Earticle

Astragalin (kaempferol-3-O-D-glucopyranoside) is one of Flavonoid. Has been contain 0.12 percent of Astragalus sinicus and plant. However these plants only the least amount of Astragalin (kaempferol-3-O-d-glucopyranoside). By the aforementioned reasons, we are production of astragalin from kaempferol in Escherichia coli BL21. Kaempferol was further modified to produce astragalin by 3-O-glucosylation utilizing the endogeneous UDP-glucose in presence of UGT78K1 from Glycine max. E. coli strain was able to produce astragalin of conversion 70 percent per 0.4 mmol of kaempferol, A 50 milliliter of 200 millilter flask size culture. Grow in the shaking incubator for 35℃ 3hr, after check O.D 0.6 direct induction(1M IPTG) inject at the flask 20㎕ and shaking incubator for 20℃ overnight. But it is not maximum limit conversion of astragalin from kaemferol. We are add 0.1 percent glycerol of fed-bath culture, increase untill conversion 90 percent. In this work deliverable (conversion of astragalin) check of TLC and HPLC.

In present study, we report two synthetic aminopyrrolic compounds that induce apoptosis in cancer. These compounds have been previously shown to act as receptors for mannosides. The extent of receptor-induced cancer cell death is greater in cells expressing a high level of high-mannose oligosaccharides than in cells producing lower levels of high-mannose glycans. The ability of synthetic receptors to induce cell death is attenuated in the presence of external mannosides. The present results provide support for the suggestion that the observed cell death reflects an ability of the receptors to bind mannose displayed on the cell surface. Signaling pathway studies indicate that the synthetic receptors of the present study promote JNK activation, induce Bax translocation to the mitochondria, and cause cytochrome c release from the mitochondria into the cytosol, thus promoting caspase-dependent apoptosis. Such effects are also observed in cells treated with mannose-binding ConA. The present results thus serve to highlight what may be an attractive new approach to triggering apoptosis via modes of action that differ from those normally used to promote apoptosis.

Multiple earlier studies have shown that interactions between glycans and carbohydrate binding proteins (lectins) displayed on cells surfaces are involved in many important physiological and pathological processes. For example, cell surface lectins in the immune system recognize glycans expressed on the exterior of pathogens and these interactions lead to stimulation of immune responses to pathogens. Mouse SIGN-R1 (SIGN-related 1) is a homolog of human DC-SIGN that is expressed largely on macrophages. This lectin recognizes mannose-rich or fucosylated glycans in a Ca2+-dependent manner. When SIGN-R1 interacts with glycans on host cells, bacteria or viruses, glycan antigens are internalized into cells via lectin-mediated endocytosis to induce immune activation. As a consequence, the lectin hasbecome fascinating markers for recognition and targeting of specific cells to regulate cellular functions and for carbohydrate-based drug discovery. To investigate glycan binding properties of cell surface lectins, we have utilized carbohydrate microarrays which allow high-throughput screening of cell-glycan interactions. Glycan microarrays used in this study were constructed by immobilizing a variety of unmodified carbohydrates onto hydrazide-coated glass slides. SIGN-R1 expressing cells pretreated with Hoechst 33342 were loaded on the glycan microarrays to evaluate the binding properties of the lectin. In addition, purified SIGN-R1 was also applied tothe microarrays for the purpose of comparison. The present study demonstrates the potential of glycan microarrays for profiling of binding specificities of cell surface lectins.

Through binding to free glycans or glycoconjugates, animal cell surface lectins are involved in a wide range of biological processes. It has been known that most animal lectins, including selectins, siglecs (sialic acid-binding immunoglobulin-type lectins), mannose receptors, mannose-6-phosphate receptors, asialoglycoprotein receptors and C-type lectins, act as signal transducers after binding to glycans although intracellular signaling events induced by lectins depend on the nature of glycans, cell type and/or species. In particular, cell-surface lectins in the immune system bind to glycans displayed on the exterior of pathogens and this recognition event stimulates the immune response. For example, mouse SIGN-R1 (SIGN-related 1), a homolog of human DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin), is highly expressed on macrophages in the splenic marginal zone and the medullar lymph nodes. This lectin binds predominantly to mannose-rich or fucosylated glycans in a Ca2+-dependent manner. Once glycans on bacterial cells or viruses interact with SIGN-R1, glycan antigens elicit SIGN-R1 mediated immune activation. To investigate immune activation by lectin mediated endocytosis, we conducted a traditional cell assay to probe six glycan-bovine serum albumin (BSA) conjugates (BSA-Man, BSA-Fuc, BSA-GlcNAc, BSA-Gal, BSA-Mana1,2Man and BSA-Lex) and S. cerevisiae mannan for their abilities to stimulate ROS generation. The results show that incubation with mannan and all of the glycoconjugates except BSA-Gal leads to increases in the intensities of fluorescence signals from cells compared to the untreated control.

Helicobacter pylori is a Gram-negative, microaerophilic bacterium found in the stomach and causes chronic gastritis and gastric ulcers. It is also associated with the development of duodenal ulcers and stomach cancer. Therefore, sensitive detection of H. pylori is crucial for diagnosis and treatment of H. pylori associated diseases. It has been known that H. pylori expresses a sialic acid-binding adhesin (SabA) and a Leb-binding adhesin (BabA) that adhere, through glycan-adhesin interactions, to the human gastric mucosa for infection. To detect H. pyroli expressing BabA, we prepared fluorescent magnetic glyconanoparticles by conjugating aminoethylated Lea, Leband H1 oligosaccharides to carboxy-containing fluorescent magnetic nanoparticles. The successful preparation of glyconanoparticles was examined by zeta potential and transmission electron microscopy (TEM). When H. pyroli J99 strain which expresses BabA was incubated with three kinds of glyconanoparticles, fluorescence microscopy analysis showed that Leb and H1 conjugated but not Lea conjugated nanoparticles bound to this strain. However, these glyconanoparticles did not recognize H. pyroli strains lacking BabA. Finally, they were employed to enrich BabA expressing H. pyroli by using a magnet. It is expected that fluorescent magnetic glyconanoparticleswill be powerful tools to sensitively detect pathogens including H. pylori.

Gangliosides has the important roles in interactions between cells and in signal transduction to regulate growth and differentiation. Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder wherein symptoms resembling aspects of aging are manifested at a very early age. Model of LMNA (Lamin A/C) mutation is known as a typical model for HPGS. However, roles of gangliosides in model of LMNA mutation (HGPS) is unclear. Therefore, this study investigated the expression patterns of gangliosides in model of LMNA-mutation. LMNA-mutation cells were primary cultured from spontaneous mouse LMNA-mutation model. Distribution of gangliosides in LMNA-mutation mouse was detected by HPTLC. We successes the primary cultured bone marrow-derived mesencymal stem cells from LMNA-mutation mouse. In addition, gangliosides in several tissues including liver, kidney and brain from LMNA-mutation mouse was various expressed compare with normal tissues. So, gangliosides may be related with LMNA in cell differentiation and proliferation. Therefore, this results suggested that gangliosides act important roles in aging and HGPS.

Several novel approaches are underway for control of plant-parasitic nematode Meloidogyne incognita. This study was aimed to compare metabolome profiles of the nematode at all its developmental stages. Metabolome analysis was performed in 5 stages (egg, J2, J3, J4 and female) and peaks detected in CE-TOFMS. Among 209 putative metabolites detected (61 anionic mode and 148 cationic mode), 110 metabolites (56 anioic and 54 cationic modes) with available standards were quantified. Principal component analysis showed that the metabolites from stages female and J2; J3 and J4 stages grouped into clusters and metabolites of egg formed a unique cluster. Quantitative analysis indicated amino acids to be the dominant group of metabolites. Further, highly expressed metabolites included osmolytes proline, betaine, hydroxyproline; antioxidant molecules putrescine and glutathione; sensory regulators gama amino benzoic acid (GABA) and β alanine; organic acids lactic and malic acids, molecules in RNA synthesis GMP, AMP and inosine. In general, concentrations of most of these metabolites were low in J3 and J4 stages compared to egg, J2 and female which may be due to the sedentary nature of the nematode in those two stages. Proline was highly expressed in egg stage, muscle tone regulator GABA abundant in J2, whereas J3 and J4 stages possessed high concentrations of alanine and female was enriched with polyamine putrescine and amino acids.

Senna tora (Cassia tora), a member of Leguminosae, is widely distributed in south-east Asian countries. The dried or roasted seed of Senna tora has been used as one of Oriental medicines for centuries for vision-improving, anti-asthenia and diuretic. Furthermore, many reports related to various biological and pharmacological activities of Senna tora have been published such as anti-inflammatory, anti-hepatotoxic, radical scavenging, anti-allergic, anti-mutagenic, antipyretic, hypoglycemic, anti-fungal and anti-bacterial effects. In this study, we have focused on characterizing and quantifying of anthraquinone compounds from Senna tora seeds during different ripening stages. Ultra-high performance liquid chromatography coupled with diode array detector and electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-DAD-ESI-Q-TOF-MS) was used to investigate the compositional changes of anthraquinone compounds that were involved in ripening of Senna tora seeds. Furthermore, non-targeted UHPLC-ESI-Q-TOF-MS data were subjected to principal component analysis and discriminant analysis to find the potential characteristic components from different ripening stages of Senna tora seeds.

Introduction High-dose intravenous immunoglobulin is a widely used therapeutic preparation. It is administered at high doses (1-2 grams per kilogram) for the suppression of autoantibody-triggered inflammation in a variety of clinical settings1. Immunoglobulin is a glycoprotein, composed of two identical heavy chains and two light chains, which in turn are composed of variable and constant domains. A minor population of IgG is glycosylated at Asn297 in the Fc domain, with glycans terminating in a-2,6 sialic acids (sFc)2-4. Recently, it has been reported that C-type lectins such as SIGN-R1 and human DC-SIGN recognize sFc of immunogloublin and mediate the suppression of autoantibody-mediated inflammation3. But the intracellular signaling mechanism of IVIG and its physiological meaning are not determined until now. Here, we identified that the binding of SIGN-R1/human DC-SIGN to IVIG rapidly generate ROS, leading to sequentially phosphorylate Protein X, P38 and NFkB. In particular, the phosphorylation of P38 and NFkB was dependent on the phosphorylation of Protein X. Also, it was verified that their binding induced anti-inflammatory cytokines such as IL-10 and IL-4 in vitro or in vivo. Currently, it is still in examination for the role of the SIGN-R1/human DC-SIGN-mediated signaling mechanism in the anti-inflammatory effects of IVIG. Although the further works are required, current results is important for unraveling a novel intracellular signaling mechanism of IVIG for its anti-inflammatory effects, which give an insight to develop a new therapy or plant-derived glycomimetic drugs to cure various inflammatory diseases or autoimmune disease.

It is well known that the antigenic vaccines shows various side effects and systhetic peptide humoral and cellular immune responses. To overcome these problems, we developed the effective antigen delivery system like antigen-antibody complex of EpCAM-Fc or EpCAM-Fc KDEL to dendritic cells (DCs) which are a representative antigen-presenting cells to T cells. First, conventional Dcs were generated by using human umbilical cord blood which is a rich source of hematopoitic stem cells, progenitor cells and immune cells including dendritic cells (DCs)at an immature stage of differentiation. When conventional DCs were stimulated with LPS (1 ㎍, 48 hours), the expression level of CD80, CD 86 and MHC Ⅱon the cellular surface was increased, confirming the molecular and cellular properties of the activated conventional DCs. Next, the antigen-antibody complexes of EpCAM-Fc (insect-EpCAM-Fc) or EpCAM-Fc KDEL (insect-EpCAM-Fc KDEL) were expressed in heterologous expression system of insect cells such as Sf9 and Hi5 cel lines in which glycosylation of glycoprotein is humanized and followed by anatlyzing their glycosylation patters and in vitro biological activities. When insect EpCAM-Fc or EpCaM-Fc KDEL (10 ㎍, 48 hours) were treated to conventional DCs, conventional DCs were not activated at all. However, when insect EpCAM-Fc or EpCAM-Fc KDEL (10 ㎍, 48 hours) were treated to conventional DCs with low dose of LPS (0.01 ng), conventional DCs were activated well showing the increased expression of CD80, CD 86 and MHC Ⅱ on their cellular surface. Taken together, the heterologous expression system using insect cells could be used to generate antigen-antibody complexes to activate DCs, providing an alternative application system to develop the ani-cancer vaccines.

SIGN-R1, a C-type lectin, is expressed on a subset of resident macrophages in spleen marginal zone. Recently, it was found that SIGN-R1 mediates complement activation pathway by interacting C1q. It is well known that C1q binds to apoptotic cells and has an important role in there clearance. Therefore, we assessed whether SIGN-R1 could be involved in the apoptotic cells clearance, too. In vitro binding assay reveals that soluble SIGN-R1 specifically bind to apoptotic thymocytes. Consistent with our in vitro results, intravenous injected apoptotic cells were uptaken by SIGN-R1-positive macrophages in the splenic marginal zone. To confirm the role of SIGN-R1, apoptotic cells were intravenously injected into control or SIGN-R1 knock-out mice(SR1KO). We observed that apoptotic cells was trapped in the marginal zone at early time point (10min) in both mice which might be due to the presence of other macrophage receptors such as MARCO or Dectin-2. However, as we expected, it was very clear that SIGN-R1 mediated C3 fixation on apoptotic cells in control mice, but not in SR1 KO. Moreover, at 1 hr time point, SR1 KO mice showed the dramatic delay of apoptotic cell clearance in spleen or liver, compared to the control mice. Considered together, our findings suggest that SIGN-R1 specifically mediates complement activation pathway in the splenic marginal zone and this leads to the efficient clearance of apoptotic cells.

In spite of beneficial roles of many flavonoids against various diseases including diabetes, cardiovascular diseases, autoimmune diseases, ulcer and inflammation, their physiochemical natures such as stability and solubility limit clinical and industrial applications. Glycosylation of natural flavonoids has been reported to influence their adsorption, distribution, metabolism, and excretion properties. The present study aimed to investigate and compare biological activities of various natural flavonoids (NF) and their glycosylated derivatives (GF) in several disease models in vitro. The results showed that anti-cancer activities of several NF in various cancer cell lines were significantly reduced or even completely abolished by glycosylation of NF. In contrast, quercetin 3-O-xyloside greatly increased cytokine (TNF- α) secretion in RAW264.7 cells whereas quercetin aglycon and it's glycosylation with rhamnose and glucose reduced TNF-α secretion. Comparing to quercetin aglycon, quercetin 3-O-glucoside significantly reduced lipid accumulation in adipocytes whereas glycosylated quercetin having xylose or rhamnose stimulated the lipid accumulation. These results showed that glycosylation to the specific position of certain flavonoid with specific sugar residue could enhance immune modulation and anti-obestiy activities.

Recently, researches on plant polysaccharides have been increasingly important topics for the development of therapeutics for various diseases due to their multiple biological properties including anti-inflammation, wound healing, anti-hepatitis, anti-ulcer, and anti-neoplastic effects. The Camellia oleifera has been used extensively as cooking oil and traditionally applied as a medicine for stomachache and burning injury in China. The objectives of this study was to investigate the anti-osteoporotic effect and immunostimulating activity of Camellia oleifera polysaccharides (COP). Polysaccharides were isolated from the defatted C. oleifera seeds mainly by water extraction and 75% ethanol precipitation. Monosaccharide composition analysis by HPEAC-PAD showed that these polysaccharides consist of rhamnose, arabinose, galactose, glucose, and xylose as the major neutral sugars. COP was not toxic at up to 500 μg/ml in RAW 264.7 cells, and increased immunostimulating cytokines (TNF-α and IL-6) secretion in a dose-dependent manner. In addition, COP inhibited the receptor activator of NF-кB ligand (RANKL)-induced tartrate-resistance acid phosphatase (TRAP) activity and the formation of multinucleated osteoclasts. These results suggest that the COP may have potential anti-osteoporotic activity.

Synthesizing the specific glycan is important for analysis and understanding of interaction with glycan-binding proteins (GBP). Especially, on-chip enzymatic synthesis of glycan can be efficient and useful tool since this method can directly provide diverse glycans to use for analyzing carbohydrate-protein interaction on the chip. However, efficient identification of yield of the synthesized glycan on the chip remains a major challenge. In this work, we prepared novel glycan microarray platform in which glycan-conjugated single-stranded DNA was synthesized using DNA synthesizer, and the synthesized carbohydrate-oligonucleotide conjugates (COCs) were immobilized onto the complementary single-stranded DNA-modified glass slide. Due to the property of DNA to be possible for reversible hybridization and denaturation under certain condition, we effectively obtained the synthesized glycan on the chip, and optimized the synthesis condition for target glycans. In our study, we anticipate that this novel glycan microarray can be successfully used in a number of glycan-related studies and applications, including analysis of diverse glycan-protein interactions, characterization of glycan-processing enzymes, and high throughput detection of target pathogens for diagnosis.

Glycosylation of monoclonal antibody (mAb) therapeutics has critical roles such as biological activity, stability, plasma half-life, and immunogenicity. Therefore, detailed glycomic analyses are important to assess quality, safety, and potency of mAbs. Although mass spectrometry (MS) has emerged as a powerful tool for qualitative glycan analysis, MS-based quantitative analysis for glycome is still limited due to the absence of analytical platform and authentic standards. This study aims to develop the method for quantitative analysis of glycan and site-specific glycoforms directly from mAbs for QA/QC assessment using multiple reaction monitoring (MRM). Here, tryptic glycopeptide on trastuzumab, representative mAb, was selected as a standard for optimization of MRM transitions and instrument parameters of C18-UHPLC/triple quadrupole (QqQ). Three major tryptic glycopeptides having the same peptide moiety (EEQYNSTYR) with three different glycans (G0F, G1F, and G2F) on mAbs were targeted and quantified using MRM. In addition, we measured the absolute amounts of major glycans (G0F, G1F, and G2F) on each mAb (adalimumab, bevacizumab, infliximab, rituximab, and trastuzumab) using FLD response of 2-aminobenzamide (2-AB) labeled glycan standards. Consequently, we found that the abundance of glycopeptide calculated by MRM has highly correlated with absolute amounts of 2-AB labeled glycan by LC-FLD. The developed MRM method can be applied to evaluate mAbs variants and/or biosimilars for both developmental and regulatory purposes.

Lytic polysaccharide monooxygenases (LPMOs) are cupper ion containing enzymes to degrade crystalline polysaccharides, such as cellulose or chitin through an oxidative mechanism. To the best of our knowledge, there is no assay method for direct characterization of LPMOs to degrade the substrates without hydrolases used as the coupled enzymes. In this study, a hydrolase-free assay method for LPMOs was developed, which is based on measuring the consumption of ascorbic acid used as an external electron donor for LPMOs. The ascorbate-based assay method yielded a pH profile with pH 8, which was consistent with the analysis of synergestic effect of BatLPMO10 for chitin degradation in the presence of a chitinase. In addition, the assay method let us know that BatLPMO10 was inhibited by sodium ion, and BatLPMO10 and the coupling chitinase cooperatively enhanced their activities for the degradation of chitin. In conclusion, the hydrolase-free assay method for the polysaccharide degradation catalyzed by LPMOs has been developed for the first time, and this assay method would be useful to search LPMOs with the same optimum pH as its coupled hydrolases or vise versa for improvement of the biomass degradation.