• BMB Reports
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• v.52 no.10
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• pp.589-594
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• 2019

Single-molecule techniques have been used successfully to visualize real-time enzymatic activities, revealing transient complex properties and heterogeneity of various biological events. Especially, conventional force spectroscopy including optical tweezers and magnetic tweezers has been widely used to monitor change in DNA length by enzymes with high spatiotemporal resolutions of ~nanometers and ~milliseconds. However, DNA metabolism results from coordination of a number of components during the processes, requiring efficient monitoring of a complex of proteins catalyzing DNA substrates. In this min-review, we will introduce a simple and multiplexed single-molecule assay to detect DNA substrates catalyzed by enzymes with high-throughput data collection. We conclude with a perspective of possible directions that enhance capability of the assay to reveal complex biological events with higher resolution.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.2
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• pp.183-190
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• 2014

This project's aim was to determine the reserpine-induced gastric ulcer preventive effect of polysaccharide of Larimichthys crocea swim bladder (PLCSB) in ICR mice. The anti-gastric ulcer effects of polysaccharide of Larimichthys crocea swim bladder was evaluated in mice model using morphological test, serum levels assay, cytokine levels assay, tissue contents analysis, reverse transcription-polymerase chain reaction (RT-PCR) analysis and western bolt assay. High concentration (50 mg/kg dose) of PLCSB reduced IFN-${\gamma}$ as compared to low concentration (25 mg/kg dose) and control mice. SS and VIP serum levels of PLCSB treated mice were higher than those of control mice, and MOT and SP serum levels were lower than control mice. Gastric ulcer inhibitory index of PLCSB treatment groups mice were much lower than control mice, and the high concentration treated mice were similar to the ranitidine treated mice. The SOD and GSH-Px activities of PLCSB treated mice were higher than control mice, close to normal mice and ranitidine treated mice. PLCSB treated mice also showed the similar contents of NO and MDA to normal group. By RT-PCR and western blot assay, PLCSB significantly induced inflammation in tissues of mice by downregulating NF-${\kappa}B$, iNOS, and COX-2, and upregulating $I{\kappa}B-{\alpha}$. These results suggest that PLCSB showed a good gastric ulcer preventive effect as the gastric ulcer drug of ranitidine. Polysaccharide of Larimichthys crocea swim bladder may be used as a drug material from marine products.

• BMB Reports
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• v.43 no.7
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• pp.474-479
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• 2010

Three assay methods for quantification of the two galactose-operon mRNAs that only differ by 5 bases in their 5'-end are presented. The 5' ends of each mRNA were extended by ligating the 3'-end of the abundant 5S rRNA. This ligation extends the 5' ends of the two gal mRNAs long enough to be distinguished by the specific PCR primers in the following quantification reactions. Quantification of the corresponding cDNAs was performed either by primer extension assay or real-time qPCR. To circumvent the problem of the RNA ligation reaction (i.e. very low ligation efficiency), we devised a new method that employs real-time qPCR directly for the quantification of the gal transcripts which differ by 5 bases in their 5'-ends.

• Mycobiology
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• v.50 no.5
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• pp.382-388
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• 2022

White mold (or Sclerotinia stem rot), caused by Sclerotinia species, is a major air, soil, or seed-transmitted disease affecting numerous crops and wild plants. Microscopic or culture-based methods currently available for their detection and identification are time-consuming, laborious, and often erroneous. Therefore, we developed a multiplex quantitative PCR (qPCR) assay for the discrimination, detection, and quantification of DNA collected from each of the three economically relevant Sclerotinia species, namely, S. sclerotiorum, S. minor, and S. nivalis. TaqMan primer/probe combinations specific for each Sclerotinia species were designed based on the gene sequences encoding aspartyl protease. High specificity and sensitivity of each probe were confirmed for sclerotium and soil samples, as well as pure cultures, using simplex and multiplex qPCRs. This multiplex assay could be helpful in detecting and quantifying specific species of Sclerotinia, and therefore, may be valuable for disease diagnosis, forecasting, and management.

• Journal of Pharmacopuncture
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• v.3 no.1
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• pp.1-19
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• 2000

To study anti-cancer effect and molecular biological mechanism of bee venom for aqua-acupuncture, the effects of bee venom on cell viability and apoptosis were analyzed using MTT assay, tryphan blue assay, $[^3H]$thymidine release assay, flow cytometric analysis, and activity of caspase-3 protease activity assay. To explore whether anti-cancer effects of bee venom are associated with the transcriptional control of gene expression, quantitative RT-PCR analysis of apoptosis-related genes was performed. The obtained results are summarized as follows: 1. The MTT assay demonstrated that cell viability was decreased by bee venom in a dose-dependant manner. 2. Significant induction of apoptosis was identified using tryphan blue assay, $[^3H]$thymidine release assay, and flow cytomet1 ric analysis of sub $G_1$ fraction. 3. In analysis of caspase-3 protease activity, the activity had increased significantly, in a dose-dependant manner. 4. Quantitative RT-PCR analysis of the apoptosis-related genes showed that Bcl-2 and Bcl-$X_L$ were down-regulated whereas Bax was up-regulated by bee venom treatment.

• The Korean Journal of Applied Statistics
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• v.25 no.4
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• pp.633-640
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• 2012

Biological methods are described for the assay of certain substances and preparations whose potency cannot be adequately assured by chemical or physical analysis. The principle applied through these assays is of a comparison with a standard preparation to determine how much of the examined substance produces the same biological effects as a given quantity (the Unit) of the standard preparation. In these dilution assays, to estimate the relative potencies of the unknown preparations to the standard preparations, it is necessary to compare dose-response relationships of standard and unknown preparations. The dose-response relationship in the dilution assay is non-linear and sigmoid when a wide range of doses is applied. The parallel line model (applied to the dose region with the steepest slope) is used to estimate the relative potency. In this paper, the statistical theory in the parallel line model is explained with an application to a dilution assay data. The parallel line method is implemented in a SAS program and is available at the author's homepage(http://cafe.daum.net/go.analysis).

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.5
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• pp.362-365
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• 2000

A rapid immunochromatographic assay kit using whole blood to screen hepatitis B surface antigen was developed and evaluated by using sera from 240 patients. The reference diagnosis was based on the results obtained with GENEDIA Anti-HBs Rapid kit which is very similar to the above kit except for the use of serum. The test demonstrated a good correlation with the reference immunochromatographic assay kit, that is, the sensitivity and the specificity of the kit was 100%, respectively. The rapid test kit using whole blood should be more convenient and useful for the diagnosis of hepatitis B virus because the kit does not need machines and time to prepare serum. In addition, this kit is safe from inadvertent infection during sample treatment because the blood is sterilized with hydrogen peroxide, eliminates the procedure required to prepare serum and reduces the possibility of exposure to infectious agents.

• Journal of Applied Biological Chemistry
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• v.57 no.4
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• pp.301-305
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• 2014

The Antioxidant activity screening identification of five kind compounds in Artemisiae annuae herba with the on-line screening high performance liquid chromatography (HPLC) $ABTS^+$ assay. The various experimental variables such as the extraction time (h) and extraction solvent composition (%) of dipping method were investigated efficiently extraction at the room temperature $25^{\circ}C$. The results, the highest yield of total extract amount (0.458 g, 15.250%) was obtained by dipping method with 100% water and extraction time to 3 h. And the on-line screening HPLC-$ABTS^+$ assay method was rapid and efficient to search for bioactivity from natural products.

• Journal of Applied Biological Chemistry
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• v.49 no.4
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• pp.143-147
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• 2006

A rapid PCR-based assay for detecting hepatitis B viral DNA(HBV DNA) in serum and plasma was developed using a new PCR instrument named GenSpector(TMC-1000, Samsung electronics). PCR was carried out using a chip-based platform, which enabled 50 PCR cycles with internal controls, and melting-curve analysis in 30 minutes. Verification of the amplified HBV DNA product and the internal control was based on specific melting temperatures(Tm) analysis, executed by the GenSpector software. Primers were designed within the region conserved through HBV genotypes A to F. The lower limit of detection was 840 copies/ml serum, conducted with serial dilutions of a HBV DNA positive control(ACCURUN 325 series 700, Boston Biomedica Inc.). The assay was also compared to another assay for HBV DNA(Versant HBV DNA 3.0 assay, Bayer HealthCare) for 200 samples(each 100 clinical negative and positive samples). The sensitivity and specificity were 100% matched. This rapid PCR-based assay is specific, reproducible, and enables qualitative detection of HBV DNA.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1646-1649
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• 2006

Protein synthesis is the ultimate outcome of gene expression which, in turn, is regulated by several translation factors. We attempted to identify substances that can inhibit the translation process in vitro when the outcome protein is luciferase. To this end, we developed a sensitive cell-free protein synthesis assay using luciferase as the reporter. The synthesis of luciferase increased proportionately as mRNA was added to a $15-{\mu}l$reaction medium in concentrations raging from 5 ng to 500 ng. The maximum amount of luciferase was synthesized when the media were incubated at $25^{\circ}C$ for 40 min. The concentration of each compound that inhibited luciferase production by 50% ($IC_{50}$) was calculated. Hygromycin, puromycin, and cycloheximide yielded an $IC_{50}$ of 0.008, 0.8, and $0.7{\mu}g/ml$, respectively. A filtrate of Streptomyces spp. isolates inhibited protein synthesis up to S-fold when added to the in vitro translation assay mixture.