• Title/Summary/Keyword: time killing assay

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Colorimetric Evaluation of the Time-Killing Assay for Citropin 1.1, Lipopeptide Palm-KK-$NH_2$, and Temporin A

  • Baranska-Rybak, Wioletta;Dawgul, Malgorzata;Bielinska, Sylwia;Kraska, Bartlomiej;Piechowicz, Lidia;Kamysz, Wojciech
    • Journal of Microbiology and Biotechnology
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    • v.21 no.5
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    • pp.536-539
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    • 2011
  • Nowadays, there are a number of colorimetric techniques available for the determination of a time killing assay in a manner much easier and faster than those previously more commonly used, which were much more time-consuming and laborious colony counting procedures. Here, an attempt has been made to test the antimicrobial peptides of Citropin 1.1, Palm-KK-$NH_2$, and Temporin A on a reference strain of Staphylococcus aureus using resazurin as the cell viability reagent. Staphylococcus aureus was exposed to the test compounds over varying periods of time and the metabolic activity measured, with a profile of antimicrobial activity then established. The results are in agreement with data from previous literature, thus confirming the relevance of the application of resazurin for the testing of antimicrobial agents.

Comparison of the Antibacterial Activity of Domestic Cirsium japonicum Collected from Different Regions (지역별 국내 자생 엉겅퀴 추출물의 항균 활성)

  • Jang, Miran;Park, Hyejin;Hong, Eunyeong;Kim, Gun-Hee
    • Korean journal of food and cookery science
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    • v.30 no.3
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    • pp.278-283
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    • 2014
  • This study was investigated the antibacterial activities of Cirsium japonicum from extracts five regions(Chungnam, Gyeonggi, Gangwon, Jeju and Jeonnam) extract against six food-borne pathogenes(Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica and Vibrio vulnificus) using the broth dilution and agar diffusion method. At concentrations between 0 and $750{\mu}g/mL$ the extracts showed an antibacterial effect against all tested bacteria. The antibacterial activities of Cirsium japonicum from Jeju and Gangwon are stronger than others. The minimum inhibitory concentration(MIC) values against the six bacteria ranged from 93.75 to $750{\mu}g/mL$. In time killing assay(cell growth curves), the tested bacteria inactivated upon exposure to the extracts for 24h. At the 24h exposure to the extracts, all bacteria were inhibited to over 70% for growth. In particular, Bacillus subtilis, Salmonella enterica and Vibrio vulnificus conveyed an inhibition of growth to almost complete. It is anticipated that Cirsium japonicum extracts may have greater potential as natural food preservatives.

CHEMOSENSITIVITY TEST OF HUMAN OSTEOSARCOMA AND EPIDERMOID CARCINOMAS USING MTT ASSAY (MTT법을 이용한 사람 골육종과 상피암 세포주들에 대한 항암제 감수성 검사)

  • Park, Sung-Oh;Shin, Hyo-Keun;Kim, Oh-Whan
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.13 no.4
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    • pp.391-404
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    • 1991
  • Three anticncer agents which are different in time or dosage dependence as well as in phase specificity, namely mitomycin and adriamycin from natural products, and widely different cancer cell lines_Four epidermoid carcinomas originated from larynx, cervix, skin and gut were used toghether with one osteosarcoma as the target cell of single and combined administration of anticancer drugs. Semiautomated tetrazolium dye assay(MTT) appears to offer an attractive option for chemosensitivity of head and neck cancers since it is a simple, valid and inexpensive method of assessing chemosensitivity for large samples in a short time. The results obtained form this study were as follows. 1. Good correlations were obtained with the results of the MTT test and those of $^3H$ thymidine uptake assay. 2. $LD_{50}$ values of HIST and St.Ca. which showed relatively high doubling time on adriamycin were $30{\mu}g/ml$ and $15{\mu}g/ml$ while those of HeLa, Hep-2 and KHOS/NP were $2.1{\mu}g/ml$, $4.8{\mu}g/ml$, and $6.8{\mu}g/ml$ respectively. 3. The $LD_{50}$ value of 5-FU on five cancer cells were very high ranging from 15mg/ml to almost indefinite number, which means 5-FU is very resistant to epidermoid carcinomas or osteosarcoma examined in this study. 4. Mitomycin was relatively effective showing 80% cancer killing effect on HeLa, 70% on St. Ca. and 50% on Hep-2 at the high concentrations used. 5. Adriamycin was the most effective showing 90% cancer cell killing effect on KHOS/NP, 98% on HeLa, 80% both on Hep-2 and St. Ca. The least susceptible cancer cells toward adriamycin was HIST having only 55% cell killing effect at the high cincentration. 6. Combined therapy of adriamycin and 5-FU was more effective than single administration in all the cases examined. Most effective synergism was observed on St. Ca. at the low concentration, showing 21 times higher than each single administration.

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Endophytic Diaporthe sp. ED2 Produces a Novel Anti-Candidal Ketone Derivative

  • Yenn, Tong Woei;Ring, Leong Chean;Nee, Tan Wen;Khairuddean, Melati;Zakaria, Latiffah;Ibrahim, Darah
    • Journal of Microbiology and Biotechnology
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    • v.27 no.6
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    • pp.1065-1070
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    • 2017
  • This study aimed to examine the anti-candidal efficacy of a novel ketone derivative isolated from Diaporthe sp. ED2, an endophytic fungus residing in medicinal herb Orthosiphon stamieus Benth. The ethyl acetate extract of the fungal culture was separated by open column and reverse phase high-performance liquid chromatography (HPLC). The eluent at retention time 5.64 min in the HPLC system was the only compound that exhibited anti-candidal activity on Kirby-Bauer assay. The structure of the compound was also elucidated by nuclear magnetic resonance and spectroscopy techniques. The purified anti-candidal compound was obtained as a colorless solid and characterized as 3-hydroxy-5-methoxyhex-5-ene-2,4-dione. On broth microdilution assay, the compound also exhibited fungicidal activity on a clinical strain of Candida albicans at a minimal inhibitory concentration of $3.1{\mu}g/ml$. The killing kinetic analysis also revealed that the compound was fungicidal against C. albicans in a concentration- and time-dependent manner. The compound was heat-stable up to $70^{\circ}C$, but its anti-candidal activity was affected at pH 2.

Rapid detection and Quantification of Fish Killing Dinoflagellate Cochlodinium polykrikoides (Dinophyceae) in Environmental Samples Using Real-time PCR

  • Park, Tae-Gyu;Kang, Yang-Soon;Seo, Mi-Kyung;Kim, Chang-Hoon;Park, Young-Tae
    • Fisheries and Aquatic Sciences
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    • v.11 no.4
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    • pp.205-208
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    • 2008
  • The mixotrophic dinoflagellate Cochlodinium polykrikoides was reported to be linked to major fish kills in Korea and Japan since the 1990s. Rapid and sensitive detection of microalgae has been problematic because morphological identification of dinoflagellates requires light microscopic and scanning electron microscopic observations that are time consuming and laborious compared to real-time PCR. To address this issue, a real-time PCR probe targeting the ITS2 rRNA gene was used for rapid detection and quantification of C. polykrikoides. PCR inhibitors in water column samples were removed by dilution of template DNA for elimination of false-negative reactions. A strong association between cell quantification using real-time PCR and microscopic counts suggests that the real-time PCR assay is an alternative method for cell estimation of C. polykrikoides in environment samples.

Temporal changes in the abundance of the fish-killing dinoflagellate Karlodinium veneficum (Dinophyceae) in Tongyeong, Korea

  • Park, Tae-Gyu;Ok, Yu-Ran;Park, Young-Tae;Lee, Chang-Kyu
    • ALGAE
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    • v.26 no.3
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    • pp.237-241
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    • 2011
  • The toxic dinoflagellate Karlodinium veneficum has been implicated in numerous fish kill events around the world. Since this species commonly co-occurs with other morphologically similar dinoflagellates, field monitoring of this species in natural waters via light microscopy only has been problematic. In this study, we investigated temporal changes in K. veneficum's abundance in the waters of Obido, Tongyeong, using a species-specific real-time polymerase chain reaction (PCR) assay. The field survey, from April to December 2010, revealed K. veneficum occurred at low densities (12 to 425 cells $L^{-1}$) during this time and that cell numbers peaked in June (early summer in Korea), indicating this species generally occurs in the warmer season (mostly at $16.9-22.3^{\circ}C$ and 33.4-34.5‰) in the Obido area.

Effects of Curcuma longa L.on Human Stomach Cancer Cells (울금(鬱金)이 위암세포(胃癌細胞)에 미치는 영향(影響))

  • Cho, Yu-Kyung;Yoon, Song-Ryub;Kim, Beong-Woo;Kim, Jin-Sung;Ryu, Ki-Won;Ryu, Bong-Ha
    • THE JOURNAL OF KOREAN ORIENTAL ONCOLOGY
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    • v.9 no.1
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    • pp.15-37
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    • 2003
  • Objective: We are aimed to identify anti-tumor effects of Curcuma longa L. on the stomach cancer cells through molecular biologic methods. Material & Methods: We used AGS as human stomach cancer cells obtained from American Type Culture Collection. The boiled extract of Curcuma longa L. $5{\mu}l$ (Sample I), $10{\mu}l$ (Sample II) was treated to cultural media(ml) for 0, 6, 12, 24, 48 hours. We measured the killing effect on stomach cancer cells through Trypan blue exclusion test and the suppressive effect on viability of stomach cancer cells via MTT assay. For identification of its anticancer mechanism, the revelation of Bcl-2, Bcl-XL, and Bax which are genes related to apoptosis using the quantitative RT-PCR, change of mitochondria membrane permeability and membrane potential via flow cytometry, the cycle of cell mitosis, caspase cleavage and annexin V staining were examined. Results: 1. showed significant killing effect on stomach cancer cell than the control group with a time(6 hours later) and density dependent manner, which was statistical significance. 2. Extract of Curcuma longa L. showed suppressive effect on viability of stomach cancer cells that each test groups had more suppressive effects on viability of stomach cancer cells than the control group with a time(6 hours later), which was statistical significance.(p<0.05) 3. In the test about the revelation of genes related to apoptosis, the revelation of Bcl-2 and Bcl-XL decreased with a density manner which was statistical significance. but the revelation of Bax was not changed with statistical significance. 4. Extract of Curcuma longa L. caused apoptosis by decreasing the absorbance of mitochondria with statistical significance, and also induced apoptosis by decreasing the membrane potential of mitochondria. 5. Extract of Curcuma longa L. destructed the cell cycle of cell mitosos. 6. Cell apoptosis was induced by extract of Curcuma longa L. certificated by method of caspase cleavage and annexin V staining. Conclusion: This experiment showed that Curcuma longa L. has anti-tumor effect with statistical significance. This is in vitro experiment and basic experiment on Curcuma longa L.. We hope more progressive research on Curcuma longa L. will go on and its anti-tumor effects will be more practically identified.

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The Effects of Duchesnea india(Audra.) Foche. on Human Stomach Cancer Cells (사매가 위암세포에 미치는 영향)

  • Ryu Bong-Ha;Kim Jin-Sung;Yoon Sang-Hyub;Ryu Ki-Won;Hong Sang-Sun
    • The Journal of Internal Korean Medicine
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    • v.24 no.2
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    • pp.290-299
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    • 2003
  • Background : Nowadays many researches about it s cure are going on world widely since cancer is one of the most human health threatening diseases. In Chinese and North Korean medicine, Duchesnea india(Audra.) Foche. is practically used to treat many kinds of cancer, but in Korea it is rarely used. So, we need to scientifically identify anti-tumor effects of Duchesnea india(Audra.) Foche. Objective : We are aimed to identify anti-tumor effects of Duchesnea india(Audra.) Foche. on the stomach cancer cells through molecular biologic methods. Material & Methods : We used AGS as stomach cancer cells from American Type Culture Collection. We added the boiled extract of Duchesnea india(Audra.) Foche. $5{\mu}l$(Sample I), $10{\mu}l$(Sample II) to cultural media(ml)for 0,6, 12, 24, 48 hours. We measured the killing effect on stomach cancer cells through Tryphan blue exclusion test and the suppressive effect on viability of stomach cancer cells via MTT assay. the quantitative RT-PCR was used to examine their effect on the revelation of Bcl-2, Bcl-XL, and Bax, which are genes related to apoptosis. We measured change of mitochondria membrane permeability and membrane potential via flow cytometry. Result : 1. The killing effect on stomach cancer cells showed that each test groups killed more stomach cancer cells than the control group with a time(6 hours later) and density dependent manner, which was statistical significance. 2. The suppressive effect on viability of stomach cancer cells showed that each test groups had more suppressive effects on viability of stomach cancer cells than the control group with a time(6 hours later), which was statistical significance. 3. In the test about the revelation of genes related to apoptosis, the revelation of Bcl-2 and Bcl-XL decreased with a density manner which was statistical significance. but the revelation of Bax was not changed with statistical significance. 4. As a result of this test, Duchesnea india(Audra.) Foche. caused apoptosis by decreasing the absorbance of mitochondria with statistical significance. and also induced apoptosis by decreasing the membrane potential of mitochondria. Conclusion : This experiment showed that Duchesnea india(Audra.) Foche. has anti-tumor effect with statistical significance. This is in vitro experiment and basic experiment on Duchesnea india(Audra.) Foche. We hope more progressive researchs on Duchesnea india(Audra.) Foche. will go on and its anti-tumor effects will be more practically identified.

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Biophysical Evaluation of Radiosensitization by AuNPs Nanoparticles Irradiated Photon beam (photon빔 조사 후 AuNPs 입자의 방사선 감수성 향상에 관한 생물물리학적 평가)

  • Choi, Eunae;Son, Jaeman
    • Journal of the Korean Society of Radiology
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    • v.10 no.7
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    • pp.483-487
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    • 2016
  • The purpose of this study is to investigate whether gold nanoparticles had radiosensitization when combined with gamma and x ray beam. Cytotoxicity was mearsured with comparing survival fraction after incubated time 6,12,18 and 24 hours. Clonogenic assay was employed to assess survival fraction of cells with and without gold nanoparticles treatment following gamma ray irradiation. The most of gold nanoparticles were distributed in the cytoplasm. And the toxicity of gold nanoparticles used this study were found to be non-cytotoxic. And we also observed enhancement by about 40% in RBE value for gamma ray irradiation of cells treated with gold nanoparticles. Dose reduction of about half for gamma ray irradiation is demonstrated for gold nanoparticles treated cells as compared to untreated cells. In cells with exposed to gamma ray, DNA damage was increased when compared to only radiation exposed cells. The study revealed a significant reduction in radiation dose for killing the cells with internalized gold nanoparticles as compared to the cells without gold nanoparticles. The gold nanoparticles treatment resulted in enhancement of radiation effect as evident from increase in relative biological effectiveness values for photon irradiated cells.

Identification of duck liver-expressed antimicrobial peptide 2 and characterization of its bactericidal activity

  • Hong, Yeojin;Truong, Anh Duc;Lee, Janggeun;Lee, Kyungbaek;Kim, Geun-Bae;Heo, Kang-Nyeong;Lillehoj, Hyun S.;Hong, Yeong Ho
    • Asian-Australasian Journal of Animal Sciences
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    • v.32 no.7
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    • pp.1052-1061
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    • 2019
  • Objective: This study was conducted to identify duck liver-expressed antimicrobial peptide 2 (LEAP-2) and demonstrate its antimicrobial activity against various pathogens. Methods: Tissue samples were collected from 6 to 8-week-old Pekin ducks (Anas platyrhynchos domesticus), total RNA was extracted, and cDNA was synthesized. To confirm the duck LEAP-2 transcript expression levels, quantitative real-time polymerase chain reaction was conducted. Two kinds of peptides (a linear peptide and a disulfide-type peptide) were synthesized to compare the antimicrobial activity. Then, antimicrobial activity assay and fluorescence microscopic analysis were conducted to demonstrate duck LEAP-2 bactericidal activity. Results: The duck LEAP-2 peptide sequence showed high identity with those of other avian species (>85%), as well as more than 55% of identity with mammalian sequences. LEAP-2 mRNA was highly expressed in the liver with duodenum next, and then followed by lung, spleen, bursa and jejunum and was the lowest in the muscle. Both of LEAP-2 peptides efficiently killed bacteria, although the disulfide-type LEAP-2 showed more powerful bactericidal activity. Also, gram-positive bacteria was more susceptible to duck LEAP-2 than gram-negative bacteria. Using microscopy, we confirmed that LEAP-2 peptides could kill bacteria by disrupting the bacterial cell envelope. Conclusion: Duck LEAP-2 showed its antimicrobial activity against both gram-positive and gram-negative bacteria. Disulfide bonds were important for the powerful killing effect by disrupting the bacterial cell envelope. Therefore, duck LEAP-2 can be used for effective antibiotics alternatives.