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Isolation and Characterization of MMP-1 Inhibitor Peptide from Crataegus pinnatifida Bunge in Fibroblast Cell Line HS68 Cells (아가위(Crataegus pinnatifida Bunge)로 부터 HS 68세포의 MMP-1에 대한 저해활성 물질의 분리)

  • Lee, Se-Young;Chun, Hyug;Cho, Hong-Yun;An, Jeung-Hee
    • Applied Biological Chemistry
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    • v.46 no.1
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    • pp.60-65
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    • 2003
  • MMP-1 inhibitory compounds were isolated from 120 Korean traditional edible plants. UP- 1 activity significantly increased linearly with increasing UVB dose in normal human foreskin fibroblast HS68 cell, showing maximum activity at approximately 35 $mJ/cm^2$, whereas in HaCaT cell, normal human keratinocyte, no increase was observed. Maximum secretion of MMP-1 after UVB treatment occurred around 36-48 k after treatment. MMP-1 inhibitory compound isolated from cold-water fraction of Cataegus pinnatifida Bunge showed the mort potent activity. The MMP-1 inhibitory compound was deduced as a peptide based on the fact that pronase digestion decreased the activity whereas periodate oxidation did not. The most potent UP- 1-inhibitory protein, CP-2Va-2, showing an activity of 88.5% against MMP-1, was isolated through sequential column chromatography on DEAE-Toyopearl 650C, Butyl-Toyopearl 650M, and Bio-Gel P-30. Molecular weight of CP-2Va-2 determined through high performance liquid chromatography and SDS PACE was 19 and 20 kDa. respectively, signifying a monomeric structure.

Detection of Mitochondrial ATP-Sensitive Potassium Channels in Rat Cardiomyocytes

  • Cuong, Dang Van;Kim, Na-Ri;Kim, Eui-Yong;Lee, Young-Suk;Kim, Hyun-Ju;Kang, Sung-Hyun;Hur, Dae-Young;Joo, Hyun;Park, Young-Shik;Hong, Yong-Geun;Lee, Sang-Kyung;Chung, Joon-Yong;Seog, Dae-Hyun;Han, Jin
    • The Korean Journal of Physiology and Pharmacology
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    • v.8 no.4
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    • pp.201-206
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    • 2004
  • Mitochondrial ATP-sensitive potassium $(mitoK_{ATP})$ channels play a role in early and late ischemic preconditioning. Nevertheless, the subunit composition of $mitoK_{ATP}$ channels remains unclear. In this study, we investigated the subunit composition of $mitoK_{ATP}$ channels in mitochondria isolated from rat cardiac myocytes. Mitochondria were visualized using the red fluorescence probe, Mitrotracker Red, while $mitoK_{ATP}$ channels were visualized using the green fluorescence probe, glibenclamide-BODIPY. The immunofluorescence confocal microscopy revealed the presence of Kir6.1, Kir6.2 and SUR2 present in the cardiac mitochondria. Western blot analysis was carried to further investigate the nature of $mitoK_{ATP}$ channels. For SUR proteins, a 140-kDa immunoreactive band that corresponded to SUR2, but no SUR1 was detected. For Kir6.2, three bands $({\sim}44,\;{\sim}46,\;and\;{\sim}30\;kDa)$ were detected, and a specific ${\sim}46-kDa$ immunoreactive band corresponding to Kir6.1 was also observed. These observations suggest that the subunits of $mitoK_{ATP}$ channels in rat myocytes include Kir6.1, Kir6.2, and a SUR2-related sulfonylurea-binding protein.

Studies on the Improvement of Shelf-life in Spicy Beef Meat Using Chitosan (키토산 첨가에 의한 양념 우육의 보존성 개선에 관한 연구)

  • 윤선경;허종현;김연주;최정수;박선미;안동현
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.33 no.1
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    • pp.207-211
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    • 2004
  • This study has been investigated the effect of different concentrations (0%, 0.05%, 0.1%, 0.5% and 1.0%) of M.W. 120 kDa of chitosan on improvement of shelf-life and quality in the spicy beef meat. The spicy beef meat without chitosan has not shown the extended effect of storage. However, concentration of more than 0.1% of chitosan have a very strong effect on shelf-life improvement of spicy beef meat. In antioxidation, spicy beef meat with 1.0% of chitosan has shown remarkable effect. The pH value and water holding capacity of these spicy beef meat revealed no significant differences among various concentration of chitosan treatments during storage periods. The value of red color was stable in samples treated with chitosan during period of storage.

Biobleaching of Softwood Kraft Pulp Using Recombinant Xylanase and Cellulase (재조합 Xylanase와 Cellulase의 연속처리에 의한 침엽수 Kraft 펄프의 Biobleaching)

  • Kim, Hyun Joo;Wi, Seung Gon;Bae, Hyeun-Jong
    • Journal of the Korean Wood Science and Technology
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    • v.35 no.6
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    • pp.166-174
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    • 2007
  • To know the effect of enzymatic pre-treatment on softwood Kraft pulp, two xylanse-encoding genes, named xynl and xynll were isolated from Thrichoderma ressei. Structural genes of xylanase (XYNI, XYNII) and cellulase (EGIV-CBDII) were isolated from T. ressei and Rumicoccus albus respectively, and expressed in E. coli. bacterial culture. The specific activity of purified recombinant XYNI is higher than XYNII. The brightness of XYNI treated softwood Kraft pulp increased to 29.9%. On further sequential treatment with EGIV-CBDII and XYNI the brightness of softwood Kraft pulp were improved to 9.1 and 73% respectively. As expected the Kappa number of softwood Kraft pulp also decreased 8.1, 4.6 and 3.2% respectively. Results further indicate that this sequential combination of enzyme treatment has synergic effect for improving bleaching of softwood Kraft pulp.

Effect of Chitosan-Ascorbate and Calcium Lactate on the Fermentation and Quality Characteristics of Squid Sikhae (Chitosan-Ascorbate 및 Calcium Lactate가 오징어 식해의 숙성과 품질 특성에 미치는 영향)

  • Lee Ye-Kyung;Park Bum-Ho;No Hong-Kyoon;Kim Soon-Dong
    • Journal of the East Asian Society of Dietary Life
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    • v.15 no.5
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    • pp.598-605
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    • 2005
  • Effects of chitosan-ascorbate(CA) and calcium lactate(CL) on fermentation and quality of squid sikhae were investigated CA and LA were added at $0.5\%$ (designated CA1 and CL1) and $1.0\%$(CA2 and CL2) concentrations, respectively and fermented for 12 days at $10^{\circ}C$. pH of CA-added sikhae was higher than that of control, while acidity of the former was lower than that of the latter. During 12 days of fermentation, CA-added sikhae showed higher protease activity than control by $2.3\~2.6$ times and CL-added sikhae by $2.8\~3.6$ times. At 12 days of fermentation, CA-added sikhae revealed higher protease activity than control by $1.2\~1.4$ times and CL-added sikhae by $1.5\~1.9$ times. CA-added sikhae also showed higher amino-nitrogen content than control by $1.4\~1.7$ times and CL-added sikhae by $1.9\~3.5$ times. In comparison of CA1 with CA2, CA2 showed all higher pH, protease and amylase activity, and amino-nitrogen content than CA1. In analysis of electrophoresis, molecular weights of major proteins in ~w squid were $116.9\~119.0$, 96.5 and 59.3kDa. However, after fermentation for 12 days, a protein band of 119.0kDa disappeared but a new protein band with below 14 kDa appeared in sikhae, especially CA-added sikhae. In sensory evaluation, the intensity of sour taste was the highest for control and the lowest for CA2. Softness of squid was the highest for control and the lowest for CA2. Overall acceptability was the best for CA2. In conclusion, these results suggest use of $1\%$ CA in sikhae preparation as addition of CA(CA2) increased the protease and amylase activity, nitrogen content of amino form, sensory acceptability as well as shelf-life of sikhae.

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Identification of Be Levels Correlated with Intrinsic Defect in p-GaSb Grown by Molecular Beam Epitaxy

  • Kim, Jun-O;Lee, Sang-Jun;Kim, Chang-Su;No, Sam-Gyu;Choe, Jeong-U;Park, Dong-U;Kim, Jin-Su;Kim, Jong-Su
    • Proceedings of the Korean Vacuum Society Conference
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    • 2010.02a
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    • pp.167-167
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    • 2010
  • 반도체는 도핑하지 않으면 대부분 n형을 나타내는 것에 반하여 GaSb는 p형을 보이는 반도체로서, 그 근원은 명확하게 규명되어 있지 않은 상태이다. GaSb의 p형 불순물인 Be은 Ga과 치환 ([$Be_{Ga}$])되므로, p형 전도의 근원으로 추정되는 잔존결함인 [$Ga_{Sb}$]와 그 복합체인 [$Ga_{Sb}-Sb_{Ga}$]와 높은 상관관계를 가질 것으로 예측된다. 본 연구에서는 Be을 도핑한 GaSb:Be 에피층을 MBE 방법으로 성장하여, PL 스펙트럼과 Hall 효과 분석을 통하여 p형 전도의 근원을 조사하였다. 도핑하지 않은 u-GaSb는 DA (deep acceptor)와 함께 A 준위를 나타낸 반면, p-GaSb:Be의 PL 스펙트럼은 Be 도핑농도가 증가함에 따라 FWHM가 줄어들면서 점차 높은 에너지 영역으로 변위하지만 농도가 가장 높은 시료에서는 PL의 FWHM가 증가하면서 에너지는 감소함이 관측되었는데, 이것은 A 피크와 Sb 관련 피크가 경쟁적으로 중첩되어 나타난 현상으로 분석된다. Hall 효과 결과는 유효 전하밀도의 증가에 따라 이동도는 감소하는 전형적인 의존성을 나타내었으며, u-GaSb의 Hall 이동도가 p-GaSb:Be의 값보다 작은 것은 u-GaSb에 잔존하는 DA에 의한 산란 때문으로 해석된다. Gaussian 형태로 분해하여 얻은 A ([$Ga_{Sb}$])와 DA ([$Ga_{Sb}-Sb_{Ga}$]) 및 Be 관련 피크로부터 특정 도핑농도 ($1.2{\times}10^{17}cm^{-3}$)의 시료를 제외한 모든 p-GaSb:Be에는 A 피크가 중첩되고 A와 Be 준위 중간에 Be과의 복합체인 중간상태(intermediate state)인 [$Be^*$]가 존재함이 관측되었는데, 특정 도핑농도에서는 [$Be_{Ga}$]이 우세하지만 더 이상 농도가 증가하면 [$Be_{Ga}$] 준위의 강도는 오히려 감소함을 관측할 수 있었다. 이것은 적정 이상의 Be을 도핑할 경우, A ([$Be_{Ga}$])와 $Be^*([Be_{Ga}-Ga_Sb}])$가 형성 ($A[Ga_{Sb}]+Be{\rightarrow}Be^*[Be_{Ga}-Ga_{Sb}]+[Be_{Ga}]$)됨을 보여 주는 중요한 결과인 것으로 분석된다. A, [Be], [$Be^*$] PL 피크 에너지는 각각 779, 787, 794 meV (오차범위 ${\pm}3\;meV$)이고, [$Be_{Ga}$]의 활성화 에너지는 ($23{\pm}3\;meV$) (20 K)임을 밝혔다.

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Enzymatic Characterization of a Thermostable 4-α-Glucanotransferase from Thermotoga neapolitana (Thermotoga neapolitana 유래 내열성 4-알파-글루칸전이효소의 효소적 특성)

  • Choi, Kyoung-Hwa;Seo, Ja-Yeong;Kim, Ji-Eun;Cha, Jae-Ho
    • Journal of Life Science
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    • v.21 no.2
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    • pp.221-226
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    • 2011
  • The gene encoding 4-$\alpha$-glucanotransferase (mgtA) from Thermotoga neapolitana was cloned and expressed in Escherichia coli in order to investigate whether this enzyme was capable of producing cycloamylose for industrial applications. MgtA was purified to homogeneity by HiTrap Q HP and Sephacryl S-200 HR column chromatographies. The size of the enzyme as determined by SDS-PAGE was about 52 kDa, which was in good agreement with its deduced molecular mass of 51.9 kDa. The optimal temperature and pH for the activity of the 4-$\alpha$-glucanotransferase was found to be $85^{\circ}C$ and 6.5, respectively. The enzyme hydrolyzed the 1,4-$\alpha$-glucosidic bonds in oligomeric 1,4-$\alpha$-glucans and transferred oligosaccharides (maltotriose being the shortest one) to acceptor maltodextrins. However, the enzymes had no activity against pullulan, glycogen, and other di- or trioligosaccharides with rare types of $\alpha$-bond. MgtA is distinguished from 4-$\alpha$-glucanotransferase from Thermotoga maritima in that it can convert maltotriose into maltooligosaccharides. The treatment of glucoamylase after the reaction of MgtA with maltotriose, maltotetraose, maltopentaose, or maltohexaose as sole substrate revealed that MgtA yielded linear maltooligosaccharides instead of cycloamylose.

Inhibitory Effect of Extracts of Platycodon grandiflorum (the Ballon Flower) on Oxidation and Nitric Oxide Production (도라지 부탄올 추출물의 항산화 및 nitric oxide 생성 저해 효과)

  • Jang, Joo-Ri;Hwang, Seong-Yeon;Lim, Sun-Young
    • Food Science and Preservation
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    • v.18 no.1
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    • pp.65-71
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    • 2011
  • We explored the effect of extracts of dried Platycodon grandiflorum on production of reactive oxygen species (ROS), glutathione (GSH) and nitric oxide (NO). To determine antioxidant activity in the presence of $H_2O_2$-induced oxidative stress, DCFH-DA (dichlorodihydrofluorescin diacetate) assay was employed. Acetone/methylene chloride (A+M) and methanolic (MeOH) extracts of P. grandiflorum reduced intracellular ROS levels. Of the various tested fractions, n-BuOH fraction showed the highest protective effect in terms of lipid peroxide production. Total GSH levels were measured after treatment of HT1080 cells with the A+M and MeOH extracts, and other solvent fractions, at various concentration. The A+M extacts and 85% (v/v) aqueous MeOH fraction significantly increased GSH levels (p<0.05). When lipopolysaccharide (LPS)-induced NO production was evaluated, all tested crude extracts, and fractions thereof, significantly reduced NO production (p<0.05), and the n-BuOH and 85% (v/v) aqueous MeOH fractions (at 0.05 mg/mL) showed the strongest inhibitory effects. The results showed that the n-BuOH fraction inhibited both cellular oxidation and NO production, and this fraction may thus contain valuable active compounds.

Effects of Electric Stimulation and Activation Conditions on the Fusion and Development of Porcine Somatic Cell Nuclear Transfer Embryos (전기적 융합과 활성화 방법이 돼지 체세포 복제수정란의 체외발달에 미치는 영향)

  • 정기화
    • Journal of Embryo Transfer
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    • v.19 no.1
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    • pp.43-51
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    • 2004
  • The present study was conducted to investigate the effects of fusion and/or activation protocol on in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine fetal fibroblast cells were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion and activation were induced simultaneous fusion/activation (SA) or delayed activation (DA) with or without cytochalasin B (CB) treatment with electic pulses in 0.28 M mannitol-based medium. The SCNT embryos were cultured in vitro for 7 days and stained with Hoechst 33342 to determine the number of nuclei. After 7 days culture, cleavage and blastocyst formation rates were 72.4% and 7.6% in SCNT and 76.3% and 20.4% in parthenotes. To examine the effect of electric field strengths on development of SCNT embryos, oocytes were fused two pulses of 110 V/mm, 130 V/mm or 150 V/mm for 30 sec post-injection. The fusion and cleavage rates in 130 V/mm group (70.2% and 72.6%) and 150 V/mm group (72.6% and 70.5%) were higher (P<0.05) than 110 V/mm group (47.1% and 48.6%), respectively. However, the rate of embryos developing to the blastocyst stage (8.1%, 9.7% and 10.7%) were not different among three groups. The cleavage rates and the blastcyst formation rates were not different among three treatment groups (SA group, 71.4% and 9.7%; SA+CB treatment group, 74.7% and 8.0%; DA+CB treatment group, 70.8% and 11.2%, respectively). And, no different in the number of cells in blastocysts was observed among the three groups (22.5$\pm$12.8, 23.3$\pm$11.2 and 21.6$\pm$10.4, respectively). These result suggest that two pulses of 130 V/mm or 150 V/mm for 30 sec with SA treatment or DA treatment are enough for fusion/activation of porcine somatic cell nuclear transfer (SCNT) embryos to develop to the blastocyst stage.

Purification, Characterization, and Gene Cloning of Chitosanase from Bacillus cereus H-l (Bacillus cereus H-1으로부터 Chitosanas리 분리와 특성연구 및 유전자 클로닝)

  • Jang, Hong-Ki;Yi, Jae-Hyoung;Kim, Jung-Tae;Lee, Keun-Eok;Park, Shin-Geon
    • Microbiology and Biotechnology Letters
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    • v.31 no.3
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    • pp.216-223
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    • 2003
  • A 1.3-kb of chitosanase gene (choA) encoding 45-kDa polypeptide was cloned, expressed, and characterized from a newly isolated Bacillus cereus H-1. The chitosanase protein (ChoA) of B. cereus H-l was purified to homogeneity by ammonium sulfate precipitation and CM-sephadex column chromatography. Optimum pH was around 7, and stable pH range in the incubation at 50 C was 4-11. Optimum temperature was around 50 C, and enzyme activity was relatively stable below 45 C. ChoA showed the activities toward carboxymethyl cellulose (CMC) in addition to soluble or glycol chitosan. Based on MALDI-TOF MS analysis of purified ChoA, the entire amino acid sequence of ChoA was interpreted by database searching of previously known Bacillus chitosanases. A 1.6 kb of PCR product of corresponding chitosanase gene was obtained and its DNA sequence was determined. The deduced amino acid of choA revealed that ChoA have a 98% homology with those of Bacillus sp. No.7-M strain and Bacillus sp. KCTC0377BP. The recombinant ChoA protein was expressed in E. coli DH5$\alpha$. Deduced amino acid comparison of choA with other chitosanases suggested that it belongs to family 8 microbial endo-chitosanase with chitosanase-cellulase activity.