• BMB Reports
• /
• 제43권7호
• /
• pp.468-473
• /
• 2010

Previously, various inhibitors of cell wall synthesis induced the drp35 gene of Staphylococcus aureus efficiently. To determine whether drp35 could be exploited in antistaphylococcal drug discovery, we cloned the promoter of drp35 ($P_d$) and developed different biological assay systems using an engineered S. aureus strain that harbors a chromosomally-integrated $P_d$ - lacZ transcriptional fusion. An agarose-based assay showed that $P_d$ is induced not only by the cell wall-affecting antibiotics but also by rifampicin and ciprofloxacin. In contrast, a liquid medium-based assay revealed the induction of $P_d$ specifically by the cell wall-affecting antibiotics. Induction of $P_d$ by sublethal concentrations of cell wall-affecting antibiotics was even assessable in a microtiter plate assay format, indicating that this assay system could be potentially used for high-throughput screening of new cell wall-inhibiting compounds.

• Applied Biological Chemistry
• /
• 제35권2호
• /
• pp.82-86
• /
• 1992

복숭아로부터 polyphenoloxidase를 추출하여 polyphenol 화합물인 caffeic acid, hydroxyhydroquinone, homocatechol 그리고 pyrogallol과 반응시켜 얻어진 4종류의 효소적 갈변반응 생성물의 생리작용을 검토한 결과 rec-assay와 돌연변이원성시험에서 돌연변이원성을 나타내지 않았다. 4종류의 시료모두 S-9Mix를 첨가한 돌연변이원성 억제활성 실험에서 B(a)p, Trp-P-1의 변이물질에 대한 Ca-PEBRP와 HCa-PEBRP가 80% 이상의 강한 돌연변이원성 억제활성을 나타내었음을 알 수 있었다. 또한 rec-assay에서도 강한 대조구인 MMC와 MNNG에 대해 Py-PEBRP경우 생육저지대차를 17 mm에서 5 mm로 감소시켜 돌연변이 억제활성이 있는 것으로 나타났고, Hca-PEBRP, HHQ-PEBRP 그리고 Py-PEBRP에 $Zn^{2+}$의 첨가로 생육저지대의 차가 5 mm로써 DNA 손상에 영향을 미치는 것으로 나타났다.

• Applied Biological Chemistry
• /
• 제51권1호
• /
• pp.79-83
• /
• 2008

본 연구에서는 신경독소일 뿐만 아니라 흥분성 신경전달물질로 잘 알려져 있는 glutamate 세포독성이 산화적 손상과 관련하고 있고, 여기에 방어효과를 보이는 제비꽃 추출물에 관하여 연구하였다. MTT reduction assay를 통하여 glutamate의 세포독성을 확인하였고 ascorbic acid와 같은 대표적인 항산화제를 처리한 후 광학 현미경을 이용한 형태학적 변화를 관찰하였다. N18-RE-105 세포주에 최종 농도 20mM의 glutamate를 처리 하면 40.8% 의 생존율을 보이는데 반하여 ascorbic acid 500 ${\mu}M$ 최종농도로 처리하였을 때 85.3%의 세포 생존율을 확인할 수 있었다. 그리고 신경세포 보호효과를 가지는 제비꽃을 methanol, ethanol, acetone 추출한 뒤 MTT reduction assay를 이용하여 활성을 확인하였으며 그 중 acetone 추출물을 최종농도 50, 100 ${\mu}g/ml$를 처리 시 76.8%, 79.4%로 가장 높은 세포 생존율을 확인할 수 있었다. 이 결과는 N18-RE-105 세포주의 형태학적 변화와 LDH release assey에서도 일치하는 결과를 확인하였다.

• Journal of Microbiology and Biotechnology
• /
• 제31권10호
• /
• pp.1358-1365
• /
• 2021

To clarify the role of long intergenic nonprotein-coding RNA 1232 (LINC01232) in the progression of gastric cancer and the potential mechanism, we analyzed the expression of LINC01232 in TCGA database using the GEPIA online tool, and the LINC01232 level in gastric cancer cell lines was detected by quantitative real time-polymerase chain reaction (qRT-PCR) as well. Cell proliferation assay, colony formation assay, transwell assay and tumor formation experiment in nude mice were conducted to observe the biological behavior changes of gastric cancer cells through the influence of LINC01232 knockdown. LncATLAS database and subcellular isolation assay were used for subcellular distribution of LINC01232 in gastric cancer cells. The interaction among LINC01232, zeste homolog 2 (EZH2) and kruppel-like factor 2 (KLF2) was clarified by RNA-protein interaction prediction (RPISeq), RNA immunoprecipitation (RIP), qRT-PCR and chromatin immunoprecipitation (ChIP) assay. Rescue experiments were further conducted to elucidate the biological function of LINC01232/KLF2 axis in the progression of gastric cancer. LINC01232 was upregulated in stomach adenocarcinoma (STAD) tissues and gastric cancer lines. LINC01232 knockdown inhibited the proliferative capacities of gastric cancer cells in vitro, and impaired in vivo tumorigenicity. LINC01232 was mainly distributed in the cell nucleus where it epigenetically repressed KLF2 expression via binding to the enhancer of EZH2, which was capable of binding to promoter regions of KLF2 to induce histone H3 lysine 27 trimethylation (H3K27me3). LINC01232 exerts oncogenic activities in gastric cancer via inhibition of KLF2, and therefore, the knockdown of KLF2 could reverse the regulatory effect of LINC01232 in the proliferative ability of gastric cancer cells.

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

• 한국수정란이식학회:학술대회논문집
• /
• 한국수정란이식학회 2006년도 The 6th Intermational Symposium on Developmental Biotechnology
• /
• pp.114-115
• /
• 2006

• Applied Biological Chemistry
• /
• 제41권4호
• /
• pp.213-217
• /
• 1998

An assay for cytochrome P450 monooxygenase activity by determination of the products of organophosphate oxidation via inhibition of acetylcholinesterase was described. Extracts from strains of Oryzaephilus surinamensis selected for resistance to chlorpyrifos-methyl (QVOS 102), fenitrothion (VOS F) and malathion (VOS 3), and a standard susceptible strain VOS 48, were incubated with an organophosphate in the presence of a NADPH-generating system and acetylcholinesterase. The degree of inhibition of the acetylchoinesterase activity was converted to manooxygenase activity using standard curves for the inhibition of acetylcholiesterase by chlorpyrifos-methyl-oxon, fenitrooxon and malaoxan. Activity was detectable in VOS 48 and was present at different increased levels with the different organophosphates in the three resistant strains, suggesting that different forms of P450 might be involved in organophosphate oxidation in these insects. The assays were carried out using crude insect homogenates and much smaller samples of insect material than the standard aldrin to dieldrin assay. It should be possible to use the method for determination of monooxygenase activity in single insert.

• Journal of Applied Biological Chemistry
• /
• 제43권4호
• /
• pp.230-234
• /
• 2000

A new method to assay the capsaicinoid synthetase (CS) activity was developed by utilizing NADHcoupled enzyme systems involving pyruvate kinase and lactate dehydrogenase. CS activities in Capsicum placenta, depending upon the kinetics of the NADH oxidation, revealed almost the same profile as compared with those shown using an HPLC-based method. When the substrates, 8-methyl nonanoic acid and vanillylamine, for the CS enzyme were employed separately or simultaneously, it appeared that the two-step reaction, acyl-CoA formation and condensation with vanillyla~ne, of the CS enzyme was a coupled reaction. Thus, this assay method of the CS enzyme can be considered as an alternative to the HPLC-based method, since it has the advantages of rapidity and simplicity as well as reliability when compared with the existing method.

• Environmental Analysis Health and Toxicology
• /
• 제17권3호
• /
• pp.245-252
• /
• 2002

Airborne pollutants in the subway facilities can be potentially harmful to the health of passengers. This study was designed to examine whether the suspended particulates have mutagenic or carcinogenic effect on the plant cell systems. Total suspended particulates were collected with a high volume air sampler, in the entrance, the waiting room, and the platform of each subway station. The biological end -points in this experiment were the pink mutations in stamen hairs and micronuclei in the pollen mother cells of Tradescantia. The exudates were collected by shaking the filter papers from the sampler in distilled water for 24 hours. All the plant cuttings exposed to the exudates resulted in positive responses. The micronucleus assay proved more reliable and sensitive to the test than the stamen hair assay. The results indicate that the air particulates can give an adverse effect on the health of subway passengers.

• 방사선산업학회지
• /
• 제4권1호
• /
• pp.39-45
• /
• 2010

The pollution of antibiotics is a major cause of spreading antibiotics resistant bacteria in the environment. Applications of ozonation, UV, and ${\gamma}-ray$ irradiations have been introduced to remove antibiotics in the effluents from wastewater treatment system. In this study, we compared the chemical (HPLC) and biological (antimicrobial susceptibility test, AMS) assays in measuring of the concentrations of residual antibiotics after ${\gamma}-ray$ and UV irradiation. Most samples were degraded by ${\gamma}-ray$ irradiation (1~2 kGy). However, lincomycin and tetracycline were not degraded by UV irradiation. The concentration of residual antibiotics, that was treated with ${\gamma}-ray$ and UV irradiation, measuring by bioassay was similar to HPLC. The concentrations of ${\gamma}-ray$ irradiated cephradine measured by AMS test were 2 times higher than that of HPLC assay, indicating AMS test is more sensitive than HPLC assay. These results indicate that ${\gamma}-ray$ irradiation technique is more useful than UV irradiation, and biological assay is more useful to detect the antibiotics and toxic intermediates in antibiotics degradation.