• BMB Reports
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• 제32권5호
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• pp.429-435
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• 1999

Unlike the mammalian glucose transporter GLUT1, little is known about the nature of the endogenous sugar transporter(s) in insect cells. In order to establish the transport characteristics and other properties of the sugar transport proteins of Sf9 cells, a series of kinetic analyses was performed. A saturable transport system for hexose uptake has been revealed in the insect cells. The apparent affinity of this transport system(s) for 2-deoxy-D-glucose was relatively high, the $K_m$ for uptake being <0.5 mM. To further investigate the substrate and inhibitor recognition properties of the insect cell transporter, the ability of other sugars or drugs to inhibit 2-deoxy-D-glucose transport was examined by measuring inhibition constants ($K_j$). Transport was inhibited by D-mannose, D-glucose, and D-fructose. However, the apparent affinity of the C-4 epimer, D-galactose, for the Spodoptera transporter was relatively low, implying that the hydroxyl group at the C-4 position may play a role in the strong binding of glucose and mannose to the transporter. The results also showed that transport was stereoselective, being inhibited by D-glucose but not by L-glucose. It is therefore concluded that insect cells contain an endogenous glucose transport activity that in several aspects resembles the human erythrocyte glucose transporter. However, the mammalian and insect transporters were different in some of their kinetic properties, namely, their affinities for fructose and for cytochalasin B.

• Nutritional Sciences
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• 제8권1호
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• pp.23-27
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• 2005

The major garlic component, Allicin [diallylthiosulfinate, or (R, S)-diallyldissulfid-S-oxide] is known for its medicinal effects, such as antihypertensive activity, microbicidal activity, and antitumor activity. Allicin and diallyldisulfide, which is a converted form of allicin, inhibited the cholesterol level in hepatocytes, in vivo and in vitro. The metabolism of allicin reportedly occurs in the microsomes of hepatocytes, predominantly with the contribution of cytochrome P-450. However, little is known about how allicin affects the genes involved in the activity of hepatocytes in vivo. In the present study, we used the short-term intravenous injection of allicin to examine the in vivo genetic profile of hepatocytes. Allicin up-regulate ten genes in the hepatocytes. For example, the interferon regulator 1 (IRF-I), the wingless-related MMTV (mouse mammary tumor virus) integration site 4 (wnt-4), and the fatty acid binding protein 1. However, allicin down-regulated three genes: namely, glutathione S-transferase mu6, a-2-HS glycoprotein, and the corticosteroid binding globulin of hepatocytes. The up-regulated wnt-4, IRF-1, and mannose binding lectin genes can enhance the growth factors, cytokines, transcription activators and repressors that are involved in the immune defense mechanism. These primary data, which were generated with the aid of the Atlas Plastic Mouse 5 K Microarray, help to explain the mechanism which enables allicin to act as a therapeutic agent, to enhance immunity, and to prevent cancer. The data suggest that these benefits of allicin are partly caused by the up-regulated or down-regulated gene profiles of hepatocytes. To evaluate the genetic profile in more detail, we need to use a more extensive mouse genome array.

• Journal of Applied Biological Chemistry
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• 제50권1호
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• pp.1-5
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• 2007

Chemical properties of spray-dried powders separated based on molecular weight from crude protein binding polysaccharide (CP-SD) of Agraricus blazei were examined. Contents of ${\beta}$-glucan in SD-1, SD-2 and SD-3 were 18.67%, 48.24%, and 37.15% respectively, and SD-2 (10-150 kDa) showed the highest molecular weight. Obtained ${\beta}$-glucans were not pure glucan, but was determined to be an acidic proteo-heteroglycan with a large amount of glucose (74.46-80.05%), galactose (8.91-15.2%), and mannose (4.9-5.46%). Composition of their amino acids was mainly aspartic and glutamic acids. FT-IR spectrum revealed SD-1, SD-2 and SD-3 were structures of ${\beta}$-1,3-glucans and ${\alpha}$-1,6-glucans at 890 and 930 $cm^{-1}$, respectively, signals of ${\alpha}$-1,6-glucans for CP-SD was not found. Useful CP-SD was recovered from A. blazei for preparation of three powder types as food materials.

• 한국어업기술학회:학술대회논문집
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• 한국어업기술학회 2000년도 춘계수산관련학회 공동학술대회발표요지집
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• pp.530-530
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• 2000

The host defense system or immune system of all modern animals has their roots in very ancient organisms. After analyzing literature data concerning properties of invertebrates and vertebrates lectins we suggest that mechanism of mannans recognition may exist in marine invertebrates, as a universal mechanism for homeostasis maintenance and host defense, and mannan-binding lectins family of vertebrates has ancient precursor, as was shown for another S-type lectins family. We carried out the screening of mannan-binding type lectin among different species of echinoderms inhabiting in Piter the Grate Bay, the sea of Japan. As a result, the C-type lectins (SJL-32) specific for high mannose glycans was isolated from the coelomic plasma of the sea cucumbers Stichopus japonicus by ion-exchange chromatography on a DEAE-Toyopearl 650M, affinity chromatography on a mannan-Sepharose 6B and gel filtration on a Sephacryl S-200. SJL-32 is homodimer with molecular mass about 32 kDa on SDS-PAGE under non-reducing conditions. Protein part of the lectin has high conteins Asn, Glu, Ser. Hemagglutination of trypsin-treated O blood group human erythrocytes by SJL-32 was competitively inhibited by high-branched -D-mannan composed of -1,2 and -1,6 linked D-mannopyranose residues. In contrast, a variety of mono-, oligo-, and polysaccharides composed of residues of galactose and fucose showed absence or little inhibitory activities. The lectin activity strong depends on Ca2+ concentration, temperature and pH. Monospecific polyclonal antibodies were obtained to the lectin. As was shown by ELISA assay, antibodies to SJL-32 cross-reacted with human serum mannan-binding lectin. This data allows making conclusion about common antigenic determinants and structural homology of both lectins. In our opinion, SJL-32 belongs to evolutionary high conservative mannan-binding lectins (MBLs) family and takes part in the host defense against pathogenic microorganisms.

• Clinical and Experimental Reproductive Medicine
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• 제29권3호
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• pp.179-186
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• 2002

Objective: Lectins are cell-agglutinating and sugar specific proteins or glycoproteins of non-immune origin that precipitate glycoconjugates having saccharides of appropriate complementarity. Because of these properties, plant lectins have been used to help characterize the carbohydrate moieties of glycoproteins in the zona pellucida (ZP) of several mammalian species including pigs. Treatment of oocytes with various lectins blocks sperm binding to the ZP in various mammalian species. This study was undertaken to examine the distribution of sugar residues in the ZP of pig oocytes matured in vitro and the ability of spermatozoa to bind to ZP and in vitro penetration in oocytes treated with fluorescein isothiocyanate (FITC)-labelled lectins. Materials and Methods: The lectins of Banderiaea simplicifolia (BS-II, bind to $\beta$-D-N-acetylglucosamine), Canavalin ensiformis (Con A, bind to $\alpha$-D-Mannose), Lens culinaris (LCA, bind to a-D-Mannose), Ricinus communis (RCA-I, bind to $\beta$-D-Galactose) and Ulex europaeus (UEA-I, bind to $\alpha$-L-Fucose) were examined for spermatozoa penetration, binding capacity to ZP and distribution of lectins. Results: The penetration rates were significantry (p<0.05) higher in control oocytes (63%) than those treated with all lectins, but penetration rates ($40{\sim}49%$) were simililar in group treated with lectins. The incidence of monospermy was similar in oocytes untreated and UEA-I, but it was higher in oocytes treated with BS-II, Con A, RCA-I and LCA. The porcine oocytes cultured for 48 h in TC-199 medium were freed from cumulus cells and treated for 30 min with fluorescein isothiocyanate-labelled lectins. When examined under fluorescein illumination, higher (p<0.001) proportions of oocytes showed fluorescein of zona pellucida after treatment with Con A (93%), LCA (93%) and RCA-I (100%) than BS-II (37%) and UEA-I (50%). All of the oocytes treated with RCA-I exhibited strong fluorescein in the outer region of the zona pellucida while those treated with LCA exhibited strong fluorescein throughout the zona pellucida. BS-II bounded mainly to the outer region and UEA-I bounded mainly to the inner region of the zona pellucida, with either strong or weak fluorescein. At 120 min after insemination in vitro, fewer spermatozoa were bound to the zona pellucida of the oocytes treated with BS-II, Con-A and RCA-I. Of the lectins, Con A most inhibited sperm binding. Conclusions: These results suggest that $\beta$-D-Galactose residues in the porcine zona pellucida may act as primary sperm receptors and inducers of the sperm acrosome reaction and these sugar residues may be involved in the block to polyspermy.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2008년도 International Meeting of the Microbiological Society of Korea
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• pp.23-25
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• 2008

Serum complement proteins comprise an important system that is responsible for several innate and adaptive immune defence mechanisms. There were three well described pathways known to lead to the generation of a C3 convertase, which catalyses the proteolysis of complement component C3, and leads to the formation of C3 opsonins (C3b, iC3b and C3d) that fix to bacteria. A pivotal step in the complement pathway is the assembly of a C3 convertase, which digests the C3 complement component to form microbial-binding C3 fragments recognized by leukocytes. The spleen clears microorganisms from the blood. Individuals lacking this organ are more susceptible to Streptococcus pneumoniae. Innate resistance to S. pneumoniae has previously been shown to involve complement components C3 and C4, however this resistance has only a partial requirement for mediators of these three pathways, such as immunoglobulin, factor B and mannose-binding lectin. Therefore it was likely that spleen and complement system provide resistance against blood-borne S. pneumoniae infection through unknown mechanism. To better understand the mechanisms involved, we studied Specific intracellular adhesion molecule-grabbing nonintegrin (SIGN)-R1. SIGN-R1, is a C-type lectin that is expressed at high levels by spleen marginal-zone macrophages and lymph-node macrophages. SIGN-R1 has previously been shown to be the main receptor for bacterial dextrans, as well as for the capsular pneumococcal polysaccharide (CPS) of S. pneumoniae. We examined the specific role of this receptor in the activation of complement. Using a monoclonal antibody that selectively downregulates SIGN-R1 expression in vivo, we show that in response to S. pneumoniae or CPS, SIGN-R1 mediates the immediate proteolysis of C3 and fixation of C3 opsonins to S. pneumoniae or to marginal-zone macrophages that had taken up CPS. These data indicate that SIGN-R1 is largely responsible for the rapid C3 convertase formation induced by S. pneumoniae in the spleen of mice. Also, we found that SIGN-R1 directly binds C1q and that C3 fixation by SIGN-R1 requires C1q and C4 but not factor B or immunoglobulin. Traditionally C3 convertase can be formed by the classical C1q- and immunoglobulin-dependent pathway, the alternative factor-B-dependent pathway and the soluble mannose-binding lectin pathway. Furthermore Conditional SIGN-R1 knockout mice developed deficits in C3 catabolism when given S. pneumoniae or its capsular polysaccharide intravenously. There were marked reductions in proteolysis of serum C3, deposition of C3 on organisms within SIGN-$R1^+$ spleen macrophages, and formation of C3 ligands. The transmembrane lectin SIGN-R1 therefore contributes to innate resistance by an unusual C3 activation pathway. We propose that in the SIGN-R1 mediated complement activation pathway, after binding to polysaccharide, SIGN-R1 captures C1q. SIGN-R1 can then, in association with several other complement proteins including C4, lead to the formation of a C3 convertase and fixation of C3. Therefore, this new pathway for C3 fixation by SIGN-R1, which is unusual as it is a classical C1q-dependent pathway that does not require immuno globulin, contributes to innate immune resistance to certain encapsulated microorganisms.

• 한국고분자학회:학술대회논문집
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• 한국고분자학회 2006년도 IUPAC International Symposium on Advanced Polymers for Emerging Technologies
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• pp.265-265
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• 2006

Multivalent interactions, which are characterized by the simultaneous binding of multiple ligands on multiple receptors, are prevalent in biological system. We have shown that it is able to make a supramolecular aggregate coated with multiple functional molecules fairly easily by simply mixing one building block. In this particular example, a mannose-coated object was able to agglutinate bacterial cells with cognate binding partners through multivalent interactions. This kind of strategy can be applied in developing materials that can selectively remove pathogens. Supramolecular assembly of this type should be very useful in exploring multivalent biological interactions.

• 약학회지
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• 제53권3호
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• pp.145-150
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• 2009

We evaluated cytotoxic effect based on the MTT assay and identified altered proteins in 5-FU(fluorouracil) treated HT29 cells using two-dimensional gel electrophoresis and MALDI-TOF/TOF-MS. As proteins inducing apoptosis, siah binding protein 1 and p47 protein isoform a were up-regulated and tumor protein translationally-controlled 1 was down-regulated by 5-FU treatment. And mannose 6 phosphate receptor binding protein 1 controls DNA mismatch repair system was increased. We suggest 5-FU promotes a cytotoxicity under the action of these proteins in colon cancer cells.

• 대한의생명과학회지
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• 제11권4호
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• pp.487-492
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• 2005

The baculovirus/insect cell expression system is of great value in the study of structure-function relationships in mammalian glucose-transport proteins by site-directed mutagenesis and for the large-scale production of these proteins for mechanistic and biochemical studies. Spodoptera frugiperda Clone 21 (Sf2l) cells grow well on TC-100 medium that contains $0.1\%$ D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, very little is known about the properties of the endogenous sugar transporter(s) in Sf2l cells, although a saturable transport system for hexose uptake has been previously revealed in the Sf cells. In order to further examine the substrate and inhibitor recognition properties of the Sf2l cell transporter, the ability of hexoses to inhibit 2-deoxy-D-glucose (2dGlc) transport was investigated by measuring inhibition constants $(K_i)$. The $K_i's$ for reversible inhibitors were determined from plots of uptake versus inhibitor concentration. Transport was effectively inhibited by D-mannose and D-glucose. Of the hexoses tested, L-glucose had the least effect on 2dGlc transport in the Sf2l cells, indicating that the transport is stereoselective. Unlike the human HepG2 type glucose transport system, D-mannose had a somewhat greater affinity for the Sf2l cell transporter than D-glucose, implying that the hydroxyl group at the C-2 position is not necessary for strong binding. However, epimerization at the C-4 position of D-glucose (D-galactose) resulted in a dramatic decrease in affinity of the hexose for the Sf2l cell transporter. Such a lowering of affinity might be the result of the involvement of the C-4 hydroxyl in hydrogen bonding. It is therefore suggested that Sf2l cells were found to contain an endogenous sugar transport activity that in several aspects resembles the human HepG2 type glucose transporter, although the insect and human transporters do differ in their affinity for cytochalasin B.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.372-379
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• 2017

The transport of lysosomal enzymes into the lysosomes depends on the phosphorylation of their chains and the binding of the phosphorylated residues to mannose-6-phosphate receptors. The efficiency of separation depends more on the phosphodiesterases (PDEs) than on the activity of the phosphorylation of mannose residues and can be determined in vitro. PDEs play important roles in regulation of the activation of lysosomes. The expression of proteins was confirmed by western blotting. All PDE4 series protein expression was reduced in high concentrations of rolipram. As a result of observing the fluorescence intensity after rolipram treatment, the lysosomal enzyme was activated at low concentrations and suppressed at high concentrations. High concentrations of rolipram recovered the original function. Antimicrobial activity was not shown in either 10 or $100{\mu}M$ concentrations of rolipram in treated HeLa cells in vitro. However, the higher anticancer activity at lower rolipram concentration was shown in lysosomal enzyme treated with $10{\mu}M$ of rolipram. The anticancer activity was confirmed through cathepsin B and D assay. Tranfection allowed examination of the relationship between PDE4 and lysosomal activity in more detail. Protein expression was confirmed to be reduced. Fluorescence intensity showed decreased activity of lysosomes and ROS in cells transfected with the antisense sequences of PDE4 A, B, C, and D. PDE4A showed anticancer activity, whereas lysosome from cells transfected with the antisense sequences of PDE4 B, C, and D had decreased anticancer activity. These results showed the PDE4 A, B, C, and D are conjunctly related with lysosomal activity.