• Microbiology and Biotechnology Letters
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• v.28 no.5
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• pp.251-257
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• 2000

Serratia marcescens purine nucleoside phosphorylase (PNP) was purfied to homogeneity by streptomycin sulfate treatment, Sephacry HR S-200 gel filtration chromatography and AMP-agarose affinity chromatography. The specific activity of the enzyme was increased 49-fold during purification with an overall yield of 7.0%. The molecular weight was 168kD as estimated by Sephadex G-150 gel filtration chromatography. The S. marcescens enzyme was composed of six identical subunits with subunit molecular weight of 28kD, as estimated by SDS-PAGE. The Km values of S. marcescens enzyme for inosine and deoxyinsoine were 0.38 and 1.20 mM, respectively. The ph optimum was near 8.0, and the enzyme was relatively heat-stable protein. The enzyme was inactivated com-pletely by 0.5 mM of $Cu^{ 2+}$.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.61-61
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• 1995

1993년 국내토양으로부터 분리동정된 Streptomyces속 균주중 항종양성이 우수한 균주 3주(DKM104, DKM117, DKM409)를 선정하고 이들을 대량 배양하여 종양억제인자를 분리하고 시험관내 종양세포 독성능 및 생체내 항암활성능과 이들 물질생산과 PLASMID DNA와의 관련성, $LD_{50}$등을 시험하여 새로운 항종양물질의 개발을 목적으로 하였다. 분리선별균주의 배양조건을 확립하였으며 배양여액을 국성이 적은 유기용매로부터 큰쪽으로 단계적으로 유효성분을 추출하여 Gel Chromatography를 이용하여 유효성분을 분획하였다. 시험관내 항종양능 시험은 MTT colorimetric검정법을 이용하여 $IC_{50}$값을 산정하였으며 생체내 항종양능은 마국의 NIC에서 권장하는 tumor panel system에 따랐다. 선정된 균주의 plasmid 분리는 alkalin lysis법을 채택하였으며 agarose gel electrophoresis로 plasmid profile을 시험하였다. Novobiocin등을 이용하여 Curing test를 시행하였고 독성실험은 $LD_{50}$량을 구해 항암효과 측정시에 Maximum dost로 투여하였고 최고용량을 기준으로 일정한 공비를 적응하여 3단계의 투여량을 설정하였다.(중략)

• Journal of Microbiology
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• v.33 no.4
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• pp.334-338
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• 1995

The electrophoretic karyotypes of 6 species in different Fusarium sections were examined by using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Intact chromosomal DNA was prepared from protoplasts and up to 9 distinct bands were separated on 0.7% or 0.8% agarose gel under several different conditions. Putative chromosome numbers varied from 6 to 9 amd polymorphic karyotypes were observed in different Fusarium sections. Using Schizosaccharomyces pombe and Saccharomyces cerevisiae chromosomes as standards, the sizes of the Fusarium spp. chromosomes were estimated. The electrophoretic karyotypes of F. moniliforme and F. subglutinans (section Liseola) were similar. Unidentified filamentour fungi, F. beomiforme was much closer to F. axysporum (section Elecgans) in karyotype and the karyotypes of F. napiforme were more similar to those of section Liseola than any other sections. F. graminearum (section Discolor) had a distinctive electrophoretic karyotype.

• Korean Journal of Veterinary Service
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• v.22 no.4
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• pp.363-370
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• 1999

Isolation of PRRSV was attempted from 646 swine sera collected from swine farms. The MARC-145 cell, which is highly permissive to PRRSV, was used for virus isolation, propagation, IFA test, and VN test. Total 36 cytopathic viruses to MARC-145 cells were isolated. The virus isolates were identified as a PRRSV by the IFA test and VN test using the reference sera prepared by experimental infection of reference PRRSV CNV-1 into 30 day-old pig. In addition to serological conformation, ORF5 of genomic RNA of 6 selected cytopathic viruses were amplified by the RT-PCR. The resulting PCR products were examined by electrophoresis in 1.2% agarose gel. An appropriate bands of about 680bp including the flanking sequence of total 80bp were seen on agarose gel.

• Toxicological Research
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• v.13 no.1_2
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• pp.39-48
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• 1997

Cyclophosphamide(25, 50 or 100 mg/kg), orally administered to male Sprague-Dawley rats, caused a time- and dose-dependent thymic atrophy. In the light microscopic examination of the atrophic thymus, thymocytes with condensed or fragmented nucleus were multifocally observed in the cortical region, started to increase 8 hr after CPA treatment and reached to the maximal level at 16 hr, although such cells were not seen after 48 hr when the severe depletion of thymocytes were marked. In agarose gel electrophoresis to analyze the DNA changes, DNA extracted from atrophic thymus showed a oligonucleosomal laddering at the corresponding time to morphological changes. In an additional supportive experiment, thymocytes showing morphological changes, nuclear condensation or apoptotic body, exhibited a positive reaction to immunoperoxidase staining using in situ apoptosis detection kit. Separately, agarose gel electrophoresis of DNA from bone .marrow cells was performed to investigate the involvement of bone marrow cells in the process of thymocyte apoptosis. Although DNA laddering was slightly increased 2 and 4 hr after treatment, no clear correlation was inferred. Taken togather, it is concluded that thymocytes showing morphological changes in thymic atrophy induced by cyclophosphamide administration represent an apoptosis having biochemical nature of programmed cell death.

• Journal of Korean Ophthalmic Optics Society
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• v.7 no.2
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• pp.189-195
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• 2002

The aim of this study was to investigate the action of benzalkonium chloride (BAC) used as a preservative in most ophthalmic topical solutions, on human corneal epithelial (HCE) cells in vitro. HCE cell line was exposed to BAC solutions at various concentrations (0.01%~0.0001%) for 15 minutes followed by 24 hours of cell recovery. Cell viability was assessed using MTT assay and chromatin condensation with a Hoechst 33342 test. The expression of membrane protein Fas and Fas ligand was examined by western blot and immunocytochemistry, and DNA fragmentation was studied by agarose gel electrophoresis. A significant decrease of membrane integrity with chromatin condensation was observed with BAC tested at concentrations of 0.005% and higher. BAC was cytotoxic preservatives in this study. An apoptotic mechanism appeared to be present at low concentrations of BAC, whereas a necrotic process appeared at higher concentrations. A functional Fas-mediated apoptotic pathway is present in cultured HCE cells and can be activated by upregulation of Fas expression with BAC.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.16
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• pp.7053-7060
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• 2015

In the present work, novel orange peel was extracted with 100%EtOH (ethanol) and fractionated into four fractions namely F1, F2, F3, F4 which were eluted from paper chromatographs using 100%EtOH, 80%EtOH, 50%EtOH and pure water respectively. The crude extract and its four fractions were evaluated for their total polyphenol content (TPC), total flavonoid content (TFC) and radical scavenging activity using DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. Their cytotoxic activity using WST assay and DNA damage by agarose gel electrophoresis were also evaluated in a human leukemia HL-60 cell line. The findings revealed that F4 had the highest TPC followed by crude extract, F2, F3 and F1. However, the crude extract had the highest TFC followed by F4, F3, F2, and F1. Depending on the values of $EC_{50}$ and trolox equivalent antioxidant capacity, F4 possessed the strongest antioxidant activity while F1 and F2 displayed weak antioxidant activity. Further, incubation HL-60 cells with extract/fractions for 24h caused an inhibition of cell viability in a concentration-dependent manner. F3 and F4 exhibited a high antiproliferative activity with a narrow range of $IC_{50}$ values ($45.9-48.9{\mu}g/ml$). Crude extract exhibited the weakest antiproliferative activity with an $IC_{50}$ value of $314.89{\mu}g/ml$. Analysis of DNA fragmentation displayed DNA degradation in the form of a smear-type pattern upon agarose gel after incubation of HL-60 cells with F3 and F4 for 6 h. Overall, F3 and F4 appear to be good sources of phytochemicals with antioxidant and potential anticancer activities.

• Journal of Life Science
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• v.18 no.8
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• pp.1036-1041
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• 2008

Methenyltetrahydrofolate synthetase extract was obtained from mouse liver and purified via $30{\sim}70%$ ammonium sulfate fractionation, Fast Q anion exchange and phenyl agarose chromatography. HPLC gel chromatography and SDS-polyacrylamide electrophoresis experiments showed that the enzyme is a monomer with molecular weight of 23 kDa. Optimum temperature and pH were $35^{\circ}C$ and 6.5, respectively. The enzyme was chemically modified only by tetranitromethane and 1-ethyl-3- (3-dimethyl aminopropyl)-carbodiimide (EDC), indicating that tyrosine and carboxylate are in the active site. pH studies showed that 2 tyrosines are involved in the binding of the substrates and a carboxylate in catalysis. Therefore, the chemical mechanism of the enzyme is likely that 2 tyrosines bind to ATP and 5-formylTHFand a carboxylate acts as a general base.

• Journal of Sericultural and Entomological Science
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• v.13 no.2
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• pp.141-143
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• 1971

By means of thin-layer electrophoresis in agarose gel, hemolymph protein of healthy silkworm larvae and of the nuclear polygedrosis virus infected larvae were studied. 1. In the 4th instar, 4 fractions moving toward anode were separated. Dye-binding Capacity of the fraction was increased according to the stage. 2. After 5th day in the 5th instar, 7 fractions moving toward anode were separated, and one fraction toward cathode was separated. 3. On the first day in the 5th instar, 5 fractions were separated, and on the 4th day of the same instar 5 fractions were separated. 4. As for the hemolymph protein fractions of the polyhedrosis virus infected larvae, on the 6th and 7th day, three fractions(D.E.F) were inclined to increase, whereas on the 8th day 4 fractions(A.B.D.E) were disappeared but F fraction was inclined to decrease.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.155-160
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• 1986

$\alpha$-Amylase gene of B. amyloliquefaciens was cloned to E. coli-yeast shuttle vector YEp-13 and expressed in E. coli. Chromosomal DNA of B. amyloliquefaciens was partially digested with Sau3Al and YEp13 plasmid was cleaved with BamH1. The hybrid plasmid, pHA28, was constructed by shotgun method and transformed to E. coli C600 and HB101. The amount of $\alpha$-amylase produced by transformants of E. coli was about 20% to 30% of that produced by B. amyloli-quefaciens. About 65% of $\alpha$-amylase produced by transformant was secreted into periplasm and the others were located in cytoplasm. $\alpha$-Amylase production was maximal when transformants were cultivated for 15hr to 20hr. As the result of agarose gel electrophoresis, pHA28 plasmid was found to be various in its size. This result suggested that pHA28 plasmid was segregated.