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Purification of TGF-$\beta$ 1 from Human Platelets by an Improved Method (개량된 방법에 의한 사람혈소판으로부터 TGF-$\beta$ 1의 분리)

  • 신충건;김상국;문병조;김평현;전계택;남상욱;김장환;이종원
    • KSBB Journal
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    • v.14 no.1
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    • pp.9-16
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    • 1999
  • Transforming growth factor $\beta$1(TGF-$\beta$1) has potentials to be used as a new therapeutic agent. However, studies with TGF-$\beta$ were hindered by its high cost. In this study, we developed an improved method to purify TGF-$\beta$1 from human platelets, for which four purification steps were used: platelet extraction, gel filtration, cation exchange chromatography, and reverse phase high performance liquid chromatography. After a final step of purification, a pure protein with a molecular weight of 25,000 corresponding to the commercially available TGF-$\beta$1 was obtained, which were confirmed by silver staining and Western blotting after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It was confirmed by the inhibitory effects of TGF-$\beta$1 on a mink lung epithelial cell line that the purified TGF-$\beta$1 had its biological activity, whose activity is slightly higher than that of the commercially available TGF-$\beta$1. About 3.7$\mug of the purified TGF-$\beta$1 was obtained from 10 units of concentrated human platelets, the final yield of which is about 21%.

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New Drug Delivery System Based on a Laser-Induced Shockwave (레이저 유도 충격파를 이용한 첨단 약물전달시스템 개발)

  • Han, Tae-Hee;Lee, Hyun-Hee;Gojani, Ardian B.;Yoh, Jai-Ick
    • Transactions of the Korean Society of Mechanical Engineers B
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    • v.34 no.1
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    • pp.67-71
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    • 2010
  • Impingement of a high power laser pulse (above 1 GW/$cm^2$) on a metal foil causes its ablation, which is characterized by a rapid expulsion of matter and the initiation of a strong shock wave inside the solid metal. The shock propagates through the foil and reverberates on the rear side, causing its deformation and microparticle ejection, which were deposited on the foil prior to ablation. Based on this principle, we are developing a new drug delivery system - Biolistic gun. Current study is focused on the controllability, stability, efficiency of the system, and characterization of the penetration shapes in various conditions. We have tested the system by applying direct and confined ablation. Several different media combinations were used for confinement-BK7 glass, water, BK7 glass with water, and succulent jelly(ultrasono jelly, RHAPAPHRM). Biological tissue was replicated by a 3% gelatin solution. Present data shows that the confinement results in enhancement of penetration shape reached by 5 um cobalt microparticles. Based on the analysis of the experimental results we observe that the penetration shape of microparticles can be controlled by adjusting the thickness of confinement media.

Effects of High Pressure and Binding Agents on the Quality Characteristics of Restructured Pork (초고압처리 및 결착제 첨가가 재구성 돈육의 품질특성에 미치는 효과)

  • Choi, Ye-Chul;Jung, Kyung-Hun;Chun, Ji-Yeon;Choi, Mi-Jung;Hong, Geun-Pyo
    • Food Science of Animal Resources
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    • v.33 no.5
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    • pp.664-671
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    • 2013
  • The objective of this study was to investigate the effect of high pressure treatment and type of binding agents on the quality characteristics of restructured pork. For binding agents, 2% (w/w) isolated soy protein (SP), 0.5% (w/w) wheat flour (WF) and 0.5% (w/w) ${\kappa}$-carrageenan (KC) were incorporated into meat batter with or without 0.5% (w/w) glucono-${\delta}$-lactone (GdL). The restructured pork was pressurized at varying pressure levels (0.1-450 MPa) for 3 min under ambient temperature and thermal treated at $75^{\circ}C$ for 30 min. As quality parameters of restructured pork, pH, water binding properties, instrumental color and texture profile analysis were determined and compared with control (C, no binder). For type of binders, SP exhibited the best water binding properties, however, the impact on textural properties were lesser than KC and WF. The addition of GdL decreased the pH of restructured pork down to 0.4 unit, while high pressure processing prevented the moisture loss caused from pH decrease by GdL. In particular, meat restructuring efficiency of SP as a binder improved under the presence of GdL. Therefore, the present study demonstrated the potential advantages of low amount of GdL (0.5%, w/w) combined with protein based binder (SP) and high pressure processing in restructuring meat particles.

Purification and Characterization of $\beta$-Xylosidase B of Bacillus stearothemophilus No.236 Produced by Recombinant Escherichia coli. (재조합 균주 Escherichia coli가 생산하는 Bacillus stearothermophilus No.236 $\beta$-Xylosidase B의 정제 및 특성)

  • 장욱진;조쌍구;최용진
    • Microbiology and Biotechnology Letters
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    • v.26 no.4
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    • pp.297-302
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    • 1998
  • $\beta$-Xylosidase B was produced by Escherichia coli HB101/pKMG12 carrying the xylB gene of Bacillus stearothermophilus No.236 on its recombinant plasmid. The $\beta$-xylosidase B produced was purified by ammonium sulfate fractionation, DEAE-Sepharose CL-6B, Sephacryl S-200 and Superdex 200 HR gel filtration. The purified enzyme showed the highest activity at pH 6.5 and 5$0^{\circ}C$. But, the enzyme was observed to be very sensitive to the pH and temperature of the reaction mixture. The enzyme was activated about 35% of its original activity in the presence of 1 mM of $Mn^{2+}$ but it was completely inhibited by $Ag^{+}$, $Cu^{2+}$and $Hg^{2+}$ions. In contrast with the $\beta$-xylosidase A, the B enzyme was found to have $\alpha$-arabinofuranosidase activity though the activity was fairly low compared with the $\alpha$-arabinofuranosidase produced from the arfI gene of the same Bacillus stearothermophilus. Therefore, $\beta$-xylosidase B is considered to be more suitable than $\beta$-xylosidase A at least for the biodegradation of arabinoxylan. The $K_{m}$ and V$_{max}$ values of the $\beta$-xylosidase B for o-nitrophenyl-$\alpha$-D-xylopyranoside were 6.43 mM and 1.45 $\mu$mole/min, respectively. Molecular mass of the enzyme was determind to be about 54 kDa by SDS-PAGE and 160 kDa by Superdex 200HR gel filtration, indicating that the functional $\beta$-xylosidase B was composed of three identical subunits.s.

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Biochemical properties of a purified protein in cystic quid of Taenia solium metacestodes (유조낭고충 낭액에서 친화성 크로마토그래피로 분리한 항원 단백질의 생화학적 성상)

  • Cho, Seung-Yull;Kim, Suk-Il;Kang, Shin-Yong;Kong, Yoon
    • Parasites, Hosts and Diseases
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    • v.26 no.2
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    • pp.87-94
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    • 1988
  • By affinity chromatography using a monoclonal antibody as ligand, Kim et at. (1986) purified a protein fraction in cystic fluid of Taenia solium metacestodes (CF) In this study, the biochemical properties of the purified protein were characterized. Discontinuous-polyacrylamide gel electrophoresis (disc-PAGE) of the protein at 4.5∼10% separating gel concentration showed its molecular weight (MW) to be 150 kilodalton (kDa) in non·denatured state, while denaturing sodium dodecyl suifate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that it was composed of 3 different subunits with respective fnw of 15, 10 and 7 kDa. Subunit of 7 kDa was shown to be linked to other subunits by disulade bonds. Isoelectric point of the protein was pH 6.8. The protein was relatively heat-stable for immunologic analysis. These properties indicated that the protein, comprising about 70% of total content in CF, had similar biochemical characters with antigen B of Oriol et at.(1971) in hydatid cyst quid (HF).

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Studies on Constituents of Higher Fungi of Korea (LXVII) -Antitumor Components of the Basidiocarp of Ganoderma lucidum- (한국산 고등 균류의 성분 연구(제67보) -영지버섯 자실체의 항암성분-)

  • Hyun, Jin-Won;Choi, Eung-Chil;Kim, Byong-Kak
    • The Korean Journal of Mycology
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    • v.18 no.2
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    • pp.58-69
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    • 1990
  • ABSTRACT: In order to find physiologically active components from higher fungi, hot-water soluble components were extracted from the basidiocarps of Ganoderma lucidum. The extract was purified and separated by DEAE cellulose ion exchange chromatography and Sepharose CL-4B gel filtration method. The separated fractions were designated CR, IN, IA, GL and GH. Fraction GL showed the highest antitumor activity among the fractions and its molecular weight was found to be 47 KD. The tumor inhibition ratio of Fr. GL was 81 % at the dose of peritoneal administration of 20 mg/kg/day for 10 days in mice. Chemical analysis of this fraction showed 82% polysaccharide, 8% protein and 0.9% hexosamine. The polysaccharide moiety consisted of 63% glucose, 27% galactose, 7% mannose and 3% fucose. Fraction IN was found to increase the amount of superoxide anion in activated macrophages to 1.6-fold and the number of plaques in hemolytic plaque assay to 6-fold, respectively. These results indicate that the antitumor activity was exerted through immunopotentiation, but not through direct cytotoxicity against the tumor.

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Characterization of Laccase Purified from Korean Trametes hirsuta S1 (한국산 흰구름버섯(Trametes hirsuta S1)으로부터 정제된 Laccase의 특성)

  • Lim, Hyung-Seon;Yoon, Kyung-Ha
    • The Korean Journal of Mycology
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    • v.32 no.2
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    • pp.112-118
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    • 2004
  • Laccase produced by Trametes hirsuta S1 isolated from Korea was partially purified and characterized using ultrafiltration, anion exchange chromatography and affinity chromatography. The laccase was produced as the predominant extracellular enzyme during primary metabolism. Neither lignin peroxidase nor veratryl alcohol oxidase (VAO) were detected in the culture fluid. Addition of 2,5-xylidine enhanced 4-fold laccase production. Purified laccase was a single polypeptide having a molecular mass of approximately 66 kDa, as determined by SDS-polyacrylamide gel electrophoresis, and carbohydrate content of 12%. $K_{m}\;and\;V_{max}$ values for laccase with ABTS [2,2-azinobis (3-ethylbenzthiazoline 6-sulfonic acid)] as a substrate (Lineweaver-Burk plot) was determined to $51.2\;{\mu}M\;and\;56.8\;{\mu}mole$, respectively. The optimal pH for laccase activity was found to be 3.0. The enzyme was very stable for 1 hour at $50^{\circ}C$. Half-life ($t_{1/2}$) of the enzyme was about 20 min at $70^{\circ}C$. Spectroscopic analysis of purified enzyme indicated that the enzyme was typical of copper-containing protein. Substrate specificity and inhibitor studies for laccase also indicated to be a typical fungal laccase. The N-terminal amino acid sequence of the T. hirsuta S1 laccase showed 100% of homology to those of laccase from C. hirsutus.

Protein Patterns of Blood Plasma in Pregnant Women by SDS/polyacrylamide Gel Electrophoresis (SDS/polyacrylamide Gel 電氣泳動에 依한 妊娠한 女子 血漿蛋白質의 패턴)

  • Park, Won-Chul;Ha, Man-Joon
    • The Korean Journal of Zoology
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    • v.28 no.4
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    • pp.237-244
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    • 1985
  • The plasma protein patterns of non-pregnant women, pregnant women, and normal male individuals were analyzed by SDS/polyacrylamide gel electrophresis. When the protein patterns of plasma of normal male individuals ranging from 10, 000 to 110, 000 daltons in molecular weights are compared to non-pregnant women, their protein patterns were the same. In this study, when the plasma of non-pregnant women are compared to pregnant women, no bands were occurred newly, but the quantity of some protein bands were increased or decreased during the pregnant periods. According to the results of measuring the molecular weights of the characteristic protein patterns, which are increasing or decreasing during the pregnancy as compared to the non-pregnant women, it was observed that the proteins over 76, 000 daltons in molecular weights were concerned in the facts mentioned above. That is, the protein of 86, 000 dalton in molecular weight was not increased in quantity until the second trimester of pregnancy, but was increased in the third trimester of pregnancy. The proteins of 91, 000-105, 000 daltons in molecular weights were gradually increased in accordance with the periods of pregnancy. On the contrary, the protein of 94, 000 dalton was rather decreased by the second trimester of pregnancy, but increased in the third trimester of pregancy. And the band of 99, 000 dalton was not changed in quantity significantly until the first trimester of pregnancy, but increased continuously from the second trimester of pregnancy to the third trimester of pregnancy. We tentatively suggest that the stages (the first, the second, and the third trimester) of pregnancy can be identified by the study on the protein patterns of the specific bands in the blood plasma of pregnant women.

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Measurements of $T_1$-and $T_2$-relaxation Time Changes According to the Morphological Characteristics of Gold Nanoparticles (GNPs) (금 나노 입자의 형태적 특성에 따른 $T_1$, $T_2$ 이완 시간의 변화 측정)

  • Jang, M.Y.;Han, Y.H.;Mun, C.W.
    • Investigative Magnetic Resonance Imaging
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    • v.15 no.1
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    • pp.48-56
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    • 2011
  • Purpose : The aim of this study is to measure the typical MR variables such as $T_1$- and $T_2$-relaxation times according to morphological characteristics of gold nanopartides as a preliminary study to perform theragnosis using local heating by gold nanopartides. Materials and Methods : Two types of gold nanoparticles were used. Spheres were synthesized by various methods and stirring speed. Rods were synthesized by adding various concentrations of sphere nanopartides. Gold nanopartides were mixed with 2% agarose gel at 1:1 ratio and then signals were acquired using a 1.5T MRI. For the measurements of $T_1$-and $T_2$-relaxation times, TR and TE were varied, respectively. The results were acquired through $T_1$ and $T_2$ curves based on the intensities of MR image using self-developed software. And Statistical analysis was performed. Results : $T_1$ times were measured 1.86 sec and 2.08 sec for sphere and rod, respectively. On the other hands, $T_2$ times were measured 57 ms and 35.45 ms for sphere and rod. Conclusion : The changes of the MR variables according to the morphological characteristics of the gold nanopartides were confirmed. Optimal MR imaging conditions can be obtained by choosing proper TR and TE according to the type of nanoparticles.

Gelation Properties and Industrial Application of Functional Protein from Fish Muscle-1. Effect of pH on Chemical Bonds during Thermal Denaturation (기능성 어육단백질의 젤화 특성과 산업적 응용-1. 가열변성 중 화학결합에 미치는 pH의 영향)

  • Jung, Chun-Hee;Kim, Jin-Soo;Jin, Sang-Keun;Kim, Il-Suk;Jung, Kyoo-Jin;Choi, Yeung-Joon
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.33 no.10
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    • pp.1668-1675
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    • 2004
  • The effect of pH on surface hydrophobicity, sulfhydryl group, infrared spectrum, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) pattern and enthalpy was investigated in recovered protein from mackerel and frozen blackspotted croaker by alkaline processing. Hydrophobic residue in myofibrillar protein exposed to the surface of protein, and hydrophobic interaction were the highest around 6$0^{\circ}C$. The surface hydrophobicity was different between myofibrillar protein and myofibrillar protein including sarcoplasmic protein (recovered protein). The peak at 1636 c $m^{-l}$ was increased with pH, and the recovered protein was unfolded in alkali pH. Difference of surface and total sulfhydryl group at pH 7.0 and 10 was comparative high, and decrease of surface sulfhydryl group indicated formation of S-S bonds. Mackerel and frozen blackspotted croaker in alkaline pH showed bands of polymerized myosin heavy chain on SDS-PAGE pattern. The transition temperatures of recovered protein were 33.1, 44.3 and 65.5$^{\circ}C$. Gelation of recovered protein from alkali processing was estimated by increase of $\beta$-sheet structure by pH treatment, S-S bonds by oxidation of surface sulfhydryl group in heating, polymerization of myosin heavy chain in order.r.