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Regulatory environment encourage pharmaceutical company to develop biosimilar product and issued relevant guideline as a general principles to be applied and addressed for development of Biosimilar and also issued product specific guideline. Celltrion is major biosimilar developing player in monoclonal antibody area and already achieved outstanding progress. Celltrion took systematic strategy with global consulting group for IND submission via scientific advisory meeting with EMA and local regulatory agencies. The presentation includes trends of biosimilar development, regulatory environment, Celltrion's approach and case study of our leading biosimilar project.

KIST 강릉분원은 지난 40년간 KIST가 성취한 연구개발의 역량과 경험을 바탕 으로, 강원지역의 과학기술 발전에 기여하기 위하여 2006년도에 강릉 과학산업단지 내에 연구소 건설을 완료하였다. KIST 강릉분원은 대관령 지역의 천연물로부터 생리활성 물질 발굴, 동해안 지 역의 해양자원 개발, 그리고 지역의 환경 보전과 같이 강릉분원이 위치한 지역적 특 성을 반영한 연구개발 활동을 활발하게 전개하고 있다. 즉, 천연의약센터, 기능성천연 물센터 및 환경연구그룹 등으로 구성되어 있으며 구체적인 연구분야 및 내용은 다음 과 같다. 천연의약센터(Natural Medicine Center)에서는 국내 육상 및 해양생물자원으 로부터 항암, 항비만, 항치매 및 항염증과 같은 다양한 질병에 효능을 보이는 신규 천 연물 의약소재를 발굴하고 실용화시키는 연구를 진행하고 있으며 이를 위해 본 연구 소의 최신 연구장비와 의약학분야의 전문인력을 바탕으로 다양한 천연물을 대상으로 생체내외 활성을 탐색하여 약효가 탁월하고 부작용이 적은 소재를 발굴하고 다양한 동물모델을 이용한 인체적용 기능성 연구도 동시에 진행하고 있다. 이러한 연구를 통 해 선정된 유용 천연물 자원을 질병 치료 및 예방을 위한 천연의약으로 개발하기 위 하여 활성성분의 탐색, 분리 최적화, 화학적 구조분석 및 대량생산기법 연구를 하고 있다. 기능성천연물센터(Functional Food Center)는 천연물의 다양한 생리활성을 탐 색하여 유용천연물을 발굴하고, 천연물 화학 및 물리적 가공 연구법을 활용함으로써 궁극적으로 기능성 식품소재 개발을 목표로 하고 있다. 천연물에 전문화된 기기도입 및 시스템구축을 통한 천연물 유효성분의 탐색, 추출, 분리 및 구조결정, 천연물의 초 고속분석법을 연구하고 있으며, 이화학적 안정성이 낮은 천연의 유효성분들을 효율적 으로 추출하고 안정화시키기 위한 신개념의 물리적 가공기술을 통하여 기능성소재의 산업화를 위한 원천기술 개발을 추진하고 있다. 환경연구그룹은환경오염정화를위한소재및시스템개발을목적으로하는연구그룹으로 강원도지역의심각한오염원인수백개의폐광산정화를위해천연미네랄과폐기물을활용하여 경제적으로오염정화 를할수있는소재를개발하고오염현장에설치및가동할수있는시스템을 개발하며토양및지하수내에존재하는중금속, 유기오염물질을 동시에 제거할 수 있는 정 화물질도 개발하고 있다. 천연물, 환경분야에서 세계적 연구기관을 지향하고 있는 KIST강릉분원은 최신 의 연구정보를 교환하고 다양한 천연물 자원을 확보하기 위하여 세계 각국의 전문연 구기관들과 활발한 국제협력을 수행하고 있으며, 국내의 여러 관련 연구기관 및 대학, 기업 등과도 활발한 교류를 통해 글로벌 시장에서 우뚝 설 수 있는 세계적인 천연물 신약 및 기능성 식품 개발에 박차를 가하고 있다.

Every cell in an organism synthesizes a heterogeneous array of glycans in the form of various structures. Cell surface glycosylation may play an important role in development and may provide important new sources of markers for differentiation. However, studies regarding the glycosylation of cell surfaces are limited due to the lack of sensitive analytical methods. A mass spectrometry-based method that enables specific detection and quantitation, and acquisition of structural information has been developed. We have mapped cell surface glycans from more than 30 human cell lines including embryonic stem cells, cancer cells, and somatic cell lines. The results represent that specific surface glycomic signatures might have functional implications as well as characteristic features of a specific cell type.

Understanding of the molecular relationships in carbohydrate-protein interactions provides useful information on biological processes in living organisms and is also helpful for development of potent biomedical agents. Herein, the interaction unbinding force between GM1 pentasaccharide and Vibrio cholera toxin (ctx) proteins was measured by using atomic force microscopy (AFM), which enabled us to determine the interaction of ctx holotoxin (ctxAB) with GM1 and the interactive From the analogue analyses, we understood how structural and binding positional differences in complex carbohydrate affect the interaction with protein and surmise that the GM1-ctxAB complex makes a “two-finger grip” formation through the conformational change of a flexible carbohydrate. Inconclusion, using AFM force analysis, we successfully quantified and characterized the interactive configuration of carbohydrate-protein molecules.

The anti-complementary substance is able to prevent hemolytic activity by the activation or inhibition of the complement system. Some anti-complementary polysaccharides have been isolated from bacteria, fungi, and plants. In order to develope new mushrooms available as functional food with excellent immuno-modulating activity, we investigated the anti-complementary activities of water soluble polysaccharides extracted from fruiting bodies of 5 mushrooms which have known as traditional medicinal mushrooms for a long time. It was observed that the crude polysaccharides of JFMF mushroom had a potent anti-complementary activity. Crude polysaccharide of JFMF was isolated into a neutral polysaccharide (JFMF-NP) and three acidic polysaccharides (JFMF-AP1, AP2, AP3) by DEAE-sepharose column chromatography. JFMF-AP2, one of these, showed the very excellent anti-complementary activity of ITCH (inhibition of total complement hemolysis) value 70% on 5 ug/ml concentrations. Its molecular weight was estimated less about 5,000 by Toyopearl HW-65F gel filtration chromatography using pullulan molecular size standard kits. Also, detection of a cleavage of C3 by immunoelectrophoresis indicated that JFMF-NP and JFMF-AP2 activates the complement via both the alternative and classical pathways.

Chitinolytic and chitosanolytic microorganisms are widely distributed in nature. Biological and enzymatic activities of these enzymes are applied to produce hydrolytic oligosaccharides which have various biological functions involved in anticancer, antimicrobial activities, immune-stimulating activities and so on. In the present study, we describe the biological characteristics including the diversity based on 16S rDNA sequence analysis, production of chitosanase and its hydrolysates, and enzyme activities. Among 15 species of chitosanolytic microorganisms isolated in this study, one named as Bacillus subtilis JH-1122 based on 16s rDNA sequence homology search has shown the highest similarity with most of sub-species of Bacillus. Various sizes of chitosan hydrolysates (Low Molecular Weight Chitosan, LMWC) ranging from 3 to 60 kDa were prepared to assess the effect of molecular weight on antimicrobial activity by enzymatic digestion of chitosan using chitosanase isolated and partially purified from the culture medium of Bacillus subtillis JH-1122. Especially it has shown the higher antimicrobial activity toward antibiotics resistant microorganisms such as Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. These results suggest that LMWC may be a predominant factor either directly or indirectly affecting on broad antimicrobial activities.

Gyeonggi-do is the center of bioengineering and pharmaceutical industries in which 30% of bioengineering and 60% of pharmaceutical companies in South Korea are located. It is home to many universities and research centers offering state-of-the-art technology and highly qualified manpower. In 2007, GyeongGi Province has found the GyeongGi Bio Center(GGBC) to support collaboration between bio and pharmaceutical industry and to promote related industrial development. We contribute to the growth of regional economy by focusing on innovative bio and pharmaceutical technology competencies, and by playing the role of a leader in the development of bio and pharmaceutical industries. GGBC takes the role of bridge in the valley of death in bio and pharmaceutical field through translational research, service for analysis & pilot production, and global networking. GGBC develops competitive lead and candidate for new drugs by utilizing high tech cell-based screening system (HCS/HTS) to cooperate between early stage techniques of university, industry, research institute. In case of supporting program of cutting-edge equipment, we have the best infrastructure for supporting research including analysis(LC-MS/MS, MALDI-TOF/TOF etc), screening(HTS, FACS Sorter etc), and pilot scale production(Fermenter/Bioreactor etc). Then we have provided various service to regional companies and institute, as analysis, screening service, and sharing equipment & facilities. Recently GGBC built high resolution mass spectrometer(LTQ-Orbitrap) and SPR Platform to offer upgraded services to support companies for the characterization of biologics and natural product resources. In addition, we will devote to build an optimal global network for importing and distributing state of the technology. Furthermore, the center is aimed to expand its support not only to the bio/pharmaceutical industries but to a broader range of related industries such as bio food, biochemical and bioenvironmental programs. All these support also goes to the start-up and fast-growing companies to ensure those who are needed enjoy our assistance.

Ginsenosides are well-known major components isolated from the radix of Panax ginseng C. A. Meyer, known as Korean ginseng, having diverse biological activities. They have recently gained much attention for their biomedical applications. In the present work, a fast and simple method for the separation and purification of 8 ginsenosides from Panax ginseng by counter current chromatography coupled with evaporative light scattering detector (CCC-ELSD) was successfully established. The crude samples for CCC separation were first purified from ginseng extract using a macroporous resin. The extract was loaded onto a Diaion-HP20 column and fractionated by methanol and water gradient elution. The ginsenosides-triols and diols fractions were subsequently eluted with 65% & 80% methanol and water gradient elution, respectively. Furthermore, these two fractions were separated by CCC-ELSD. The two phase solvent system used for separation was composed of chloroform-methanol-water-isopropanol at a volume ratio of 4:3:2:1. Each fraction obtained was collected and dried, which yielded eight ginsenosides namely, Rg1, Re, Rf, Rh1, Rb1, Rc, Rb2 & Rd. The purity of these ginsenosides was more than 97% assessed by HPLC-ELSD system, and their structures were characterized by electrospray-ionization mass spectrometry (ESI-MS), 1H NMR and 13C NMR spectroscopy. This is the first report regarding the CCC separation of ginsenosides Rh1, Rb2 & Rc from Panax ginseng.

Previously, we reported the a-directing effect due to the remote participation of 3-O-acyl and 6-O-acetyl groups of donors in mannosylations. Herein we report the a-directing effect by remote participation of 4-O-acyl groups of galactose donors in galactosylations. Galactosyldonors possessing benzylsulfonyl, benzoyl, p-methoxybenzoyl, and acetyl groups were prepared. The outcome of the stereochemistry in glycosylations of various acceptors with these donors indicated that 4-O-acyl groups of galactose donors participated during glycosylations. The outcome of the stereochemistry of the galactosylations could be interpreted either by the remoteparticipation of acylgroups or by the reaction between the acceptor and the galactosyl oxocarbeniumion in 4H3 or 3H4 comformation. Detection by the mass spectrometry of the bicyclic product with a seven-membered trichlorooxazepine ring, resulting from the activation of phenyl 1-thio-4-trichloroacetimidoyl galactoside with BSP and Tf2O, is provided a further evidence for the participation of 4-O-acyl groups in the galactosylation. Galactosylations were conducted by treatment of thioglycosides possessing electron-withdrawing groups at O-4 with BSP and Tf2O at -60℃.

Previously, we reported the a-directing effect due to the remote participation of 3-O-acyl and 6-O-acetyl groups of donors in mannosylations. Herein we report the a-directing effect by remote participation of 3-O-acyl groups of galactose donors in galactosylations. Galactosyl donors possessing benzylsulfonyl, benzoyl, acetyl, and benzyl groups were prepared. The outcome of the stereochemistry in glycosylations of various acceptors with these donors indicated that 3-O-acyl groups of galactose donors participated during glycosylations. Detection by the mass spectrometry of the bicyclic product with a six-membered trichlorooxazepine ring, resulting from the activation of phenyl 1-thio-3-trichloroacetimidoyl galactoside with BSP and Tf2O, is provided a further evidence for the participation of 3-O-acyl groups in the galactosylation.Galactosylations were conducted by treatment of thioglycosides possessing electron-withdrawing groups at O-3 with BSP and Tf2O at -60 .

O-GlcNAcylation is a dynamic posttranslational modification which controls many nuclear and cytosolic protein activity. It features attachment and removal of O-linked β-N-acetylglucosamine on ser/thr residues. Cycling of O-GlcNAc is known to be regulated by two enzymes, O-GlcNAc transferase (OGT) involved in addition of O-GlcNAc and O-GlcNAcase (OGA) which catalases removal of O-GlcNAc. O-GlcNAc modification is concerned with many cellular events such as transcription, proteasomal degradation, apoptosis, proliferation and cell cycle, however, there is a limitation to produce a large amount of O-GlcNAc modified proteins in mammalian cells. So in this study, we make an attempt to establish E.coli plant which enables us to get much O-GlcNAcylated targets in more efficient way. Expected impact of this research is making it easy to generate site-specific antibody, which will lead to gather momentum to study various functions of this modification on target proteins.

β-O-linked N-acetylglucosamine (O-GlcNAc) is dynamic post-translational modification in nucleus and cytosol. O-GlcNAc transferase (OGT) and O-GlcNAcase are involved in O-GlcNAc modification. Many proteins such as transcription factors, cytoskeletal proteins and proteins of variable signal transduction pathway are modified with O-GlcNAc at their Ser/Thr residues and their functions are affected by O-GlcNAc. The hexosamine biosynthesis pathway (HBP) consumes about 5% of glucose in total glucose flux, and it's final product is UDP-GlcNAc, which is source of O-GlcNAc modification. It is reported that O-GlcNAc modification is involved in skeletal muscle metabolism and development process. We studied whether O-GlcNAc modification is involved in differentiation in myoblast C2C12 cell lines. Myoblast C2C12 is differentiated to myotube in differentiation media for 4-5 days. We observed that total O-GlcNAc modification level was dynamically changed during myogenesis. During first 24 hours after induction of myogenesis, total O-GlcNAc modification level decrease and increase again. Also, we treated NAG-thiazoline derivative, specific O-GlcNAcase inhibitors and observed the block of the decrement of total O-GlcNAc modification and inhibition of myotube formation and expression of myogenic markers. Especially, after treatment NAG-thiazoline derivative, we observed that myogenin expression level decreased. So, we checked the myogenin protein stability using MG132 and mRNA level of myogenin using RT PCR. Even after treatment of MG132, the result of the experiment is same as previous experiment, thus myogenin protein stability is not involved with decrease of myogenin expression after treatment NAG-thiazoline derivative. And using RT PCR, we found that mRNA level of myogenin decrease after treatment NAG-thiazoline derivative. So we conclude that O-GlcNAc modification affect the transcription of myogenin.

Ganglioside are ubiquitous membrane component in mammalian cells and suggested to play important roles in various cell functions such as cell-cell recognition, differentiation and transmembrane signaling. These compounds are localized in a glycosphingolipid-enriched microdomain on the cell surface and regulated by the glycosphingolipid composition. However, the role that gangliosides play in the adhesive interaction response by xenotransplantation is not yet clearly understood. In this study, we investigated expression patterns of gangliosides in tight contact between human umbilical vein endothelial cells (HUVECs) and micro-pig aortic endothelial cells (MPAECs). Analysis of ganglioside expression pattern showed that differential expression patterns of ganglioside in tight contact between HUVECs and MPAECs. Furthermore, we confirmed that cell adhesion molecule expression in tight contact between HUVECs and MPAECs. Taken together, these results suggested that differential expression of gangliosides in tight contact between HUVECs and MPAECs may play a role in adhesive interaction response in successful clinical xenotransplantation.

Gangliosides, sialic acid-conjugated glycosphingolipids, are involved in cell proliferation and differentiation. This study investigated the possible role of gangliosides in the dopaminergic neuron-like cells of human mesenchymal stem cells (hMSCs). We have reported the in vitro conditions for inducing hMSCs into dopaminergic neuron-like cells. hMSCs were induced in vitro to become dopaminergic neuron-like cells in 12 days by using a cocktail that includes sonic hedgehog and fibroblast growth factors. First, we investigated the characterization of hMSCs through FACS analysis. The mesenchymal stem cell specific markers, CD44 and CD105, were expressed in hMSCs while the hematopoetic markers, CD45 and CD117 were not. The cells developed a neuronal morphology expressing the neuronal markers Map2 and βⅢtubulin. RT-PCR analysis revealed the expression of dopamine specific genes such as TH, Pitx3, Nurr1 and DA transporter. Immunocytochemistry showed that hMSCs expressed GM3, GM2, GD1a, GD3 and GT1b based on tyrosine hydroxylase (TH) expression. Specifically, a significant decrease in GM3 and GT1b expression was observed during dopaminergic neuron differentiation. These results suggest that ganglioside GT1b may play a role in the dopaminergic neuron differentiation and development process of hMSCs.

Glycosylation, the attachment of sugars to proteins, is one of the most abundant post translational modifications in all living cells. It plays very important roles in protein folding, stabilization, embryogenesis, neuronal development, cell-cell adhesion and recognition. Glycosylation produces huge structural diversity in proteins which is necessary for the functionalization of nascent proteins. While peptides are an attractive class of molecules to synthesize glycopeptides, they possess undesirable drawbacks including sensitivity to proteases, limited cell permeability and poor bioavailability. Thus, there have been increasing needs for new peptidomimetics with improved pharmacokinetic characteristics. Among them, peptoids, N-alkylated glycine oligomers where side chains are directly attached to nitrogen atom rather than α-carbon of peptide, possess many advantages over peptides. Attachments of sugars to peptoid backbone generate glycopeptoids which have interesting features by mimicking glycopeptides. Our strategy was to install sugar derivatives to alkenyl moiety-containing peptoids through solid-phase cross metathesis approach. The peptoids were prepared on bead having strategically positioned alkenyl moieties in different length. O-linked sugar-alkene derivatives based upon glucose, mannose, and galactose monomers were also made. Initially, we screened reaction condition parameters such as catalyst, concentration of sugar derivative, temperature, solvent, and reaction time. HG2 was proved to be a better catalyst than G1 and G2. The length of alkenyl moieties was critical on cross metathesis. The cross metathesis was facilitated easily with 3-butenyl group over allyl group. We have successfully synthesized glycopeptoids on solid-phase through cross metathesis in very good yields. This systematic study into the use of solid-phase cross metathesis for the synthesis of glycopeptoids provides a particularly valuable tool for easy access to the molecular sources of glycopeptidomimetics and the rapid generation of glycopeptoid libraries.

Silver nanoparticles (AgNPs) are nanoparticles of silver that between 1 nm and 100 nm in size and are prepared by reduction of Ag+ ions to Ag metal by chemical reducing agents as a common method. It has been reported that the bactericidal effects of AgNPs are dependent on size with preferential size of ~1-10 nm. In recent days biological molecules instead of chemical reducing agents have been reported to synthesize AgNPs including plant extracts, polysaccharide, fungi, DNA, yeast, and bacteria. Recently, research articles report the use of polysaccharides for the synthesis of AgNPs. Among polysaccharides, glycosaminoglycans (GAGs: Chondroitin sulfate (CS) and Acharan sulfate (AS)) exhibit diverse biological activities through interactions between physiological proteins. Owing to biological activities of GAGs, we could expect synergistic effects of GAGs-derived nanomaterials from GAGs and nanoparticles. AgNPs-GAGs represented several activities such as anti-oxidant, anti- bacterial, and wound healing. AgNPs by using the CS exhibited antioxidative, where as CS only remained almost inactive. AgNPs has no cytotoxicity on fibroblast cells. When cells were treated with AgNPs by using GAGs for 24 hr, there were no cytotoxicity effects on fibroblast cells. AgNPs-GAGs displayed significant inhibitory action against both antibiotic-susceptible and resistant strains of many strains with MIC ranging from 1 to 8 mg/ml. AgNPs-CS showed the highest inhibitory activity against Enterobacter cloacae 1321E with MICs of 1 mg/ml. At animal model, after excision skin, wound size of AgNPs-CS and AgNPs-AS ointment group was smaller than not treated and Vaseline group. Time to closure was defined as the time at which the wound bed was completely filled in with new tissue. Therefore, the utilization of such biological entities and environmentally benign solvents, and renewable materials are some of the key issues that merit important consideration in a green synthetic strategy.

This experiment was carried out to prove the efficacy of Rhynchosia volubilis (RV) extract in ovariectomized rats. Forty rats were divided into 4 groups, administrated saline after sham operation(sham-op), saline after ovariectomy (control), RV 500 mg/kg after ovariectomy and 0.042 mg/kg Livial after ovariectomy (positive control). We examined the water extract of RV that is capable of affects osteoblast proliferation using MG-63 and HOS-TE85. Dual energy X-ray absorptiometry (DEXA) was used to measure the tibia bone mineral density of the ovariectomized rat and sham-op groups. Serum was collected for analysis of Ca and phosphorous. The results showed that body weight gain, Tibia BMD and BMC was significantly different among groups. These results suggest that RV has therapeutic effect on ovariectomized rats.

Polyketides are a large class of natural products that include clinically important antibiotics, antitumor compounds, immunosuppressants, pigments and herbicides. Herboxidiene is a polyketide natural product that was found to control several important biannual weeds at relatively low application rates (<250 g/hectare) without damaging wheat. Early study indicated that the epoxide and the C-18 hydroxyl groups are important for the activity of herboxidiene. While Sakai et al., 2002 reported that herboxidiene showed antitumor activity against human tumor cell lines, Koguchi et al., 1997 reported that herboxidiene up-regulated the gene expression of low density lipoprotein receptors. The interesting biological profile of this natural product has prompted us to produce its glycosylated analogues so as to alter its activity. It is well known that bioactive natural products are often complexed with sugar moieties which are essential for activity of the compound. In this study, we carried out in vitro experiments to generate glucosyl-herboxidiene and galactosyl-herboxidiene.

Steryl glucosides play important roles in many physiological and biochemical process in organism such as the heat shock, enhancement of immunological system, etc. The alignment of a putative sterol glucosides isolated from S. tropica CNB-440 has been shown 34%, 42% and 57% in homology with the corresponding ones from Arabidopis thaliana, Avena sativa and Salinispora arenicola CNS-205, respectively. Engineered E. coli host-high level production of the UDP-glucose was used for whole-cell bioconversion of free sterol (cholesterol and β-sistosterol) and the production of glycosylated product was only detected with β-sitosterol as substrate.

Glycosylation of small molecule based therapeutics and natural products influences biological activities by altering molecular and cellular specificities. E. coli has been engineered by expressing unnatural TDP-4-amino-4, 6-dideoxy-D-galactose biosynthetic pathway genes to produce pools of activated TDP-aminodeoxysugar, and glycosyltransferase gene from A. thaliana. Quercetin and Kaempferol Deoxyaminosugar conjugates were produced by whole cell biocatalysis and the products were analysed by TLC and Proton NMR. This strategy of in vivo glycosylation offers vast combinatorial biosynthesis potential to produce glycorandomized natural products of pharmaceutical importance by simple fermentation.

In the present study, we investigated structure and immune modulating activity of a polysaccharide (tentatively named JS-MP-1), previously purified from Mulberry fruits (Morus bombycis Koidz) pericarp. Based on HPAEC-PAD analysis, major monosaccharides of JS-MP-1 were shown to be galactose (37.6% in mole percentage), arabinose (36.3%) and rhamnose (18.4%), respectively. EA analysis did not show the presence of sulfur (0.07%). Biochemical analysis showed neutral sugars (35.25%), uronic acid (11.25%), sulfate (0.16%) and proteins (0.3%). FT-IR spectra of JS-MP1 confirmed the presence of carboxyl groups (uronic acid – α-GalAp in RG1) and COC, CO and CC stretching vibrations at 948 – 1150 cm-1 corresponding to polysaccharides. Peak at 894 cm-1 indicates the presence of β-Galp units. 1H-NMR spectrum (in D2O) confirmed the presence of α-1,2-Rhap, α-Araf, β -Galp and α-1,4-GalAp units. The most intense HMQS signals were observed for terminal α-Araf. The presence of methylester groups in the residues of α -1,4-GalAp was confirmed by the HMQS peak at C 54.3/H 4.01 ppm (OCH3). Taken collectively, the JS-MP-1 is probably a rhamnogalacturonan type 1 (RG1), which contains: (a) partially methyl esterified α-1,4-D-GalAp and α -1,2-L-Rhap units in backbone, and (b) α-L-Araf and β-Galp are bound to Rha as terminal units or more complex side chains (arabinan, galactan and/or arabinogalactan). The ELISA assay showed that JS-MP-1 remarkably stimulates the release of the proinflammatory cytokines, TNF-α and IL-6, in a dose-dependent manner, from the RAW 264.7 cells. RT-PCR analysis also showed a significant induction of the iNOS and COX-2 gene expression. These results suggest that JS-MP-1 isolated from the Mulberry fruits has immune modulating activities.

Anthocyanins belong to a group of flavonoid compounds and are well known for their various health beneficial effects including antioxidative activities. Among them, the major anthocyanins isolated from the seed coat of black soybean (Glycine max L.) were previously characterized as glycosides having glucopyranose. Asthma is an allergic disease that is strongly associated with various immune cells including basophils and mast cells. Eosinophils, basophils and mast cells play important roles in asthma through the release of inflammatory mediators such as athma-specific T-helper (Th)2 cytokines and subsequent amplification of asthma symptoms by their degranulation. Rat basophilic leukemia RBL-2H3 cells was the most common in vitro models used for evaluating asthmatic reactions. We examined the effects of anthocyanin from the seed coat of black soybean (Glycine max L.) on the antigen-stimulated degranulation and Th2 cytokines production in RBL-2H3. Cell degranulation was evaluated by detecting the release of β -hexosaminidase. The β-hexosaminidase release and Th2 cytokine production in RBL-2H3 cells upon stimulation with IgE-antigen complex was much higher those that in untreated control cells. Anthocyanins significantly suppressed the IgE-antigen complex-induced degranulation of RBL-2H3 and inhibited IgE-antigen complex-mediated interleukin (IL)-4, IL-13 and TNF-α production in RBL-2H3 cells. These findings suggest that anthocyanins from the seed coat of black soybean (Glycine max L.) effectively inhibit asthmatic reactions and may have beneficial effects against allergic asthma.

Natural compounds offer interesting pharmacological perspectives for antiviral drug development. In this study, we obtained low-molecular weight fuco-oligosaccharides (LMFOs), ranging from 1,389 to 3,749 Da from the fucoidan of Korean Undaria pinnatifida sporophyll by crude enzyme preparation of Sphingomonas paucimobilis PF-1 (KCTC 11130BP). The fucoidan and LMFOs had potent antiviral activity against herpes simplex virus types 1 (HSV-1) any detectable level of cytotoxicity on Vero cells. Fucoidan and LMFOs contained strong anti-HSV activity with 50% inhibitory (IC50) at 2.43 and 2.65 μg/ml concentration. Interestingly, they also showed inhibitory activity directly against viral particles as observed in a virucidal assay (88.4%, and 70.5% at 5 μg/ml concentration). Respectively, LMFOs exerted anti-HSV activity when pre-incubated with host cells prior to viral infection, suggesting that a main inhibitory could be mediated via prevention of viral entry. The antiviral activity was significantly affected depending on the degree of sulfation ; antiviral activity was significantly reduced upon removal of sulfate groups. From the FT-IR and NMR analysis, sulfate groups were shown to be located at C-4 of (1→3)-linked fucopyranosyl units, and appeared to be very important for the anti-HSV activity of this polymer.

Yeasts have been extensively studied as model organisms to elucidate cellular processes and their mechanism in lower eukaryotes. Consequently, several powerful genetic tools have been developed to engineer yeast and improve its utility. Although native hyper-mannose yeast glycosylation is not suitable, high-mannose core glycan producing yeast is an attractive for production of viral vaccines containing Man9GlcNAc2 (Man9) and Man8GlcNAc2 (Man8) structures. To study the production of glycoproteins for putative viral vaccine target and the screening system for novel glycosidase and glycosyltransferase, we generated a mutant yeast strain and constructed glycosylated glycoprotein substrates. Both och1 and mnn1 genes of Saccharomyces cerevisiae, which are involved in biosynthesis of outer high mannose chains in the N-glycosylation pathway, were deleted. Glycan profiling revealed that N-glycans produced by this mutant were almost exclusively Man8GlcNAc2. In addition, we are constructing to glycosylated protein substrates, based on yeast expression system. In this presentation, our resources, including glycosyltransferase and glycosidase libraries as well as enzymatic tools for N-glycanengineering, will be also presented for glycoscience and glycoengineering.

The development of cancer biomarker is of a great promise to conquer cancer since it leads to an early detection and provide an opportunity for better cancer treatment. Validation is a time-consuming step for biomarker developments requiring investigation of biomarker candidates using thousands or more of biosamples, which is why a sensitive, multiplexing validation method is necessary. To address this challenge, we are developing as antibody-based validation method, in which antibody with oxidized N-glycans was covalently linked to an identifiable DNA tag for use of the template in the transcription by T7 polymerase. The transcripts will be quantified and used for an indicator of the amounts of the antigen bound to the DNA-tagged antibody. This strategy will be multiplexed for an establishment of a novel validation method.

The development of cancer biomarker is of a great promise to conquer cancer since it leads to an early detection and provide an opportunity for better cancer treatment. Validation is a time-consuming step for biomarker developments requiring investigation of biomarker candidates using thousands or more of biosamples, which is why a sensitive, multiplexing validation method is necessary. To address this challenge, we are developing as antibody-based validation method, in which antibody with oxidized N-glycans was covalently linked to an identifiable DNA tag for use of the template in the transcription by T7 polymerase. The transcripts will be quantified and used for an indicator of the amounts of the antigen bound to the DNA-tagged antibody. This strategy will be multiplexed for an establishment of a novel validation method.

In this study, we will attempt to analyze the ER localization, glycosylation pattern and anti-tumor activity of KDEL-fused monoclonal antibody. Advantages of the baculovirus insect cell expression system for production of recombinant proteins include high capacity, flexibility, and glycosylation capability. In this study, this expression system was exploited to produce anti-cancer monoclonal antibody (mAb) CO17-1A, which recognizes the antigen GA733 that is highly expressed on the surface membrane of colorectal carcinoma cells. The heavy chain (HC) and light chain (LC) genes of KDEL-fused mAb CO17-1A were cloned under the control of P10 and PPH promoters in the pFastBacTM dual vector, respectively. Gene expression cassettes carrying the HC and LC genes were transposed into a bacmid in Escherichia coli (DH10Bac). The transposed bacmid was transfected to Sf9 insect cells to generate baculovirus expressing mAb CO17-1A. In future, we will study the expression and localization of KDEL-fused mAb CO17-A in baculovirus infected cells by PCR, western blot, enzyme linked immunosorbent assay (ELISA), confocal immunofluorescence, fluorescence-activated cell sorting analysis (FACs) and antibody dependent cell cytotoxicity (ADCC).

Colorectal cancer is the third leading cause of cancer-related death in the Korea. It is eager to find a method for preventing and curing colorectal cancer. The tumor-associated antigen GA733 is a cell surface glycoprotein highly expressed in colorectal carcinoma. In this study, GA733 was fused to Fc fragment of human IgG as a vaccine candidate in plant expression system. The fusion protein GA733-Fc can improve their expression in plant cells and induce cellular and humoral immune responses. GA733-Fc is glycosylated, however the N-glycan structures of plant-derived GA733-Fc differ from their parental counterparts. Plant-derived specific N-glycoforms have the potential to induce immunogenicity and allergenic responses in human therapy. Thus, oligomannose glycan structure was tried to be generated through retaining GA733-Fc and GA733 in ER with KDEL sequence. Agrobacterium-mediated transformation was conducted to generate transgenic plant expressing GA733-Fc and GA733. Recombinant protein expression, accumulation and biological activity of GA733-Fc with KDEL (GA733-FcK), GA733-Fc without KDEL (GA733-Fc), and GA733 with KDEL (GA733K) in transgenic plants were compared by western blot, confocal immunofluorescence analysis, ELISA and mouse immunization, respectively.

Cancer is one of serious human diseases that cause angiogenesis, metastasis, and abnormal cell divisions including genetic change. Thus, lots of researchers have been investigated to inhibit cancer. Human 90K (h90K) protein has very crucial role for inhibition of cancer metastasis. h90K is a glycoprotein having potential 7 N-glycosylation sites (4 sites are clear but remaining 3 sites are not yet). In the present work, we performed expression of recombinant h90K glycoprotein in insect Drosophila S2 cell system. We constructed stably-transfected S2-h90K cells by antibiotic selection and confirmed h90K expression using Western blot and in vitro bioactivity analyses. We found that recombinant h90K has anti-cancer activity. We also identified N-glycan patterns of recombinant h90K glycoprotein using HPLC and MALDI-TOF MS. Based on the identified N-glycan structures, we next engineered N-glycosylation pathway to develop advanced N-glycan patterns using multiple expression platform strategy with recombinant baculoviruses. We found that infection with recombinant baculovirus having galactosyltransferase resulted in advancement of bi-antennary galactosyl form of N-glycans in recombinant h90K glycoprotein.