• Toxicological Research
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• 제32권1호
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• pp.81-87
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• 2016

Aflatoxin B1 (AFB1) is produced by Aspergillus flavus growing in feedstuffs. Early detection of maize contamination by aflatoxigenic fungi is advantageous since aflatoxins exert adverse health effects. In this study, we report the development of an optimized conventional PCR for AFB1 detection and a rapid, sensitive and simple screening Real-time PCR (qPCR) with SYBR Green and two pairs of primers targeting the aflR genes which involved aflatoxin biosynthesis. AFB1 contaminated maize samples were divided into three groups by the toxin concentration. Genomic DNA was extracted from those samples. The target genes for A. flavus were tested by conventional PCR and the PCR products were analyzed by electrophoresis. A conventional PCR was carried out as nested PCR to verify the gene amplicon sizes. PCR-RFLP patterns, obtained with Hinc II and Pvu II enzyme analysis showed the differences to distinguish aflatoxin-producing fungi. However, they are not quantitative and need a separation of the products on gel and their visualization under UV light. On the other hand, qPCR facilitates the monitoring of the reaction as it progresses. It does not require post-PCR handling, which reduces the risk of cross-contamination and handling errors. It results in a much faster throughout. We found that the optimal primer annealing temperature was $65^{\circ}C$. The optimized template and primer concentration were $1.5{\mu}L\;(50ng/{\mu}L)$ and $3{\mu}L\;(10{\mu}M/{\mu}L)$ respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at $88.0^{\circ}C$, $87.5^{\circ}C$, $83.5^{\circ}C$, and $89.5^{\circ}C$ respectively. Consequently, it was found that the four primers had elevated annealing temperatures, nevertheless it is desirable since it enhances the DNA binding specificity of the dye. New qPCR protocol could be employed for the determination of aflatoxin content in feedstuff samples.

• Biomolecules & Therapeutics
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• 제23권5호
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• pp.486-492
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• 2015

Drug metabolism mostly occurs in the liver. Cytochrome P450 (CYP) is a drug-metabolizing enzyme that is responsible for many important drug metabolism reactions. Recently, the US FDA and EU EMA have suggested that CYP enzyme induction can be measured by both enzymatic activity and mRNA expression. However, these experiments are time-consuming and their interassay variability can lead to misinterpretations of the results. To resolve these problems and establish a more powerful method to measure CYP induction, we determined CYP induction by using luminescent assay. Luminescent CYP assays link CYP enzyme activity to firefly luciferase luminescence technology. In this study, we measured the induction of CYP isozymes (1A2, 2B6, 2C9, and 3A4) in cryopreserved human hepatocytes (HMC424, 478, and 493) using a luminometer. We then examined the potential induction abilities (unknown so far) of mesalazine, a drug for colitis, and mosapride citrate, which is used as an antispasmodic drug. The results showed that mesalazine promotes CYP2B6 and 3A4 activities, while mosapride citrate promotes CYP1A2, 2B6, and 3A4 activities. Luminescent CYP assays offer rapid and safe advantages over LC-MS/MS and qRT-PCR methods. Furthermore, luminescent CYP assays decrease the interference between the optical properties of the test compound and the CYP substrates. Therefore, luminescent CYP assays are less labor intensive, rapid, and can be used as robust tools for high-throughput CYP screening during early drug discovery.

• 식물조직배양학회지
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• 제25권5호
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• pp.341-346
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• 1998

색소유전자의 전이를 통하여 새로운 색소발현체계를 가진 품종을 육종하기 위한 기초연구로 stock (Matthiola incana R. Br.)의 꽃봉오리로부터 cDNA-library를 합성하였고 screening을 통하여 anthocyanin 합성경로의 중요효소의 하나인 DFR (dihydroflavonol 4-reductase) 유전자를 분리하였다. 염기서열분석을 수행하여 분리유전자의 크기가 1450bp 이며 이중 coding region은 1029 bp 임을 확인하였다. 이미 밝혀진 다른 식물체의 DFR 유전자와 서로 염기서열의 일치성을 비교해 본 결과 외자엽식물인 옥수수와 보리와는 각각 61%를 보였으며, 쌍자엽식물인 페튜니아, 금어초, 거베라, 과꽃 그리고 카네이션 등 과는 66%-67%의 일치성을 나타내었다. 아울러 염기서열의 G/C 함량분석을 통하여 쌍자엽식물의 G/C 함량은 외자엽식물의 그것에 비해 매우 낮은 수치를 나타내었다. 분리유전자의 발현을 확인하기 위하여 인위적으로 기내에서의 전사와 해석을 수행한 결과 42-44 kd 크기의 단백질을 확인하였다. Southern blot 분석의 결과 DFR 유전자는 stock의 genome에 다른 대부분의 식물체와 유사하게 한 개가 존재하며 야생종과 돌연변이종의 stock을 분리 DFR 유전자를 probe 로 Northern blot 분석을 수행하여 돌연변이종인 lineK17b가 DFR 돌연변이임을 확인하였다.

• Journal of Plant Biotechnology
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• 제38권2호
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• pp.162-168
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• 2011

고염 스트레스는 식물의 성장과 수확량에 치명적인 영향을 야기한다. 그와 같은, 환경 스트레스에 의하여 식물은 다양한 유전자의 발현으로 저항성을 가지게 하는 기작이 발달되어 있다. 본 연구에서는 애기장대에서 다양한 환경 스트레스에 관여하는 유전자를 분리할 목적으로 GGM(Graphical Gaussian Model) program을 사용한 후, BLH8(BEL1-Like Homeodomain Gene 8) 유전자의 돌연변이 식물체를 구축하였다. atblh8-1 돌연변이체는 고농도의 $Na^+$과 $K^+$ 이온에 특이적으로 백화현상을 보이지만, 뿌리 성장에는 변화를 보이지 않았다. 그러므로, BLH8 단백질은 $Na^+$과 $K^+$과 같은 환경스트레스 저항성에 관여하는 중요한 인자임을 시사한다. 이와 같이, GGM program은 환경 스트레스에 관여하는 유전자를 분리하기 위한 유용한 도구일 것으로 사려된다.

• 대한수의학회지
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• 제32권4호
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• pp.641-647
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• 1992

This study was conducted to determine the serum antibodies against toxoplasma in the artificially infected dogs, pet and street dogs by latex agglutination (LA) and indirect fluorescent antibody (IFA) test. LA test was carried out with commercial Toxo-MT kit (Eiken chemical Co.) and IFA test was carried out with rabbit-anti-dog IgG labelled with FITC (Cappel Co.) and toxo-antigen slides prepared in laboratory. The results obtained were as follows ; 1. Antibodies to Toxoplasma gondii in the artificially infected dogs were detected firstly at the Day 8 in IFA and Day 9 in LA test after inoculation. Positive antibody reactions by these tests were declined gradually afterward but maintained up to 12 weeks. 2. In LA test serum antibody titers in 310 test sera were shown as 10 cases(32%) in 1 : 32.5(1.0%) in 1 : 64, 4(1.3%) in 1 : 128 and 2(0.7%) in 1 : 256. In IFA test serum antibody titers 310 test sera were shown as 17 cases(5.5%) in 1 : 64, 8(2.6%) in 1 : 128 and 5(1.6%) in 1 : 256. 3. In the total of 310 sera from pet and street dogs toxoplasma antibody positive rates were 21 cases (6.8%) by LA and 30 cases (9.7%) by IFA test and the positive detection rates between these two groups by LA and IFA test were not significant(p<0.05). 4. In the total of 115 sera from pet dogs toxoplasma antibody positive rates were 12 cases(10.4%) by LA and 15(13.0%) by IFA test. And in the 195 street dogs the positive rates were 9 cases(4.6%) by LA and 15(7.7%) by IFA test. Also, the positive detection rates between these two groups by LA and IFA test were not significant(p<0.05). 5. Agreement of reactivity between LA and IFA test for 310 sera was 91.3% in total of 283 cases consisting of 12 cases(3.9%) of both LA and IFA positive and 271 cases(87.4%) of LA and IFA negative. 6. LA test was almostly equivalent to the IFA test in producibility and proved to be a simple tool for the screening of toxoplasma antibody in laboratory.

• 한국약용작물학회지
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• 제6권4호
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• pp.277-285
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• 1998

50종의 약용식물 추출액을 공시하여 생체외(in vitro) 및 생체내 (in vivo)에서 몇 가지 주요 종자전염성병 억제를 위한 항균활성식물을 선발하고 선발된 식물에 대한 항균활성을 검정한 결과는 다음과 같다. 가) 공시된 50종의 약용식물추출액을 공시병원균(벼도열병균, 참깨검은무늬병균, 고추탄저병균, 무·배추검은무늬병균)을 접종한 종자에 대한 항균활성을 습지법과 물한천법으로 정정한 결과 마늘, 주목, 대황, 무슬, 감초, 달맞이꽃 등에서 항균활성이 우수한 것으로 나타났다. 나) 1차 선발된 식물추출액을 종자에 희석 처리하여 감자한천법으로 검정한 결과, 마늘 추출액이 10배에서도 항균활성을 보였고 종자침적 시간에 따른 효과를 보면 긴 시간 침적이 가장 높은 항균활성을 나타냈으며 종자침적온도에 따른 항균효과를 보면 $25^{\circ}C$ 처리가 대체적으로 우수했다. 다) 식물 추출액의 종자발아및 초기생육에 미치는 영향은 마늘 추출액의 경우 무처리에 비하여 종자발아에 차이가 없었고 초기생육은 오히려 양호하였다. 주목의 추출액은 무와 배추에서 약간의 영향이 있었고 우슬, 감초, 달맞이꽃 등에서는 종자 발아 및 초기 생육을 심하게 저해했다.

• 한국약용작물학회지
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• 제10권2호
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• pp.109-115
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• 2002

황칠나무 잎의 추출물의 면역활성증진 실험에서 인간정상 간세포의 경우 모든 추출물이 1.0 Mg/ml의 농도에서 최고 26%이하의 세포독성을 나타내었다. 서로 다른 4가지의 암세포주(MCF7, A549, Hep3B, AGS)에서 50%이상의 저해율을 나타냈고, 정상 세포의 결과와 암세포의 저해율을 비로 나타낸 selectivity의 측정에서 모든 암세포주가 1.5이상의 사멸도를 나타내었고 전체적으로 에탄올 추출물의 효과가 가장 좋았다. 에탄올 추출물의 경우에서 인감 유방암 세포주(MCF7)와 인간 간암세포주(Hep3B)의 경우에서 1.0 mg/ml농도에서 각각 65%와 67%의 저해율을 기록했다. 면역세포 실험에서 에탄올 추출물이 1.0 mg/ml의 농도에서 B세포는 1.22배, T세포는 1.27배의 촉진 활성을 보였고, 6일 동안측정한 $cytokines(IL-6,\;TNF-{\alpha})$의 양도 에탄올 추출물의 경우 T cell의 경우 IL-6은 94pg/m1, $TNF-{\alpha}$은 75pg/ml로 증가하는 것을 알 수 있었다. 이상의 결과로 볼 때 추출 열과 추출용매 등에 의해 황칠나무 잎에 존재하는 여러 가지 유용성분들이 풍부하게 존재하며, crude추출물 중에 에탄올 추출물이 면역활성에서 좋은 효과를 보였다는 것을 알 수가 있었고, 이를 통해서 황칠나무 잎을 이용한 기능성 식품으로의 연구 개발이 통해서 충분히 그 가치가 있다는 것을 알 수 있었다.

• 한국약용작물학회지
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• 제11권2호
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• pp.161-166
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• 2003

순계 분리된 인삼의 우수 계통으로부터 염류내성 계통을 선발하기 위하여 배배양으로부터 유기된 인삼 개체를 염류 종류와 농도별 차이에 따라 엽병의 생장율과 생존율을 조사한 결과 N, P, K 그리고 Na와 Fe의 복합처리구에서는 10배 이상의 농도로 첨가된 처리구 모두에서 엽병의 생장과 개체의 생존율이 전혀 이뤄지지 않는 것으로 나타났으나 K 처리구에서만 다수의 생존개체를 확인할 수 있었다. 또한 Ca 10배 처리구와 Mg 20배 처리구까지는 비교적 엽병의 생장과 개체의 생존이 유지되는 것으로 나타났다. 7가지 염류 모두를 복합처리한 결과에서도 1.25와 2.5배 처리구에서만 약간의 생장을 보였을 뿐 5배 이상 처리한 고농도에서는 생장과 생존을 모두 불가능한 것으로 나타나 인삼의 염류내성 계통을 선발하기 위한 종합염류 농도는 2.5배 수준으로 처리하는 것이 적절하리라 사료된다.

• 한국산업보건학회지
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• 제30권2호
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• pp.205-212
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• 2020

Objectives: The purpose of this study was to identify the needs and improvement measures of the risk assessment system through a survey of asbestos abatement companies. Methods: This study prepared a questionnaire that includes improvement measures for the risk assessment system(six questions) and the necessity of a risk assessment system for asbestos abatement(one question). The questionnaire was sent to 2,170 asbestos abatement companies and returned by 83 companies(return rate = 3.8%). We conducted frequency analysis, ANOVA, and Chi-squared testing at the 5% significance level. Results: This study analyzed the survey results and identified six main opinions on the risk assessment system and improvement measures. First, giving an advantage to companies with a high grade(S and A grades) in public bidding and/or qualification screening showed a similar preference(agree: 50.6%, disagree: 49.4%). Second, 57.6% of the respondents wanted to allow air showers along with water showers for low risk asbestos work. However, 23.2% of the respondents asserted that only a water shower should be allowed since there is no scientific evidence supporting the removal of asbestos by air shower. Third, in order to prevent missing the enrollment of workers, simply submitting a change report should be allowed when any worker is changed (40.0%). Fourth, 43% of the respondents answered that they did not know how to calculate the proper number of negative pressure units. The reasons given were a lack of guidelines or standards(38.9%), inconvenience (36.1%), and absence of education(25%). Fifth, the respondents who were favorable toward the necessity of a risk assessment system gave higher scores compared to unfavorable respondents on the necessity of the management of workers' work history(favorable respondent = 3.96 out of 5, unfavorable respondent = 2.68) and the necessity of professional training for workers(favorable respondent = 3.71, unfavorable respondent = 2.56). Finally, the respondents favorable toward a risk assessment system showed higher scores on the awareness of calculating the proper number of negative pressure units(4.79) compared to unfavorable respondents(3.3). Conclusions: The opinions of asbestos abatement companies identified through the survey in this study can be usefully utilized as fundamental information to improve the risk assessment system.

• Journal of Microbiology
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• 제44권4호
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• pp.423-431
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• 2006

Among 53 Acinetobacter baumannii isolates collected in 2004, nine imipenem-resistant isolates were obtained from clinical specimens taken from patients hospitalized in Busan, Korea. Nine carbapenemase-producing isolates were further investigated in order to determine the mechanisms underlying resistance. These isolates were then analyzed via antibiotic susceptibility testing, microbiological tests of carbapenemase activity, pI determination, transconjugation test, enterobacterial repetitive consensus (ERIC)-PCR, and DNA sequencing. One outbreak involved seven cases of infection by A. baumannii producing OXA-23 ${\beta}-lactamase$, and was found to have been caused by a single ERIC-PCR clone. During the study period, the other outbreak involved two cases of infection by A. baumannii producing IMP-1 ${\beta}-lactamase$. The two clones, one from each of the outbreaks, were characterized via a modified cloverleaf synergy test and an EDTA-disk synergy test. The isoelectric focusing of the crude bacterial extracts detected nitrocefin-positive bands with pI values of 6.65 (OXA-23) and 9.0 (IMP-1). The PCR amplification and characterization of the amplicons via direct sequencing showed that the clonal isolates harbored $bla_{IMP-1}$ or $bla_{oxA-23}$ determinants. The two clones were characterized by a multidrug resistance phenotype that remained unaltered throughout the outbreak. This resistance encompassed penicillins, extended-spectrum cephalosporins, carbapenems, monobactams, and aminoglycosides. These results appear to show that the imipenem resistance observed among nine Korean A. baumannii isolates could be attributed to the spread of an IMP-lor OXA-23-producing clone. Our microbiological test of carbapenemase activity is a simple method for the screening of clinical isolates producing class D carbapenemase and/or class B $metallo-{\beta}-lactamase$, in order both to determine their clinical impact and to prevent further spread.