• Title/Summary/Keyword: isolation techniques

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Expression of Polyhistidine-Containing Fusion Human HepG2 Type Glucose Transport Protein in Spodoptera Cells and Its Purification Using a Metal Affinity Chromatography

  • Lee, Chong-Kee
    • Biomedical Science Letters
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    • v.16 no.3
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    • pp.201-206
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    • 2010
  • In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

Chemical Constituents from the Stony Coral Alveopora japonica

  • Youn, Ui-Joung;Lee, Yoo-Jin;Jeon, Ha-Rim;Shin, Hyun-Ji;Son, Young-Min;Nam, Joo-Won;Han, Ah-Reum;Song, Jun-Im;Won, Yong-Jin;Seo, Eun-Kyoung
    • Natural Product Sciences
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    • v.17 no.1
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    • pp.1-4
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    • 2011
  • Chemical investigations of the stony coral Alveopora japonica Eguchi (Poritidae) resulted in the isolation of four known compounds (1 - 4). The structures of 1 - 4 were identified as a sterol, ergosta-5,24(28)-dien-$3{\beta}$-ol (1), a mixture of monoacyl glycrols (2), eicosanoic acid and tetracosanoic acid, and two nucleosides, thymine (3) and 2'-deoxythymidine (4), respectively, on the basis of spectroscopic and physicochemical analyses including 1D- and 2D- NMR techniques as well as by comparison of their data with the published values. Compounds 1 - 4 were isolated from this species for the first time. Moreover, these compounds (1 - 4) were found in the genus Alveopora and the family Poritidae for the first time.

Study on germline transmission by transplantation of spermatogonial stem cells in chicken

  • Lee, Young-Mok;Han, Jae-Yong
    • Proceedings of the Korea Society of Poultry Science Conference
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    • 2006.11a
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    • pp.43-58
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    • 2006
  • As a bioreactor, bird has proved to be most efficient system for producing useful therapeutic proteins. More than half of the egg white protein content derives from the ovalbumin gene with four other proteins(lysozyme, ovomucoid, ovomucin and conalbumin) present at levels of 50 milligrams or greater. And the naturally sterile egg also contains egg white protein at high concentration allowing for a long shelf life of recombinant protein without loss in activity. In spite of these advantages, transgenic procedures for the bird have lagged far behind because of its complex process of fertilized egg and developmental differences. Recently, a system to transplant mouse testis cells from a fertile donor male to the seminiferous tubules of an infertile recipient male has been developed. Spermatogenesis is generated from transplanted cells, and recipients are capable of transmitting the donor haplotype to progeny. After transplantation, primitive donor spermatogonia migrate to the basement membrane of recipient seminiferous tubules and begin proliferating. Eventually, these cells establish stable colonies with a characteristic appearance, which expands and produces differentiating germ cells, including mature spermatozoa. Thus, the transplanted cells self-renew and produce progeny that differentiate into fully functional spermatozoa. In this study, to develop an alternative system of germline chimera production that operates via the testes rather than through developing embryos, the spermatogonial stem cell techniques were applied. This system consisted of isolation and in vitro-culture of chicken testicular cells, transfer of in vitro-maintained cells into heterologous testes, production of germline chimeras and confirmation of germline transmission for evaluating production of heterologous, functional spermatozoa.

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Module-Type Switching Rectifier for Cathodic Protection of Underground and Maritime Metallic Constructions (지하매설 및 해양 금속구조물 음극방식용 모듈 타입 스위칭 정류기)

  • 문상호;김보경;김인동;노의철;권영원;정성우;임헌호
    • The Transactions of the Korean Institute of Power Electronics
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    • v.7 no.6
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    • pp.570-578
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    • 2002
  • Cathodic protection is widely used to prevent corrosion of steel materials buried in the underground and sea. As a rectifier for cathodic protection, the conventional phase-controlled rectifiers have been used so far in spite of such shortcomings as large volume, heavy weight and floor power factor. In order to overcome such disadvantages, this paper proposes a new module-type switching rectifier for cathodic protection, which is composed of two parts, namely, AC/DC converter and module- type DC/DC converter. The AC/DC converter is a single-phase IGBT PWM rectifier, thus resulting in almost unity power factor and controlled DC output voltage. The module-type DC/DC converter operates under ZVS/ZCS switching condition to permit high frequency switching operation. It enables to use high-frequency transformer for electrical isolation, thus reducing volume and weight of overall system and improving system efficiency. It should be anticipated that the proposed rectifier techniques apply to the similar technical areas.

A Study on the Process for Web Domain Applications Development Based on the UML+Navigation Diagrma (UML+Navigation Diagram 기반 웹 도메인 응용 개발 프로세스에 관한 연구)

  • Kim, Haeng-Kon;Shin, Ho-Jun
    • The Transactions of the Korea Information Processing Society
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    • v.7 no.9
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    • pp.2846-2856
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    • 2000
  • Recently, according to the rapid development of web technology, a lot of applications based on web techniques have been developed. However, most of web applications have been developed relying on knowledge and experiences of the developer without systematic process. Web Applications are seldom developed in isolation. For web application designers, the simple and semantically rich methodology is needed to improve design and development process. In this paper, we propose a new development process methodology to improve low level technology based application development process which do not provide high level abstraction. We also suggest a new methodology to construct applications based on web. We describe a systematic web application development process by using Navigation Diagram as a analysis, design modeling method to develop web application with productivity and Quality. We apply the new development process to the EPBS(Electronic Problem Bank System) as examples. Web application development process proposed in this thesis can be maintained through reverse development, because it can be defined as high level abstraction for modeling. It is very easy to be understood as a process based on models. Also, analysis and design models can be reused as useful component whenever similar web application is developed.

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Isolation of Candida albicans Chitin Synthase 1 Inhibitor from Streptomyces sp. A6705 and Its Characterization

  • KIM NA RAE;HWANG EUI IL;YUN BONG SIK;LEE SANG HAN;MOON JAE SUN;LIM CHI HWAN;LIM SE JIN;KIM SUNG UK
    • Journal of Microbiology and Biotechnology
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    • v.15 no.4
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    • pp.895-898
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    • 2005
  • In the course of searching for potent chitin synthase 1 inhibitors from natural resources, Streptomyces sp. A6705 was found to exhibit potent inhibitory activity against the chitin synthase 1 from C. albicans (CaCHS1p). As a result, the inhibitor was isolated and identified using a series of chromatographies. Through chemical analyses with UV spectrophotometry, MS spectrometry, and various NMR techniques, the inhibitor was identified as N,N-bis(2-phenylethyl)urea. The compound exhibited strong inhibitory activity against the chitin synthase 1 from C. albicans with an $IC_{50}$ of 14 ${\mu}g$/ml, representing a similar inhibitory activity to that of the well-known chitin synthase inhibitor, polyoxin D ($IC_{50}$ 15 ${\mu}g$/ml). However, the compound showed no inhibitory activity against the chitin synthase 2 of Saccharomyces cerevisiae up to 280 ${\mu}g$/ml, which is structurally and functionally analogous to CaCHS 1 p. In addition, the compound exhibited weak antifungal activities against Cryptococcus neoformans and Rhizoctonis solani.

Studies on The Microbial Utilization of Agricultural Wastes (Part 5) Isolation and Selection of Cellulase Producing Fungi (농산폐자원의 미생물학적 이용에 관한 연구 (제오보) 섬유소분해효소 생산 곰팡이의 분리 및 선별)

  • Bae, Moo;Kim, Byung-Hong;Lee, Gye-Jun
    • Microbiology and Biotechnology Letters
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    • v.4 no.3
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    • pp.105-110
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    • 1976
  • In the studies of microbiological utilization of cellulosic wastes, cellulolytic fungi were isolated and screened out. At the first stage, 221 cellulolytic fungi were isolated from different sources such as soils, humus, composts and rotten wood debris by enrichment culture techniques. In the second stage, 36 strains of fungi out of those previously isolated were selected for their cellulase activities estimated by means of filter paper degradation, carboxy methyl cellulose liquefaction and cup method. Activities of C$_1$-cellulase, C$\sub$x/-cellulase and filter paper activity were adopted on the final screening stage and five different strains which are tentatively identified as Aspergillus sp.(strain No. AS-9), Penicillium sp. (strain No. KNI-1-2), Trichoderma, sp. (strain No. KI-7-2, KI-7-5, KI-4-1-1B) were selected for their high potency of C$_1$ and C$\sub$x/-cellulase activities. When rice straw milled and treated with NH$_4$OH was hydrolyzed with the crude enzyme Prepared from the culture broth of Trichoderma sp. (strain No. KI-4-1-1B), saccharification rate was obtained up to 26%.

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Isolation and Characterization of α-glucosidase Inhibitors from Euonymus alatus (화살나무(Euonymus alatus)로 부터 α-glucosidase 저해 물질의 분리 및 동정)

  • Kim, Shin-Duk
    • Microbiology and Biotechnology Letters
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    • v.45 no.4
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    • pp.311-315
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    • 2017
  • ${\alpha}$-glucosidase inhibitory compounds (1-4) in a 50% methanol extract of Euonymus alatus were isolated by activity-based fractionations and the structures determined on the basis of chemical and spectral characterization techniques such as $^1H$ and $^{13}C$ nuclear magnetic resonance spectroscopy, $^1H-^1H$ correlation spectroscopy (COSY), and heteronuclear multiple bond correlation (HMBC). The compounds 1-4 belong to flavonols and exhibited potent inhibitory activities against ${\alpha}$-glucosidase, with $IC_{50}$ values of 25.3, 17.1, 47.3, and $35.1{\mu}M$, respectively. All the isolated compounds were more potent than the positive control acarbose. This is the first report describing the potential hypoglycemic effect of Euonymus alatus through ${\alpha}$-glucosidase inhibition and identification of its active components.

Identification of 2-methylbutyric Acid as a Nematicidal Metabolite, and Biocontrol and Biofertilization Potentials of Bacillus pumilus L1

  • Lee, Yong-Seong;Cho, Jeong-Yong;Moon, Jae-Hak;Kim, Kil-Yong
    • Korean Journal of Soil Science and Fertilizer
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    • v.49 no.4
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    • pp.401-408
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    • 2016
  • The present study described the isolation of 2-methylbutyric acid (2-MBA) produced from Bacillus pumilus L1, to subsequently investigate its nematicidal activity for the control of the root-knot nematode. The results showed that 2-MBA could be purified by chromatographic techniques and was identified using nuclear magnetic resonance and liquid chromatography-mass spectrometry. Crude extract and partially purified compounds had a significant effect on the inhibition of egg hatchability and second-stage juvenile (J2) mortality. A dose-dependent effect of 2-MBA was observed for J2 mortality and egg hatchability. Egg hatchability was 69.2%, 59.9%, 32.7%, and 0.0% at 125, 250, 500, and $1000{\mu}g\;mL^{-1}$ of 2-MBA after 4 d of incubation, respectively. Meanwhile, J2 mortality was in the range of 24.4%-100.0% after 2 d of incubation, depending on the concentrations of 2-MBA used. A pot experiment also demonstrated that treatment of B. pumilus L1 culture caused a significant reduction in the number of galls, egg masses, and J2 population than that of the tap water (TW) control. However, as the B. pumilus L1 culture concentration was decreased, the efficacy of nematode control by treatment of B. pumilus L1 culture was reduced compared to that of TW. B. pumilus L1 inoculation at different concentrations also promoted cucumber plant growth. Therefore, our study demonstrated the potential of 2-MBA from B. pumilus L1 as a biocontrol agent against the root-knot nematode and a plant growth promoter for cucumber plants.

Antioxidant, antinociceptive activity and general toxicity study of Dendrophthoe falcata and isolation of quercitrin as the major component

  • Hasan, Md Shihab;Ahmed, Md Iqbal;Mondal, Sukla;Masud, Mohammad Methedi;Sadhu, Samir Kumar;Ishibashi, Masami;Uddin, Shaikh Jamal
    • Advances in Traditional Medicine
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    • v.6 no.4
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    • pp.355-360
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    • 2006
  • Plants have been used in traditional medicinal system for centuries. Bangladeshi medicinal plants have received considerable attention from the researchers for evaluation of their bioactivity. As a part of our ongoing research of screening the Bangladeshi medicinal plants, the ethanolic extract of Dendrophthoe falcata have been chosen for the present study. The ethanolic extract of the leaves of the plant have been assessed for their antioxidant, antinociceptive, and general toxicity. The extract showed potent antioxidant activity ($IC_{50}5.1{\mu}g/ml$) using DPPH radical scavenging assay, which is comparable to the standard ascorbic acid ($IC_{50}4.6{\mu}g/ml$). The extract significantly and dose dependently inhibited the acetic acid induced writhing in mice (71.2%, P < 0.001 and 28.0%, P < 0.05 for 500 and 250 mg/kg body weight, respectively). A general toxicity was assessed by a simple and low cost assay using brine shrimp lethality as an indicator. The extract showed low level of toxicity ($LC_{50}100{\mu}g/ml$). Using different chromatographic techniques, quercitrin (quercetin 3-O-${\alpha}$-rhamnoside) was separated as the major component from the extract. The structure was elucidated by detailed 1D and 2D NMR and mass spectral analysis.