• Bulletin of the Korean Chemical Society
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• 제35권5호
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• pp.1279-1284
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• 2014

The binding of TATA-binding protein (TBP) to the TATA-box containing promoter region is aided by many other transcriptional factors including TFIIA and TFIIB. The mechanistic insight into the assembly of RNA polymerase II preinitation complex (PIC) has been gained by either directly altering a function of target protein or perturbing molecular interactions using drugs, RNAi, or aptamers. Aptamers have been found particularly useful for studying a role of a subset of PIC on transcription for their ability to inhibit specific molecular interactions. One major hurdle to the wide use of aptamers as specific inhibitors arises from the difficulty with traditional assays to validate and determine specificity, affinity, and binding epitopes for aptamers against targets. Here, using a technique called the bio-layer interferometry (BLI) designed for a label-free, real-time, and multiplexed detection of molecular interactions, we studied the assembly of a subset of PIC, TBP binding to TATA DNA, and two distinct classes of aptamers against TPB in regard to their ability to inhibit TBP binding to TFIIA or TATA DNA. Using BLI, we measured not only equilibrium binding constants ($K_D$), which were overall in close agreement with those obtained by electrophoretic mobility shift assay, but also kinetic constants of binding ($k_{on}$ and $k_{off}$), differentiating aptamers of comparable KDs by their difference in binding kinetics. The assay developed in this study can readily be adopted for high throughput validation of candidate aptamers for specificity, affinity, and epitopes, providing both equilibrium and kinetic information for aptamer interaction with targets.

• 한국환경성돌연변이발암원학회지
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• 제24권2호
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• pp.99-107
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• 2004

The validation of many synthetic chemicals that may pose a genetic hazard in our environment is of great concern at present. Since these substances are not limited to the original products, and enter the environment, they have become widespread environmental pollutants, thus leading to a variety of chemicals that possibly threaten the public health. In this respect, the regulation and evaluation of the chemical hazard playa very important role to environment and human health. The clastogenicity of 11 synthetic chemicals was evaluated in Chinese hamster lung (CHL) fibroblast in vitro. Benzoyl chloride (CAS No. 98-88-4) induced chromosomal aberrations with statistical significance at the concentration of 31-123 $\mug/ml$ and 43 $\mug/ml$ in the absence and presence of S-9 metabolic activation system, respectively. 2-Propyn-l-o1 (CAS No. 107-19-7) and 2-Phenoxy ethanol (CAS No. 122-99-6) revealed clastogenicity only at the highest concentration in the presence of S-9 mixture. However, 1-naphthol (CAS No. 90-15-3) which is one of the most cytotoxic chemical among 11 chemicals tested revealed no clastogenicity both in the presence and absence of S-9 metabolic activation system. From the results of chromosomal aberration assay with 11 synthetic chemicals in CHL fibroblast in vitro, Benzoyl chloride (CAS No. 98-88-4), 2-Propyn-l-01 (CAS No. 107-19-7) and 2-Phenoxy ethanol (CAS No. 122-99-6) revealed positive clastogenic results in this study.

• Journal of Biosystems Engineering
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• 제32권6호
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• pp.440-447
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• 2007

In this study, a biosensor was developed using an enzyme-linked immunosorbent assay (ELISA) to rapidly measure the fungicide iprovalicarb residues in agricultural products. The biosensor was designed to include micro-pumps and solenoid valves for fluid transport, a spectrophotometer cuvet as a reaction chamber, a photodiode with a light-emitting diode for optical density measurement, and a control microcomputer to implement assay. The rate of change in optical density of the cuvet was read as final signal output. Micro-pumps were evaluated to investigate their delivery capability, the highest values of the error and the coefficient of variation were 4.3% and 4.6% respectively. As the incubation period was reduced from 15 minutes to 11 minutes to shorten the total processing time, the sensor sensitivity was decreased as the antibody dilution ratio was reduced to a half. The maximum usable period of the coated cuvet was found to be two days with 1% error limit. To predict the concentration of the iprovalicarb residue in agricultural products, a linear calibration model was obtained with r-square values of 0.992 for potato and 0.985 for onion. In validation test for the samples of potatoes and onions against the high performance liquid chromatography, very high correlation values were obtained as 0.996 and 0.993 respectively. Using the cuvet immobilized with antigen, it took 21-minutes for the biosensor to complete the measuring process of the iprovalicarb residues.

• 분석과학
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• 제30권1호
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• pp.32-38
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• 2017

In attempt to contribute in official monographs of Korean Pharmacopoeia, an HPLC method was developed and fully validated for the determination of tolperisone hydrochloride in tablets which have never been published in other forgein Pharmacopoeia. Analysis was carried out in an ODS column ($250{\times}4.6mm$ I.D., $5{\mu}m$) with common solvents include acetonitrile and ammonium hydrophosphate buffer as mobile phase. The assay was validated according to International Conference on Harmonization (ICH) guidelines. The method has good linearity in the range of $5-200{\mu}g/mL$ tolperisone. Intra-day precision varied between 0.04 and 0.10 %. Relative standard deviations of inter-day precision ranged between 0.43 and 1.24 % for peak area. The percentage recovery of the tolperisone ranged between 99.8 and 101.2 % in material. Recoveries in tablets were ranged between 98.7 and 100.8 %, thus confirmed the suitability of method for estimation of tolperisone hydrochloride in tablet dosage form.

• Environmental Analysis Health and Toxicology
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• 제21권3호
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• pp.291-299
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• 2006

The experiments investigated whether early exposure to testosterone propionate (TP) during prepuberty alters testis development in Sprague-Dawley male rats. We performed Hershberger assay using the stimulated weanling male rats by OECD protocols, cDNA microarray, and Western blot. TP was subcutaneously injected to uncastrated Sprague-Dawley male rat of 22 days old for 10 consecutive days at doses of 0.4, 0.8, 1.0, 1.2, 1.6 mg/kg per day. At necropsy, the following tissues were removed and weighed: combined testes, epididymides (Epi), Cowper's glands (COW), levator am, and bulbocavernosus muscles (LABC), seminal vesicles, together with coagulating gland (SV) and ventral prostate (VP). We found that TP increased the weights of Epi, VP, SV, COW, and LABC, while testis was decreased in a dose-dependent manner. In cDNA microarray analysis of testis, there were significant reductions in the expression of cytochrome P450 11A (CYP11A), the rate-limiting enzyme of steroidogenesis. Taken together these results, TP exposure before puberty in male rats may produce the delay in testis development by inhibiting the CYP11A gene expression.

• Journal of Microbiology and Biotechnology
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• 제31권12호
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• pp.1716-1721
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• 2021

Chikungunya fever is an arboviral disease caused by the Chikungunya virus (CHIKV). The disease has similar clinical manifestations with other acute febrile illnesses which complicates differential diagnosis in low-resource settings. We aimed to develop a rapid test for CHIKV detection based on the nucleic acid lateral flow immunoassay technology. The system consists of a primer set that recognizes the E1 region of the CHIKV genome and test strips in an enclosed cassette which are used to detect amplicons labeled with FITC/biotin. Amplification of the viral genome was done using open-source PCR, a low-cost open-source thermal cycler. Assay performance was evaluated using a panel of RNA isolated from patients' blood with confirmed CHIKV (n = 8) and dengue virus (n = 20) infection. The open-source PCR-NALFIA platform had a limit of detection of 10 RNA copies/ml. The assay had a sensitivity and specificity of 100% (95% CI: 67.56% - 100%) and 100% (95% CI: 83.89% - 100%), respectively, compared to reference standards of any positive virus culture on C6/36 cell lines and/or qRT-PCR. Further evaluation of its performance using a larger sample size may provide important data to extend its usefulness, especially its utilization in the peripheral healthcare facilities with scarce resources and outbreak situations.

• 대한화장품학회지
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• 제17권1호
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• pp.64-80
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• 1991

The SKINTEX Method is based on a two-compartment physico-chemical model which includes a Biomembrane Barrier in compartment one and an organized macromolecular matrix in compartment two. Test samples absorb onto or permeate through the keratin/collagen Biomembrane Barrier and then can interact with the organized macromolecular matrix. Changes in the integrity of the barrier release a dye indicator: Changes in the matrix can alter its transparency. The sum of these two responses is read spectrophotometrically at 470nm. An early investigation of 950 chemicals and formulations in the SKINTEX System produced results which were 89% concordance to in vivo Draize dermal irritation results obtained with 24-hour occluded application of test samples with-out abrasion and standard scoring. Alkaline materials were analyzed in a specialized SKINTEX AMA Protocol. In this early study, the model did not distinguish nonirritant test materials and formulation with PDII(Primary Dermal Irritation Index)in the range from 0 to 1.2, A High Sensitivity Assay Protocol(HSA)was developed to amplify the changes in both compartments of this model and provide more accurate calibration of these changes. A study of 60 low irritation test samples including cosmetics, household products, chemicals and petro-chemicals distinguished nonirritants with PDII $\leq$ 0.7 for 26 of 30 nonirritants. A second protocol was developed to evaluate the SKINTEX model predictability with respect to human irritation. The Human Response Assay (HRA )has been optimized based on differences in penetration and irritation responses in humans and rabbits. An additional 32 test materials with different mechanisms and degrees of dermal toxicity were evaluated by the HRA. These in vitro results were 86% concordant to human patch test results. In order to further evaluate this model, a Standard Chemical Labelling (SCL) Protocol was developed to optimize this system to predict Draize dermal irritation results after a 4-hour application of the test material. In a study of 52 chemicals including acids, bases, solvents, salts, surfactants and preservatives, the SCL results demonstrated 85% concordance to Draize results for a 4-hour application of test samples on non-abraded rabbit skin. The SKINTEX System, including three specialized protocols, provided results which demonstrated good correlation to the endpoint of dermal irritation in man and rabbits at different application times.

• 생명과학회지
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• 제29권6호
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• pp.653-661
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• 2019

본 연구는 siRNA 기반 치료제등의 핵산치료제 개발에 있어서 필수적인 약물의 생체내 흡수, 분포, 대사, 배설에 대한 동태의 확인을 위해 stem-loop RT-qPCR 법을 이용하여 보다 더 정확한 시험법을 확립하고자 하였다. siRNA에 특이적인 primer와 probe를 선별하여 siRNA 정량검출 시험법을 최적화하였다. siRNA 표준시료를 이용하여 최적화된 시험법을 적용하였을 때 siRNA 표준시료에 대한 Cp 값(y)간의 선형분석 결과, 기울기 평균 -3.3, 결정계수 $R^2$>0.99으로 확인되어 siRNA 표준시료와 Cp 값 간의 회귀성이 매우 높아 정량 분석이 가능한 시험법임을 확인하였고, 같은 표준시료를 이용한 stem-loop RT-qPCR의 검출한계(LOD)는 10 fM, 최소정량한계(LLOQ)는 100 fM이었다. 확립된 시험법의 신뢰성을 확인하기 위해 시험자를 다르게 하고, 시험법을 3회 반복하여 각각 진행한 결과, siRNA 표준시료에 대한 Cp 값(y)간의 선형분석 결과 기울기와 결정계수 $R^2$의 재현성(slope ${\pm}-3.2$, 결정계수 $R^2$>0.99)을 확인하였고, 표준 곡선으로부터 환산된 siRNA 표준시료의 회수율(recovery ${\pm}20%$)과 완건성이 우수함을 확인하였다. 확립된 stem-loop RT-qPCR을 생체내 존재하는 약물 검증에 적용할 수 있는지 확인하기 위하여 시험동물에 siRNA를 주입 후 시간별 혈액을 채취하여 확립된 시험법으로 시험을 진행하였고 약물 동태학적 분석을 통해 siRNA치료제의 혈액내의 안정성을 확인하였다. 따라서 본연구에서 개발된 stem-loop RT-qPCR 분석법은 정확성, 정밀성 및 민감도가 높은 분석법으로 핵산치료제 개발 연구의 다양한 생체시료 분석 연구에 적용할 수 있을 것으로 기대한다.

• 대한두경부종양학회지
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• 제33권1호
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• pp.21-29
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• 2017

Methylation of CpG islands in the promoter region of genes acts as a significant mechanism of epigenetic gene silencing in head and neck squamous cell carcinoma (HNSCC). DNA methylation markers are particularly advantageous because DNA methylation is an early event in tumorigenesis, and the epigenetic modification, 5-methylcytosine, is a stable mark. In the present study, we assessed the genome-wide preliminary screening and were to identify novel methylation biomarker candidate in HNSCC. Genome-wide methylation analysis was performed on 10 HNSCC tumors using the Methylated DNA Isolation Assay (MeDIA) CpG island microarray. Validation was done using immunohistochemistry using tissue microarray of 135 independent HNSCC tumors. In addition, in vitro proliferation, migration/invasion assays, RT-PCR and immunoblotting were performed to elucidate molecular regulating mechanisms. Our preliminary validation using CpG microarray data set, immunohisto-chemistry for HNSCC tumor tissues and in vitro functional assays revealed that methylation of the Homeobox B5 (HOXB5) and H6 Family Homeobox 2 (HMX2) could be possible novel methylation biomarkers in HNSCC.

• Natural Product Sciences
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• 제11권3호
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• pp.174-178
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• 2005

Oxidative stress is an important causative factor in several human chronic diseases, such as atherosclerosis, cardiovascular disorders, mutagenesis, cancer, several neurodegenerative disorders, and the aging process. Phenolics and tannins are reported to be good antioxidants. Anogeissus latifolia (Combretaceae) bark has been used in the Indian traditional systems of medicine for curing a variety of ailments, but scientific validation is not available till date. Hence the present study was undertaken to isolate antioxidant compounds by activity-guided isolation. Inhibtion of diphenyl picryl hydrazyl (DPPH) and Xanthine oxidase along with photochemiluminescence assay were used as bioassay for antioxidant activity. Activity guided isolation was carried out using silica column and the compounds were quantified using HPLC. Ethyl acetate and butanol fraction exhibited potent antioxidant activity. Bioassay-guided isolation led to isolation of ellagic acid (1) and dimethyl ellagic acid (2) as the main active compounds, which along with gallic acid were quantified by HPLC. Thus we conclude that these three major tannoid principles present in A. latifolia, are responsible for the antioxidant potential and possibly their therapeutic potential.