• BMB Reports
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• 제43권2호
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• pp.110-114
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• 2010

The yeast three-hybrid system (Y3H), a powerful method for identifying RNA-binding proteins, still suffers from many false positives, due mostly to RNA-independent interactions. In this study, we attempted to efficiently identify false positives by introducing a tetracycline operator (tetO) motif into the RPR1 promoter of an RNA hybrid expression vector. We successfully developed a tight tetracycline-regulatable RPR1 promoter variant containing a single tetO motif between the transcription start site and the A-box sequence of the RPR1 promoter. Expression from this tetracycline-regulatable RPR1 promoter in the presence of tetracycline-response transcription activator (tTA) was positively controlled by doxycycline (Dox), a derivative of tetracycline. This on-off control runs opposite to the general knowledge that Dox negatively regulates tTA. This positively controlled RPR1 promoter system can therefore efficiently eliminate RNA-independent false positives commonly observed in the Y3H system by directly monitoring RNA hybrid expression.

• Journal of Microbiology and Biotechnology
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• 제12권3호
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• pp.514-517
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• 2002

To investigate oleic acid biodegradation, 47 fungal strains were tested with modified Czapek Dox broth media containing oleic acid, and their biodegradative activities were assayed by measuring the release of $[^14C]CO_2$ from the $^14C-$labeled oleic acid. After 72 h of cultivation, Aspergillus flavus, Aspergillus ochraceus, and Alternaria species metabolized approximately $25\%\;to\;35\%$ of the supplied oleic acid. The relationship between the fungal degradation of oleic acid and the fungal growth was also examined using 7 strains of Aspergillus niger. A. niger. YMC 0100 and YMC 0322 degraded about $26\%$ of the oleic acid after 72 h, while their germination ratios were more than $30\%$.

• Applied Biological Chemistry
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• 제27권3호
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• pp.151-157
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• 1984

Penicillium islandicum은 Czapek-Dox 배지를 이용하여 어두운 곳에서 배양되었을때 황적색 색소인 skyrin을 생합성 하였다. $[^{14}C]$-acetate 배지에 이 균주를 접종 배양하여 얻어진 대사산물로부터 radioactive skyrin을 순수 정제 분리할 수 있었고 이를 sodium dithionite 처리하여 Emodin이 합성되는 것을 동정할 수 있었다. 한편, Penicillium islandicum의 배양액에서 추출 정제된 효소를 이용한 균체의 효소실험에서 $[^3H]$-emodin과 $[^3H]$-emodin은 skyrin생합성의 유력한 전구체물질임이 밝혀졌으며, 효소반응결과 $[^3H]$ emodinanthrone(4.8%)이 $[^3H]$-emodin보다 radioactive skyrin으로 주입되는 비율이 더 크다는 것을 알 수 있었다. 균체외 효소실험결과, skyrin이 생합성되는 경로는 $emodinanthrone{\rightarrow}emodin{\rightarrow}skyrin$으로 진행되었다.

• KSBB Journal
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• 제11권4호
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• pp.470-475
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• 1996

썩은 나무 잎에서 세포외 생물고분자 응집제를 생 산하는 흰 곰팡이를 분리하여 Pestalotiopsis sp. M01로 동정하였다. 이 균주가 생산하는 생물고분자 응집제의 응집활성에 대한 배양 조건을 무기염 배지인 Czapek-Dox 배지를 기초로 하여 탄소원, 질소 원, 배지의 pH멸, 그리고 옹도별로 조사하였다. 그 결과로서 응집활성 조건은 3 % sucrose, 0.3 % $KN0_3$, pH 7, 그리고 $25^{\circ}C$에서 최대 응집활성을 나타내었다. 반면에 이 균주의 생육 조건은 3% su-crose, 0.3 % $KN0_3$, pH 5, 그리고 $25^{\circ}C$에서 최대 생육을 나타내었다.

• Journal of the Korean Wood Science and Technology
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• 제33권5호통권133호
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• pp.92-98
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• 2005

This study was performed to get the basic information concerned to the optimum culture condition of Inonotus obliquus. Several solid media, PDA, MEA and Czapek-Dox, and three liquid media were adopted for the in vitro cultivation. Some main features of the fungal morphological characteristics under cultivation conditions were observed and described. Preliminary results showed that appearance of the mycelial mat, hyphal size and substrate pigmentation differed according to the media. The PDA medium was the most favorable substrate for the growth on solid culture, followed by MEA and Czapek-Dox media. Concerned to the addition of amino acids, 5 amino acids, such as alanine, alginine, isoleucine, leucine and threonine, enhanced to the mycelial growth. Isoleucine was shown the best fungal growth. An important morphological hyphal structure for the fungus, the setae, was found in abundance and diverse its shape and size. In liquid culture, fresh potato broth was the best growth stimulant of the fungus, followed by Malt extract and potato broth. Addition of yeast extract to the liquid media had improved the biomass, but not laccase production.

• Molecules and Cells
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• 제38권11호
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• pp.959-965
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• 2015

Inducible and reversible gene silencing in desired types of cells is instrumental for deciphering gene functions using cultured cells or in vivo models. However, efficient conditional gene knockdown systems remain to be established. Here, we report the generation of an inducible expression system for short hairpin RNA (shRNA) targeted to PTEN, a well-documented dual-specificity phosphatase involved in tumor suppression and ontogenesis. Upon induction by doxycycline (DOX), the reverse tetracycline transcriptional activator (rtTA) switched on the concomitant expression of GFP and a miR-30 precursor, the subsequent processing of which released the embedded PTEN-targeted shRNA. The efficacy and reversibility of PTEN knockdown by this construct was validated in normal and neoplastic cells, in which PTEN deficiency resulted in accelerated cell proliferation, suppressed apoptosis, and increased invasiveness. Transgenic mice harboring the conditional shRNA-expression cassette were obtained; GFP expression and concurrent PTEN silencing were observed upon ectopic expression of rtTA and induction with Dox. Therefore, this study provides novel tools for the precise dissection of PTEN functions and the generation of PTEN loss of function models in specific subsets of cells during carcinogenesis and ontogenesis.

• Journal of Ginseng Research
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• 제19권2호
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• pp.181-187
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• 1995

Cylindvocarpon destmtalns isolate CY-92-01, pathogen of root-rot of Panax ginseng showed t the maximum mycelial growth on the Czapek solution agar among the thirteen kinds of media. Five isolates (Isolate CY-92-01, CY-92-03, CY-92-07, CY-94-01, CY-94-02) of C. destructan from various growth stages of p. ginseng recovered from several geographical sites also showed maximum growth in the Czapek-Dox broth compared with potato dextrose broth and V-8 juice broth. Rapid growth rate was maintained until 12 days after inoculation on the Czapek-Dox broth and mycelial weight was somewhat constant until 20 days. After 30 days of incubation, the mycelial weight began to decrease. The fungal growth occurred from 5$^{\circ}C$ to $25^{\circ}C$ and optimum temperature for growth was 2$0^{\circ}C$. Mycelial weight orderly decreased at 15, 25, 10, and 5$^{\circ}C$. Quantitative measurement was impossible at 5$^{\circ}C$. No fungal growth was occurred at the temperature higher than 3$0^{\circ}C$. Growth was observed at all tested pH ranges from 2.8 to 8.0. Optimum pH for growth was 4.0~5.0 followed by pH 3.3~3.5 and 5.4~6.0. The least growth occurred at pH 2.8.

• 센서학회지
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• 제32권6호
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• pp.412-417
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• 2023

There is increasing interest in the rapid and highly sensitive monitoring of cell viability in biological and toxicological research. Conventional methods depend on optical assays using Water Soluble Tetrazolium-8 (WST-8) or 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, which requires a large volume of samples and special instruments, necessitating shipment of clinical samples to laboratories. This paper reports on the development of a rapid and sensitive electrochemical (EC) sensor using screen printed electrode (SPE) and surface modification using 4'-mercapto-N-phenylquinone diamine (4'-NPQD), as double electron mediators, for monitoring cell viability via the measurement of nicotinamide adenine dinucleotide (NADH). We used the sensor to observe the viability of MCF-7 and doxorubicin (Dox)-treated cells. The oxidation current of NADH was measured via chronoamperometry (CA), and the EC results showed a good linear relationship when compared with NADH quantification using WST-8 assay. The analysis time was only 10 s and limit of detection (LOD) of NADH was 1.78 µM. Our EC method has the potential to replace conventional WST assays for cell viability and cytotoxicity experiments.

• Asian Pacific Journal of Cancer Prevention
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• 제16권8호
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• pp.3213-3222
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• 2015

Background: Cancer metastasis depends on cell motility which is driven by cycles of actin polymerization and depolymerization. Reactive oxygen species (ROS) and metabolic oxidative stress have long been associated with cancer. ROS play a vital role in regulating actin dynamics that are sensitive to oxidative modification. The current work aimed at studying the effects of sub-lethal metabolic oxidative stress on actin cytoskeleton, focal adhesion and cell migration. Materials and Methods: T47D human breast cancer cells were treated with 2-deoxy-D-glucose (2DG), L-buthionine sulfoximine (BSO), or doxorubicin (DOX), individually or in combination, and changes in intracellular total glutathione and malondialdehyde (MDA) levels were measured. The expression of three major antioxidant enzymes was studied by immunoblotting, and cells were stained with fluorescent-phalloidin to evaluate changes in F-actin organization. In addition, cell adhesion and degradation ability were measured. Cell migration was studied using wound healing and transwell migration assays. Results: Our results show that treating T47D human breast cancer cells with drug combinations (2DG/BSO, 2DG/DOX, or BSO/DOX) decreased intracellular total glutathione and increased oxidized glutathione, lipid peroxidation, and cytotoxicity. In addition, the drug combinations caused a reduction in cell area and mitotic index, prophase arrest and a decreased ability to form invadopodia. The formation of F-actin aggregates was increased in treated T47D cells. Moreover, combination therapy reduced cell adhesion and the rate of cell migration. Conclusions: Our results suggest that exposure of T47D breast cancer cells to combination therapy reduces cell migration via effects on metabolic oxidative stress.

• 생명과학회지
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• 제24권7호
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• pp.798-803
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• 2014

Cathepsin D (CtsD)는 아스파르트산 단백질 분해효소로서 cytochrome C의 방출을 유도하여 apoptosis 기전을 활성화시킨다. 본 연구에서는 3T3-L1 지방전구세포에서 CtsD 발현 조절에 관여하는 microRNA에 대해 조사하였다. 먼저 지방전구세포 사멸시 CtsD 발현 변화를 관찰하기 위하여 DNA damage agent인 doxorubicin을 3T3-L1 세포주에 노출시켜 CtsD 발현이 증가함을 확인하였다. 또한 지방전구세포주에서 CtsD가 과발현되면 세포 생존율이 감소하였다. miRanda program을 이용하여 CtsD 유전자를 표적으로 하는 microRNA를 탐색하여 miR-145를 선발하였다. Luciferase reporter assay에 의해 miR-145가 CtsD 유전자의 3' UTR 부위에 결합하여 luciferase 활성을 감소시킴을 관찰하였다. 3T3-L1 세포주에 miR-145 mimic을 도입한 결과 CtsD mRNA 발현과 단백질 수준이 감소하였다. 또한 세포주에 doxorubicin을 처리한 결과 CtsD 유전자 발현 증가와 상반되게 miR-145 발현이 감소하였다. 이외에도 miR-145 inhibitor을 세포에 도입하면 세포 생존율이 감소하였다. 이러한 결과는 지방전구세포의 세포사멸에 CtsD가 관여할 수 있으며, miR-145에 의해 CtsD 발현이 직접 조절되고 있음을 나타낸다. 따라서, 지방전구세포의 사멸을 유도하기 위해서는 miR-145 발현 제어가 주요한 표적이 될 수 있을 것으로 생각된다. 본 연구결과는 향후 비만 예방 및 치료를 위한 지방세포 사멸기전 규명에 중요한 기초 자료를 제공할 수 있을 것으로 기대한다.