• Environmental Analysis Health and Toxicology
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• 제14권1_2호
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• pp.1-12
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• 1999

Recently, several new methods for the detection of genetic damages in vitro and in vivo based on molecular biological techniques were introduced according to the rapid progress in toxicology combined with cellular and molecular biology. Among these methods, mouse lymphoma thymidine kanase (tk) gene forward mutation assay, single cell gel electrophoresis (comet assay) and transgenic animal and cell line model as a target gene of lac I (Big Blue) and lac Z (Muta Mouse) gene mutation are newly introduced based on molecular toxicological approaches. The mouse lymphoma tk$\^$+/-/ gene assay (MOLY) using L5178Y tk$\^$+/-/ mouse lymphoma cell line is one of the mammalian forward mutation assays, and has many advantages and more sensitive than hprt assay. The target gene of MOLY is a heterozygous tk$\^$+/-/ gene located in 11 chromosome, so it is able to detect the wide range of genetic changes like point mutation, deletion, rearrangement, and mitotic recombination within tk gene or deletion of entire chromosome 11. The comet assay is a rapid, simple, visual and sensitive technique for measuring and analysing DNA breakages in mammalian cells, Also, transgenic animal and cell line models, which have exogenous DNA incorporated into their genome, carry recoverable shuttle vector containing reporter genes to assess endogenous effects or alteration in specific genes related to disease process, are powerful tools to study the mechanism of mutation in vivo and in vitro, respectively. Also in vivo acridine orange supravital staining micronucleus assay by using mouse peripheral reticulocytes was introduced as an alternative of bone marrow micronucleus assay. In this respect, there was an International workshop on genotoxicity procedure (IWGTP) supported by OECD and EMS (Environmental Mutagen Society) at Washington D. C. in March 25-26, 1999. The objective of IWGTP is to harmonize the testing procedures internationally, and to extend to finalization of OECD guideline, and to the agreement of new guidelines under the International Conference of Harmonization (ICH) for these methods mentioned above. Therefore, we introduce and review the principle, detailed procedure, and application of MOLY, comet assay, transgenic mutagenesis assay and supravital staining micronucleus assay.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2003년도 추계국제학술대회
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• pp.159-159
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• 2003

Comet assay is a potential monitoring tool because DNA strand breakage may be produced by a wide range of agents. The comet assay, also called the single-cell gell electrophoresis (SCGE) assay, is rapid and sensitive method for the detection of DNA damage in cells. This study was performed for the identification of DNA damage in the cells from flounders and clams exposed to PAHs. As a control experiments, flounder and clam cells were exposed to $H_2O$$_2$. The cells exposed to $H_2O$$_2$ were displayed a typical nuclei movement DNA damage of cells were significantly increased when the isolated cells from the blood of flounders and the tissue of clams were in vitro exposed to the different concentrations (5, 10, 50, 100 ppb) of five kinds of PAHs (benzo[a]pyrene, pyrene, fluoranthene, anthrancene, and phenanthrene). For the in vivo test, flounders and clams were exposed to the different concentrations of BaP for 4 days. The results showed that DNA strand breakage was effected by the concentration of BaP and the duration of exposure. In high concentration of BaP, the mean tail lengths of nuclei was longer than it In low concentration, while the mean size of head DNA decreased. In this research, both in vitro and in vivo genotoxicity of PAHs could be biomonitored by the comet assay. Especially, clams and flounders seem to be useful as materials for monitoring genotoxic damage by comet assay.

• Clinical and Experimental Reproductive Medicine
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• 제8권1호
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• pp.1-12
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• 1981

면역학적인 방법을 사용하여 가돈 여포액내의 특이단백질의 존재여부와 이들을 추출하여 난자성숙에 미치는 영향을 조사한 결과 다음과 같았다. 1. 가돈 혈청 및 혈장에는 존재하지 않는 면역학적으로 특이한 두 단백질이 가돈 여포액내에서 검출되었다. 2. 이들 두 단백질은 전기영동적으로 각각 fast alpha-I globulin 과 beta-globulin 의 이동성을 나타냈다. 3. 이들 두 단백질은 생쥐 여포난자의 성숙을 억제시켰다. 본 실험의 결과로 미루어 가돈 여포액내에는 난자성숙을 억제시키는 특이단백질이 존재하는 것으로 사료된다.

• Journal of Ginseng Research
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• 제45권4호
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• pp.519-526
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• 2021

Background: This study aimed to investigate the effect of cold plasma treatment on the improvement of seed germination and surface sterilization of ginseng seeds. Methods: Dehisced ginseng (Panax ginseng) seeds were exposed to dielectric barrier discharge (DBD) plasma operated in argon (Ar) or an argon/oxygen mixture (Ar/O₂), and the resulting germination and surface sterilization were compared with those of an untreated control group. Bacterial and fungal detection assays were performed for plasma-treated ginseng seeds after serial dilution of surface-washed suspensions. The microbial colonies (fungi and bacteria) were classified according to their phenotypical morphologies and identified by molecular analysis. Furthermore, the effect of cold plasma treatment on the in vitro antifungal activity and suppression of Cylindrocarpon destructans in 4-year-old ginseng root discs was investigated. Results: Seeds treated with plasma in Ar or Ar/O₂ exhibited a higher germination rate (%) compared with the untreated controls. Furthermore, the plasma treatment exhibited bactericidal and fungicidal effects on the seed surface, and the latter effect was stronger than the former. In addition, plasma treatment exhibited in vitro antifungal activity against C. destructans and reduced the disease severity (%) of root rot in 4-year-old ginseng root discs. The results demonstrate the stimulatory effect of plasma treatment on seed germination, surface sterilization, and root rot disease suppression in ginseng. Conclusion: The results of this study indicate that the cold plasma treatment can suppress the microbial community on the seed surface root rot in ginseng.

• Parasites, Hosts and Diseases
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• 제58권2호
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• pp.173-179
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• 2020

Leishmaniasis is a prevalent cause of death and animal morbidity in underdeveloped countries of endemic area. However, there is few vaccine and effective drugs. Antimicrobial peptides are involved in the innate immune response in many organisms and are being developed as novel drugs against parasitic infections. In the present study, we synthesized a 5-amino acid peptide REDLK, which mutated the C-terminus of Pseudomonas exotoxin, to identify its effect on the Leishmania tarentolae. Promastigotes were incubated with different concentration of REDLK peptide, and the viability of parasite was assessed using MTT and Trypan blue dye. Morphologic damage of Leishmania was analyzed by light and electron microscopy. Cellular apoptosis was observed using the annexin V-FITC/PI apoptosis detection kit, mitochondrial membrane potential assay kit and flow cytometry. Our results showed that Leishmania tarentolae was susceptible to REDLK in a dose-dependent manner, disrupt the surface membrane integrity and caused parasite apoptosis. In our study, we demonstrated the leishmanicidal activity of an antimicrobial peptide REDLK from Pseudomonas aeruginosa against Leishmania tarentolae in vitro and present a foundation for further research of anti-leishmanial drugs.

• 생명과학회지
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• 제14권1호
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• pp.26-31
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• 2004

본 연구는 redox regulator로 알려 진 Ref-1 (Redox factor-1)과 결합하는 새로운 단백질을protein-protein interaction의 원리를 이용한 방법인 yeast two-hybrid assay로 검색, 동정하고, 검색된 단백질의 in vitro, in vivo 기능을 규명하는 데 그 목적을 두고, mouse 11-day Embryo cNA library를 prey로, full length REF-1을 bait로 하여 yeast strain 인 HF7C에 cotransformatiom시킨 후 histidine, leucine, tryptophan이 결핍된 SD plate에서 키워 자란 yeast transformants를 $\beta$-galactosidaseassay하여 screening하여 분리한 세 개의 clone중 한 clone이 DNA sequencing으로 확인한 결과 mouse thioredoxin임을 확인하였다.

• 농약과학회지
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• 제3권2호
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• pp.37-46
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• 1999

침투성 살충제 carbofuran의 작물체중 잔류성과 그 안전성을 평가하기 위하여 carbofuran을 살포했을 것으로 간주되는 감자와 땅콩 및 마늘 시료를 주산지 부근의 대규모 시장에서 수집한 후 carbofuran과 carbofuran의 식물체중의 주 대사산물로 보고된 3-hydroxycarbofuran의 잔류량을 조사하고 carbofuran의 각 작물체내 대사 양상을 구명하기 위하여 각 작물의 phesphate buffer 추출액중 carbofuran의 분해실험을 수행하였다. 여러 지역의 시장으로부터 수집한 총 20 점의 완숙마늘 시료중 2점 (M-12와 M-16)의 시료에서 0.13과 0.07mg/kg의 carbofuran이 검출되어 10%의 검출빈도를 보였으나, 그 잔류량은 잔류허용기준인 0.5 mg/kg 보다 훨씬 작았다. 3-Hydroxycarbofuran은 총 20점의 시료중 1점 (M-12)의 시료에서 0.13 mg/kg이 검출되어 5%의 검출빈도를 보였으며, 풋마늘과 감자 및 땅콩 시료중 carbofuran과 3-hyoxycarbofuran의 잔류량은 분석결과 모든 시료에서 검출한계 미만이었다. 완숙마늘의 1일 섭취량에 근거한 carbofuran의 최대 섭취추정량은 0.0013mg으로 1일 최대섭취허용량인 0.55 mg의 0.24%이었으며, 마늘과 감자 및 땅콩시료에서 carbofuran이 검출되지 않아 마늘과 감자 및 땅콩 경작시 carbofuran을 사용하여도 생산물은 안전한 것으로 평가되었다. Carbofuran은 각 작물의 phosphate buffer 추출액중에서 주로 가수분해에 의하여 주 대사산물인 carbofuran phenol (m/z 164)을 생성하였으며, 그 양은 배양기간에 비례하여 증가하였다.

• KSBB Journal
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• 제22권2호
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• pp.67-72
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• 2007

형광을 이용한 암 진단을 위해 배양된 정상 폐세포 및 폐암 세포주에 광민감제인 5-ALA를 투여하고 세포 내 외에서 생성된 protoporphyrin IX (PpIX)의 형광을 측정하여 5-ALA 투여의 최적 농도를 조사하였다. 정상 폐세포주 (Hel299) 및 폐암 세포주 (A549, NCI-H460)에 5-ALA를 $0\sim800{\mu}g/mL$ 농도별로 투여하여 24시간 동안 배양한 다음 MTT assay로 세포증식 저해율 및 이때 생성되는 PpIX의 양을 형광의 강도로 측정하였다. 그 결과 Hel299 및 A549에서는 5-ALA의 처리농도가 증가할수록 세포 증식의 저해율이 증가하였으나 NCI-H460에서는 세포 증식이 저해되지 않았다. 그리고 폐암세포인 A549와 NCI-H460에 대한 5-ALA의 최적농도는 $100{\mu}g/mL$이며, 이때의 형광 (emission) 스펙트럼은 여기 파장이 410 nm일 때 세포 외에서는 615.8 nm와 660.8 nm, 616.7 nm와 660.2 nm, 세포 내에서는 603.2 nm와 661.4 nm, 603.5 nm와 661.4 nm에서 각각 형광 봉우리가 관찰되었다. 또한 PpIX를 형광 강도로 측정하면, PpIX는 정상세포에서는 낮은 농도로 축적이 되는 반면에 암세포에서 높은 농도로 축적되었으며, 세포 외보다는 세포 내에서 더 높은 농도로 축적됨을 알 수 있었다.

• Biomaterials and Biomechanics in Bioengineering
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• 제3권3호
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• pp.129-140
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• 2016

This article reports the development of biodegradable photoluminescent polymer (BPLP)-based nanoparticles (NPs) incorporating either magnetic nanoparticles (BPLP-MNPs) or gadopentate dimeglumine (BPLP-Gd NPs), for cancer diagnosis and treatment. The aim of the study is to compare these nanoparticles in terms of their surface properties, fluorescence intensities, MR imaging capabilities, and in vitro characteristics to choose the most promising dual-imaging nanoprobe. Results indicate that BPLP-MNPs and BPLP-Gd NPs had a size of $195{\pm}43nm$ and $161{\pm}55nm$, respectively and showed good stability in DI water and 10% serum for 5 days. BPLP-Gd NPs showed similar fluorescence as the original BPLP materials under UV light, whereas BPLP-MNPs showed comparatively less fluorescence. VSM and MRI confirmed that the NPs retained their magnetic properties following encapsulation within BPLP. Further, in vitro studies using HPV-7 immortalized prostate epithelial cells and human dermal fibroblasts (HDFs) showed > 70% cell viability up to $100{\mu}g/ml$ NP concentration. Dose-dependent uptake of both types of NPs by PC3 and LNCaP prostate cancer cells was also observed. Thus, our results indicate that BPLP-Gd NPs would be more appropriate for use as a dual-imaging probe as the contrast agent does not mask the fluorescence of the polymer. Future studies would involve in vivo imaging following administration of BPLP-Gd NPs for biomedical applications including cancer detection.

• 한국약용작물학회지
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• 제12권6호
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• pp.448-453
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• 2004

Transformed roots of Schisandra chinensis were obtained following co-cultivation of in vitro cultivated plantlet segments with Agrobaterium rhizogens ATCC15834. This root was examined for its growth and gomisin J contents under various culture conditions. Among the six basal culture media tested, WPM (Lloyd & McCown, 1980) medium supplemented with 5% sucrose was the best roots growth 6.2 (g D.W/flask) and gomisin J accumulation 1.56 $(X10^{-3}\;ug/g\;D.W)$. Initial inoculum size correlated with the yield of biomass while gomisin J contents was not affect. Gomisin J production was influenced by the initial sucrose concentration and the highest production yield was achieved at the concentration of 7%. The optimal shaking speeds for roots growth and gomisin J production was 120 and 140 rpm, respectively.