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Hydrogen Bonds in GlcNAc( β1,3)Gal( β)OMe in DMSO Studied by NMR Spectroscopy and Molecular Dynamics Simulations

  • Shim, Gyu-Chang;Shin, Jae-Min;Kim, Yang-Mee
    • Bulletin of the Korean Chemical Society
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    • v.25 no.2
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    • pp.198-202
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    • 2004
  • Hydrogen bond is an important factor in the structures of carbohydrates. Because of great strength, short range, and strong angular dependence, hydrogen bonding is an important factor stabilizing the structure of carbohydrate. In this study, conformational properties and the hydrogen bonds in GlcNAc( ${\beta}$1,3)Gal(${\beta}$)OMe in DMSO are investigated through NMR spectroscopy and molecular dynamics simulation. Lowest energy structure in the adiabatic energy map was utilized as an initial structure for the molecular dynamics simulations in DMSO. NOEs, temperature coefficients, SIMPLE NMR data, and molecular dynamics simulations proved that there is a strong intramolecular hydrogen bond between O7' and HO3' in GlcNAc( ${\beta}$1,3)Gal(${\beta}$)OMe in DMSO. In aqueous solution, water molecule makes intermolecular hydrogen bonds with the disaccharides and there was no intramolecular hydrogen bonds in water. Since DMSO molecule is too big to be inserted deep into GlcNAc(${\beta}$1,3)Gal(${\beta}$)OMe, DMSO can not make strong intermolecular hydrogen bonding with carbohydrate and increases the ability of O7' in GlcNAc(${\beta}$1,3)Gal(${\beta}$)OMe to participate in intramolecular hydrogen bonding. Molecular dynamics simulation in conjunction with NMR experiments proves to be efficient way to investigate the intramolecular hydrogen bonding existed in carbohydrate.

Overexpression of Protein Kinase $C{\beta}_1$ Restores Mitogenic Responses of Enterocytic Differentiated Colon Carcinoma Cells to Diacylglycerol and Basic FGF

  • Lee, Han-Soo
    • BMB Reports
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    • v.30 no.3
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    • pp.194-199
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    • 1997
  • Previous studies have shown that the HD3 human enterocytic differentiated colon carcinoma cell lines having low $PKC{\beta}$ activity did not respond to diacylglycerol and basic FGF by growth and by activation of pp57 MAP kinase, but undifferentiated cell lines exhibiting high $PKC{\beta}$ activity did. To confirm a role of $PKC{\beta}$ in colonocyte mitogenesis, derivatives of HD3 cell line that stably overexpress a full-length of cDNA encoding the ${\beta}_1$ isoform of human PKC were generated. The abundance and activity of $PKC{\beta}$ in two of the these cell lines, PKC3 and PKC8 were much higher than those in the C1 control cell line that carries the vector lacking the $PKC{\beta}_1\;cDNA$ insert. Following exposure to diacylglycerol or basic FGF, proliferation of PKC3 and PKC8 cells increased about 50%; but this effect was not seen with the control C1 cells. Also, in contrast to the control cells, the $PKC{\beta}_1-overproducing$ cells displayed activation of pp57 MAP kinase when treated with diacylglycerol and basic FGF as undifferentiated cell lines did. These results provide direct evidence that $PKC{\beta}_1$ which plays a key role in mitogenic responses of colon carcinoma cells to diacylglycerol and basic FGF is down-regulated in enterocytic differentiation of colon cells.

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Preparation and Evaluation of Inclusion Complex of Muscone with ${\beta}-Cyclodextrin$ (무스콘의 ${\beta}$-시클로덱스트린 포접 복합체의 제조 및 평가)

  • Kwack, Eun-Sun;Cho, In-Sook;Lee, Gye-Won;Jee, Ung-Kil;Park, Dae-Kyu
    • Journal of Pharmaceutical Investigation
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    • v.27 no.4
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    • pp.265-269
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    • 1997
  • An inclusion complex of muscone with ${\beta}-cyclodextrin$ (CD), as a solid form of muscone, was prepared to increase the solubility of muscone. The molar ratio of muscone to ${\beta}-CD$ in complex was in the range of $1:1{\sim}1:5$ when prepared by freeze-drying method. The interaction of muscone with ${\beta}-CD$ in solid state was investigated by Infrared (IR) spectroscopy and differential scanning calorimetry (DSC). IR and DSC studies between $muscone-{\beta}-CD$ inclusion complex and physical mixture showed that $muscone-{\beta}-CD$ inclusion complex was prepared stably. From the amount of muscone incorporated in the inclusion complex, it was found that the molar ratio of muscone : ${\beta}-CD$ was 1:1. Relative spatial position of muscone and ${\beta}-CD$ was observed by Hyperchem molecular modelling program.

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Cloning of \beta-mananase gene from Aeromonas sp. in E. coli (토양에서 분리한 Aeromonas sp 로 부터 \beta-mannanase 유전자의 클로닝)

  • 박봉환;강대경;김하근
    • Microbiology and Biotechnology Letters
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    • v.29 no.4
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    • pp.201-205
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    • 2001
  • A bacteria strain producing extracellular $\beta$-mannanase was isolated from soil and was identified as Aeromonas sp. A genomic DNA library constructed from Aeromonas, sp that secrets a $\beta$-mannanase was screened for mannan hydrolytic acticity. Recombinant $\beta$-mannanase activity was detercted on the basis of the clear zones around Escherichia coli colonies grown on a LB medium supplemented locust bean gum, EcoRI restriction analysis of plasmid prepared from recombinant E. coli which showed a $\beta$-mannanase activity revealed 10 kb DNA insert, The optimum pH and temperature for the activity of reconmbinant $\beta$-mannanase were 6.0 and $50^{\circ}C$ respectively and were identical to those of the native enzyme.

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Thermus caldophilus GK24로부터 내열성 $\beta$-galactosidase의 최적 생산

  • Yoo, Jinsang;Kim, Hyunkyu;In, Man-Jin;Kim, Min-Hong;Kwon, Suk-Tae
    • Microbiology and Biotechnology Letters
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    • v.25 no.3
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    • pp.298-304
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    • 1997
  • Thermus caldophilus GK24 was selected as sources of thermostable $\beta$-galactosidase from a survey of genus Thermus. T. caldophilus GK24 (Tca) $\beta$-galactosidase was found to be inducible. The enzyme was optimally active at 75$\circ$C. Enzyme induction was achieved by addition of lactose, galactose and cellobiose to basal media. The addition of glucose to culture media had a repressive effect on further enzyme synthesis. T caldophilus GK24 was tested for production of $\beta$-galactosidase by addition of various concentration of lactose, galactose and cellobiose to standard media. Cellobiose was found to be effective for the $\beta$-galactosidase induction. The optimal induction medium for production of $\beta$-galactosidase was composed of 0.2% cellobiose, 0.3% bactotryptone, 0.3% yeast extract, basal salts and Tris/HCI(pH 7.8). The activity of the enzyme in the optimal induction medium increased nearly 16.5-fold compared to the standard medium. Tca $\beta$-galactosidase was detected when cell extracts was subjected to electrophoresis in a nondenaturing polyacryamide gel and stained for activity with 6-bromo-2-naphtyl-$\beta$-D-galactopyranoside(BNG).

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Seed-Conjugated Polymer Bead for ${\beta}2$-Microglobulin Removal at Neutral pH

  • Kim, Mi-Ra;Kang, Sung-Soo;Myung, Eun-Kyung;Ahn, Min-Koo;Choi, Jeong-Hyun;Paik, Seung-R.;Lee, Yoon-Sik
    • Journal of Microbiology and Biotechnology
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    • v.19 no.9
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    • pp.960-965
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    • 2009
  • ${\beta}2$-Microglobulin (${\beta}2m$) is known to be a major factor for dialysis-related amyloidosis. We have studied ${\beta}2m$ removal through an aggregation process, which was initiated by a ligand that could catch the ${\beta}2m$ monomer and promote its aggregation into fibril. As a ligand, we have prepared ${\beta}2m$ fibril fragments and used them as a seed. The seed was coupled to PEGylated-PS beads to remove the monomeric ${\beta}2m$ from solution. The ${\beta}2m$ seed-conjugated resin effectively adsorbed the ${\beta}2m$ monomers with a capacity of 3.6 mg/ml via promoting their aggregation into fibrils on the resin at pH 7.4.

Sorting Nexin 17 Interacts Directly with Kinesin Superfamily KIF1B${\beta}$ Protein

  • Seog, Dae-Hyun;Han, Jin
    • The Korean Journal of Physiology and Pharmacology
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    • v.12 no.4
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    • pp.199-204
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    • 2008
  • KIF1B${\beta}$ is a member of the Kinesin superfamily proteins (KIFs), which are microtubule-dependent molecular motors that are involved in various intracellular organellar transport processes. KIF1B${\beta}$ is not restricted to neuronal systems, however, is widely expressed in other tissues, even though the function of KIF1B${\beta}$ is still unclear. To elucidate the KIF1B${\beta}$-binding proteins in non-neuronal cells, we used the yeast two-hybrid system, and found a specific interaction of KIF1B${\beta}$ and the sorting nexin (SNX) 17. The C-terminal region of SNX17 is required for the binding with KIF1B${\beta}$. SNX17 protein bound to the specific region of KIF1Bf3 (813-916. aa), but not to other kinesin family members. In addition, this specific interaction was also observed in the Glutathione S-transferase pull-down assay. An antibody to SNX17 specifically co-immunoprecipitated KIF1B${\beta}$ associated with SNX17 from mouse brain extracts. These results suggest that SNX17 might be involved in the KIF1B${\beta}$-mediated transport as a KIF1B${\beta}$ adaptor protein.

Crosslinking of $\beta$-Cyclodextrin on Cholesterol Removal from Milk

  • Kim, S.H.;Ahn, J.;Kwak, H.S.
    • Archives of Pharmacal Research
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    • v.27 no.11
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    • pp.1183-1187
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    • 2004
  • This study was designed to develop crosslinking of $\beta$-cyclodextrin ($\beta$-CD), and determine the optimum conditions of different factors (mixing time, mixing temperature, and mixing speed) on cholesterol reduction from milk. Crosslinked $\beta$-CD was prepared with epichlorohydrin. When milk was treated with different conditions, the cholesterol removal rate was in the range of 79.4 to 83.3% with 1 % crosslinked $\beta$-CD addition, which were not significantly different among treatments. After cholesterol removal from milk, the used crosslinked $\beta$-CD was washed for cholesterol dissociation and reused. For recycling study, the cholesterol removal rate in first trial was 81.8%, which was mostly same as that using new crosslinked $\beta$-CD. With five trials repeatedly using the same sample, the mean cholesterol removal rate was 81.2%. The present study indicated that the optimum conditions on cholesterol removal using crosslinked $\beta$-CD were 10 min mixing with 400 rpm speed at $5^{\circ}C$ with about 80% cholesterol removal. In addition, crosslinked $\beta$-CD resulted in the effective recycling efficiency almost 100%.

Purification and Characteristics of ${\beta}$-Galactosidase from Strawberry (딸기 ${\beta}$-Galactosidase의 정제 및 생화학적 특성)

  • Lee, Kwang-Hee;Yoon, Kyung-Young;Kim, Kwang-Soo;Kim, Nam-Woo;Shin, Seung-Ryeul
    • Food Science and Preservation
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    • v.7 no.2
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    • pp.201-206
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    • 2000
  • ${\beta}$-Galactosidase was extracted and purified from strawberry. The purified ${\beta}$-Galactosidase from strawberry was investigated their physicochemical characteristics. ${\beta}$-Galactosidase was purified 25.74 fold from strawberry. The purification procedure include ammonium sulfate fraction, acetone powder treatment and gel and ion exchange chromatography. Yield of the enzyme purification was 18.11%. The purified enzyme has native molecular weight of 116,000 dalton. Vmax value and Km value of ${\beta}$-Galactosidase were 0.077 mM ONPG/ml/15mim and 1.75x10-2mM, respectively. The optimum temperature and pH of ${\beta}$-Galactosidase were 43$^{\circ}$C and pH 4.0, respectively. The ${\beta}$-Galactosidase activity was stable below 50$^{\circ}$C and at pH 4.0 to pH 6.0. Among the metal ions Ca and Mg were did not affect, whereas K, Cu and Zn show a little effect on the enzyme activity. The ${\beta}$-Galactosidase activities were inhibited by treatment with EDTA and SDS.

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Immunomodulating Activity of Fungal ${\beta}-Glucan$ through Dectin-1 and Toll-like Receptor on Murine Macrophage

  • Kim, Ha-Won
    • 한국약용작물학회:학술대회논문집
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    • 2006.11a
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    • pp.103-115
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    • 2006
  • [ ${\beta}-Glucan$ ] is a glucose polymer that has linkage of ${\beta}-(1,3)$, -(1,4) and -(1,6). As exclusively found in fungal and bacterial cell wall, not in animal, ${\beta}-glucans$ are recognized by innate immune system. Dendritic cells (DC) or macrophages possesses pattern recognition molecule (PRM) for binding ${\beta}-glucans$ as pathogen-associated molecular pattern (PAMP). Recently ${\beta}-glucans$ receptor was cloned from DC and named as dectin-l which belongs to type II C-type lectin family. Human dectin-l is consisted of 7 exons and 6 introns. The polypeptide of dectin-l has 247 amino acids and has cytoplasmic, transmembrane, stalk and carbohydrate recognition domains. Dectin-l could recognize variety of beta-l,3 and/or beta-l,6 glucan linkages, but not alpha-glucans. In our macrophage cell line culture system, dectin-l mRNA was detected in RA W264.7 cells by reverse transcription-polymerase chain reaction (RT-PCR). Dectin-l was also detected in the murine organs of spleen, thymus, lung and intestines. Treatment of RA W264.7 cells with ${\beta}-glucans$ of Ganoderma lucidum (GLG) resulted in increased expression of IL-6 and $TNF-{\alpha}$ in the presence of LPS. However, GLG alone did not increase IL-6 nor $TNF-{\alpha}$ These results suggest that receptor dectin-l cooperate with CD14 to activate signal transduction that is very critical in immunoresponse.

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