Earticle

Sulfated polysaccharides extracted from Agarum cribrosum and its fractionated using ion-exchange chromatography (crude, F1 and F2) were investigated to determine their molecular characteristics and biological activities. The crude and fractionated polysaccharides (F1 and F2) contained mostly of carbohydrates (52.4-56.0%), sulfates (12.7-23.0%) and uronic acid (14.1-21.8%) with small amount of proteins (3.9-9.3%). The monosaccharides levels of sulfated polysaccharides were mainly included fucose (43.1-44.2%), mannose (17.9-29.2%) and galactose (5.6-33.0%) with minor amount of rhamnose (2.3-4.6%) and glucose (0.9-3.5%). The sulfated polysaccharides contained one or two subfractions with average molecular weights (Mw) ranging from 123 × 103 to 765 × 103 g/mol. The sulfated polysaccharides, especially the F1 and F2 fractions, strongly stimulated mouse macrophage cells, Raw 264.7 cells, producing considerable amount of nitric oxide (NO), tumor necrosis factor-α (TNF-a) and various cytokines (IL 1-β, COX-2 and iNOS) which suggested that they could be strong immunostimulators. The overall results implies that the strong immunomodulatory activity of sulfated polysaccharides from A. cribrosum might be good sources of natural healthcare products, such as supplements and nutraceuticals.

O-GlcNAcylation, the addition of β-N-acetylglucosamine(O-GlcNAc) to serine or threonine, is one of post-translational modification occurs on myriad proteins in nucleus and cytosol. It cycles on proteins with a time-scale similar to protein O-phosphorylation, and has surprisingly extensive cross talk with O-phosphorylation, where is serves as a nutrient/stress sensor to modulate signaling, transcription, and cytoskeletal functions. O-GlcNAcylated proteins in Drosophila were analyzed using 2D gel electrophoresis and MALDI/TOF-MS, and ATP synthase β was identified as a novel O-GlcNAcylated protein in Drosophila SL2 cell. F1F0 ATP synthase complex produces ATP from ADP in the presence of a proton gradient across mitochondrial membrane which is generated by electron transport of the respiration, and β subunit has ATPase activity. By immunoprecipiation and immunoblotting, O-GlcNAcylation of ATP synthase β was confirmed. Interestingly, we found that O-GlcNAcylation of ATP synthase β is increased by nucleocytoplasmic O-GlcNAc transferase. Hence we will concentrate on demonstrating how the ATP synthase β is modified with O-GlcNAc and what the functional roles of O-GlcNAcylation on ATP synthase β are.

The purpose of this study is to optimize sandwich ELISA conditions to quantify the colorectal cancer antigen GA733 linked to the Fc antibody fragment complex protein fused to KDEL, an ER retention signal (GA733-FcK) expressed in the transgenic plant. Variable conditions of capture antibody, blocking buffer, and detection antibody for ELISA were optimized with application of leaf extracts from the transgenic plant, expressing GA733-FcK. In detection antibody, anti-EpCAM/CD362 IgG conjugated to HRP recognizing the GA733 did not detect any GA733-FcK whereas anti-human Fc IgG conjugated to HRP recognizing the human Fc detected GA733-FcK distinguish the presence of GA733-FcK in leaf extract. For blocking buffer conditions, 3 % BSA buffer properly clearly blocked the ELISA plate, compared to the 5 % skim milk buffer. For the capture antibody, mAb CO17-1A was applied to coat the 96 well ELISA plate with different amounts (1, 0.5, and 0.25 μg /well). Among the different amounts of the capture antibody, 1 and 0.5 μg/well of the capture antibody showed similar absorbance; whereas, 0.25 μg/well of the capture antibody showed significantly less absorbance. Taken together, the optimized sandwich ELISA conditions to quantify plant-derived GA733-FcK were 0.5 μg/well of mAb CO17-1A per well for the capture antibody, 3 % BSA for blocking buffer, and anti-human Fc conjugated HRP. To confirm the optimized ELISA conditions, correlation analysis was conducted between the quantified amount of GA733-FcK in ELISA and its protein density values of different transgenic plant leaf samples in Western blot. The coefficient value R2 between the ELISA quantified expression value and protein density was 0.85 (p<0.01), which indicates that the optimized sandwich ELISA conditions feasibly provides quantitative information of GA733-FcK expression in the transgenic plant leaves.

To date, many efforts have been made to detect lectins in cells by using single imaging techniques. However, only a few dual-labeled glycan-based probes, which integrate advantageous features of two imaging methods to enhance the visualization of biological processes associated with lectins in cells, have been reported. Herein we describe the synthesis of dual fluorescence and magnetic resonance imaging agent conjugated neoglycopeptides and their application in the simultaneous imaging of lectins in mammalian cells. The dual-labeled neoglycopeptides bind to lectins on cell surfaces and subsequently enter the cells via lectin-mediated endocytosis. The results of these efforts show that the novel dual-labeled neoglycopeptides are effective fluorescence and MR imaging agents for monitoring biological processes associated with lectins.

Carbohydrates have been known to play biologically important roles such as cell adhension, cell recognition, fertilization and embryogenesis though interactions with proteins. Importantly, these biomolecular interactions are also involved in tumor metastasis and inflammation. In addition, some pathogens infect human though interactions between the pathogenic proteins and human glycans. For example, human influenza virus produce proteins that preferentially adhere to NeuNAcα2,6Gal residues on epithelial cells of the lungs and upper respiratory tract. Some of Helicobacter pyroli which express Leb binding adhesin (BabA) and sialyl Lex-binding adhesin (SabA) recognize the human gastric mucosa expressing these glycans. To detect pathogens as well as mammalian cells, various glycoconjugates, including Lea, Leb, H1, Lex and Ley, were synthesized.

N-glycosylation is one of the major post-translational protein modifications, which alters physicochemical properties of the protein, affecting the folding, distribution, stability and thus biological function and efficiency of protein. Although the earlier steps in the N-glycosylation pathway leading to the formation of oligomannosidic structures are conserved in plants and mammals, the later steps in the formation of complex N-glycans are quite different from each other. In particular, plant type complex N-glycans are distinctive from those found in mammalian because they contain β 1,2-xylose and core α1,3-fucose residues attached to pentasaccharide (Man3GlcNAc2) core structure but no sialic acid residues. The presence of β1,2-xylose and core α1,3-fucose residues on plant type complex N-glycans has long been an irritating limitation in the use of plant-made pharmaceuticals (PMPs) in human therapy, as these N-glycan epitopes are potentially immunogenic in mammals. In this study, to remove the plant specific sugar residues and humanize the N-glycosylation in plant, we isolated mutants of the corresponding plant specific glycosyltransferase genes and used for multiple-mutants construction. The resulting mutants will be transformed by human glycosyltransferases genes to accomplish humanized N-glycosylation in plant.

N-linked Glycosylation is enzymatic process that attaches sugars residue to tripeptide sequence Asparagine-X-Serine /Threonin of nascent polypeptide, where X could be any amino acid except Proline. Extracellular and endomembrane protein are mostly glycosylated by N-linked oligosaccharides in eukaryote. The maturation of N-glycan is modified by glycosidases and glycosyl transferases in the ER and golgi of the secretory pathway. The GNUT is a glycosyltransferases that responsible for the transfer core sugar residues to N-glycans of glycoprotein. To explore the physiological role of alpha GNUT, the mutant gnut1 was isolated in rice. Genetic analysis shows that T-DNA was inserted in the first intron of the GNUT gene. The T-DNA insertion causes loss-of-function mutation of this gene. Biochemical analysis have confirm that sugar residue was absent from core N-glycan in this mutant. The gnut1 shows internodes length retardation in the late vegetative growth. In generative growth, the gnut1 mutant also exhibit short panicles length, grain filling defective and small grains. The result indicates that GNUT is important for normal growth of rice in the generative phase.

Environmental or physiological influences that induce accumulation of unfolded proteins in the lumen of endoplasmic reticulum (ER) cause ER stress and activate signaling pathway called unfolded protein response (UPR). An ER-located transmembrane receptor protein kinase/ribonuclease called Ire1 plays an essential role in the UPR in yeasts and mammals. However, it has been unclear whether a similar mechanism is applicable to Arabidopsis. To elucidate the role of Arabidopsis IRE1, we performed functional analyses by isolating loss-of-function mutants of IRE1A and IRE1B. We found that a double mutant of Arabidopsis IRE1A and IRE1B (ire1a ire1b) is more sensitive to the ER stress inducer tunicamycin than the wild-type. ire1a ire1b result in a delayed induction of BiP3 that is well known ER chaperone by tunicamycin treatment, whereas induction of several other ER chaperones in ire1a ire1b was similar with that of WT. Our results indicate that IRE1A and IRE1B are implicated in unfolded protein response signaling in plants.

Gangliosides are sialic acid-containing glycosphingolipids that are abundant in neurons and have a variety of functions in developing and mature tissue. Mini-pig neural stem cells contained GM2 and GM3 as major gangliosides. In order to study their distribution in mini-pig neural stem cells and its possible changes during the differentiation of neuronal cells, cells were stained with specific monoclonal antibodies against 11 ganglio-series gangliosides including those mentioned above. GM2 and GM3 were expressed in a spatio-temporally different manner during differentiation of mini-pig neural stem cells, but other gangliosides were not immunohistochemically detected. These results suggest that ganglioside GM2 and GM3 play an important role in the regulation of neuronal differentiation of mini-pig neural stem cells through the activation of retionic acid (RA) and basic fibroblast growth factor (bFGF).

The human colorectal carcinoma-associated GA733 antigen epithelial cell adhesion molecule (EpCAM) was initially described as a cell surface protein selectively expressed in some myeloid cancers. The glycoprotein was originally defined by anti-GA733, anti-CO17-1A, and anti-EpCAM mAbs, which bind to different epitopes on this antigen. We demonstrated that treatment with plant-derived multiple antibodies (mAbPC×B; anti-colorectal cancer mAb, mAbp CO17-1A and anti-breast cancer mAb, BR 55 mAb) with RAW264.7 cells significantly inhibited cell growth in SW 620 and MCF 7 cells. Expression of p53 and p21 increased, whereas the expression of G1 phase-related proteins, cyclin D1, CDK4, cyclin E and CDK, decreased. In addition, plant-derived multiple antibodies with RAW264.7 cells treatment decreased the expression of anti-apoptotic proteins such as Bcl-2, but the expression of pro-apoptotic proteins Bax, TNF-a, caspase-8, caspase-9, caspase and caspase-6 increased. We observed that plant-derived multiple antibodies significantly inhibited the growth of colon tumors, as determined by a decrease in tumor volume and weight. These results suggest that plant-derived multiple monoclonal antibodies could have an anti-cancer effect for colorectal and breast cancer. Further clinical investigation should be conducted on plant-derived multiple monoclonal antibodies to determine its possible chemopreventive and/or therapeutic efficacy for the treatment of human colon and breast cancer.

Drug targeting using colloidal carriers such as nanoparticles or colloidal carriers have been extensively investigated for the treatment of various disease in the biomedical field [1-3]. Especially, nanoparticles can be simply decorated with tumor-specific ligand and then used for site-specific delivery of anticancer agents to tumor cells since cancer cells have various kind of receptors [3]. Among them, hyaluronic acid (HA) receptor such as CD44 is especially over-expressed on the surfaces of highly malignant tumor cells. Then, HA nanoparticles can be used to target CD44 receptor of tumor cells [4]. Furthermore, HA can be considered as a ideal candidate for tumor-specific drug targeting because HA is a fully biocompatible material. In this study, we synthesized block copolymer composed of hyaluronic acid and oly(e-caprolactone) (HAPCL) to prepare nanoparticles for targeting of CD44 receptor. Chemical composition of HAPCL was confirmed using 1H NMR spectroscopy. Doxorubicin (DOX) as a model drug was incorporated into nanoparticles of HAPCL copolymer. The diameters of HAPCL nanoparticles were around 100nm and their morphology was spherical shape. CD44 receptor targeting of HAPCL nanoparticles was confirmed with MDA-MB231 cells. The treatment with DOX-incorporated nanoparticles revealed strong fluorescence intensity at MDA-MB231 cells. However, fluorescence intensity was significantly reduced when CD44 receptor was blocked by pretreatment with free HA, indicating that HAPCL nanoparticles have targetability aginst CD44 receptor.

Since chitosan, a natural and polycationic polysaccharide, is a biocompatible, biodegradable, and low immunogenic properties, it has been extensively investigated in biomedical field such as drug delivery, dene delivery, and tissue engineering [2,3]. Chitosan is known to accelarate drug delivery the mucosal layer without tissue damage [4]. Furthermore, chitosan has been frequently employed for conjugation of bioactive chemicals or formation of polyelectrolyte complexes with anionic molecules since chitosan has cationic amine functional grous [3]. For this study, we synthesized hydrophobically modified chitosan using phenethyl isothiocyanate (ChitoPEITC) for photosensitizer delivery to tumor cells. Chlorin E6 (Ce6) was used as a photosensitizer and Ce6-incorporated ChitoPEITC nanoparticles were prepared by simple mixing of Ce6 and ChitoPEITC. Nanoparticles have small particle sizes less than 300 nm. When Ce6-incorporated nanoparticles were treated to HuCC-T1 cholangiocarcinoma cells, nanoparticles improved Ce6 uptake into tumor cells compared to free Ce6. Furthermore, nanoparticles also enhanced ROS generation in tumor cells. Furthermore, nanoparticles revealed higher phototoxicity against tumor cells than free Ce6. We suggest that ChitoPEITC is a promising vehicles for photosensitizer delivery.

β-O-linked N-acetylglucosamine(O-GlcNAc) is dynamic post-translational modification in nucleus and cytosol and it is regulated by O-GlcNAc transferase(OGT) and O-GlcNAcase. Many proteins, such as transcription factors, enzymes, cytoskeletal proteins, ribosomal proteins and chaperones have been identified to be modified with O-GlcNAc. O-GlcNAc modification modifies at their Ser/Thr residues and affects their functions. The hexosamine biosynthesis pathway takes about 5% of glucose in total glucose flux, and it’s final product is UDP-GlcNAc, which is source of O-GlcNAc modification. O-GlcNAc modification is reported to involve in skeletal muscle metabolism and development process. We studied whether O-GlcNAc modification is involved in differentiation in myoblast C2C12 cell. We observed that total O-GlcNAc modification level was dynamically changed during myogenesis. After treatment NButGT, we observed that myogenin expression level decreased. Using RT PCR, we found that mRNA level of myogenin decrease after treatment NButGT. And we found that the promoter activity of myogenin decreases after NButGT using reporter gene assay. For finding the promoter region affected O-GlcNAc , we did reporter gene assay using shorter length promoter and found at least 169 base pair upstream region of myogenin is affected by O-GlcNAc. And using this region, we found that Mef2c protein can be important transcription factor regulated by O-GlcNAc using avidin-biotin complex DNA binding assay. We confirm that Mef2c is O-GlcNAcylated using immunoprecipitation and we found several O-GlcNAcylated sites on Mef2c using mass spectrometry. Based on the result, we made the Mef2c point mutants converted from Serine, Threonine of O-GlcNAcylated sites to Alanine. Additionally, we found O-GlcNAc modification could regulate DNA binding affinity and transcriptional complex formation.