• Molecular & Cellular Toxicology
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• v.1 no.4
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• pp.230-236
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• 2005

Formaldehyde is a common environmental contaminant found in tobacco smoke, paint, garments, diesel and exhaust, and medical and industrial products. Formaldehyde has been considered to be potentially carcinogenic, making it a subject of major environmental concern. However, only a little information on the mechanism of immunological sensitization and asthma by this compound has been known. So, we performed with Jurkat cell line, a human T lymphocyte, to assess the induction of DNA damage and to identify the DEGs related to immune response or toxicity by formaldehyde. In this study, we investigated the induction of DNA single strand breaks by formaldehyde using single cell gel electrophoresis assay (comet assay). And we compared gene expression between control and formaldehyde treatment to identify genes that are specifically or predominantly expressed by employing annealing control primer (ACP)-based $GeneFishing^{TM}$ method. The cytotoxicity ($IC_{30}$) of formaldehyde was determined above the 0.65 mM in Jurkat cell in 48 h treatment. Based on the $IC_{30}$ value from cytotoxicity test, we performed the comet assay in this concentration. From these results, 0.65 mM of formaldehyde was not revealed significant DNA damages in the absence of S-9 metabolic activation system. And the one differentially expressed gene (DEG) of formaldehyde was identified to zinc finger protein 292 using $GeneFishing^{TM}$ method. Through further investigation, we will identify more meaningful and useful DEGs on formaldehyde, and then can get the information on the associated mechanism and pathway with immune response or other toxicity by formaldehyde exposure.

• Journal of Marine Life Science
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• v.9 no.1
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• pp.9-21
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• 2024

Paralytic shellfish poisoning (PSP) including Saxitoxin (STX) is caused by harmful algae, and poisoning occurs when the contaminated seafood is consumed. The mouse bioassay (MBA), a standard test method for detecting PSP, is being sanctioned in many countries due to its low detection limit and the animal concerns. An alternative to the MBA is the Neuro-2a cell-based assay. This study aimed to establish various test conditions for Neuro-2a assay, including cell density, culture conditions, and STX treatment conditions, to suit the domestic laboratory environment. As a result, the initial cell density was set to 40,000 cells/well and the incubation time to 24 hours. Additionally, the concentration of Ouabain and Veratridine (O/V) was set to 500/50 μM, at which most cells died. In this study, we identified eight concentrations of STX, ranging from 368 to 47,056 fg/μl, which produced an S-shaped dose-response curve when treated with O/V. Through inter-laboratory variability comparison of the Neuro-2a assay, we established five Quality Control Criteria to verify the appropriateness of the experiments and six Data Criteria (Top and Bottom OD, EC₅₀, EC₂₀, Hill slop, and R² of graph) to determine the reliability of the experimental data. The Neuro-2a assay conducted under the established conditions showed an EC₅₀ value of approximately 1,800~3,500 fg/μl. The intra- & inter-lab variability comparison results showed that the coefficients of variation (CVs) for the Quality Control and Data values ranged from 1.98% to 29.15%, confirming the reproducibility of the experiments. This study presented Quality Control Criteria and Data Criteria to assess the appropriateness of the experiments and confirmed the excellent repeatability and reproducibility of the Neuro-2a assay. To apply the Neuro-2a assay as an alternative method for detecting PSP in domestic seafood, it is essential to establish a toxin extraction method from seafood and toxin quantification methods, and perform correlation analysis with MBA and instrumental analysis methods.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.111-111
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• 2005

• Proceedings of the Korean Society of Life Science Conference
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• 2005.04a
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• pp.89-89
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• 2005

• Restorative Dentistry and Endodontics
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• v.45 no.1
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• pp.2.1-2.7
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• 2020

Objectives: This study aimed to evaluate the cell viability and migration of Endosequence Bioceramic Root Canal Sealer (BC Sealer) compared to MTA Fillapex and AH Plus. Materials and Methods: BC Sealer, MTA Fillapex, and AH Plus were placed in contact with culture medium to obtain sealers extracts in dilution 1:1, 1:2 and 1:4. 3T3 cells were plated and exposed to the extracts. Cell viability and migration were assessed by 3-(4,5-dimethylthiazoyl)-2,5-diphenyl-tetrazolium bromide (MTT) and Scratch assay, respectively. Data were analyzed by Kruskal-Wallis and Dunn's test (p < 0.05). Results: The MTT assay revealed greater cytotoxicity for AH Plus and MTA Fillapex at 1:1 dilution when compared to control (p < 0.05). At 1:2 and 1:4 dilutions, all sealers were similar to control (p > 0.05) and MTA Fillapex was more cytotoxic than BC Sealer (p < 0.05). Scratch assay demonstrated the continuous closure of the wound according to time. At 30 hours, the control group presented closure of the wound (p < 0.05). At 36 hours, only BC Sealer presented the closure when compared to AH Plus and MTA Fillapex (p < 0.05). At 42 hours, AH Plus and MTA Fillapex showed a wound healing (p > 0.05). Conclusions: All tested sealers demonstrated cell viability highlighting BC Sealer, which showed increased cell migration capacity suggesting that this sealer may achieve better tissue repair when compared to other tested sealers.

• Korean Journal of Microbiology
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• v.55 no.3
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• pp.199-205
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• 2019

Yeast two-hybrid (Y2H) technique has been used to study protein-protein interactions, but its application particularly to a large-scale analysis of protein interaction networks, is limited by the fact that the technique is labor-intensive, based on scoring colonies on plate. Here, we develop a new reporter for the measurement of the protein-protein interactions by flow cytometry. The yeast harboring interacting proteins can also be enriched by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). When two interacting proteins are present in the same yeast cell, a reporter protein containing 10 tandem repeats of c-myc epitope becomes localized on the surface of the cell wall, without affecting cell growth. We successful measured the surface display of c-myc epitope upon interacting p53 with SV40 T antigen by flow cytometry. Thus, the newly developed Y2H assay based on the display of c-myc repeat on yeast cell wall could be used to the simultaneous analysis of multiple protein-protein interactions without laborious counting colonies on plate.

• Biomedical Science Letters
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• v.16 no.4
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• pp.279-285
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• 2010

Endogenous cannabinoids (endocannabinoids) display various pharmacological effects including pain control, anti-inflammation, and neuroprotection. The synthesis and release of endocannabinoids are regulated under both physiological and pathological conditions. The main degrading enzyme of endocannabinoid is fatty acid amide hydrolase (FAAH). Therefore we have developed the fluorescence-based assay system for FAAH. We established stable CosM6 cell lines expressing human FAAH. We also synthesized 2-oxo-2H-chromen-7-yl decanoate (DAEC) as a fluorogenic substrate for FAAH. When crude membrane extracts stably expressing FAAH was incubated with DAEC at $25^{\circ}C$, FAAH reacted specifically to DAEC and catalyzes the hydrolysis of DAEC into decanoic acid and highly fluorescent coumarin. Furthermore, the serin hydrolase inhibitor, phenylmethanesulfonylfluoride, inhibited the coumarin release to the reaction buffer in concentration dependent manner. This assay system is suitable for high-throughput screening since this system has simple experimental procedure and measurement method.

• Journal of Life Science
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• v.15 no.5 s.72
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• pp.779-782
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• 2005

Cell-free expression is a powerful tool for rapid protein analysis, enabling an efficient identification of gene without cumbersome procedure of transformation and cell culture. Epoxide hydrolase (EH) gene of Rhodotorula glutinis was simply amplified by PCR, and the resultant gene was expressed in vitro using a coupled Transcription/translation system. The cell-free expressed EH protein mixture exhibited the enantioselective hydrolysis activity toward (R)-styrene oxide, representing that cell-free protein synthesis system can be used for the rapid expression of an enantioselective enzyme for an efficient identification of the chiral activity.

• KSBB Journal
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• v.21 no.3
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• pp.212-219
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• 2006

The purpose of this study was to develop a assay system of host cell-derived residual proteins in final pharmaceutical products. Accurate and simple assay system for host cell-derived proteins(HCPs) is very important test item in pharmaceutical qualification control. In this study, methods for quantification of residual HCPs in recombinant anti-GPIIbIIIa antibody were developed using a process-specific immunoligand assay which was based on the Enzyme linked Immunosorbent assay(ELISA) system. The assay had a detection limit of 10.8 ng/ml of HCPs with a product concentration of 1 mg/ml. The practical implication of these results is that the developed ELISA system can be used for HCPs qualification control and this system will be applicable to develop another ELISA system of different antibody drug.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.2
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• pp.689-692
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• 2012

Objective: To investigate correlations between adenosine triphosphate chemotherapy response assay (ATP-CRA) and clinical outcomes after ATP-CRA-based chemotherapy for drug selection in patients receiving intravesical chemotherapy to prevent recurrence of superficial bladder cancer after surgery. Methods: The chemosensitivities of 12 anticancer drugs were evaluated, including 5-Fu ADM, and EPI, using ATP-CRA and primary tumor cell culture in 54 patients. In addition, a further 58 patients were treated according to clinical experience. Differences in post-chemotherapeutical effects between drug sensitivity assay and experience groups were compared. Results: The evaluable rate of the test was 96.3%, the clinical effective rate was 80.8%, the sensitivity rate was 97.6% (41/42), the specificity was 20%, the total predicting accuracy was 74.3%, the positive predictive value was 83.7% (41/49), the negative predictive value was 66.7% (2/3); in the drug sensitivity test group, the clinical effective rate was 80.8%, the experience group response rate was 63.8%, with a significant difference in clinical effects between the ATP-based sensitivity and experience groups (${\chi}^2$=7.0153, P<0.01). Conclusion: ATP-CRA is a stable, accurate and potentially practical chemosensitivity test providing a predictor of chemotherapeutic response in patients with superficial bladder cancer.