porcine epidemic diarrhea virus (PEDV) causes enteritis inswine of all ages, and is fatal in neonatal piglets. The spikeprotein of PEDV is a primary target antigen for developingan effective vaccine against coronaviruses, since it mediatesessential biological functions. Sweetpotato [Ipomoeabatatas(L.)Lam.] is an attractive root plant producing plant-basededible vaccine as functional feed for swine. In this study,in order to develop the transgenic sweetpotato plants thatproduce an effective antigen protein against PEDV, weconstructed the transformation vectors using partial fragmentof PEDV spike protein (SP). Transgenic sweetpotatoplants were successfully developed by Agrobacterium-mediated transformation and confirmed by PCR, Southernblot and RT-PCR analysis. (3th China-Japan-KoreaWorkshop on Sweetpotato : Beijing (China), 2008)

Keywords : Agrobacterium-mediated transformation,edible vaccine, porcine epidemic diarrheavirus, sweetpotato

B-21Comparison of prediction models for cyanobacterialbloom in a eutrophic reservoir (Korea)

Chi-Yong Ahn1, Young-Seuk Park2, and Hee-Mock Oh1


1Environmental Biotechnology Research Center, KRIBB2Department of Biology, Kyung Hee University

Cyanobacterial bloom is a common issue in eutrophicfreshwaters. Some cyanobacteria produce toxins, threateninghuman and livestock health. Microcystins, the representativecyanobacterial hepatotoxin, are frequently detected inmost Korean lakes and reservoirs. Predicting the bloomoccurrence can help to cope with bloom-related problemsin advance. In this study, we tried to develop predictionmodels for cyanobacterial bloom in Daechung Reservoir,Korea. Three modelling techniques were compared, suchas multiple linear regression (MLR), self-organizing map(SOM), and classification and regression tree (CART).Data measured weekly from spring to autumn for fiveyears (2001, 2003-2006) were used in the model. Thepredicted values were compared with the measured valuesfor each model. MLR model exhibited a fairly goodprediction power for cyanobacterial cell density, threeweeks earlier. Water temperature was the major determiningfactor for cyanobacteria. However, it did not show acomparable prediction power for chlorophyll-a concentration.SOM technique was inferior to MLR in predicting properpeaks in bloom seasons. CART model exhibited goodresults for training data, but did not succeed to predictcyanobacterial blooms accurately with new data set. Whilethe three prediction models showed different performancewith respective merits and demerits, the common conclusionwas that water temperature primarily determines cyanobacterialbloom 3 weeks later, in Korean eutrophic reservoir. (12thInternational Symposium on Microbial Ecology (ISME12) :Cairns (Australia), 2008)

Keywords : bloom, chlorophyll-a, cyanobacteria, model,prediction

B-22Cloning, sequencing, and expression of reductivedehalogenase from the anaerobic PCE to VCdechlorinating enrichment culture

Kyung-Hwa Baek1, Byung-Hyuk Kim1, Youl-Boong Sung2,Hee-Mock Oh1, and Hee-Sik Kim1

1Environmental Biotechnology Research Center, KRIBB,2Gwangyang Environment Research Department, Environment& Energy Research Center, RIST

An anaerobic PCE to VC dechlorinating enrichment culturewas derived from creek sediment. The results of PCR-DGGE and 16S rRNA gene library showed that Geobactersp. Dehalococcoides sp., Desulfovivrio sp. are involved inreductive dechlorination of PCE. In the metagenome of theenrichment, reductive dehalogenase genes (pceA, tceA,bvc, and vcrA gene) were amplified and sequenced. Their

nucleotide sequences were identified with Desulfitobacteriumsp. CR1 95% (1200/1259) Dehalococcoides ethenogenes 97%(1636/1685), Dehalococcodies sp. Strain BAV1 99%(837/839),s train VS 99% (1476/1482), respectively. Usingthe pGEX and pET system, tceA gene was transferred intoE. coli BL21(DE3)/pLysS. After induction with IPTG,TceA dehalogenase with a molecular mass of approximately62 kDa was expressed in crude extracts of the respectiverecombinant bacteria. Dehalogenase activity for TCEcould not be detected in the cell extracts of recombinant E.coli. Further research will focus on cofactors, O2 sensitivity,and folding effected on the RDase activity. (108thGeneral Meeting of American Society for Microbiology: Boston (USA), 2008)

Keywords : anaerobic enrichment, PCE, RDase, reductivedechlorination

B-23A study on quorum sensing system for theconservation of biodiversity in a eutrophicwater

Young-Ki Lee1, Chi-Yong Ahn1, Gang-Guk Choi1, Hee-Sik Kim1, Jung-Kee Lee2, and Hee-Mock Oh1

1Environmental Biotechnology Laboratory, KRIBB2Department of Life Science and Genetic Engineering, PaiChai University

Microcystis aeruginosa is an ubiquitous cyanobacteriumthat causes ecological and economic damage to freshwaterecosystems when it is abundant. While blooms are unpleasantboth visually and due to released odor and taste factors, toxicmetabolites pose a more serious problem for humans. Recentstudies focused on cell to cell communications in ecogenomics.This cell to cell communication has become known as"quorum sensing (QS)". In this study, to investigate therelationship between cell to cell communication andbloom, we carried out experiments with Microcystis sp.Mi0601 isolated from scum during the summer, 2006 inDaechung reservoir and Microcystis sp. KW isolated fromWhang-Song reservoir. Both of two isolated Microcystissp. were identified as M. aeruginosa by 16S rRNA geneand cpc-IGS region PCR. Concentrated cell free extractswere separated by thin-layer chromatography andbioassayed with Agrobacterium tumefaciens NT1 (traR,tra::lacZ749). Two signal molecules could be detected inTLC plate which was not different from C10-homoserinelactone standard. In each of the two spots extracts, signalmolecules were identified by GC-MS. From GC-MS analysis,we assume that these molecules of MW 390.5 are differentwith the homoserine lactones series in gram negativebacteria. (International Symposium & Annual Meeting: Seoul, 2008)


Keywords : bloom, cell to cell communication, cyanobacteria,Microcystis sp., quorum sensing

B-24Selection of microalgae for lipid productionunder high-level carbon dioxide

Chan Yoo, So-Young Jun, Jae-Yon Lee, Chi-Yong Ahn,and Hee-Mock Oh


One of the various strategies to mitigate CO2 is the masscultivation of photosynthetic microalgae using CO2 as thecarbon source. Thus, the purpose of this study was toselect microalgae that can be cultivated under a high levelof CO2. To screen microalgae with a high growth rate andlipid content, Botryococcus braunii, Chlorella vulgaris,and Scenedesmus sp. were cultivated with 10% CO2 at 0.3v/v/m. The biomass and lipid productivity were estimatedbased on the dry weight and total lipid content. The biomassyield and productivity of Scenedesmus sp. were 3.13 g dwL-1 and 217.50 mg dw L-1 d-1, respectively, while the lipidcontent for B. braunii increased up to 25%. Among thefatty acids, oleic acid occupied the highest proportion in B.braunii. Therefore, the present results suggested thatScenedesmus sp. is appropriate for mitigating CO2, due toits high biomass productivity and C-fixation ability, whereas B. braunii is appropriate for producing biodiesel, due toits high lipid content and proportion of oleic acid. (RenewableEnergy 2008 : Busan, 2008)

Keywords : 10% CO2, fatty acids, microalgae, photosyntheticparameter, total lipid

B-25Comparison of several methods for effectivelipid extraction from microalgae

Jae-Yon Lee, Chan Yoo, So-Young Jun, Chi-Yong Ahn,and Hee-Mock Oh


Lipid extraction is a key process in biodiesel productionfrom mass-cultured microalgae, and the efficiency of thelipid extraction depends greatly on which cell disruptionmethod is used. However, the best cell disruption methodfor microalgae has not yet been established. Therefore, thisstudy tested various methods including autoclaving, bead-beating, microwaves, sonication, and a 10% NaCl solution,to identify the most effective cell disruption method. The

total lipids from Botryococcus sp., Chlorella vulgaris, andScenedesmus sp. were extracted using a mixture ofchloroform and methanol (1:1). The lipid contents from the3species were 5.4 - 11.9, 7.9 - 8.1, 10.0 - 28.6, 6.1 - 8.8, and6.8 - 10.9 g L-1 when using autoclaving, bead-beating,microwaves, sonication, and a 10% NaCl solution, respectively.Botryococcus sp. showed the highest oleic acid productivityat 5.7 mg L-1 d-1 when the cells were disrupted using themicrowave oven method. Thus, among the tested methods,the microwave oven method was identified as the mostsimple, easy, and effective for lipid extraction frommicroalgae. (Renewable Energy 2008 : Busan, 2008)

Keywords : biodiesel, cell disruption, lipid extraction,microalgae

Members

Director Sang-Soo Kwak ([email protected])

Principal Researcher Haeng-Soon Lee ([email protected])Il-Gin Mok ([email protected])Hee Mock Oh ([email protected])Hee-Sik Kim ([email protected])

Senior Researcher Chi-Yong Ahn ([email protected])

Post-DocCha Young Kim ([email protected])Young Ock Ahn ([email protected])Myung Duck Kim ([email protected])Yun-Hee Kim ([email protected])So-Young Jun ([email protected])Young-Ki Lee ([email protected])Kyung-Hwa Baek ([email protected])

Researcher Eun-Kyung Kim ([email protected])Kyoung-Sil Yang ([email protected])Byung-Hyuk Kim ([email protected])Dae-Hyun Cho ([email protected])Chan Yoo ([email protected])Jae-Yon Lee ([email protected])Sun Ha Kim ([email protected])Jung-Mae Liu ([email protected])

Division of Biotechnology R&DB

Biological Resources Center

Korean Bio Information Center

Daejeon-KRIBB-FHCRC Research Cooperation Center


A-17Study on cleavage by various proteases andtheir applications to purification of a fusion-tagged protein

Jung Eun Baek, Byung Lip Kim, Eun Gyo Lee, Jung OhAhn, Hong Weon Lee, and Joon Ki Jung

Biomedical Process Development Department, KRIBB

Expression of heterologous protein in bacterial system is acommon technology to obtain valuable proteins in a highexpression-level. Especially Escherichia coli(E.coli) facilitatesrecombinant protein expression by relative simplicity andhigh yield. A frequently encountered problem is theaggregation of recombinant protein which requires laboriousand inefficient refolding step prior to purification. In orderto avoid inclusion body formation a specific fusion partneris generally adopted to enhance the solubility and stabilityof a desired protein. This study reports the method toproduce the bioactive and highly purified human protein ina large quantity using the fusion partner of maltose-bindingprotein, which acts as afolding enhancer and purificationtag. MBP/human protein system was expressed in asolublefraction more than 90% in fed-batch culture. Followingfermentation MBP/human protein was purified by one-stepaffinity chromatography. To remove N-terminus MBP fusionprotein, the specific proteases such as enterokinase, FactorXa and thrombin cleavage were used, but non-specificcleavage was observed due to further degradation. To overcomethis undesirable cleavage reaction,the various cleavagelinkers were introduced. As a result, the human protein wassuccessfully purified by one-step reversed-phase chromatography.MBP fusion and factor Xa cleavage system efficientlyallows the protein of interest to be expressed in a high yieldand can be purified through relatively simple chromatographysteps in high purity and yield. (The Korean Society forMicrobiology and Biotechnology 2008 InternationalSymposium : Seoul, 2008)

Keywords : cleavage, interleukin-6, maltose binding protein

A-18Signal enhancement of surface plasmonresonance using carbon nanotube-antibodycomplex

Kyung Mi Park1, Seung Hui Lee1, Joon Ki Jung2, Eun GyoLee2, and Bong Hyun Chung1

1Bionanotechnology Research Center, KRIBB2Biomedical Process Development Department, KRIBB

Surface Plasmon Resonance (SPR) is an affinity opticalsensor based on the detection of changes in mass concentration

at a biospecific interface. A major impediment to SPRbiosensor technology, however, is a lack of sensitivity at lowconcentration of protein of interest. Nano-materials have beenused to improve the sensitivity of SPR, caused by theirhigh molecular weight to strengthen the SPR signal.Recently, SPR-based immnunoassay has been examined toexpand its application to the clinical diagnosis for the detectionof tumor biomarkers in human blood. Nanoparticles havebeen reported for the sensitive detection of biomolecules.We have improved a sandwich immunoassay between anantigen and two kinds of antibodies such as monoclonaland polyclonal antibodies by using carbon nanotubes (CNTs),which are composed solely of carbon atoms, arranged inbenzene rings forming graphene sheets, rolled up to giveseamless cylinders, and are applied for the visualization,transportation, biocatalysts, and sensing components ofbiomolecules. The SPR signal was amplified over 50 foldsby using CNTs conjugation, enabling to be detectable evenat lower concentration range of 2 ng/mL. This study presentsan alternative techniqueto enhance SPR measurementprecisely and sensitively, and it can be applicable to otherSPR-based immunoassays. (The Korean Society forMicrobiology and Biotechnology International Symposium& Annual Meeting : Seoul, 2008)

Keywords : carbon nanotube, SPR, surface plasmonresonance

A-19Purification of recombinant human erythropoietinin milk of transgenic pigs

Seung Hui Lee1, Kyung Mi Park1, Jung Eun Baek2, Jin KiPark3, Won Kyong Chang3, Joon Ki Jung2, Eun Gyo Lee2,and Bong Hyun Chung1

1Bionanotechnology Research Center, KRIBB2Biomedical Process Development Department, KRIBB3Animal Biotechnology Division, National LivestockResearch Institute

We developed an efficient method to purify recombinanthuman erythropoietin (rhEPO), which is a hydrophobicacidic glycoprotein responsible for the regulation of redblood cell production, from the milk of transgenic pigs. TherhEPO was purified by heparin chromatography, reverse-phase chromatography, and gel filtration chromatography,resulting in a 16.5% yield and >98% purity. The combinationof these well-connected steps could produce transgenicpig-derived rhEPO with a high yield and purity with nochanges in the sialic acid content. Although the 6 N-terminalamino acid sequences of transgenic pig-derived rhEPOmatched that of commercial rhEPO derived from the CHOcell culture, their isoforms and extent of glycosylation weredifferent. The rhEPO purified from the milk of transgenic


Division of Biotechnology R&BD

pigs had less acidic isoforms and was underglycosylated incontrast to CHO-derived rhEPO. Cell proliferation of theEPO-dependent cell line was proportional to the dose oftransgenic pig-derived rhEPO, demonstrating thattransgenic pig-derived rhEPO has a potency similar to thatof CHO-derived rhEPO. (The Korean Society forMicrobiology and Biotechnology InternationalSymposium & Annual Meeting : Seoul, 2008)

Keywords : erythropoietin, purification, transgenic

Members

Director Joon Ki Jung ([email protected])

Principal Researcher Hong Weon Lee ([email protected])Eun Gyo Lee ([email protected])Heung Chae Jung ([email protected])

Senior Researcher Jung Oh Ahn ([email protected])

Researcher Chun Sug Kim ([email protected])Hyeok Won Lee ([email protected])Joo Hwan Lee ([email protected])Jung Eun Baek ([email protected])Kyung Mi Park Seung Hui Lee

B-1Pontibaca methylaminivorans gen. nov., sp. nov., anovel member of the family Rhodobacteraceae

Kwang Kyu Kim, Jung-Sook Lee, Keun Chul Lee, Hee-MockOh, and Song-Gun Kim

Biological Resource Center, KRIBB

Novel alphaproteobacteria, strains GRP21T and PH34,were isolated from the East Sea and were subjected to apolyphasic taxonomic investigation. The strains comprisedGram-negative, non-motile, non-spore-forming, oval-shaped rods, produced creamy white colonies on trypticsoy agar, required NaCl for growth, contained Q-10 as thepredominant ubiquinone, contained 19:0 cyclo 8c, 16:0and 18:1 7c as the major fatty acids, and had polar lipidprofiles consisting of phosphatidylcholine andphosphatidylglycerol. Phylogenetic analysis, based on 16SrRNA gene sequencing, showed that the strains were mostclosely related to Donghicola eburneus KCTC 12735T

with 94.1 % sequence similarity and formed a separatelineage within the family Rhodobacteraceae. The combinedgenotypic and phenotypic data supported the conclusionthat the strains represent a novel genus and species, forwhich the name Pontibaca methylaminivorans gen. nov.,sp. nov. is proposed. The type strain is GRP21T (= KCTC22497T = DSM 21219T). (International Meeting of theFederation of Korean Microbiological Societies : Seoul,2008)

Keywords : alphaproteobacteria, bacteria, Donghicolaeburneus, identification, PH34, Pontibacamethylaminivorans, strains GRP21

B-2Candida alkalitolerans sp. nov., a novel alkalitolerantyeast from industrial waste water

Kee-Sun Shin, Kang Hyun Lee, Byeoung Hwan Park, andHee-Mock Oh


Two undescribed anamorphic yeast strains of ascomycetousaffinity were isolated from industrial sewage. Thetaxonomic position of the yeast strains 98Y75 and 98Y78were examined. Sequence analysis of D1/D2 domain ofthe 26S rRNA cording gene as well as physiological andchemosystematic studies on strains 98Y75 and 98Y78placed them within the genus Candida, including Candidarugosa and Candida catenulata. However, there wereseveral differences in some physiological characteristicsand 26S partial sequence, which are making the strainsrecognizable from other Candida species. The results of


Biological Resource Center

the present study indicated that strains 98Y75 and 98Y78should be placed under the genus Candida as a newspecies. The type strain of the new species is strain 98Y78(= KCTC 17119T). (International Symposium & AnnualMeeting of the Korean Society for Microbiology andBiotechnology : Seoul, 2008)

Keywords : alkalitolerant yeasts, Candida alkalitolerans,new yeast species

B-3Candida nymphaea sp. nov., a novel yeast in theKodamaea clade from water

Kee-Sun Shin, Kang Hyun Lee, Byeoung Hwan Park, andHee-Mock Oh


Two novel flower-associated ascomycetous yeasts wereisolated from water lily collected in the Gungnamji,Buyeo, Korea. Phylogenetic trees based on the D1/D2domains of the large-subunit (26S) rDNA sequencesshowed that the isolates belonged to the Kodamaea cladeand they were related closely to Kodamaea ohmeri, Candidarestingae and Candida leandrae. But They were clearlydistinct from these species and the other species of theKodamaea clade that have been reported by more than2.4% sequence divergence, indicating that it should beclassified as a novel species. Candida nymphaea sp. nov.The type culture of C. nymphaea is a strain 186-1T (= KCTC17414T = NCAIM Y.01856T). (International Symposium& Annual Meeting of the Korean Society for Microbiologyand Biotechnology : Seoul, 2008)

Keywords : Candida nymphaea, flower-associatedascomycetous yeasts, Kodamaea clade, newyeast species

B-4Gordonia kroppenstedtii sp. nov., a phenol-degrading actinomycete isolated from apolluted stream

Kwang Kyu Kim1, Keun Chul Lee1, Hans-PeterKlenk2,Hee-Mock Oh1, and Jung-Sook Lee1

1Biological Resource Center, KRIBB2Deutsche Sammlung von Mikroorganismen und Zellkulturen,Braunschweig, Germany

A phenol-degrading actinomycete, strain NP8-5T, was

isolated from a polluted stream in Gumi, Korea. The strainwas aerobic, Gram-positive, non-motile and non-spore-forming, displayed a rod-coccus growth cycle, exhibitedwhite opaque colonies on complex media, and showedchemotaxonomic markers that were consistent with theclassification in the genus Gordonia, i.e. meso-diaminopimelicacid, arabinose and galactose in whole-cell hydrolysates, N-glycolylmuramic acid in the peptidoglycan wall, unbranchedsaturated and monounsaturated fatty acids plustuberculostearic acid (TBSA), mycolic acids that comprised56-60 carbon atoms, MK-9(H2) as the predominantmenaquinone and a DNA G+C content of 68.5 mol%.Phylogenetic analysis, based on 16S rRNA gene sequencing,also showed that strain NP8-5T belonged to the genusGordonia, which shared the highest levels of sequencesimilarity with Gordonia araii IFM 10211T, Gordoniahydrophobica DSM 44015T and Gordonia sinesedisNCIMB 13802T (96.4, 96.0 and 95.9 %, respectively) andformed a separate lineage with G. sinesedis. Combinedphylogenetic and phenotypic data supported that strainNP8-5T represents a novel species of the genus Gordonia,for which the name Gordonia kroppenstedtii sp. nov. isproposed; the type strain is NP8-5T (= KCTC 19360T =DSM 45133T). (International Meeting of the Federation ofKorean Microbiological Societies : Seoul, 2008)

Keywords : Gordonia kroppenstedtii sp. nov., phenol-degrading actinomycete, taxonomy

B-5Solirhabdus panacisegetis gen. nov., sp. nov.,isolated from soil of a ginseng field

Jung-Sook Lee, Keun Chul Lee, Kwang Kyu Kim, MiJeong Kim, Mi Kyung Eom, and Hee-Mock Oh


A novel Gram-positive, short-rod bacterial strain, P4-7T,was isolated from soil of a ginseng field, Korea. The strainhad MK-8(H4) as the major menaquinone, glucose,mannose, xylose and rhamnose as whole-cell sugar,diphosphatidylglycerol as major polar lipid and meso-diaminopimelic acid as the diagnostic diamino acid in thepeptidoglycan. It contained iso-C16:0 and anteiso-C15:0as the predominant cellular fatty acids and DNA G+Ccontent of 69.2mol%. Phylogenetic analysis, based on 16SrRNA gene sequencing, showed that the isolate P4-7T wasmost closely related to the genus Humicoccus, Humicoccusflavidus (with 96.4% sequence similarity). However, strainP4-7T showed distinct differences from Humicoccusflavidus incontent of the major menaquinone, cell-wallsugar, major polarlipid and fatty acid profile. On the basisof combined phenotypic, chemotaxonomic and phylogeneticdata, it is proposed that strain P4-7T represents a new


genus and a novel species, Solirhabdus panacisegetis gen.nov., sp. nov. The type strain is P4-7T (= KCTC 19426T).(XII International Congress of Bacteriology andApplied Microbiology : Istanbul (Turkey), 2008)

Keywords : gram-positive, P4-7, short-rod bacterial strain,Solirhabdus panacisegetis gen. nov., sp. nov.,taxonomy

B-6Patulibacter ginsengiterrae sp. nov., isolatedfrom soil of a ginseng field



A novel Gram-positive, short-rod bacterial strain, P4-5T,was isolated from soil of a ginseng field, Korea. The strainhad glucose, galactose, mannose, arabinose and rhamnoseas whole-cell sugar, and meso-diaminopimelic acid as thediagnostic diamino acid in the peptidoglycan. It containedC18:1w9c, summed feature 7 (C19:0cyclow10c/19w6)and C18:0 as the predominant cellular fatty acids and DNAG+C content of 75.0mol%. Phylogenetic analysis, basedon 16S rRNA gene sequencing, showed that the isolateP4-5T was most closely related to the genus Patulibacter,Petulibacter minatonensis (with 97.2% sequence similarity).On the basis of combined phenotypic, chemotaxonomicand phylogenetic data, it is proposed that strain P4-5T

represents a novel species, Patulibacter ginsengiterrae sp. nov.The type strain is P4-5T (= KCTC 19427T). (XII InternationalCongress of Bacteriology and Applied Microbiology :Istanbul (Turkey), 2008)

Keywords : gram-positive, P4-5, short-rod bacterial strain,Patulibacter ginsengiterrae sp. nov., taxonomy

B-7A polyphasic investigation on some novelHalomonas species isolated from a renal carecenter



A total of 14 Halomonas strains were isolated from twopatients' blood and from dialysis machines of a renal carecenter. The strains were Gram-negative, moderately halophilic,

motile, produced cream-coloured colonies and containedQ-9 as the predominant ubiquinone and C18:1 7c,C16:0 andC12:03-OHas the major fatty acids. Phylogenetic analysis,based on 16S rRNA gene sequencing, showed that thefourteen isolates were most closely related to Halomonasmagadiensis NCIMB 13595T with 98.1-98.9% of sequencesimilarity and formed three separate lineages amongthemselves. Combined phenotypic data and DNA-DNAhybridization data supported the conclusion that theyrepresent three novel species of the genus Halomonas, forwhich the names H.stevensii sp. nov. (type strain S18214T

= KCTC 22148T = DSM 21198T), H.hamiltonii sp. nov. (typestrain W1025T = KCTC 22154T = DSM 21196T) and H.johnsoniaesp. nov. (type strain T68687T = KCTC 22157T = DSM 21197T)are proposed. (International Symposium & AnnualMeeting of the Korean Society for Microbiology andBiotechnology : Seoul, 2008)

Keywords : Halomonas stevensii sp. nov., Halomonashamiltonii sp. nov., Halomonas johnsoniaesp. nov., polyphasic investigation, taxonomy

Members

Director : Hee-Mock Oh ([email protected])

Principal Researcher Jung-Sook Lee ([email protected])Ho-Yong Park ([email protected])Kyung Sook Bae ([email protected])

Senior Researcher Suk Weon Kim ([email protected])Young Hyo Chang ([email protected])Kee-Sun Shin ([email protected])Jin-Woo Bae ([email protected])Song-Gun Kim ([email protected])Hyun-Woo OH ([email protected])Doo-Sang Park ([email protected])Keun Chul Lee ([email protected])Kang-Hyun Lee ([email protected])Moon-Soo Rhee ([email protected])In Soon Park ([email protected])Yong Jae Lee ([email protected])

Post-DocGang-Guk Choi ([email protected])Do Young Kim ([email protected])Kyung-Ho Kim ([email protected])Magay Elena ([email protected])Jung-Hye Choi ([email protected])


D-1SNP@WEB: a web-based catalog of SNP (SingleNucleotide Polymorphism) databases and tools

KOBIC Variome Team

Korean BioInformation Center(KOBIC), KRIBB

Single Nucleotide polymorphisms (SNPs) can affect genefunctions and expressions from DNA to protein levels.Predicting and understanding the effects of SNPs usingbioinformatics are becoming important. To guide researchers, wedeveloped SNP@WEB, a web-based catalog of databases andtools for SNP studies. Currently, SNP@WEB collected ~90SNP resources classified by eight categories (SNP acquisition,annotation, tagSNP, haplotype, population, mutability, database,and SNP effect). For each resource, information such aslinks, description, web popularity, and server status isavailable. Since SNP@WEB is developed as a Wiki (real-time editable website) system, it supports fully open curationand communication among SNP researchers. SNP@WEB isavailable from online: http://variome.kobic.re.kr/SNPatWEB/or http://www.variome.net. (Korean Society for Bioinformatics: Daejeon, 2007)

Keywords : catalog of database, expression, gene function,SNP, SNP@WEB

D-2In silico identification of gene-related patents

Byungwook Lee and Woo-Yeon Kim

Korean BioInformation Center(KOBIC), KRIBB

The number of patent applications containing gene sequencesincrease each year, with gene sequences appearing inpatents that may be broadly applied to many aspects ofbiotechnology and drug development. Nonetheless, relativelyfew reports have focused on gene-related patents, and therehas been no prior attempt to study patent-related sequencesfrom a biological perspective. We herein used bioinformaticsanalysis to identify patent-associated genes. The biologicalsequences disclosed in published patents were comparedwith RefSeq database, and the patent-associated hits weremapped to Entrez Gene. Mapping analysis revealed that24,273 (61%) of known human genes were related topatenting. We have consolidated the association resultsand the patent sequence data to a relational database andimplemented a web-based user interface to provide searchservice. (Seventh International Conference on Bioinformatics(InCob) : Taipai (Taiwan), 2008)

Keywords : Entrez Gene, gene sequence, gene-patent,RefSeq database

Members

Director Jong Bhak ([email protected])

Principal Researcher Gee Chan Ryu ([email protected])

Senior Researcher Nam Shin Kim ([email protected])Byung Sook Lee ([email protected])Bo Kyung Her ([email protected])Sung Hoon Lee ([email protected])Seng Woo Hwang ([email protected])Jin Ok Yang ([email protected])Sung Soo Kang ([email protected])

Post-DocDae Soo Kim ([email protected])Chul Hong Kim ([email protected])

Researcher Woo Yeon Kim ([email protected])Sung Hyun Kim ([email protected])Kyung A Lee ([email protected])Jeong Heui Lim ([email protected])Byoung-Chul Kim ([email protected])Sang Yun Kim ([email protected])Soo Ahn Cho ([email protected])Hana Byun ([email protected])Tae Hui Hong ([email protected])Gun Hwan Ko ([email protected])Young Seok Lee ([email protected])Shin Ho Kim ([email protected])Daeui Park ([email protected])Sang Ho Oh ([email protected])Sung Jin Park ([email protected])Ji Han Kim ([email protected])Je Wun Ryu ([email protected])Jang Gyu Lim ([email protected])Nam Cheol Kim ([email protected])Jae Min Kim ([email protected])Gwang Sik Shin ([email protected])Jin Man Ryu ([email protected])Ui Seok Jung ([email protected])Ji Hye Park ([email protected])Yeon Ju Jo ([email protected])Je Keun Kwon ([email protected])Sung Woo Yang ([email protected])Ho Young Kang ([email protected])Sung ung Cho ([email protected])Lohit Reja ([email protected])


Korean BioInformation Center(KOBIC)

D-27Functional proteomic study reveals that GnT-Vreinforces the invasive/metastatic potential ofcolon cancer through aberrant glycosylation onTIMP-1

Yong-Sam Kim, Sun Hee Kim, Hyang Sook Yoo, and JeongHeon Ko

Daejeon-KRIBB-FHCRC Research Cooperation Center,KRIBB

N-acetylglucosaminyltransferase-V (GnT-V) has beenreported to be up-regulated in invasive/metastatic cancercells, but a comprehensive understanding of how thetransferase correlates with the invasive/metastatic potentialis not currently available. Through a glycomic approach,we identified 36 proteins including tissue inhibitor ofmetalloproteinase-1 (TIMP-1) as a target protein for GnT-V in human colon cancer cell WiDr. TIMP-1 is aberrantlyglycosylated as characterized by addition of ß1,6-N-acetylglucosamine, polylactosaminylation, and possiblysialylation in GnT-V-overexpressing WiDr cells. Theaberrant glycosylation of TIMP-1 is responsible for themitigated inhibition on both matrix metalloproteinase (MMP)-2 and MMP-9, and this aberrancy is closely associated withcancer cell invasion and metastasis in vivo as well as invitro. Integrated data both of TIMP-1 expression level andaberrant glycosylation could provide important informationto aid to improve clinical outcome of colon cancer patients.(3rd ICBC Annual Meeting : Hawaii (USA), 2008)

Keywords : aberrant glycosylation, GnT-V, invasion, MMP,TIMP-1

D-28Glycomics-based biomarker discovery for liver& colon cancer

Yong-Sam Kim, Ok Lye Son, Hyang Sook Yoo, and JeongHeon Ko

Daejeon-KRIBB-FHCRC Research Cooperation Center,KRIBB

Colon cancer is often difficult to early diagnose, but earlydiagnosis is a crucial factor for good outcome in the cancer.N-acetylglucosaminyltransferase V catalyzes an additionof b1,6-N-acetylglucosamine (GlcNAc) to the core N-glycan, many lines of evidence have demonstrated the roleof N-acetylglucosaminyltransferase V (GnT-V) in thepathogenecity of colon cancer cell, and thus identificationof target proteins for GnT-V has been an important subjectfor cancer biomarker discovery. We developed a protocolin which L-PHA, a lectin recognizing ß1,6-GlcNAc, is

conjugated to avidin-agarose complex to capture ß1,6-GlcNAc-carrying serological glycoproteins and the capturedglycoproteins were identified using an LTQ-FTICR massspectrometer. Candidate proteins showing differentialamounts between normal and cancer sera were confirmedby western blot analysis and will be subject to validationfor valid biomarker for colon cancer. (3rd ICBC AnnualMeeting : Hawaii (USA), 2008)

Keywords : Aberrant Glycosylation, Cancer biomarker,GnT-V, L-PHA

D-29Profiling of tumor associated antigens directedautoantibodies of cancer patient

Chang-Kyu Heo1, Yang-Shin Park1, Ju-Youn Lee2, Jong-Shin Yoo2, Hyang-Sook Yoo1, Jong-Young Choi3,Jhingook, Kim4, Jeong-Heun Ko1, and Eun-Wie Cho1

1Daejeon-KRIBB-FHCRC Cooperation Research Center,KRIBB

2Analysis & Measurement Division, Korea Basic ScienceInstitute

3Colleage of Medicine, Catholic University of Korea 4Department of Thoracic and Cardiovascular Surgery,Samsung Medical Center

An ideal cancer marker would allow a simple blood test todetect cancer and its level would correlate with the stage oftumor progression. Recently, based upon the observationof increased incidence of autoantibodies in malignancies,there are attempts to develop cancer diagnostic method bythe detection of autoantibodies against tumor antigens inpatient sera. Compared to serum-derived proteins,antibodies in serum are stable, highly specific and readilydetected with well-validated secondary reagents, makingthem ideal for diagnosis. If we could identify a set oftumor antigens directedautoantibodies which are specificfor the detection of tumor progression, the autoantigenmicroarray composed of these antigens would be a powerfultool for the diagnosis of cancer. But it is not easy to identifyautoantigens against autoantibodies in cancer patient serabecause it is not well-known about the characteristics ofautoantibodies in cancer patients. In our laboratory, we usedspleen cells derived from HCC model mouse to characterizeautoantibodies generated in hepatocellular carcinoma(HCC).The spleen cells from HCC model mouse werefused with myeloma cells and stable hybridoma clonesproducing antibodies reactive to hepatocellular carcinomacells were selected and characterized. One interesting result isthat the subtypes of most autoantibodies characterizedareIgM. So we also tested IgM level in cancer patient sera and,as expected, in the sera of malignancies IgM level werehighly increased. So we purified immunoglobulins


Daejeon-KRIBB-FHCRC Research Cooperation Center

containing IgM from cancer patient sera, prepared affinitysupport,and used it for the purification of autoantigensfrom cancer cell lysates. Then the purified proteins wereidentified by mass spectroscopy. The profile of identifiedproteins showed many of cancer-related proteins and severalproteins already known as autoantigens. We anticipate thata set of autoantigens among the identified proteins wouldbe proposed as a diagonistic kit for cancer. (KHUPO, 8thAnnual International Proteomics Conference : Jeju, 2008)

Keywords : autoantibody, tumor-associated antigens, tumormarker

Members

Director Hyang Sook Yoo ([email protected])

Principal Researcher Jeong Heon Ko ([email protected])Eun Wie Cho ([email protected])

Senior Researcher Sang Jick Kim ([email protected])Yong-Sam Kim ([email protected])

Researcher Chang Kyu Heo ([email protected])Yun Mi Seo ([email protected])Sun Hee Kim ([email protected])Hyun Joo Ahn ([email protected])Hyun Jung Kim ([email protected])Su Jin Jung ([email protected])Ji Ae Jung ([email protected])


Division of Ochang R&D

Korea National Primate Research Center

Bio Evaluation Center


A-1In silico analysis and experimental validationof recently exonized Alu in Macaca fascicularis

Young-Hyun Kim, Jae-Won Huh, Hyoungwoo Kim,Sang-Rae Lee, Myeong-Su Kim, and Kyu-Tae Chang

National Primate Research Center (NPRC), KRIBB

Crab-eating monkey (Macaca fascicularis) and rhesusmonkey (Macaca mullata) are frequently used and valuableprimate model species. Although they most commonprimate model organism for biomedical approaches, theirgenetic information is not yet applicable except for rhesusmonkey. In this study, we tried to analyze genomic diversityof closely related two macaca species with recently integratedAlu elements. First, the Macaca fascicularis mRNAsequences (10221 mRNA) were collected from Genebankdatabase, and 'young' Alu-exonized mRNA sequenceswere sorted by repeatmasker program (216 mRNA).Second, for avoiding the false positive data (avoiding thegenomic contaminated cDNA sequences), manualcorrection were conducted. Third, ten genes were chosen,and eight genes contained young Alu element were identified.Finally, for the verification of exonized young Aluelement, PCR amplification and sequencing procedurewere conducted using various human and primate DNAsamples. Intriguingly, two genes (C9orf6 and NOLC1gene) harbor the insertional polymorphic Alu element intheir transcript. Although, we did not use the whole genomeinformation of Macaca fascicularis, genome wide surveycould be a useful tool for understanding the useful primatemodel organism. (Annual Meeting of the Society ofMolecular Biology and Evolution (SMBE) : Barcelona(Spain), 2008)

Keywords : Alu element, exonization, in silico analysis,Macaca fascicularis, primate

A-2Identification of novel retromer complexes inthe mouse testis

Ekyune Kim1, Sang- Hyun Kim1, Sang-Rae Lee1, Myeong-Su Kim1, Kazuhiko Imakawa2, and Kyu-Tae Chang1

1National Primate Research Center (NPRC), KRIBB2Graduate School of Agricultural and Life Sciences, TheUniversity of Tokyo, Japan

The Vps proteins (Vps26, Vps29, and Vps35) have beenreported to function in many biological processes,such asearly embryonic development, vacuolar transportation ofhydrolases, and transcytosis of cell membrane proteins.However, little is known about the diversity of the VPS-

mediated retromer complex in various tissues of mouse. In thisstudy with a view to exploring the functional characterizationof the retromer complex, we first have examined theexpression pattern of these retromer components in thevarious mouse tissues. The expression of Vps26b waspredominantly observed in brain, in contrast to theubiquitous expression of Vps26a in the mouse. Moreover,Vps26aT was expressed exclusively in the testis in a tissue-specific manner, compared with Vps26a that was ubiquitouslyexpressed in all mouse tissues tested. These results suggestthat at least in the mouse, these Vps26 isoforms may havedifferential role in various tissues. Next, we tried to showthe formation of the retromer complex consisted ofVps26a, Vps26b, Vps29, and Vps35 at in vivo level.Immunoprecipitation analysis with mouse brain extractsclearly demonstrated the presence of the retromer complexin the mouse brain. The retromer component Vps26a hasbeen reported to interact directly with Vps35 through theVps35 binding motif. Unexpectedly, Vps26a also interactswith Vps26b as well as Vps29 directly or indirectly. Theseresults suggest that the retromer complex is formed withcomplicated binding of the component proteins of Vps26a,Vps26b, Vps29, and Vps35. In the subsequent experimentwith yeast two-hybrid system, we could provide evidencesthat Vps26a interacts with Vps26b as well as Vps29 andVps35. Here we now demonstrate that interactions betweenVps26a and Vps29, Vps26b-Vps29, and Vps26a-Vps26bare all Vps35-independent. These findings provide someinsights not only into the functional association among theretromer complex among the components, but also into thespecialized roles of Vps proteins in the brain functionsand/or spermatogenesis. (Society for the Study ofReproduction 41st Annual Meeting : Hawaii (USA), 2008)

Keywords : retromer complex, tissue-specific manner, Vpsproteins

A-3Pig ADAM3 disintegrin domain has animportant rolein the sperm-egginteraction

Ekyune Kim1, Ji-Su Kim2, Dong chul Baek1, Jae-WoongLee1, Sang-Rae Lee1, Myeong-Su Kim1, Sang-Hyun Kim1,Chan-Shick Kim3, Deog-Bon Koo2, and Kyu-Tae Chang1

1National Primate Research Center (NPRC), KRIBB2Center for Regenerative Medicine, KRIBB, 3Faculty of Biotechnology, Cheju National University

The A Disintegrin and Metalloprotease (ADAM) proteinfamily has important roles in various biological processes,such as fertilization, neurogenesis, myogenesis, andinflammation. Most ADAM proteins have a uniqueorganization, containing an N-terminal signal sequence,followed by a pro-domain, metalloprotease, disintegrin, Cys-


National Primate Research Center

rich, epidermal growth factor (EGF)-like, transmembrane,and cytoplasmic tail domains. The metalloprotease domainpossesses shaddase activity toward the ectodomain ofmembranous precursor proteins, whereas the cytoplasmicdomain is related to the interaction with Src family proteintyrosine kinases. It is known that the disintegrin domain isinvolved in cell-to-cell adhesion. Although the roles ofseveral testis-specific ADAMshave been identified inknockout (KO) mice, they were expressed in a species-specific manner. Thus, the specific role of testis-specificADAM molecules in the sperm-egg interaction remains tobe determined. Cyritestin, known also as ADAM3, is amember of the ADAM family, and is expressed specificallyin male germ cells. It is suggested that cyritestin is able tobind to the ZP through the disintegrin domain because thepretreatment of eggs with peptides of the ADAM3 disintegrindomain inhibited IVF in mouse. However, little is knownabout ADAM3 in other species, such as pigs. We haveidentified and characterized porcine ADAM3 in an attemptto elucidate its role(s) in the interaction with the ZP. In thisstudy, we found two isoforms of porcine ADAM3, ADAM3aand ADAM3b, which are present on the surface of sperm,and are involved in the interaction with the ZP. Thesesresults indicate that both ADAM3a and ADAM3b couldhave important roles in fertilization. (수정란이식학회 : 안성,2008)

Keywords : ADAM3, ADAM family, cyritestin, sperm-egginteraction

A-4Identification of a binding domain in non-human primate (Macaca fascicularis) ADAM2for hetero complex

Ekyune kim1, Jae-Woong Lee1,2, Myeong-Su Kim1, Sang-Rae Lee1, Sang-Hyun Kim1, Zae-Young Ryoo2, and Kyu-Tae Chang1

1National Primate Research Center (NPRC), KRIBB2School of Life Science and Biotechnology, KyungpookNational University

Family members of ADAM (A Disintegrin And Metalloprotease)are multidomain proteins, composed of metalloprotease,disintegrin, cystein-rich, EGF-like, and cytoplasmic domains.Among about 40 members of this family that distributed toa variety of tissues, approximately half members areexclusively or predominantly expressed in the testis. Malemice lacking ADAM1a, ADAM2 and ADAM3 are infertiledue to following features; 1) the sperm of these mice havedefects in migration from the uterus into the oviduct. 2)cauda epididymal sperm from these mutant mice can notbind to the egg zona pellucidia. 3) level of ADAM3 on thesperm surface was severely reduced. In contrast, only

ADAM2 is known to be expressed in the non-humanprimate. In this study, to identify the role(s) of testis-specific ADAMs in the primate fertilization, we first haveproduced specific antibody against monkey ADAM2carrying recombinant proteins of cys/EGF like domains ofmonkey ADAM2. As a result of Western blot analysisusing against anti-ADAM2 cys/EGF antibody, two 100-and 40- kDa proteins and a single 40- kDa protein wererecognized in germ cells and sperm. By conducting biotinlabeling of sperm surface and trypsin treatment, monkeyADAM2 was identified to be localized on the sperm surface.Moreover, hetero-dimerization experiments suggest thatthe monkey ADAM2 may form complex with unknownprotein in both of germ cells and sperm. To identify a novelprotein interacted with ADAM2 in non-human primatesperm, we first tested which domains play an importantrole in association with sperm plasma membrane protein.Each recombinant protein of metalloprotease, disintegrin,cystein-rich, EGF-like, and cytoplasmic domain wassubjected to SDS-PAGE and then blotted with the spermsurface proteins biotinylated using EZ-link-sulfo-NHSbiotin and anti-biotin antibody. The band only detected inthe cystein-rich domain. This implies that the cystein-richdomain is essential to form hetero complex. From theseresults, we concluded that the primate ADAM2 wasexpressed on the monkey sperm surface, forming heterocomplex with unknown protein. And cystein-rich domainhas an important role of interaction with unknown ADAM2partner. Due to the absence of ADAM1 and ADAM3, inthe case of primate, it seems that ADAM2 might be in acomplex with a new as-yet unknown partner for its stability.(Society for the Study of Reproduction 41st Annual Meeting: Hawaii(USA), 2008)

Keywords : ADAM2, ADAM family, fertilin, sperm-egginteraction, hetero complex

Members

Director Kyu-Tae Chang ([email protected])

Senior Researcher Myeong-Su Kim ([email protected])Ekyune Kim ([email protected])Sang-Hyun Kim ([email protected])Sang-Lae Lee ([email protected])

Post-DocJae-Won Huh ([email protected])

Researcher Jong-Man Kim ([email protected])Kang-Jin Jeong ([email protected])Hee-Jong Eom ([email protected])Young-Hyun Kim ([email protected])Jae-Woong Lee ([email protected])


Hyoung-Woo Kim ([email protected])Keun-Su Kim ([email protected])Sung-Jin Kim ([email protected])Dong-Chul Baek ([email protected])Hye-Rim Kim ([email protected])

A-20In vitro absorption and metabolism characteristicsof 2'-benzoyloxycinnamaldehyde, an antitumordrug candidate

Kang-Jeon Kim1, Soo Jin Oh1, Kye Sook Lee1, Je KyoungRyu1, Byoung-Mog Kwon2, Dong Cho Han2, Young-MinHan2, Jong Soon Kang1, Song-Kyu Park1, Kiho Lee1, andHwan Mook Kim1

1Bio-Evaluation Center, KRIBB2Molecular Cancer Research Center, KRIBB

Background. 2'-Benzoyloxycinnamaldehyde (CB-Ph) is asynthetic analog of 2'-hydroxycinnamaldehyde (CB-OH)which was originally isolated from the stem bark ofCinnamomun cassia. CB-Ph has been shown to inhibit growthof various tumor cells in vitro and in vivo, currently beingdeveloped as an antitumor drug. Purpose. The aim of thisstudy was to characterize the absorption and metabolism of CB-Ph, as part of our efforts to support its preclinical development. Methods. The transport and metabolism of CB-Ph werestudied using Caco-2 cell monolayers grown in Transwellsystems for three weeks. The in vitro metabolism was examinedalso in human and rat sera and liver microsomes. Results. CB-Ph was not detected in both the apical (AP)and basolateral (BL) sides during the absorptive transportacross Caco-2 cell monolayers instead, its metabolites, CB-OH and 2'-hydroxycinnamic acid (CB-OH acid) weredetected in both the AP and BL sides. CB-Ph was degradedrapidly in both rat and human sera, with half life <1 min.In rat serum, both CB-OH and CB-OH acid were producedrapidly and then CB-OH was degraded in short time. However,CB-OH acid was not produced in human serum. CB-Ph wasdegraded rapidly also in rat and human liver microsomesin the presence and absence of NADPH, being rapidlyconverted to CB-OH and CB-OH acid. These resultsindicate that the degradation of the CB-Ph occurred in both ratand human liver microsomes were mediated by enzymatichydrolysis, distinct from CYP450. Conclusions. Based on the results of our Caco-2 study, CB-Phis likely to be rapidly converted to and, thus absorbed as, itsactive metabolite, CB-OH. CB-Ph is likely rapidly converted toCB-OH in the serum and liver microsomes. (SpringInternational Convention of the Pharmaceutical Societyof Korea : Jeju, 2008)

Keywords : 2'-benzoyloxycinnamaldehyde, 2'-hydroxycinnamaldehyde, absorption, metabolism

A-21Transport and metabolism of JAC106, an NODonor-containing tubulin binding agent, inCaco-2 cells

Kye Sook Lee1, Kang-Jeon Kim1, Soo Jin Oh1, Myung-


Bio-Evaluation Center

Hwa Kim2, Kwang-Woo Chun2, Jong-Hee Choi2, Jong SoonKang1, Song-Kyu Park1, Kiho Lee1, and Hwan Mook Kim1

1Bio-Evaluation Center, KRIBB2R&D Center, Jeil Pharmaceutical Co.

Purpose. JAC106 is a novel NO donor containing tubulinbinding agent, currently under preclinical development as anantitumor agent. The aim of this study was to characterizethe transport and metabolism of JAC106 in Caco-2 cells. Methods. Caco-2 cells, grown to confluency for 3 weeks onTranswell filter membranes, were used. The permeabilityrate of JAC106 and its active metabolite, JAC096,acrossCaco-2 cell monolayers was determined at its initialconcentration of 5 µM for 2 hr. Transport of JAC106 andJAC096 were studied from apical (AP) side to basolateral(BL) side or from BL side to AP side. The studies of JAC106metabolism were performed to evaluate the role of cytochromeP450 (CYP) and glutathione S-transferase (GST)usingCYP inhibitor (1-aminobenzotriazole; ABT) and GSTinhibitors such as 1-chloro-2, 4-dinitrobenzene (CDNB),7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), andethacrynic acid (EA). JAC106 and JAC096 were quantitatedsimultaneously using liquid chromatography-tandem massspectrometry (LC-MS/MS). Results. The Caco-2 data has shown that JAC106 is highlypermeable with an apparent permeability of 23.5 ±2.510-6 cm/s. JAC106 was produced active metabolite, JAC096,in Caco-2 cells. JAC096 showed a moderate permeability(11.2 ±0.6 10-6 cm/s) across Caco-2 cells. The efflux ratioof JAC106 and JAC096 was 1.3 and 2.9, respectively. CDNBand NBD-Cl were concentration-dependently inhibitedbiotransformation of JAC106. However, ABT had nosignificant effect on the metabolism of JAC106. Conclusions. JAC106 has highly permeability and it wastransformed to active metabolite, JAC096 in Caco-2 cells.These results indicated that JAC106 converted to JAC096by not CYP-mediated but GST-mediated metabolism inCaco-2 cells. (Spring International Convention of thePharmaceutical Society of Korea : Jeju, 2008)

Keywords : Caco-2 cells, GST, JAC106, JAC096, metabolism,transport

A-22Plasma pharmacokinetics of 2'-benzoyloxy-cinnamaledhyde, an antitumor drug candidate,in rats

Je Kyung Ryu1, Kang-Jeon Kim1, Soo Jin Oh1, Kye Sook Lee1,Byoung-Mog Kwon2, Dong Cho Han2, Young-Min Han2, JongSoon Kang1, Song-Kyu Park1, Kiho Lee1, and Hwan MookKim1

1Bio-Evaluation Center, KRIBB2Molecular Cancer Research Center, KRIBB

2'-Benzoyloxycinnamaldehyde (CB-Ph) is a syntheticanalog of 2'-hydroxycinnamaldehyde (CB-OH) which wasoriginally isolated from the stem bark of Cinnamomumcassia. CB-Ph has been shown to inhibit growth of varioustumor cells in vitro and in vivo, currently being developedas an antitumor drug. To investigate plasma pharmacokineticsof the antitumor drug candidate, CB-Ph in rats. Thepharmacokinetics of CB-Ph was evaluated in male Sprague-Dawley rats following a single intravenous (50 mg/kg) ororal (100 mg/kg) administration. Concentrations of CB-Phand its metabolites, CB-OH and 2'-hydroxycinnanic acid(CB-OH acid) were determined by an HPLC/UV and LC-MS/MS method, respectively. Following i.v. injection,CB-Ph was not detected in the plasma for 24 hr post dosing.On the other hand, the metabolites, CB-OH and CB-OHacid, were detected above the quantitation limit for 4-8 hr,with tmax of 5 min (the first sampling time point). The half-lives of CB-OH and CB-OH acid were estimated to be 1.7hr and 0.6 hr, respectively. Likewise, CB-Ph was notdetected in the plasma following the oral administration.Its metabolites, CB-OH and CB-OH acid, were detectedcontinuously over 24 hr post dosing, with their tmax occurringat 15 min (the first sampling time point). The half-lives ofCB-OH (10.4 hr) and CB-OH acid (9.6 hr) were muchlonger compared to the i.v. administration. These resultssuggest that CB-Ph was metabolized rapidly to CB-OHand CB-OH acid in the body, which was consistent withits low serum and microsomal stability demonstrated inour separate study. (Spring International Convention ofthe Pharmaceutical Society of Korea : Jeju, 2008)

Keywords : 2'-benzoyloxycinnamaldehyde, 2'-hydroxycinnamaldehyde, 2'-hydroxycinnanic acid, pharmacokinetics

A-23Gene expression profiling of KBH-A42 in humanleukemia and bladder cell lines

Moo Rim Kang1, Jong Soon Kang1, Jang hyun Kim1, SeoJi-min1, Kiho Lee1, Hwan Mook Kim1, Lee Ki Hoon1, JongSeong Kang2, Gyoonhee Han3, and Song-Kyu Park1

1Bio-Evaluation Center, KRIBB2Chungnam National University3Yonsei University

We investigated the anti-tumor activity of KBH-A42, anovel synthetic histone deacetylase (HDAC) inhibitor.KBH-A42 significantly suppressed the proliferation of 26human cancer cell lines tested. Among them, the humanleukemia cell line K562 was the most sensitive, whereasthe bladder cancer cell lines UM-UC-3 was the least sensitive.Growth inhibition of K562 cells by KBH-A42 was, in part,mediated by induction of apoptosis but that of UM-UC-3


cells was not. To gain insights into the mechanism by whichKBH-A42 inhibits cell growth, mRNA expression profiles ofK562 and UM-UC-3 cells were analyzed. In a human tumorxenograft model using Balb/c nude mice, KBH-A42significantly inhibited the growth of K562 tumors, butslightly inhibited that of UM-UC-3 tumors. Taken together,our in vitro and in vivo results suggest that KBH-A42 hasan anti-cancer activity but different cells may be regulateddifferentially by KBH-A42. (The 9th InternationalCongress on Cell Biology : Seoul, 2008)

Keywords : apoptosis, HDAC, microarray, xenograft

A-24KBH-A42 overcomes multi-drug resistance andinduces cell death in P-glycoprotein expressingcells

Moo Rim Kang1, Jong Soon Kang1, Jang hyun Kim1, JeongWook Yang1, Chang Woo Lee1, Kiho Lee1, Jong Seong Kang2,Gyoonhee Han3, Hwan Mook Kim1, and Song-Kyu Park1

1Bio-Evaluation Center, KRIBB2Chungnam National University3Yonsei University

We investigated the anti-tumor activity of KBH-A42, anovel synthetic histone deacetylase (HDAC) inhibitor, onmulti drug resistance cell lines. KBH-A42 induced equivalentcell death in a dose-dependent manner in both P-gp-positive and P-gp-negative cells. In both P-gp-positive andP-gp-negative K562 cells, KBH-A42 caused the accumulationof G0/G1 by inhibition of CDK2, CDK4 and CDK6, andinduction of p21waf1 protein level. In both P-gp-positiveand P-gp-negative MCF7 cells, KBH-A42 caused theaccumulation of G2/M by inhibition of cyclin A andinduction of p21waf1 protein level. Furthermore, in K562P-gp-positive cells, KBH-A42 induced apoptosis byenhancing the levels of cleaved caspase-3, caspase-9,PARP and gelsolin. In MCF7 P-gp-positive cells, theexpression levels of Bcl-xL were suppressed while Bad andBax were increased. These data demonstrates that KBH-A42 strongly inhibits the growth of both P-gp-positive andP-gp-negative cell by cell cycle arrest at G0/G1 or G2/Mphase and partially by caspase-3 mediated apoptosis. (The9th International Congress on Cell Biology : Seoul, 2008)

Keywords : apoptosis, cell cycle arrest, HDAC, P-glycoprotein

A-25The effect of inserted cucumber mosaic virus-coat protein gene chili pepper ((CMV-cp (line

7)) on the non-target insects, green peachaphids (Myzus persicae Sulzer, Homoptera)

Ji Eun Park1, Hoonbok Yi1, Kuang Sik Oh1, Min ChulKwon2, Chang-Gi Kim1, and Hwan mook Kim1

1Bio-Evaluation Center, KRIBB 2Namhae Betterfly Park

To assess the environmental risks of transgenic chilipepper with CMV-cp (line 7) on non-target organismsbefore it exposes to the agro-ecosystem environments, weconducted the three sets of green peach aphids (Myzuspersicae S.) life table experiment under laboratory conditions(Temp. 25°C, R.H. 50-70%, Photoperiod L16:D8) in seriesduring 2006-2008. We measured the net reproductive rate(R0)*, the intrinsic rate of increase (rm), the mean generationtime (TC), fecundity*, life span, and reproduction periodbetween non-transgenic chili peppers and transgenic chilipeppers, respectively. The life span of green peach aphidsfrom three sets was 24, 28, 32 days, and the period of lifespan was similar to the general average length of greenpeach aphids, 25-29 days. Although the first reproductionof transgenic pepper was similar to the non transgenicpepper (P>0.05), the fecundity and the net reproductiverate (Ro) by using Jackknife method of transgenic pepperwere similar of non transgenic pepper (P>0.05). Conclusively,we observed the adverse effect from our results but weshould execute further experiments to confirm the resultsat the fields with the similar way. (The 63th Annual Meetingof the Korean Association of Biological Sciences : Muan,2008)

Keywords : chili pepper, environmental risk, green peachaphids, transgenic

A-26

The soybean mosaic virus resistance Locus, Rsv3,is cosegregating with a cluster of genes thatencode a nucleotide binding site and a leucine-rich repeat region

Kiwoung Yang, Namhee Jeong, Woo Kyu Lee, and Soon-Chun Jeong

Bio-Evaluation Center, KRIBB

The soybean mosaic virus resistance locus, Rsv3, wasmapped to the soybean molecular linkage group B2. One endsequence of a marker, M1a, closely linked but notcosegregating to the Rsv3 locus revealed the consensussequence to a leucine rich repeat (LRR) characteristic ofthe extracellular LRR superfamily of resistance genes. Ouranalysis of the preliminary soybean wholegenome shot-gun sequencing data, which was released in the early


2008, indicated that the LRR-containing sequence wasrepeated at least 10 times in the vicinity of the Rsv3 locus.By mapping markers developed from the LRR-containingsequences as well as investigating full-length genestructures of the LRR-containing sequences, we showed thatthe Rsv3 locus is cosegregating with a cluster of genes thatencode a nucleotide binding site (NBS) and a LRR region.The results suggested that the Rsv3 gene likely encodes amember of the NBS-LRR gene cluster. (The 9th

International Congress on Cell biology & The 20th AnnualConference of the Korean Society for Molecular andCellular Biology : Seoul, 2008)

Keywords : LRR, soybean mosaic virus, Rsv3

A-27Towards confirmation of a major quantitativetrait loci for isoflavones content in soybeanseed

Kiwoung Yang1, Namhee Jeong1, Jung-Kyung Moon2,Hyo-Kon Chun1, Kyoungwhan Back3, and Soon-ChunJeong1

1Bio-Evaluation Center, KRIBB 2National Institute of Crop Science3Chonnam National University

Soybean seeds frequently contain high levels ofisoflavones. Breeding soybean seeds with desirableisoflavone content would be beneficial to the food andhealth industries, but the environmental sensitivity of thetrait complicates phenotypic selection. This study was toidentify quantitative trait loci (QTL) associated withisoflavones contents in soybean seeds. A population of113 F12 recombinant inbred lines (RILs) generated via across between a cultivar 'Hawngkeum' and a wild soybean'IT182932' was used. The population was phenotyped atthree locations in Suwon, Daejeon and Ochang in Korea,and genotyped by means of 434 polymorphic sequence-based markers. Interval mapping and composite intervalmapping identified regions associated with isoflavonescontents linkage groups (LGs) A1, A2, B1, B2, C1, C2,D1b, E, F, H, J, K, M, N, and O. The 15 LGs detected 20QTLs (LOD score > 2.5). Particularly, the QTL controllingdaidzein, genistein, and total isoflavone on a telomericregion of LG A1 appeared to be a major QTL. Fine mappingof the telomeric region on th LG A1 where the major QTLis located was attempted using preliminary soybean wholegenome sequence release. (International Symposium onPlant Genetic Resources and Biotechnology : Yongpyong,2008)

Keywords : isoflavones, quantitative trait loci, soybean

A-28Genetic analysis and event-specific detection ofa CGMMV-CP transgenic watermelon rootstockline

Eun Soo Youk1, In Soon Pack1, Yu-Jin Kim1, Won KeeYoon1, Chang-Gi Kim1, Stephen B. Ryu1, Chee Hark Han2,Soon-Chun Jeong1, and Hwan Mook Kim1

1Bio-Evaluation Center, KRIBB 2Biotechnology Center, NongWoo Bio

Transgenic plants that over express virus coat proteingenes have attracted particular interest from researchers, byvirtue of their tolerance to virus infection. The transgenicwatermelon rootstock analyzed in this study was establishedby introducing CGMMV coat protein (cp) under the controlof CaMV 35S promoter and NOS terminator (Park et al.,(2005) Plant Cell Rep. 24: 350-6). The primary objectiveof this study was to determine the copy number andintegration site of the transgene element, in order to developdetection techniques required for monitoring of thetransgenic watermelon rootstock. The Southern blot analysisindicated that a single copy of CGMMV-cp gene wasinserted into the genome of transgenic watermelon rootstock.We also identified the genomic sequences flanking theintegration site of the transgene by inverse PCR analysis.In an effort to find a sequence usable as an internal positivecontrol for the screening of the watermelon and watermelonrootstock, we found that the Sat and DIP-1 genes appearsas one copy within their genomes and is watermelonrootstock- and watermelon-specific. The information ofthe integrated site and the internal positive control sequencewas used to establish a new event-specific PCR-baseddetection method. In addition, mRNA and protein expressionlevel of the transgene in the transgenic watermelonrootstock and grafted watermelon were investigated. Theexpression of both mRNA and protein of CGMMV-CP wasnot detected in the transgenic watermelon rootstocks andwatermelons, suggesting that the movement of transgeneproducts from transgenic rootstock to watermelon does notoccur at our detection level. (Symposium TowardEnhancing Agricultural Biodiversity for Future CropProduction : Gyeongju, 2008)

Keywords : chili pepper, environmental risk, green peachaphids, transgenic

A-29Agronomic evaluation of transgenic Capsicumannuum L.resistant to anthracnose fungus

Eun-Mi Ko1, Do Young Kim1, Kee Woong Park1, Chang-Gi Kim1, Bumkyu Lee1, Jeum Kyu Hong1, Hoonbok Yi1,


Heungtae Kim2, and Hwan Mook Kim1

1Bio-Evaluation Center, KRIBB 2Department of Plant Medicine, Chungbuk NationalUniversity

Somaclonal variation caused by tissue culture proceduresmay produce undesirable characteristics in transgenicplants. The objective of the present study was to evaluatevariability in agronomic characteristics and diseasesusceptibility of chili pepper transformed for resistance toanthracnose fungus in the field. Seed germination of thetransgenic chili pepper was compared with that of its non-transgenic parent line at 20, 28, and 35°C. Initialgermination time of transgenic line was slightly slowerthan that of non-transgenic line at 28 and 35°C. Themaximum cumulative germination between the transgenicand non-transgenic lines was not different at all temperatureregimes. Significant differences in agronomic traits andyield components were detected between the transgenicand non-transgenic lines. Fruit production of the transgenicline was 75%, when compared with the non-transgenicline. We also observed occurrence of three chili pepperdiseases mainly infested in Korean fields bacterial leafspot, powdery mildew, and virus disease in the transgenicand non-transgenic lines in the fields in 2006 and 2007.No significant differences in disease occurrences wereobserved between transgenic and non-transgenic lines.Although the fitness of the transgenic line was less thanthe non-transgenic line, this penalty could be overcome bybackcrossing a transgenic line with its parents. (The 63thAnnual Meeting of the Korean Association of BiologicalSciences : Muan, 2008)

Keywords : anthracnose fungus, chili pepper, diseases,germination

Members

Director Hwan Mook Kim ([email protected])

Principal Researcher Hyoung Chin Kim ([email protected]) Song-Kyu Park ([email protected])Ki Ho Lee ([email protected])Soon-Chun Jeong ([email protected])Won Kee Yoon ([email protected])Ki Hoan Nam ([email protected])

Senior Researcher Jong Soon Kang ([email protected])Chang-Gi Kim ([email protected])Young Suk Won ([email protected])

Post-DocMoo Rim Kang ([email protected])Kiwoung Yang ([email protected])

Researcher Ki Hoon Lee ([email protected])Chang Woo Lee ([email protected])In Soon Pack ([email protected])Eun-Mi Ko ([email protected])Kang-Jeon Kim ([email protected])Kye Sook Lee ([email protected])Je Kyung Ryu ([email protected])Ji Eun Park ([email protected])Eun Soo Youk ([email protected])


Jeonbuk Branch Institute

Bioindustry Research Center

Molecular Bioprocess Research Center


A-39Analysis of ß-(1 3)(1 6)-glucan produced byAureobasidium pullulans IMS-822

Seog June Lee, Keug-Hyun Ahn, Chan-Sun Park, and Byung-Dae Yoon

Bioindustry Research Center, KRIBB

The exo-polysaccharide producing microorganism,Aureobasidium pullulans IMS-822, was isolated andidentified from soil. The viscosity-average molecularweight of exo-polysaccharide was calculated as 8.9 X 105

by Mark-Houwink equation. The sugar component of exo-polysaccharide was determined as glucose by HPLC analysis.The IR spectra indicated that the exo-polysaccharide hasan absorption peak at 890 cm-1 for the ß-configuration ofD-glucan. The 13C NMR signal at 86.62 ppm arose fromthe substituted C-3 of glucose. The signal at 72.11 ppmwas assigned to C-6 of branched ß-(1 3)-D-glucosylresidues. Viscosity and Congo red reaction indicated thatß-(1 3)(1 6)-glucan produced by A. pullulans IMS-822has a highly ordered hydrogen-bond dependent conformationin aqueous solution, which collapses in strong alkalinesolution. (Under preparation)

Keywords : aqueous conformation, Aureobasidium sp., ß-(1 3)(1 6)-glucan, viscosity-average molecularweight

A-40Real-time assays for the detection of Bacilluscereus strains in fermented foods

Seong Bin Kim, Sun Yi Lee, Min Soo Kim, and Byung DaeYoon


Bacillus cereus is widespread in nature and frequently isolatedfrom soil and growing plants, but it is also well adapted forgrowth in the intestinal tract of insects and mammals. It iseasily spread to foods, where it may cause an emetic or adiarrhoeal disease that is becoming increasingly important inthe industrialized world. According to the increasingnumber of reports of food-borne disease, fast detectionmethods are required for diagnostic purpose as well as for theprevention of food contamination and food-borne outbreaks. We performed DNA isolation from Korean fermentedfood and established real-time assays were used to analyzefood poisoning. Real-time PCR assay was developed toprovide a rapid and sensitive method for the specificdetection of B. cereus in food using the specific primers totarget of food poisoning hemolysin genes. In conclusion,the proposed real-time PCR is a reliable, simple, rapid, and

efficient method for the simultaneous identification of B.cereus group bacteria from food samples in a single tube.(Under preparation)

Keywords : Bacillus cereus, fermented foods, real-timeassays

A-41Neuraminidase inhibitory activities offlavonoids from Rhodiola rosea

Hyung Jae Jeong1, Young Bae Ryu1, Jang Hoon Kim1, HyungJun Kwon1, Jin Hyo Kim2, Su Jin Park1, Ki Hun Park2, andWoo Song Lee1*

1Bioindustry Research Center, KRIBB2Division of Applied Life Science (BK21 Program),Gyeongsang National University

Neuraminidase exiting on surface of avian influenza (AI)plays an important role in viral proliferation and is stablypresent in both influenza virus A and B. AI antiviral drugsto be neuraminidase inhibitor are Tamiflu (Oseltamivir)and Relenza (Zanamivir), but they have several side effects. Inorder to solve these side effects, flavones (1, 4-9) wereisolated by bioassay-guided fractionation from the ethylacetate-soluble extracts of Rhodiola rosea root and commercialavailable flavones (2, 3, 10-16) were prepared. When sixteenflavones 1-16 were evaluated for their neuraminidaseinhibitory effect, the IC50 values of all tested compound (1-16) showed a range of 0.78-60 M, respectively. Among thetested flavone derivatives, gossypetin 3 exhibited the mostpotent inhibitor and has proven to be depending upon thenumber of the hydroxy group on flavone backbone. In kineticstudies, all isolated compounds (1, 4-9) screened wereexhibited to be noncompetitive inhibitor. (InternationalSymposium and Annual Neeting of the KSABC : Daegu,2008)

Keywords : flavonoid, neuraminidase, noncompetitiveinhibitor, Rhodiola rosea

A-42The strain development of Aureobasidiumpullulans for pure beta-glucan elaboration

Byung-Kwan Kang, Seug-June Lee, Min-Soo Kim, Keug-Hyun Ahn, Hee-Jong Yang, and Byung-Dae Yoon

Bioindustry Research Center, KRIBB

Production of pure ß-glucan exopolysaccharide was



engineered in A. pullulans IMS822 KCTC11179BP. ThePullulan cDNA which is participated in synthesis ofpullulan exopolysaccharide was disrupted by homologousrecombination and then mutant was named by A. pullulansNP1221. Through enzyme reaction, which is pullulanseand laminarinase treatment, an elimination of pullulan andproduction of pure ß-glucan in NP1221 mutant wasconfirmed by a type of liberated carborhydrates. And bothparent strain and mutant strain were fermented in Czapek(Difco Laboratory) medium and compared with fermentationkinetics. The substrate uptake rate and yield of ß-glucanwere not significantly different, but the case of biomassand pullulan synthesis were very different in mutant strain.The mutant strain was not produce pullulan and yield ofbiomass was more high 2.3 folds then wild type in 9.2 g·l-1. Insecond approach, the mutant strain was cultured withvarious agitation (150, 250, 350, 500 rpm) in 5L JARfermentorincrease ß-glucan productivity. At 500 rpm, theproductivity of pure ß-glucan was 10 g·l-1. (Underpreparation)

Keywords : A. pullulans NP1221, Aureobasidium pullulans,beta-glucan, exopolysaccharide,

Members

Director Byung-Dae Yoon ([email protected])

Principal Researcher Woo Song Lee ([email protected]) Mun Chual Rho ([email protected])

Senior Researcher Min-Soo Kim ([email protected])

Post-DocSeog June Lee ([email protected])Seong Bin Kim ([email protected])

Researcher Chan-Sun Park ([email protected])Keug-Hyun Ahn ([email protected])Sun Yi Lee ([email protected])Byung-Kwan Kang ([email protected])

A-30Molecular cloning, nucleotide sequence andexpression of levansucrase from Bacillusamyloliquefaciens type 1 in Escherichia coli

Dina Rairakhwada, Mi Young Seo, Jeong-Woo Seo, andChul Ho Kim

Molecular Bioprocess Research Center, KRIBB

Levansucrase is a family of bacterial enzymes with ß-fructofuranosyl fructotransferase activity (E.C. 2.4.1.10)which produce different type of D-fructose polymer (levan)and oligomers (1-kestose and nystose) from sucrose. Levanobtained from wild-type Bacilllus amyloliquefaciens type 1at a concentration of 0.5% was used as an immunostimulantin aquaculture for common carp, Cyprinus carpio. Theproduction of levan using the wild-type strain is not cost-effective due to the multiple purification steps required toproduce purified levan, hence in the present study a novellevansucrase from B. amyloliquefaciens type 1 was isolatedfollowed by the nucleotide sequence analysis. B.amyloliquefaciens type 1 SacB consisted of 473 aminoacids including the 29 amino acids of an N-terminal signalpeptide. The kinetic parameters were determined, whichgave the Km value of 47.81 mM sucrose for hydrolysismeasured for the levansucrase from B. amyloliquefacienstype 1. The optimized conditions gave the sucrosehydrolysis activity of 37.32 µmole glucose/min mg proteinand levan yield of 50.6 gms /L of the reaction mixture wasobtained from 10% sucrose. (The Thirteenth InternationalBiotechnology Symposium and Exhibition : Dalian(China), 2008)

Keywords : Bacillus amyloliquefaciens type 1, expression,immunostimulant, levansucrase

A-31Heterologous expression and characterizationof L-Amino acid deaminases from Proteusmirabilis in Escherichia coli

Jin-Oh Baek1,2, Jeong-Woo Seo1, Ohsuk Kwon4, Su-IlSeong3, Ik-Hwan Kim2, and Chul Ho Kim1

1Molecular Bioprocess Research Center, KRIBB2School of Lifesciences and Biotechnology, Korea University3R&D center, Biotopia Co., Ltd. 4Omics and Integration Research Center, KRIBB

Phenyllactic acid (PLA) has a very good antimicrobialactivity against not only gram positive and gram-negativebacteria but also fungi of a few mycotoxigenic species.The PLA is known to be produced from phenylalanine(Phe) in many of lactic acid bacteria, in which the Phe is


Molecular Bioprocess Research Center

first conversed to phenylpyruvic acid (PPA) by action oftransaminase using -ketoglutarate as an acceptor of aminogroup, and then the PPA is transited to the PLA by actionof dehydrogenase. The transaminase reaction is the rate-limiting step in the catalytic pathway because of thelimited supply of -ketoglutarate, by which the commercialapplication of the PLA as an alternative of antibiotics hasbeen hampered. Therefore, new catatlytic route to producePPA need to be explored. Here, two L-amino aciddeaminases (AADs), Pma and Pm1, from Proteusmirabilis werecloned, expressed in Escherichia coli, andcharacterized. Both of AADs were well expressed in E.coli in active forms that are associated with cytoplasmicmembrane. The production of PPA from Phe by the actionof enzymeswas confirmed by HPLC and LC/MS analyses.As determined by enzymatic kinetics for Pma and Pm1,the Vmax and Km values were found to be 152.6 (gPPA/mg min), 8.66 (mM Phe) and 119.7, 31.55, respectively.As per the kinetic results, the Pma is more specific foraromatic amino acids than the Pm1, suggesting that thePma is more useful for the development of industrialprocess to produce PLA. (The Thirteenth InternationalBiotechnology Symposium and Exhibition : Dalian(China), 2008)

Keywords : L-amino acid deaminase, phenyllactic acid,phenylpyruvic acid, PLA, Proteus mirabilis

A-32Enhanced production of eicosapentaenoic acidby reengineering of EPA biosynthesis genecluster in Escherichia coli

Su-Jin Lee1,2, Jeong-Woo Seo1, Byung-Ki Hur2, and ChulHo Kim1

1Molecular Bioprocess Research Center, KRIBB2Department of Biological Engineeringand Institute ofBiotechnological Industry, Inha University

We isolated the eicosapentaenoic acid (EPA) biosyntheticgene cluster from Shewanella oneidensis MR-1 by thelong-PCR method. The production of EPA was observedin the recombinant Escherichia coli clones, of which themaximum yield was 0.69% of the total fatty acids in a clonedesignated 9704-23. In order to enhance the heterologousproduction of EPA, genetic engineering of the EPA biosyntheticgene cluster was carried out. Firstly, the native promoter ofEPA biosynthetic gene cluster was replaced by strong lacpromoter on pBluescript SK(+) and Secondly, the probabletranscription repressor gene was omitted from the gene

cluster to enhance the expression level of the EPAbiosynthetic genes in E. coli. In addition, espression of 4-phophopantetheine transferase that plays a pivotal role inthe activation of enzymes in biosynthetic machinery was alsostimulated under lac promoter. E. coli clone harboring theengineered plasmid, pBSKEPA-MR1 showed remarkableincrease in the production of EPA up to 11-fold higherlevels (7.5%). (The Thirteenth International BiotechnologySymposium and Exhibition : Dalian (China), 2008)

Keywords : biosynthesis gene, E. coli, eicosapentaenoicacid, EPA

A-33Screening and characterization of a novelmetalloprotease from a metagenomic library

Nack-Shick Choi, Bo-Hye Kim, Chang-Su Park, Yun-JonHan, Won-Bok Joo, Joong Su Kim, Sukhoon Koh, and JaeJun Song*


Proteases are ubiquitous enzymes with important roles inphysiological process and they constitute two thirds of thetotal enzymes used in industry and their industrialdemands expected to be continuously increased. Using ametagenomic approach, a novel metalloprotease gene wasisolated. The metagenomic library was constructed withtotal metagenomic DNA extracted from soil in theJeongeup-si of Korea and was used together with a fosmidvector, pCC1FOS. Five active clones from approximately20,000 fosmid library clones were selected on the skimmed-milk plate. One clone of them was subcloned into pUC19and sequenced. Nucleotide sequence analysis revealed asingle open reading frame (ORF) of 2,070 base pairs encoding689 amino acids. The amino acid sequence was 52%identical to a zinc-metalloprotease gene from Salinivibriosp. strain. The deduced amino acid sequence includes azinc-metalloprotease His-Glu-X-X-His (H-E-X-X-H)consensus motif which is highly conserved in the M4family of proteases, suggesting that the enzyme is ametalloprotease. The recombinant protease was detected onan apparent molecular mass of 60 kDa by fibrin zymographyanalysis. (International Symposium & Annual Meetingof the Korea Society for Microbiology and Biotechnology :Seoul, 2008)

Keywords : fosmid library, metagenomics, metalloprotease,screening, soil


A-34Isolation and identification of microorganismsfor cellulytic activity from soil

Sung Suk Cho, Jae Jun Song, Sukhoon Koh, and Joong Su Kim*


A lignocellulose, one of the major polymers in plant cellwall is the most abundant renewable biological resourse.Bioenergy from less costly renewable lignocellulose isimportant for the sustainable development of humanbeings. The widely accepted mechanism for enzymaticcellulose hydrolysis involves synergistic action byendoglucanase, cellobiohydrolase, and ß-glucosidase. Inthis study, For the first step, we prepared the dye-labelledsubstrates by dyeing carboxy-methyl cellulose(CMC) andxylan with Remazol Brilliant Blue R (RBB) and CibacronBrilliant Red 3BA (CBR) to screen endo-ß-1,4-glucanaseand endo-ß-1,4-xylanase, respectively. 309 positivemicroorganisms showing high endoglucanase activitieswas obtained on RBB-O-CMC agar plate from soils. Forthe second step, the production of reducing sugar wasmeasured from culture broth using CMC, Avicel and xylan assubstrates. ß-Glycosidase activity was determined by usingp-NPG as a substrate. Among the micro-organisms screened,two microorganisms showing high endoglucanase activitywere found and subjected to 16S ribosomal RNA analysis.The 16S rRNA analysis revealed that two isolates weresimilar to Paenibacillus polymyxa and Bacillus subtiliswith 99% and 100% homologies, respectively. (InternationalSymposium & Annual Meeting of the Korea Societyfor Microbiology and Biotechnology : Seoul, 2008)

Keywords : ß-glucosidase, cellobiohydrolase, endoglucanase,lignocellulose

A-35Screening and PCR amplification ofglucansucrases from soil microorganisms

Hyun Jung Cho, Min Ji Yeon, Jae Jun Song, Suk Hoon Koh,and Joong Su Kim*


Microbial glucansucrases (GH-family 70) are exclusivelyrelated to synthesize glucan polymers. An interestingdiversity exists in the GH-family 70, where enzymes cansynthesize various types of glucosidic linkages. Amongthe enzymes in the GH-family 70, the glucansucrases are largeenzymes with an average molecular mass of 160kDa andcatalyze the synthesis of glucose polymer including dextran,mutan, alternan. In this study, we tested 800 microorganisms

collected from soils of Naejangsan, Gapyeong and Imsil areato find the glucansucrases which catalyze the synthesis ofglucan from sucrose. Among them, sixty three bacteriawere selected as the glucan producing bacteria by using thephenol-sulfuric acid method. To clone the gene encodingglucansucrase from selected bacteria, degenerate primerswere designed based on various glucansucrase amino acidsequences from lactic acid bacteria and applied to PCRusing genomic DNA of isolates as template. The 670 bp ofDNA fragment produced from PCR were cloned into T-vector and sequenced. The comparison of the deducedamino acid sequences of the amplified DNA fragmentswith those of the published glucansucrase genes showedhigh identities to the sequences of Leuconostoc mensenteroidesglucansucrase. (International Symposium & AnnualMeeting of the Korea Society for Microbiology andBiotechnology : Seoul, 2008)

Keywords : GH-family 70, glucansucrase, Leuconostocmensenteroides, microbial glucansucrases

A-36Optimization of culture conditions for 1,3-propanediol production from crude glycerol byKlebsiella pneumoniase using response surfacemethodology

Baek-rock Oh, Jeong-Woo Seo, Min Ho Choi, and ChulHo Kim


To produce 1,3-propanediol (1,3-PD) from crude glycerol,cultivation conditions were optimized by response surfacemethodology (RSM) based on a 25 factorial central compositedesign(CCD). RSM was adopted to derive a statisticalmodel for the individual and interactive effects of crudeglycerol, (NH4)2SO4, pH, cultivation time and temperatureon the productionof1,3-PD. Optimal conditions formaximum 1,3-PD production were as follows: crudeglycerol, 35 g/l; (NH4)2SO4, 8g/l; pH,7.37; cultivationtime,10.8h; temperature,36.88˚C. Under these optimal conditions,the design expert presented the maximal numerical solutionwith a predicted 1,3-PD production level of up to 13.74 g/l.The experimental production of 1,3-PD yielded 13.8 g/l,which was in close agreement with the model prediction.(International Symposium & Annual Meeting of theKorea Society for Microbiology and Biotechnology :Seoul, 2008)

Keywords : 1,3-propanediol, central composite design,crude glycerol, Klebsiella pneumoniase, responsesurface methodology


A-37Expression and purification of the humanpapillomavirus type 11 L1 capsid protein inEscherichia coli

Sun-Yeon Heo, Pil-Soo Seo, Eun Jong Han, Jeong-WooSeo, and Chul Ho Kim


The major capsid protein L1 of human papillomavirus(HPV) having the immunodominant neutralization epitopesof the virus can be assembled into virus-like particles(VLPs), which is useful as a vaccine cadidate to preventthe infection of the HPV. In this study, we isolated HPVtype 11 L1 from Korean patient and cloned and expressedthe L1 gene in Escherichia coli. The L1 protein washomogeneously purified by Ni-NTA affinity chromatographyand analyzed with western blotting using rabbit anti-denatured papillomavirus polyclonal antibodies. Auto-assembly of L1 proteins into VLPs in vivo or in vitro wasexamined by transmission electron microscopy. (InternationalSymposium & Annual Meeting of the Korea Societyfor Microbiology and Biotechnology : Seoul, 2008)

Keywords : capsid protein L1, human papillomavirus,HPV, VLPs

A-38Isolation and identification of anti-microbialagent from volatile oils

Lianhua Luo, Min-ho Choi, Jeong-Woo Seo, and Chul HoKim


According to the increasing awareness of healthy lifestyle, finishing textiles with antimicrobial agents havegained in popularity over recent years. However, consumersand manufacturers still remain unsatisfied with someproblems such as costs, safety and permanence etc.Essential oils, also named as volatile oils, are obtainedfrom plant materials and have been shown to possessantimicrobial properties. So far, 24 volatile oils fromplants are chosen as candidates for developing novelagents. In addition, in the study, compound EO-1 wasseparated successfully from sample D by following theactivity-isolation guidance. The inhibitory effect of thecompound was examined against a gram-positive bacteriaStaphylococcus aureus and a gram-negative bacteriaklebsiella pneumonia by disc diffusion method. The clearzone of inhibition above 20mm in diameter was exhibitedon both of species. It may be used in functional textileindustry. (Spring KSBB Meeting and International

Symposium : Chonju, 2008)

Keywords : anti-microbial agent, functional textile,klebsiella pneumonia, taphylococcus aureus,volatile oils

Members

Director Chul Ho Kim ([email protected])

Principal Researcher Hyo Kon Chun ([email protected])Joong Su Kim ([email protected])Jae Jun Song ([email protected])

Senior Researcher Jeong Woo Seo ([email protected])Jong Hyun Choi ([email protected])

Post-DocMin Ho Choi ([email protected])Pil Soo Seo ([email protected])Nack Shick Choi ([email protected])Sun Hwa Kwon ([email protected])Dina D. Rairakhwada ([email protected])

Researcher Dong Min Chung ([email protected])Ji Young Park ([email protected])Eun Jong Han ([email protected])Sun Yeon Heo ([email protected])Soo Jin Lee ([email protected])Jin Oh Baek ([email protected])Beak Rock Oh ([email protected])LUO LIANHUA ([email protected])Mi Young Seo ([email protected])Won Kyung Hong ([email protected])Bo Hye Kim ([email protected])Hye Won Lee ([email protected])Eun Hee Lee ([email protected])Won Bok Joo ([email protected])Yun Jon Han ([email protected])Yong Mo Kim ([email protected])Dong Ho Son ([email protected])Min Ji Yeon ([email protected])Sung Sook Cho ([email protected])Hyun Jung Cho ([email protected])


79 A-1 In silico analysis and experimental validation of recently exonized Alu in Macaca fascicularis79 A-2 Identification of novel retromer complexes in the mouse testis 79 A-3 Pig ADAM3 disintegrin domain has an important rolein the sperm-egginteraction 80 A-4 Identification of a binding domain in non-human primate (Macaca fascicularis) ADAM2 for hetero complex41 A-5 New type of FabI-directed antibacterial agent from microbial metabolites 41 A-6 New anti-MRSA agent from Penicillium sp. FR0011 42 A-7 Cordyformamide and chrysophanol, phenolic antioxidative metabolites from the fungus Eupenicillium

shearii F80695 42 A-8 New lactone compounds from the Basidiomycete Stereum ostrea 42 A-9 Chemical and biological properties of the pine bark extract of Pinus densiflora Siebold et Zuccarini 43 A-10 Endoplasmic reticulum stress induction by honokiol isolated from Magnolia obovata43 A-11 Repression of E-cadherin by oncogenic K-ras involving DNA methyltransferase activity 43 A-12 DNA methylation and moderately decreased CBR1 gene expression in K-ras overexpressed prostate cancer

cell lines 44 A-13 Isolation and identification of Micromonospora sp. showing nematocidal activity against Pine wood

nematode 44 A-14 Diversity of culturable prokaryotes in Bigeum Island, established by phylogenetic approach 44 A-15 Clitocybin A, a new antioxidative compound from the culture broth of Clitocybe aurantiaca45 A-16 Etoposide-resistant HT-29 human colon carcinoma cells during glucose deprivation are sensitive to

Piericidin A, a GRP78 down-regulator 69 A-17 Study on cleavage by various proteases and their applications to purification of a fusion-tagged protein 69 A-18 Signal enhancement of surface plasmon resonance using carbon nanotube-antibody complex 69 A-19 Purification of recombinant human erythropoietin in milk of transgenic pigs 81 A-20 In vitro absorption and metabolism characteristics of 2'-benzoyloxycinnamaldehyde, an antitumor drug

candidate 81 A-21 Transport and metabolism of JAC106, an NO Donor-containing tubulin binding agent, in Caco-2 cells 82 A-22 Plama pharmacokinetics of 2'-benzoyloxycinnamaledhyde, an antitumor drug candidate, in rats 82 A-23 Gene expression profiling of KBH-A42 in human leukemia and bladder cell lines 83 A-24 KBH-A42 overcomes multi-drug resistance and induces cell death in P-glycoprotein expressing cells83 A-25 The effect of inserted cucumber mosaic virus-coat protein gene chili pepper ((CMV-cp (line 7)) on the non-

target insects, green peach aphids (Myzus persicae Sulzer, Homoptera) 83 A-26 The soybean mosaic virus resistance Locus, Rsv3, is cosegregating with a cluster of genes that encode a

nucleotide binding site and a leucine-rich repeat region 84 A-27 Towards confirmation of a major quantitative trait loci for isoflavones content in soybean seed 84 A-28 Genetic analysis and event-specific detection of a CGMMV-CP transgenic watermelon rootstock line 84 A-29 Agronomic evaluation of transgenic Capsicum annuum L.resistant to anthracnose fungus 90 A-30 Molecular cloning, nucleotide sequence and expression of levansucrase from Bacillus amyloliquefaciens

type 1 in Escherichia coli90 A-31 Heterologous expression and characterization of L-Amino acid deaminases from Proteus mirabilis in

Escherichia coli91 A-32 Enhanced production of eicosapentaenoic acid by reengineering of EPA biosynthesis gene cluster in

Escherichia coli 91 A-33 Screening and characterization of a novel metalloprotease from a metagenomic library 92 A-34 Isolation and identification of microorganisms for cellulytic activity from soil 92 A-35 Screening and PCR amplification of glucansucrases from soil microorganisms 92 A-36 Optimization of culture conditions for 1,3-propanediol production from crude glycerol by Klebsiella

pneumoniase using response surface methodology 93 A-37 Expression and purification of the human papillomavirus type 11 L1 capsid protein in Escherichia coli 93 A-38 Isolation and identification of anti-microbial agent from volatile oils 89 A-39 Analysis of ß-(1 3)(1 6)-glucan produced by Aureobasidium pullulans IMS-822 89 A-40 Real-time assays for the detection of Bacillus cereus strains in fermented foods 89 A-41 Neuraminidase inhibitory activities of flavonoids from Rhodiola rosea89 A-42 The strain development of Aureobasidium pullulans for pure beta-glucan elaboration 52 A-43 Spectrum of proton irradiation-induced mutations in the tonB gene of Escherichia coli


Poster Board Number Index

52 A-44 Genome sequence of the probiotic bacterium Bifidobacterium bifidum BGN4 52 A-45 Identification of a new gene cluster for type II secretion in Escherichia coli BL21(DE3) 53 A-46 Genome sequence analysis of the marine microbe Donghaeana dokdonensis DSW-6 53 A-47 Multidimensional comparative omics analysis of Escherichia coli B and K-12 53 A-48 Enzyme platform based on yeast TFP technology for the production of cellulosic bioethanol 54 A-49 Development of FRET-based biosensors for rapid and quantitative analysis of sugars in biological samples 54 A-50 Rapid cloning and expression of target genes by homologous recombination system, pRMT-iTGR 54 A-51 Catalytic protein particles based on cellulose-binding domain fusion 55 A-52 Protein engineering supported by homology modeling and docking simulation 55 A-53 Novel cold-adapted alkaline lipase from intertidal flat metagenome and proposal of a new family of

bacterial lipase 56 A-54 Mycotoxins, a group of fungal secondary metabolites, can induce motility of bacterial cells 56 A-55 Functional analysis of the fusaricidin biosynthetic gene of Paenibacillus polymyxa E681 and a possible role

of fusaricidin in biofilm formation 56 A-56 Airborne induction and priming of plant defenses against a bacterial pathogen 57 A-57 Insight into plant defense signalings against Ralstonia solanacearum in Nicotiana benthamiana 57 A-58 Plant innate immunity by bacterial genetic materials 57 A-59 Role of priming resistance genes on bacilli-elicited induced systemic resistance 58 A-60 Improved thermostability and acetic acid tolerance of Escherichia coli by directed evolution of MetA58 A-61 Autoinduction expression system in Escherichia coli59 A-62 Crystal structure of fully oxidized human thioredoxin1 containing disulfide between Cys62 and Cys69 59 A-63 Genomic basis of the two E. coli workhorse strains for their improved capacity to synthesize membrane

proteins at high levels 59 A-64 Characterization and molecular modeling of a thermophilic and alkaliphilic galactokinase from Thermus

caldophilus GK24 27 A-65 Reconstruction of sulfur metabolism pathway in Hansenula polymorpha based on transcriptome and

metabolite analysis 27 A-66 Analysis of the unfolded protein response pathway in the methylotrophic yeast Hansenula polymorpha27 A-67 Functional characterization of Yarrowia lipolitica homologues to Saccaromyces cerevisiae MNN4p and

MNN6p. 28 A-68 Global gene expression profiles of the capnophilic rumen bacterium Mannheimia succiniciproducens

grown under anaerobic conditions 28 A-69 Genome-wide transcriptional responses of different Escherichia coli strains to recombinant protein

overproduction 29 A-70 Characterization of the YgiXY two-component signal transduction system of the capnophilic rumen

bacterium Mannheimia succiniciproducens29 A-71 Tomato chromosome 2: BAC assemble and gene annotation 31 A-72 Comparative proteomic analysis of peripheral blood mononuclear cells from atopic dermatitis patients

and healthy donors 31 A-73 Identification of proteins involved in hepatocellular carcinogenesis using HBx transgenic mouse 31 A-74 Role of autophagy in oxidative stress-induced cytotoxicity in HT22 cells 32 A-75 A proteome profiling of differentiating human embryonic neural stem cells 32 A-76 Comparative proteome analysis of 3T3-L1 cell differentiation into adipocytes 32 A-77 Role of Hsp70 Co-chaperones in the degradation of neurodegenerative proteins 11 A-78 Generation and characterization of a large human Fab library 11 A-79 Isolation and characterization of human antibodies that recognize both hL1cam and mL1cam 11 A-80 Panning strategies for screening of anti-L1CAM human monoclonal antibodies by phage display 12 A-81 TMPRSS4 promotes invasion, migration and metastasis of human tumor cells by facilitating an

epithelial mesenchymal transition 12 A-82 PAUF is a metastasis factor of human pancreatic cancer 12 A-83 Therapeutic potential of a monoclonal antibody against PAUF in human pancreatic cancer 13 A-84 CTHRC1 promotes adhesion and motility of pancreatic cancer cells


70 B-1 Pontibaca methylaminivorans gen. nov., sp. nov., a novel member of the family Rhodobacteraceae70 B-2 Candida alkalitolerans sp. nov., a novel alkalitolerant yeast from industrial waste water 71 B-3 Candida nymphaea sp. nov., a novel yeast in the Kodamaea clade from water 71 B-4 Gordonia kroppenstedtii sp. nov., a phenol-degrading actinomycete isolated from a polluted stream 71 B-5 Solirhabdus panacisegetis gen. nov., sp. nov., isolated from soil of a ginseng field 72 B-6 Patulibacter ginsengiterrae sp. nov., isolated from soil of a ginseng field 72 B-7 A polyphasic investigation on some novel Halomonas species isolated from a renal care center 4 B-8 Osteopontin promotes the development of natural killer cells from hematopoietic stem cells 4 B-9 Characterization of Annexin II as a tumor associated marker of hepatocellular carcinoma 4 B-10 Hypoxia-induced IL-18 increases hypoxia-inducible factor-1alpha expression trough a Rac1-dependent

NFkappaB pathway 5 B-11 Cystatin SN(CST1), a cystein protease inhibitor, was upregulated in gastric tumors 5 B-12 Upregulation of the Cyclin-dependent kinase subunit CKS2 in gastric cancer 61 B-13 Transgenic crops with enhanced tolerance to multiple environmental stresses for sustainable agriculture 61 B-14 Expression of sweetpotato IbMYB1 transcription factor gene induced anthocyanin accumulation in

plants 61 B-15 Characterization of sweetpotato orange gene involved in carotenoid biosynthesis 62 B-16 Enhanced tolerance of transgenic potato plants expressing 2-Cys peroxiredoxin in chloroplasts against

oxidative stress and high temperature 62 B-17 Transgenic sweetpotato plants expressing a cold-inducible zinc finger protein, SCOF-162 B-18 Transgenic sweetpotato plants overexpressing nucleoside diphosphate kinase 2 showed increases of

antioxidant enzymes activities and enhanced tolerance to multiple environmental stresses 63 B-19 Differential response of thirteen sweetpotato peroxidase genes under various abiotic stresses 63 B-20 Transgenic sweetpotato plants expressing spike protein of porcine epidemic diarrhea virus 63 B-21 Comparison of prediction models for cyanobacterial bloom in a eutrophic reservoir (Korea) 64 B-22 Cloning, sequencing, and expression of reductive dehalogenase from the anaerobic PCE to VC

dechlorinating enrichment culture 64 B-23 A study on quorum sensing system for the conservation of biodiversity in a eutrophic water 65 B-24 Selection of microalgae for lipid production under high-level carbon dioxide 65 B-25 Comparison of several methods for effective lipid extraction from microalgae 23 B-26 Activation of PI2-K and p38 MAPK signal pathways is required for lipopolysaccharide-induced microglial

phagocytosis 23 B-27 Antihypertensive effect of MB12066 compound associated with e modulation in spontaneously

hypertensive rats 24 B-28 Acceleration of K-rasG12D-driven tumor progression by deleted mutation of Peroxiredoxin I in mouse embryonic

fibroblasts

3 C-1 Protein tyrosine phosphatases for targeted proteomics research 14 C-2 Sophora flavescens attenuates atherosclerosis and hyperlipidemia in LDL receptor-deficient mice 14 C-3 Antioxidant property of eupatilin attenuates atherosclerosis and improves adipokine profiles in LDL receptor-

deficient mice 14 C-4 The ethanolic extracts of Rhodiola rosea prevent atherosclerosis and obesity in LDLr-/- mice via

stimulation of lipid decomposition pathway 15 C-5 Ethanolic extracts of Brassica campestris spp rapa roots prevent high-fat diet-induced obesity via beta3-

adrenergic regulation of white adipocyte lipolytic activity49 C-6 Repression of BiP, an ER-resident protein, prevents R-gene-mediated hypersensitive response (HR) but

enhances non-host pathogen-induced necrosis in N. benthamiana. 49 C-7 In silico screening and promoter analysis of pathogen-induced genes in pepper 49 C-8 Silencing of an NbLytB gene involved in the plastid nonmevalonate pathway compromised hypersensitive

response 50 C-9 Increased salinity tolerance by carbon metabolite production in chloroplast transgenic Nicotiana tabacum50 C-10 Expression of RNAi suppressor codon-optimized for Chlamydomoans reinhardtii 50 C-11 Idenification and characterization of the PAMPs and effectors of Burkholderia glumae, the causative agent

of bacterial grain rot of rice 25 C-12 Expression patterns of protein kinase A subunits during meiotic maturation periods in porcine oocytes


25 C-13 Porcine XBP-1 is a transcription factor involved in UPR response 25 C-14 Characterizations of seven Drosophila insulin-like peptide genes 26 C-15 Isolation of differentially expressed genes in rosette-enriched neuroectodermal spheres derived from

human embryonic stem cells 38 C-16 Diacylglycerol acyltransferase inhibitory effect by sesquiterpenoids isolated from the flower buds of

Tussilago farfara38 C-17 Synthesis and biological evaluation of benzoimidazole derivatives as inhibitors of DGAT 39 C-18 ACAT inhibitors from the fruits of Piper longum and Piper nigrum 39 C-19 Tiarellic acid and randianin induce apoptosis in clone 15 HL-60 cells via caspase-3- and mitochondria-

mediated pathway 39 C-20 ERK1/2 signaling pathway is involved in verproside-mediated anti-inflammation in a mouse model of

allergic asthma 40 C-21 Nicotine attenuates iNOS expression and contributes to neuroprotection in a compressive model of spinal

cord injury 40 C-22 Anti-influenzavirus acitivity of quercetin 7-rhamnoside isolated from Korean medicinal plants

73 D-1 SNP@WEB: a web-based catalog of SNP (Single Nucleotide Polymorphism) databases and tools 73 D-2 In silico identification of gene-related patents 6 D-3 Paramagnetic gold nanostructures for dual modal bioimaging and phototherapy of cancer cells 6 D-4 Toxicity of gold nanoparticles functionlized with cationic, neutral and anionic side chains 7 D-5 Non-invasive imaging of dendritic cell migration into lymph nodes using near-infrared fluorescent

semiconductor nanocrystals 7 D-6 Mixed self-assembly of polydiacetylenes for highly specific and sensitive strip biosensors 7 D-7 Peptide antibody transducer 8 D-8 Kinetic characterization of artificial cleavable caspase-3 8 D-9 Development of cascade enzyme-linked immunosorbent assay(CELISA) 8 D-10 Immunization with HPV16L1 VLP expressed in Lactobacillus induces systemic and mucosal neutralizing

antibosies in Balb/C mice 9 D-11 Oral administration of poly-gamma-glutamic acid improves dermatitis in NC/Nga mice, a model of human

atopic dermatitis 9 D-12 Poly(g-glutamic acid)-chitosan nanoparticles induces antigen specific humoral and cellular immunity9 D-13 Nobel peptides with a specific binding to the Fc domain of immunoglobulin G 10 D-14 Label-free detection of p53 point mutation using field effect transistor 37 D-15 Characterization of tailoring genes involved in the modification of geldanamycin polyketide in Streptomyces

hygroscopicus JCM4427 37 D-16 Cryptotanshinone inhibits constitutive STAT3 function through blocking the dimerization in DU145 prostate

cancer cells 37 D-17 YJ-1 induces apoptosis through inhibition of HSF1 in human cancer cells 19 D-18 Functional and clinical evidence for NDRG2 as a candidate suppressor of liver cancer metastasis 19 D-19 The genome-wide effect of calsequestrin 2 (Casq2) knockdown on mouse myoblast differentiation 19 D-20 Genome-wide analysis of DNA methylation patterns in gastric cancer cells using genomic tiling microarrays 20 D-21 Aberrant promoter methylation profile in gastric carcinoma by pyrosequencing of sample pools 20 D-22 number variations (CNVs) identified in normal Korean individuals 21 D-23 LRRC3B, encoding a leucine-rich repeat-containing protein, is a putative tumor suppressor gene in gastric

cancer 21 D-24 Stroke web portal based on Korean oriental medicine 21 D-25 Genome deletion project in S. pombe22 D-26 Fission yeast-based screening to identify putative HDAC inhibitors using a telomeric reporter strain74 D-27 Functional proteomic study reveals that GnT-V reinforces the invasive/metastatic potential of colon cancer

through aberrant glycosylation on TIMP-1 74 D-28 Glycomics-based biomarker discovery for liver & colon cancer 74 D-29 Profiling of tumor associated antigens directed autoantibodies of cancer patient


2nd KRIBB Poster Festival

발 행 인

발 행 처

디자인·인쇄

| Abstracts |

박 훈

한국생명공학연구원305-806 대전광역시 유성구 어은동 과학로 111Tel 042.860.4114 Fax 042.860.1759

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-1Contents Division of Leading Research 3 Systemic Proteomics Research Center C-1 Protein tyrosine...

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