• BMB Reports
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• 제46권10호
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• pp.495-500
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• 2013

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus. We have modified TYMV coat protein (CP) by inserting a c-Myc epitope peptide at the N- or C-terminus of the CP, and have examined its effect on assembly. We introduced the recombinant CP constructs into Nicotiana benthamiana leaves by agroinfiltration. Examination of the leaf extracts by agarose gel electrophoresis and Western blot analysis showed that the CP modified at the N-terminus produced a band co-migrating with wild-type virions. With C-terminal modification, however, the detected bands moved faster than the wild-type virions. To further examine the effect, TYMV constructs producing the modified CPs were prepared. With N-terminal modification, viral RNAs were protected from RNase A. In contrast, the viral RNAs were not protected with C-terminal modification. Overall, the results suggest that virion assembly and RNA packaging occur properly when the N-terminus of CP is modified, but not when the C-terminus is modified.

• Asian Pacific Journal of Cancer Prevention
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• 제16권15호
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• pp.6423-6428
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• 2015

The present study was designed to evaluate in vitro anti-proliferative potential of extracts from four Indian medicinal plants, namely Anogeissus latifolia, Terminalia bellerica, Acacia catechu and Moringa oleiferna. Their cytotoxicity was tested in nine human cancer cell lines, including cancers of lung (A549), prostate (PC-3), breast (T47D and MCF-7), colon (HCT-16 and Colo-205) and leukemia (THP-1, HL-60 and K562) by using SRB and MTT assays. The findings showed that the selected plant extracts inhibited the cell proliferation of nine human cancer cell lines in a concentration dependent manner. The extracts inhibited cell viability of leukemia HL-60 and K562 cells by blocking G0/G1 phase of the cell cycle. Interestingly, A. catechu extract at $100{\mu}g/mL$ induced G2/M arrest in K562 cells. DNA fragmentation analysis displayed the appearance of a smear pattern of cell necrosis upon agarose gel electrophoresis after incubation of HL-60 cells with these extracts for 24h.

• 대한의생명과학회지
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• 제11권3호
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• pp.281-287
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• 2005

Although actinomycin D (AMD) is known to induce apoptotic cell death to various cell lines, the mechanism of apoptosis induced by AMD is still unclear. Understanding this mechanism may improve its therapeutic efficacy. The present study has been performed to elucidate expression of Bcl-2 and Caspase-3 proteins related to apoptosis in human leukemia K-562 cells. Five different assays were performed in this study; DNA fragmentation analysis by agarose gel electrophoresis, quantitative assay of fragmented DNA, morphological assessment of apoptotic cells, quantification of apoptosis by annexin V (AV) and propidium iodide (PI) staning, and expression of Bcl-2 and Caspase-3 proteins by the western blot analysis. The number of apoptotic cells and amount of fragmented DNA in this cell line treated with AMD was increased at 6 hour. DNA ladder pattern was also appeared at 6 hour. The expression of Bcl-2 was decreased, and disappeared from 12 hours after AMD treatment. Precursor of Caspase-3 was degraded, and 20 kDa cleavage products were detected. These results suggest that AMD induced apoptosis of K-562 cells is Caspase-3-dependent fashion, and this apoptosis is related to the degradation of Bcl-2 proteins.

• 한국미생물·생명공학회지
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• 제13권3호
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• pp.251-255
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• 1985

국내 논의 흙으로부터 벼과식물에 관련된 질소 고정 미생물을 238주 분리하여 비교적 활성이 높은 4주의 질소고정활성 미생물을 선별하여 이중 3주는 K. pneumoniae, 1주는 E. agglomerans로 동정하였다. NFB3, NFB264, NFB278의 3주는 1.7-84Mdal. 의 plasmid들을 가지고 있었으나 이들 plasmid에 질소고정유전자 단편을 함유하고 있지는 않았다. 선별미생물의 chromosomal DNA상에 존지하는 질소고정 유전자는 K. pneumoniae M5al의 질소고정유전자와 homology를 나타냈으며, 이들의 질소고정유전자는 K. pneumoniae M5al의 질소고정유전자와 제한효소 절단부위가 상이하였다.

• BMB Reports
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• 제31권2호
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• pp.183-188
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• 1998

2-chloroethylethyl sulfide (CEES) is an alkylating agent that readily reacts with a wide variety of biological molecules causing metabolic abnormality. The mechanism of cell death during CEES injury is poorly understood. We have examined the effect of exposure of thymocytes with various concentrations of CEES to determine the pattern of cell death in thymocytes injury induced by CEES. In the present study, we show that two patterns of cell death occurred by either one of two mechanisms: apoptosis and necrosis. Exposure to low level of CEES (100 ${\mu}M$) for 5 h caused an induction of apoptosis on thymocytes, as identified by the following criteria: DNA fragmentation visualized by the characteristic "ladder" pattern was observed upon agarose gel electrophoresis and morphological features were revealed by microscopical observations. In contrast, exposure to high levels of CEES (500 ${\mu}M$) induce necrotic features such as cell lysis. Thus, depending on the concentrations, CEES can result in either apoptotic or necrotic cell damage. Our findings suggest that thymocytes which are not killed directly, but merely injured by low levels of CEES, are able to activate an internally-programmed cell death mechanism, whereas thymocytes receiving severe damages apparently can not.

• Journal of Microbiology
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• 제34권4호
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• pp.363-369
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• 1996

As basic study to evaluate the treatability of oil-contaminated environment with bacteria, isolation and characterization of crude oil-degrading bacterium were carried out. A bacterial strain SH-14 capable of degrading crude oil was isolated from contaminated soils by enrichment culture technique and identified as Acinetobacter sp. by morphological, cultural and biochemical characteristics, and so named Acinetobacter sp. SH-14. The optimal medium composition and cultural conditions for the growth and emulsification of crude oil by Acinetobacter sp. SH-14 used were crude oil of 2.0%, $KNO_3$ of 0.2%, $K_2HPO_4$ of 0.05%, and $MgSO_4\;{\cdot}\;7H_2O$ of 1.0%, along with initial pH 7.0 at $30^{\circ}C$. Acinetobacter sp. SH-14 showed to be resistant to chloramphenicol and utilized various hydrocarbons such as dodecane, hexadecane, isooctane, cyclo-hexane etc., as a sole carbon source. Acinetobacter sp. SH-14 harbored a single plasmid. By agarose gel electrophoresis and curing experiment it was found that the genes for crude oil components degradation were encoded on the plasmid.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.777-782
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• 2004

The arylsulfatase gene (astA, 984 bp ORF) from the P. carrageenovora genome was amplified by PCR and subcloned into the pET21a vector. When the constructed plasmid pAST-A1 (6.4 kb) was introduced into E. coli BL21(DE3), the transformant on the LB plate containing IPTG showed a hydrolyzing activity for 4-methylumbelliferyl sulfate and p-nitrophenyl sulfate. The highest arylsulfatase activity (2.1 unit/ml) was obtained at 10 mM IPTG. Most arylsulfatase activity was found in the cell lysate, whereas no significant activity was detected in the culture supernatant. The molecular weight of the recombinant enzyme was estimated to be 33.1 kDa by SDS-PAGE. After the reaction of agar with arylsulfatase for 12 h at $40^{\circ}C$, the gel strength of the agar increased by 2-fold, and 73% of the sulfate in the agar had been removed. This result suggests that arylsulfatase expressed in E. coli could be useful in the production of electrophoretic grade agarose.

• 동의생리병리학회지
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• 제17권2호
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• pp.338-345
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• 2003

The purpose of this research was to investigate the anticancer effects of Dojeokseungki-tang(DJSKT) on the various leukemia cell lines. DJSKT treatment suppressed proliferation of cultured-HL60, Jurkat, L1210 cells and increased apoptosis of cultured-L1210, HL60, Molt4, Jurkat cells. DJSKT treatment induced apoptosis of Jurkat cells including the morphologic changes such as the 'ladder pattern' revealed by agarose gel electrophoresis of DNA in a dose-dependent manner. Administration of DJSKT induced apoptosis of transplanted-L1210 cells in vivo, and decreased of mitochondrial transmembrane potential of L 1210 and Jurkat cells in vitro. DJSKT treatment reduced the expression of bcl-2 proteins in Jurkat cells and increased ICE, c-myc, p53 mRNA expression in Molt4 cells. In conclusion, these results suggest that DJSKT might be usefully applied for anti-carcinogenic agent of leukemia.

• Biomolecules & Therapeutics
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• 제4권4호
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• pp.402-407
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• 1996

Effects of oxidative stress on the induction of apoptosis and the activity of antioxidant enzymes were investigated in HL-60 cells using $H_2O$$_2$and cisplatin which generate oxygen species in the cell. Various concentrations of oxidants were treated to cells and at different incubation time, cells were harvested for assays. Cell viability, morphology by propidium iodide staining and DNA fragmentation by agarose gel electrophoresis were observed to determine whether they induce apoptosis. The activity of antioxidant enzymes such as superoxide dismutase and catalase was also measured to evaluate the cellular response to the oxidative damage. The results are as follows: $H_2O$$_2$ induced apoptosis at 10 $\mu$M after 6h incubation, while it took 12h for cisplatin. Both oxidants induced the superoxide dismutase activity at a tolerable low concentration. However, at a concentration which causes apoptotic cell death, the enzyme level was dropped markedly at first and then recovered to the normal level after which it declined again, probably due to cell death. On the other hand, changes in the activity of catalase were not significant at most concentrations except the statistically significant decrease at 24h after 10 $\mu$M-$H_2O$$_2$treatment. In this study, $H_2O$$_2$- and cisplatintreated cells showed similar results in apoptotic response and enzyme activities, suggesting that anticancer activity of cisplatin may be related, at least in part, to the production of oxygen free radicals.

• 한국식물병리학회지
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• 제11권1호
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• pp.73-79
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• 1995

Complementary DNAs (cDNAs) to the coat protein gene of an ordinary Korean strain of potato virus Y (PVY-OK) isolated from potato (cv. Superior) were synthesized and cloned into a plasmid pUC119 and sequenced. The RNA of the virus propagated in tobacco (Nicotinaa sylvestris) was extracted by the method of phenol extraction. The first strand of cDNAs to the coat protein penomic RNA of the virus was made by Moloney murine leukemia virus reverse transcriptase. The cDNA were synthesized and amplified by the method of polymerase chain reaction (PCR) using a pair of oligonucleotide primers. PVYCP3P and PVYCP3M. The size of cDNAs inserted in pUC119 plasmid was estimated as about 840 bp upon agarose gel electrophoresis. Double stranded cDNAs were transformed into the competent cell of E. coli JM109. Sequence analysis of cDNAs was conducted by the dideoxynucleotide chain termination method. Homology of cDNAs of the PVY-OK coat protein genomic RNA with those of PVY-O (Japan), PVY-T (Japan), PVY-TH (Japan), PVYN (The Netherlands),and PVYY (France) was represented as 97.3%, 88.9%, 89.3%, 89.6% and 98.5%, respectively. Homology at the amino acid level turned out to the be 97.4%, 92.5%, 92.9%, 92.9% and 98.5%, respectively.