• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.350-355
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• 2004

Sulforhodamine B (SRB) assay is a rapid, sensitive, and inexpensive method for measuring cell proliferation and chemosensitivity. However, the lactate dehydrogenase (LDH) release assay is generally used to measure cytototoxicity of infectious microorganisms against host cells. In this study, we investigated the possibility of applying the SRB assay to determine cytotoxicity for infectious microorganisms, and compared the results with those obtained by the LDH release assay. We used Vibrio vulnificus as a model of infectious microorganisms. The SRB assay showed that V vulnificus strongly induced cytotoxic activity against human intestinal cells, Caco-2 and INT-407 cells. The degree of cytotoxicity closely correlated with infection time and number ratios of V. vulnificus to intestinal cells (MOI, multiplicity of infection). Furthermore, cytotoxicity values obtained by SRB assay correlated well with results obtained by the LDH release assay, and both assays gave a linear response with respect to MOI Heat-inactivation of V. vulnificus for 35 min at $60^{\circ}C$ did not induce cytotoxic activity, indicating that viability of V. vulnificus is crucial for cytotoxic activity against intestinal cells. Although both assays are suitable as cytotoxicity endpoints, the SRB assay is recommended for measuring cytotoxicity of infectious microorganisms against host cells because of its significantly lower cost and more stable endpoint than the LDH release assay.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.968-974
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• 2008

Proteomic analysis led to identification of the proteins of Vibrio vulnificus that were induced upon exposure to INT-407 cells, and 7 of which belong to the functional categories such as amino acid transport/metabolism, nucleotide transport/metabolism, posttranslational modification/protein turnover/chaperones, and translation. Among the genes encoding the host-induced proteins, disruption of purH, trpD, tsaA, and groEL2 resulted in reduced cytotoxicity. The purH, trpD, and tsuA mutants showed impaired growth in the INT-407 lysate; however, the growth rate of the groEL2 mutant was not significantly changed, indicating that the possible roles of the host-induced proteins in the virulence of V. vulnificus are rather versatile.

• BMB Reports
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• 제32권3호
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• pp.273-278
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• 1999

Cytolysin produced by Vibrio vulnificus has been known to be lethal to mice by increasing vascular permeability and neutrophil sequestration in the lung. In the present study, a cytotoxic mechanism of V. vulnificus cytolysin on the neutrophil was investigated. Cytolysin rapidly bound to neutrophils and induced cell death, as determined by the trypan blue exclusion test. V. vulnificus cytolysin caused the depletion of cellular ATP without the release of ATP or lactate dehydrogenase. Formation of transmembrane pores was evidenced by the rapid efflux of potassium and 2-deoxy-D-[$^3H$]glucose from cytolysin-treated neutrophils. It was further confirmed by the rapid flow of monovalent ions in the patch clamp of cytolysin-treated neutrophil membrane. The pore formation was accompanied by the oligomerization of cytolysin monomers on the neutrophil membrane as demonstrated by immunoblot, which exhibited a 210 kDa band corresponding to a tetramer of the native cytolysin of $M_r$ 51,000. These findings indicate that V. vulnificus cytolysin rapidly binds to the neutrophil membrane and oligomerizes to form small transmembrane pores, which induce the efflux of potassium and the depletion of cellular ATP leading to cell death without cytolysis.

• 한국식품과학회지
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• 제52권1호
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• pp.89-93
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• 2020

본 연구에서는 패혈증비브리오균에 대한 PEM, RCM, RRM과 CTM 메탄올 추출물의 항균 활성을 평가하였다. 96-well microplates를 사용한 액체 배양법 실험 결과 4가지 천연물은 200 ㎍/mL 농도에서 80%이상의 항균 효과를 나타내었다. 또한 페이퍼디스크 확산법을 사용하여 항균 활성을 평가한 결과 4가지 천연물은 20, 40 mg/disc의 농도에서 농도 의존적으로 패혈증비브리오균의 성장을 억제하였다. 다음으로 4가지 천연물의 안전성을 평가하기 위해 MTS assay를 통해 HeLa 세포에 대한 독성을 확인한 결과 100, 200 ㎍/mL의 농도에서 세포 독성을 나타내지 않았다. 또한 LDH assay를 사용하여 패혈증비브리오균의 HeLa 세포에 대한 세포막 손상 억제 효과를 평가한 결과 PEM, RCM, RRM과 CTM 메탄올 추출물은 200 ㎍/mL 농도에서 세포막 손상억제 효과를 나타내었다. 다음으로 바닷물과 조개를 사용한 수족관 모델에서 4가지 천연물의 메탄올 추출물 혼합액 200 ㎍/mL 농도는 colony 생성을 현저하게 감소시켜 패혈증비브리오균의 증식을 억제 하는 결과를 나타내었다. 이와 같은 결과를 통해 4가지 천연물의 메탄올 추출물은 바닷물과 해산물에 존재하는 패혈증비브리오균에 대해 뛰어난 항균효과를 나타내면서 인체에는 무해한 천연소독제로 응용할 수 있을 것으로 판단된다.

• Biomolecules & Therapeutics
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• 제16권4호
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• pp.356-363
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• 2008

A bacterial strain with good antibacterial activities against Staphylococus aureus and Escherichia coli was isolated from a marine sponge Stelleta sp., and it was identified as a Psychrobacter sp. by comparative 16S rDNA sequence analysis. In our search for bioactive secondary metabolites from this psychrophillic and halotolerent bacterium, sixteen cyclic dipeptides (1-16) were isolated and their structures were identified on the basis of NMR analysis. In the test of the compounds for the protective effect against Vibrio vulnificusinduced cytotoxicity in human intestinal epithelial cells, cyclo-(L-Pro-L-Phe) (5) exhibited significant protective effect. Compounds 2, 6, and 11, which contain D-amino acid, were first isolated from bacteria.