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Development of efficient and stereoselective glycosylation methodologies has been a major concern in synthetic organic chemistry over the past decade due to important roles of complex oligosaccharides in many fundamental life-sustaining processes. The selection of an appropriate glycosyl donor is one of the key processes for the successful glycosylation in terms of efficiency and stereoselectivity. We reported previously a new direct glycosylation method with anomeric hydroxy sugars as donors and phthalic anhydride and triflic anhydride as promoters, and the result of investigation of intermediates in the new glycosylation by low temperature NMR [1]. Although our direct glycosylation method works nicely in various glycosylations including stereoselective β-mannosylations, in certain cases, it gives undesired self-condensed esters of donors. In the first part of the lecture is presented a modified, improved method for the direct glycosylation with anomeric hydroxy sugars by employing phthalic anhydride derivatives in the place of phthalic anhydride itself. One of the most useful stereoselective glycosylation strategy utilizing protecting groups has been the 1,2-trans glycoside synthesis. Although the acyl groups at O-2 of donors give 1,2-trans glycosides by the neighboring group participation, their electron-withdrawing effects on the glycosylation stereochemistry might not have been properly recognized because of the overwhelming effect of the neighboring group participation. In fact, the effect of the non-participating electron-withdrawing groups such as methanesulfonyl and benzylsulfonyl groups at the O-2 positions of the donors on the glycosylation stereochemistry has been observed. Remote electron-withdrawing groups at O-3, O-4, and O-6 positions of pyranosyl donors or at O-3 and O-5 positions of furanosyl donors, which would reduce the reactivity of glycosyl donors, might also exert directing effects on the outcome of the stereochemistry of glycosylations. In fact, we reported previously that mannosylations of various acceptors with donors possessing an electron-withdrawing sulfonyl or acyl groups at O-3, O-4, or O-6 positions were found to be β-selective except when donors had 3-O-acyl and 6-O-acetyl groups, which afforded α-mannosides as major products [2]. The α-directing effect of 3-O-acyl and 6-O-acetyl groups was attributed to their remote participations while the origin of the β-directing effect was ascribed to the SN2-like reaction of the acceptor and the α-triflate intermediate, which is stabilized by remote electron-withdrawing groups. In the second part of the lecture is concerned with the directing effect of electron-withdrawing groups at the remote positions of donors on the stereochemistry in not only mannosylations but also other glycosylations. Directing effect of remote electron-withdrawing groups of other glycosyl donors was found to be quite different from the directing effect of those of mannosyl donors.

Modified nucleosides are regarded as important templates in view of being extensively utilized as biodrugs as well as biological tools. They can be developed as viral and cellular polymerase inhibitors, antimetabolites, or modulators of receptor or enzyme functions. Also after being converted into oligonucleotides, they can be used in studying genome, siRNA, micro RNA, and so on. Especially, they have been used as drugs of choice for the treatment of viral diseases such as AIDS, hepatitis B/C virus (HBV/HCV), and herpetic virus (HSV). Despite of many clinically used drugs, the first generation nucleosides suffer from many drawbacks such as appearance of drug-resistance, toxicity, and metabolic unstability. To solve these problems, we have developed the 2nd generation nucleosides in which furanose oxygen was substituted with sulfur or methylene (CH2), among which several compounds are being developed as antitumor, anti-glaucoma, and anti-inflammatory agents. Recently, we discovered the novel template as next generation nucleosides, 4'-selenonucleosides with various antiviral and antitumor activities. In this symposium, the first, second, and third generation nucleosides developed in our laboratory will be presented in detail.

In this seminar I will briefly introduce about current research progress in our Lab related with glycoside synthesis using glycansucrases and carbohydase inhibitor screening using Grid. We are synthesizing various glycosides of the hydroquinone for the improvement of antioxidant activity. Hydroquinone (HQ) functions as a skin whitening agent, but it has the potential to cause dermatitis. We synthesized a novel HQ fructoside (HF), HQ galactoside and HQ glucoside as potential skin whitening agents by reacting glycansucrases from Leuconostoc mesenteroides with HQ as an acceptor and sucrose as a donor. The product was purified using butanol partition and silica-gel column chromatography. One of them HF synthesis was determined using a response surface methodology and the final optimum condition was 350-mM HQ, 115-mM sucrose, and 0.70 U/ml levansucrase, and the final HF produced was 1.09 g/l. HF showed anti-oxidation activities and inhibition against tyrosinase. The IC50 of DPPH scavenging activity was 5.83-mM, showing higher anti-oxidant activity compared to β-arbutin (IC50 = 6.04-mM). The Ki value of HF (0.67-mM) against tyrosinase was smaller than that of β-arbutin (Ki = 2.8-mM). The inhibition of lipid peroxidation by HF was 108.12% that of HQ (100%) and much higher than that of β–arbutin (0.81% of HQ). Virtual screening (VS) was applied for discovery of new inhibitors for the human intestinal maltase (HMA) enzyme. VS of 308 307 compounds was performed with HMA using 4700 CPUs and AutoDock 3.0.5 in a WISDOM (Wide In Silico Docking On Malaria) production environment. The 42 best ranked compounds containing hydrogen bond interaction with key residues from VS were tested in vitro for their inhibitory activities against the recombinant HMA from Pichia pastoris. Compounds 17 and 18 were identified as competitive inhibitors for enzyme inhibition with Ki values of 19.8 and 19.6 μM, respectively. In contrast to acarbose, the two compounds showed no inhibition on human pancreatic α-amylase, suggesting potential inhibitors with fewer side effects, including abdominal discomforts.

Previously, we showed that simple paucimannosidic N-glycan structures in insect Drosophila S2 cells arise mainly because of β-N-acetylglucosaminidase (GlcNAcase) action, which removes terminal N-acetylglucosamine (GlcNAc) residues. In an earlier report, we suppressed GlcNAcase activity and clearly demonstrated that more complex N-glycanswithtwo terminal GlcNAc residues were synthesized. In the present work, we investigated synergistic effects of N-acetylglucosaminyltransferase II and/or β -1,4-galactosyltransferase co-expressions (by recombinant baculoviral infection) and GlcNAcase suppression (by knock-out screening) on N-glycan patterns. Using HPLC and MALDI-TOF MS analyses, we found that the N-glycosylation pattern of human erythropoietin secreted by engineered S2 cells expressing glycosyltransferase but not GlcNAcase was complete except for sialylation; N-glycan structures had two terminal galactose residues. Therefore, it will be possible to express complete functional human therapeutic glycoprotein in engineered Drosophila cells by suppressing GlcNAcase and co-expressing additional glycosyltransferases of N-glycosylation pathway.

Glycans are now attracting much attention in the fields of biomarkers, stem cells, and biosimilar drugs. The combination of evanescent-field fluorescence scanner "GlycoStation™ Reader 1200" and lectin microarray "LecChip™" creates a highly sensitive platform for monitoring interactions between lectins and glycans among different technologies. Because of the high sensitivity and simple procedure, GlycoStation™ can be positioned as the most powerful differential analysis tool in screening novel biomarkers focusing on changes of glycan structures. Moreover, antibody-overlay, lectin microarray methodology applied to GlycoStation™, makes it more precise analysis tool. In this talk, the basic strategy of biomarker discovery using GlycoStation™ will be explained, introducing some examples of paraffin-embedded colon tissue arrays, and antibody-overlay lectin microarrays for detecting prostate cancer. In order to make the biomarker development process more efficient, an automated enrichment device of target biomarkers from serum (using antibodies and lectins) is under development, and will also be introduced. In another example, application of cell surface glycome profiling using GlycoStation™ will be described as a powerful method of characterizing stem cells. This work is a joint development project between three groups: GP Biosciences, National Institute of Advanced Industrial Science and Technology, and National Center for Child Health Development.

Collectins are a family of collagenous calcium-dependent defense lectins in animals. Their polypeptide chains consist of four regions: a cysteine-rich N-terminal domain, a collagen-like region, an alpha-helical coiled-coil neck domain and a C-terminal lectin or carbohydrate-recognition domain. These polypeptide chains form trimers that may assemble into larger oligomers. The best studied family members are the mannan-binding lectin, which is secreted into the blood by the liver, and the surfactant proteins A and D, which are secreted into the pulmonary alveolar and airway lining fluid. The collectins represent an important group of pattern recognition molecules, which bind to oligosaccharide structures and/or lipid moities on the surface of microorganisms. Collectin placenta 1 (CL-P1), a recently discovered scavenger receptor, mediates the uptake of oxidized low density lipoprotein and microbes. In this study, we investigated CL-P1-mediated binding and ingestion of yeast-derived zymosan bioparticles using Chinese hamster ovary (CHO) cells stably expressing human CL-P1 (CHO/CL-P1) and human vascular endothelial cells constitutively expressed CL-P1. The uptake of zymosan by CHO/CL-P1 was dependent upon the level of CL-P1 expressed on the membrane and was inhibited by cytochalasin D and wortmannin. The binding of zymosan was also inhibited by ligands of other scavenger receptors such as poly(I) and dextran sulfate. Real time reverse transcription-PCR analyses showed that other scavenger receptors, namely LOX-1, Stabilin-2, or macrophage receptor with collagenous structure (MARCO), were not expressed in human umbilical vein endothelial cells isolated from different individuals. Nonopsonic zymosan ingestion was inhibited in three primary cultured vascular endothelial cells, including different human umbilical vein endothelial cells from nine individuals treated with CL-P1 small interfering RNAs (siRNAs), although small interfering RNAs of other scavenger receptors had no effect on zymosan uptake in these cells. Furthermore, we confirmed that CL-P1 is expressed in human and murine vascular endothelial layers. Our results demonstrated that CL-P1 predominantly mediated phagocytosis for fungi in vascular endothelia.

The O-GlcNAc modification is a quite different fro m conventional glycosylation in two aspects. First, it occurs in cytoplasm and nucleus and does not in endoplasmic reticulum or Golgi apparatus. Second, this is a single sugar modification and is not a long chain oligosaccharide modification. O-GlcNAc is covalently modified on hydroxyl group of serine and threonine and usually this modification affects or competes with phosphorylation. Thus this modification might modulate many cellular events due to inhibiting or sometimes accelerating phosphorylation. O-GlcNAc transferase and O-GlcNAcase are two important enzymes for modifying proteins with O-GlcNAc. More than 800 proteins have been identified as O-GlcNAcylated proteins. Today I am going to summarize results obtained last 10 years and discuss about future aspects in O-GlcNAc biology.

UDP-D-glucose is then used as a glucosyl donor for the biosynthesis of various carbohydrates, such as the cell envelope of Escherichia coli, lipopolysaccharide, capsular polysaccharide, and membranederived oligosaccharides. In addition, it is an essential intermediate for the growth on galactose and trehalose, including the synthesis of rehalose. UDP-D-glucose is also considered to be the glucosyl donor for glycogen synthesis in mammalian cells, but ADP-D-glucose or UDP-D-glucose can serve as glucosyl donors in eukaryotic microorganisms and plants. So to synthesize UDP-glucose from UMP via UDP and UTP, three enzymes TMK (thymidine monophosphate kinase), ACK (acetate kinase) and Gal-U (Glucose-1-phosphate uridylyltransferase gene) are needed. a one-pot reaction system with ATP regeneration was designed, in which easily available UMP and glucose-1-phosphate were used as starting materials. Enzyme immobilization is a method to keep enzyme molecules cofined or localized in a certain defined region of space with retention of their catalytic activities. In comparison with their native form, immobilized enzymes offer several advantages, such as enhanced stability, easier product recovery and purification, the possibility of repeated usage, and continuous process technology. so UDP-glucose synthase was immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and cross-linking with glutaraldehyde. The effects of precipitation and cross-linking on CLEA activity were investigated and the immobilized enzymes were characterized.

Flavonoids are important natural pharmaceutical compounds found in almost all plants showing antioxidant, anti-inflammatory, antifungal, and anti-carcinogenic properties. Biosynthesis of glycosylated natural compound results in the formation of an overwhelming number of natural glycosides with numerous applications like solubility, stability, and bioavailability. To produce glycosylated flavonoids, the engineered E.coli cells were fed with Quercetin and Keampferol, the flavonol(or3-hydroxyflavone) exogenously and product was analyzed to be 3-O-rhamnosyl-quercetin and 3-O-rhamnosyl-kaempferol. Hence, based on the above strategy, the same engineered E.coli BL21(DE3, △pgi) cells generated by deleting the glucose-phosphate isomerase(Pgi) gene and heterologously expressing TDP-glucosesynthase, TDP-glucose4,6-dehydratase, TDP-4-keto-6-deoxyglucose3,5-epimerase, TDP-glucose 4-ketoreductase and arGt-3 (3-O-glycosyltransferase) from various sources were used as whole cell biotransformation reaction by feeding with fisetin exogeneously. The product was isolated and analyzed to be glycosylated fisetin by HPLC, ESI-MS and LC/MS, ESI-MS/MS. Since the glycosylated flavonoids have shown enormous pharmaceutical properties, the recombinant host generated can be utilize to discover glycosylated flvonoids as well as glycosylated quercetin and kaempferol as drugs.

Insect cell expression system using baculovirus has several benefits from high capacity, flexibility, safety to humans and glycosylation capability. Thus, the baculovirus insect cell system has been widely used for production of recombinant protein. In this study, the insect cell expression system was designed to produce anti-cancer mAb CO17-1A, which recognizes the antigen GA733 highly expressed on the surface membrane of colorectal carcinoma cells. The heavy (HC) and light chain (LC) genes of the mAb CO17-1A were cloned under the control of two different promoters, P10 and PPH, respectively, on baculovirus expression pFastBacTM Dual vector. The gene expression cassettes carrying HC and LC genes were transferred into a parent bacmid in E.coli (DH10Bac). The bacmid was transfected to Sf9 insect cells to generate the baculovirus expressing mAb CO17-1A. Western blot and immunoflouorescence analyses confirmed expression and secretion of mAb CO17-1A in the virus infected insect cells. The optimum condition for mAb expression was optimized at 24, 48 and 72hr after the virus infection with MOI (optimum virus concentration) ranging (0.2, 1 and 5). Expression of mAb CO17-1A in insect cells significantly increased at both 48 and 72hr after transfection with the MOI 1. HPLC chromatography revealed that the insect-derived mAb CO17-1A had insect specific glycan structures. Cell ELISA showed that the purified mAb from insect cell cultured media had a specific binding activity to SW948 human colorectal cancer cell. These results indicated that the baculovirus insect cell system is able to express, assemble, and secrete functional full size monoclonal antibody with insect specific glycosylation.

Gangliosides are complex glycosphingolipids that contain one or more sialic acids, the major components of cytoplasmic cell membranes. these are tought to play a role in the control of biological processes including cell surface interaction, cell differentiation and transmembrane signaling. Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be achieved by overexpression of Oct-4, Sox-2, Klf-4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. In this study, we demonstrated that differential expression patterns of gangliosides in iPS cells. First, we investigated characterization of iPS using Alkaline phosphate (AP) and stage-specific embryonic antigen-1 (SSEA-1) straing and RT-PCR. The iPS cells expressed AP and SSEA-1. In RT-PCR analysis, iPS cells also expressed Oct-4, Sox-2 and Nanog mRNA. Immunocytochemistry and high-performance thin-layer chromatography analysis showed that mouse embryonic fibroblast (mEF) expressed GM3, GM2 and GD3, but not GM2 in iPS cells. Furthermore, ganglsiode expression pattern were equal to mouse embryonic stem cell and iPS. In RT-PCR analysis confirmed that GM3 and GD3 synthase mRNA expressed in iPS, whereas mEF also expressed GM2 synthase mRNA. These results suggest that ganglioside GM2 play a important role in regulating the reprogramming of somatic cells into iPS.

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 immune rejection response by xenotransplantation is not yet clearly understood. In this study, differential expression patterens of gangliosides of Micro-Pig solid organs, which are heart, kidney, liver, pancreas and spleen. We examined expression level of high-performance thin-layer chromatography (HPTLC) showed that all of solid organs contained GM3, GM2, GM1 and GD1a as major gangliosides. Especially, ganglioside GD1b expressed in liver and pancreas, not in heart, kidney and spleen. Also, GT1b expressed in liver, kidney and spleen, not in heart and pancreas. As a results suggest that differential expression patterens of gangliosides of Micro-Pig solid organs are related immune rejection response in xenotransplantation by biomarker.

Human adipose-derived stem cells (hADSCs) and dental pulp-derived stem cells (hDPSCs) have been considered alternative sources of adult stem cells because of their potential to trans-differentiate into multiple cell lineages. This study investigated the possible role of gangliosides in the osteoblast differentiation of hADSCs and hDPSCs. First, we investigated characterization of hADSCs and hDPSCs using FACS analysis. Mesenchymal stem cell specific markers, CD44 and CD105, were expressed but not hematopoetic markers, CD45 and CD117 in both of hADSCs and hDPSCs. High-performance thin-layer chromatography analysis showed that increased gangliosides were associated with differentiation of hADSCs and hDPSCs into osteoblasts. RT-PCR analysis confirmed that osteoblast specific genes, ALP, BMP-2, collagen were expressed in differentiated osteoblasts, however, the another osteoblast specific gene, osteocalcin, was not expressed. When hADSCs and hDPSCs were cultured under osteoblast- differentiation conditions, alkaline phosphatase (ALP) activity was increased in comparison to hADSCs and hDPSCs. Furthermore, specifically both ALP activity and ganglioside expression increased more in hDPSCs-derived osteoblasts than hADSCs-derived osteoblasts. These results suggest that gangliosides play a more important role in regulating the osteoblast-differentiation of hDPSCs compared to hADSCs.

Gangliosides are complex glycosphingolipids that contain one or more sialic acids, the major components of cytoplasmic cell membranes. these are tought to play a role in the control of biological processes including cell surface interaction, cell differentiation and transmembrane signaling. Particularly, ganglioside GM3 is abundant in the kidney tissue. Diabetes mellitus is one of the most serious metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both, associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessel. In this study, we demonstrated that gangliosides expression patterns on kidney of mulberry-treated to STZ-induced diabetic rats. High-performance thin-layer chromatography analysis and immunostating showed that ganglioside GM3, GM2, GM1, GD1a, GD1b and GT1b were expressed in normal rats kidney, whereas ganglioside expression were strongly reduced in the kidney of diabetic rats. In 1.0% mulberry administration to diabetic rats kidney, ganglioside GM3, GM2 and GM1 expression were not different compared to normal rats kidney, whereas the other bands were not seen. Therefore, alteration of ganglioside expression in kidney of STZ-induced diabetic rats seem to be importantly involved in the development of glomerular hypertrophy and proteinuria.

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 immune rejection response by xenotransplantation is not yet clearly understood. In this study, differential expression patterens of gangliosides between human vein endothelial cells (ECV304) and micro-pig aortic endothelial cells (MPAECs, primary cultured) was investigated. we examined expression level of high-performance thin-layer chromatography (HPTLC) showed that ECV304 cells contained GM3 and GM2 as major gangliosides. However, MPAECs contained only GM3 as major ganglioside. Furthermore, we confirmed that human serum induces cell death of MPAECs. As a results suggest that differential expression patterens of gangliosides of ECV304, MPAECs are related immune rejection response and cell death with xenogeneic serum in xenotransplantation by biomarker. This study was supported by a grant from the Korean Rural Development Administration Agenda Program, 2010030106112200103) and a research grant from the Ministry of Education, Science and Technology (KGC5401011), Republic of Korea.

With the completion of the human genome projects, one of the most important scientific issues is to investigate the biological function of each protein. In higher organisms, the functional outputs of the numerous gene products seem to be much more complex when compared to the number of genes, emphasizing the importance of post-translational modifications such as glycosylation and phosphorylation. Despite the fact that glycoproteins are involved in various biological phenomena, the systematic studies of the effects of protein glycosylation have been limited due to the heterogeneity and structural complexity of carbohydrates. In order to access glycopeptides and glycoproteins of defined structure, various chemical, enzymatic and genetic approaches have been developed. As glycopeptide mimetics, we envisioned to develop glycopeptoids because peptoids have many advantages over peptides, including easy synthesis of libraries, a myriad of chemical diversity, proteolytic resistance and improved cell permeability. For the synthesis of glycopeptoids, we have employed the well established ‘submonomer strategy’ on the solid-phase using many commercially available primary amines and synthetically prepared sugar amines. In addition, diastereomeric sugar monomers were differentiated by using different amine linkers, resulting in successful sequencing by tandem mass spectrometry (MS/MS). Glycopeptoids should be useful chemical tools for various biological applications including studies for binding affinity to specific proteins or lectins, development of therapeutics such as antibiotics, and profiling studies of diseases.

Since the completion of the human genome project, development of new therapeutic and diagnostic proteins have been exponentially increased, but widespread use of these molecules has been hampered by production bottlenecks such as low yields, poor and inconsistent product quality and a shortage of production capacity. Especially, we are facing a growing demand for protein diagnostics and therapeutics, but lack the capacity to meet those demands using established facilities. Over the last decade, plants have emerged as a convenient, safe and economical alternative to mainstream expression systems which are based on the large-scale culture of microbes or animal cells, or transgenic animals. The production of plant-made pharmaceuticals and technical proteins is known as Molecular Farming. The objective is to harness the power of agriculture to cultivate and harvest plants or plant cells producing recombinant therapeutics, diagnostics, industrial enzymes and green chemicals. Molecular Farming has the potential to provide virtually unlimited quantities of recombinant antibodies, vaccines, blood substitutes, growth factors, cytokines and enzymes for use as diagnostic and therapeutic tools in health care, the life sciences and the chemical industry. The overall aim of our research is to develop new plant molecular farming systems which are economic, efficient, stable and safe. To accomplish the purpose, we are performing specific research objectives in regards to plant molecular farming.

Platycosides, the saponins found in the roots of Platycodon grandiflorum (PlatycodiRadix), are typically composed of oleanene backbones with two side chains. Of the platycosides, platycodin D (glucose unit at C-3) is a major component and it has several pharmacological activities. Because of the high demand for this compound, we attempted to enzymatically covert platycodin D3 and platycoside E, having two and three glucose units at C-3, respectively, into platycodinD. In this study, we tested the ability of several glycosidases to transform platycosides, or morespecifically, the ability to transform platycoside E and platycodin D3 into platycodin D. To get pure platycodin D on a preparative scale, high-speed counter-current chromatography (HSCCC) with a solvent system ethyl acetae-n-butanol-water (1.2:1:2, v/v/v) was used for separation of the enzymatically-transformed product from the reaction mixture. 40.5 mg of platycodin D (99.8% purity) was obtained from 200 mg of the productin one-step seapartion.

The by-products of marine organisms including salmon, skate, flatfish, and yellow goosefish were investigated to search for new source of chondroitin sulfate. Agarose gel electrophoresis with chondroitinase depolymerization showed that purified chondroitin sulfate did not contain any other glycosaminoglycans. 1H-NMR spectra were acquired to confirm the structure and purity. The average molecular weight ranging from 22 to 64 kDa was determined by high-performance size exclusion chromatography. Disaccharide compositions and purities were determined by strong anion exchange-high performance liquid chromatography after chondroitinase ABC depolymerization. Strong anion exchangehigh- performance size exclusion chromatography data exhibited that the purity was from 81.7±1.3 to 114.2±2.5% and the yield was from 1.3 to 12.5%. All analytical results indicate that salmon cartilage, skate cartilage, and yellow goosefish bone could be promising sources of chondroitin sulfate to substitute shark cartilage chondroitin sulfate in commercial neutraceuticals.

Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, is a novel glycosaminoglycan, consisting primarily of the repeating disaccharide structure a-D-N-acetylglucosaminyl (1!4) 2-sulfoiduronic acid. AS shows anti-tumor activity in vitro and in vivo. Despite this activity, AS is only weakly cytotoxic towards cancer cells. We examine the interactions between AS and cell-surface proteins in an effort to explain this anti-tumor activity. Using flow cytometry and affinity column chromatography, we confirm that AS has strong affinity to specific cell-surface proteins including nucleolin (NL) in A549 human lung adenocarcinomas. Surprisingly, we found the translocation of NL from nucleus to cytoplasm under the stimulation of AS (100 mg/ml) in vitro. Also, as NL exits the nucleus, the levels of growth factors such as bFGF and signaling cascade proteins, such as p38, p53, and pERK, are altered. These results suggest that the communication between AS and NL plays a critical role on signal transduction in tumor inhibition.

The mycolyl arabinogalactan-peptidoglycan complex is one of the major polysaccharide segments of the cell wall of Mycobacterium tuberculosis and plays an essential role in the survival and pathogenicity of Mycobacterium tuberculosis. The synthesis of mAGP motifs is significant because it helps to understand their potential as antitubercular drugs as well as their biological significance as substrates for enzymes involved in the biosynthesis of mycobacterial cell wall. We previously synthesised an octaarabinofuranoside of the arabinan,1 a tetrasaccharide phosphate of the linkage region,2 and cyclic galactooligofuranosides3 from cell wall of Mycobacterium tuberculosis. In this work, the synthesis of a suitably protected fragment 1 of the subunit from the arabinogalactan of Mycobacterium tuberculosis is reported. Nine monosaccharide building blocks were assembled by stereoselective glycosylations employing 2'-carboxybenzyl glycosides and glycosyl fluorides as glycosyl donors.

The mad cow disease, bovine spongiform encephalopathy, is a deadly, neurodegenerative disease in cattle, which causes spongy in the brain and spinal cord. Many scientists think the disease as transmittable to human beings by taking the brain and spinal cord of infected cattle. In an effort to study important biological functions of the mad cow disease and to develop the chemotherapy agents, it is essential to synthesize the glycosyl phosphatidyl inositol (GPI) anchor, which connects scrapie prion protein via their C-terminus to eukaryotic cell membranes. In the present work, the synthesis of {a-D-Manp-(1→2)-a-D-Manp-(1→2)-a-D-Manp-(1→6)-[b-D -GalNAcp-(1→4)]-a-D-Manp}, a suitably protected form of pentasaccharide unit of GPI anchor of scrapie prion protein was achieved by assembling of five monosaccharide building blocks with careful stereo- and position-manipulations. The ready accessibility of all starting materials, the high yields obtained in all reaction steps, including the glycosylation reactions which were carried out with trichloroacetimidates, provide an excellent basis for further successful syntheses in this field.

Hydroquinone galactoside (HQ-Gal) as a potential skin whitening agent was synthesized by the reaction of lactase (β-galactosidase) from Kluyveromyces lactis, Aspergillus oryzae, Bacillus circulans and Thermus sp. with lactose as a donor and HQ as an acceptor. Among these lactases, the acceptor reaction involving HQ and lactose with K.lactis lactase showed higher conversion ratio to HQ-Gal(60.27%). HQ-Gal was purified using butanol partitioning and silica-gel column chromatography. The structure of the purified HQ-Gal was determined by nuclear magnetic resonance and the ionic product was observed at m/z 295(C12H16O7Na)+ using matrix assisted laser desorption ionization time-of-flight mass spectrometry. HQ-Gal was identified as 4-hydroxyphenyl-β-D-galactopyranoside. The optimum conditions for HQ-Gal synthesis by K.lactis determined using response surface methodology were 50 mM HQ, 60 mM lactose, and 20 U mL-1 lactase. These conditions produced a yield of 2.01 g L-1 HQ-Gal. The half maximal inhibitory concentration (IC50) of diphenylpicryl-hydrazyl radical scavenging activity was 3.31 mM indicating a similar anti-oxidant activity compared to β-arbutin(IC50=3.95 mM). The Ki value of HQ-Gal(0.75 mM) against tyrosinase was smaller than that of β-arbutin (Ki=1.97 mM),indicating superiority as an inhibitor.

Baculovirus expression system is considered to have several benefits for production of recombinant protein such as high capacity, flexibility, and similar human glycosylation pattern. An accurate titering of recombinant baculovirus is required to ensure the most appropriate expression conditions in baculovirus insect cell. BaculoELISA Titer Kit (Clontech, Mountain Biew, CA, USA) is one of baculovirus titration methods. In this method, the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) type baculovirus can be measured by a standard ELISA. However, control virus of this kit applied to this method is highly expensive. In this study, expression of monoclonal antibody (mAb) CO17-1A genes by baculovirus infection was optimized at concentrations of baculovirus, passages of baculovirus and different insect cell harvesting times. Immunoblot analysis confirmed expression of mAb CO17-1A in the infected cells. mAb CO17-1A expressed significantly at the highest expression level under the conditions of 400㎕ of baculovirus passages 3(P3) in 5 ml insect cells. In addition, mAb CO17-1A expressed with a higher level when the infected cells were harvested after 72h after baculovirus infection. The results propose that baculovirus concentration, virus passage, harvesting times after virus infection conditions of can be optimized to enhance the mAb production in insect cell system without BaculoELISA assay.

Colorectal cancer is the third leading cause of cancer-related death with 655,000 deaths worldwide per year in the Western world. The tumor-associated antigen GA733 is a glycoprotein highly expressed on colorectal carcinoma cell surface. We have fused GA733 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 host immune responses. GA733-Fc is glycosylated, but the N-glycanstructures of plant-derived GA733-Fc differ from their parental counterparts. Plant-derived specific N-glycoforms can potential to induce immunogenicity and allergenic responses in human therapy. Thus, we have tried to generate oligomannose glycan structure by retaining GA733-Fc and GA733 in ER with KDEL sequence. Agrobacterium-mediated transformation was conducted to generate transgenic plant expressing GA733-Fc and GA733. We have compared protein expression, accumulation and biological activity of GA733-Fc with KDEL, GA733-Fc without KDEL, GA733 with KDEL and GA733 without KDEL in transgenic plants by western blot, confocal immunofluorescence analysis, ELISA and mouse immunization, respectively.

Baculovirus expression system is considered to have several benefits for production of recombinant protein such as high capacity, flexibility, and similar human glycosylation pattern. Typically, before any large scale expression work was commenced, an accurate titering of recombinant baculovirus is required to ensure the most appropriate expression conditions. One of them, the method have been developed by BaculoELISA Titer Kit (Clontech, Mountain Biew, CA, USA). Stocks of any Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) type baculovirus can be titered using this kit. However, control virus of this kit is high cost and relatively small quantity in kit. In this study, production of mAb CO17-1A by baculovirus infection was performed at different times, concentrations and passages. Immunoblot analysis confirmed expression of mAb CO17-1A in the infected cells. mAb CO17-1A expressed significantly high level under the condition of baculovirus passages 3, 72h and 400㎕ in 5ml insect cells after infection. The results indicated that it is not necessary to use BaculoELISA assay.

Plant genetic engineering has led to production of plant-derived mAb (mAbP), which can provides a safe and economically effective alternative to the conventional antibody expression methods. In this study, the expression levels of mAbP SO57 with or without ER-retention peptide extensions signal (Lys-Asp-Glu-Leu; KDEL) in transgenic tobacco plants were analysed in transgenic plant. The expression levels of mAbP SO57 with KDEL were significantly higher than that without KDEL regardless of transcription level. mAbP SO57 with KDEL localized surround to the nucleus suggesting that the mAbP with KDEL is localized in ER. The mAbP without KDEL and mAbH had mainly Golgi type glycans, whereas the ER-localized mAbP with KDEL had glycan profile with both oligomannose type (47.6%) and Golgi type (52.4%). The Fc domains of both purified mAbP (with and without KDEL) and human-derived mAb (mAbH) had similar bingding activity to the Fcγ RI receptor (CD64). Both mAbP (with and without KDEL) had a shorter half-life than mAbH. However, both mAbP with and without KDEL was as effective at neutralizing activity of the rabies virus CVS-11 as the mAbH. These results suggest that ER localization of recombinant mAbP by KDEL reprograms glycosylation and enhances production of the functional antivirus therapeutic antibody in the plant.

Baculovirus has been widely used for production of numerous recombinant proteins in insect cells. Baculovirus expression has the following advantages: proper post-translational modification, a high capacity for expression of multiple genes or a large insert, biosafety, and high yield driven by the strong promoters polyhedrin or p10. Recently, recombinant proteins have been expressed in both insect cell and mammalian cells by baculovirus system. To date, however, there has been no report of recombinant protein expressed by baculovirus infection in plant. In this study, this expression system was exploited to express GFP (green fluorescence protein). To investigate the baculovirus capability to express the recombinant protein in plant cell, the GFP was cloned under the control of polyhedrin (PPH) promoter and 35S (Cauliflower Mosaic Virus) promoter into the pFastBacTM dual vector, respectively. Gene expression cassetees carrying the GFP genes were transposed into a bacmid in Escherichiacoli (DH10Bac). Bacmid DNA containing GFP gene expression cassette was isolated from transformed DH10Bac using Bac-to-Bac system. The existence of GFP gene in bacmid was confirmed by PCR with GFP specific primers. Both bacmid and baculovirus generated from bacmid-infected Sf9 cells was transfected NicotianaBenthamiana plant leaves, respectively. Confocal immunoflouorescence and fluorescence microscopic analyses confirmed expression of GFP in baculovirus-infected plant leaves and protoplasts isolated from baculovirus–infected plant leaves. In additon, reverse transcription-polymerase chain reaction (RT-PCR) confirmed transcription of GFP gene. In this study, taken together, these results suggest that the baculovirus is able to express recombinant protein gene in plant cells.

When cellular glucose concentrations fall below normal levels, in general the extent of protein O-GlcNAc modification (O-GlcNAcylation) decreases. However, recent reports demonstrated increased O-GlcNAcylation by glucose deprivation in HepG2 and Neuro-2a cells. Here, we report increased O-GlcNAcylation in non-small cell lung carcinoma A549 cells and various other cells in response to glucose deprivation. Although the level of O-GlcNAc transferase was unchanged, the enzyme contained less O-GlcNAc, and its activity was increased. Moreover, O-GlcNAcase activity was reduced. The studied cells contain glycogen, and we show that its degradation in response to glucose deprivation provides a source for UDP-GlcNAc required for increased O-GlcNAcylation under this condition. This required active glycogen phosphorylase and resulted in increased glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting enzyme in the hexosamine biosynthetic pathway. Interestingly, glucose deprivation reduced the amount of phosphofructokinase 1, a regulatory glycolytic enzyme, and blocked ATP synthesis. These findings suggest that glycogen is the source for increased O-GlcNAcylation but not for generating ATP in response to glucose deprivation and that this may be useful for cancer cells to survive.