• 대한수의학회지
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• 제43권2호
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• pp.239-246
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• 2003

Polymerase chain reaction (PCR) was evaluated for the early detection of Aujeszky's disease virus (ADV) DNA from virus-infected cell cultures. For the purposes, the Korean ADV NYJ1-87 was propagated in swine kidney (SK) cells and subjected to the amplification of DNA (217 bp) by PCR using sense and antisense primers specific to gp50 gene of the ADV. In detection of cell-associated viral DNA, reliable PCR conditions were determined as 30 cycles of reaction consisting 1 minute each of denaturation at $94^{\circ}C$, annealing at $55^{\circ}C$ and polymerization at $72^{\circ}C$. The PCR encountered best results with reagent mixtures of $50{\mu}l$ containing $200{\mu}M$ dNTPs, $0.2{\mu}M$ each sense and antisense primers, 1 mM $MgCl_2$ and 10% (v/v) template DNA in the final concentrations. ADV-specific DNAs were detected as early as 6, 6, and 9 hours post-infection, respectively, from lysates of the SK cells infected with ADV of $10^3$, $10^2$ and $10^1\;TCID_{50}/ml$ by this condition. In culture supernatant, the DNAs were detected from ADV of as low infectivity as $10^ {-3}\;TCID_{50}/ml$ by the reduced reagent concentrations and 30 cycles of 1 minute each of denaturation at $94^{\circ}C$ and annealing at $55^{\circ}C$, and 2 minutes of polymerization at $72^{\circ}C$. The lowest amount of detectable ADV DNA was 1 fg. In conclusion, the PCR condition established in the present study was recognized as a feasible alternative to time-consuming procedures in isolation and characterization of the virus.

• 한국입자에어로졸학회지
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• 제8권2호
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• pp.89-95
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• 2012

Spherical mesoporous silica particles, of which main pore diameter was 3.8 nm, were successfully prepared by spray pyrolysis from aqueous silicic acid. The effect of precursor concentration, reaction temperature, and the addition of urea and PEG on the particle diameter and pore properties such as pore diameter, total pore volume, and specific surface area were investigated by using FE-SEM, particle size analyzer, and nitrogen absorption-desorption analysis. With an increase of the precursor concentration from 0.2 M to 0.7 M, the average particle diameter, total pore volume, and specific surface area of the porous silica particles increased from 0.56 to $0.96\;{\mu}m$, 0.434 to $0.486\;cm^3/g$, 467.8 to $610.4\;m^2/g$, respectively. Within the temperature range $(600\;^{\circ}C{\sim}800\;^{\circ}C)$, there was no significant difference in the pore diameter, total pore volume, and specific surface area. In addition, the addition of urea as an expansion aid led to slight increases in particle diameter, pore diameter, and specific surface area. However, when the polyethylene glycol (PEG) as an organic template was used, the total pore volume of porous particles increased dramatically.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2013년도 제45회 하계 정기학술대회 초록집
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• pp.88-89
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• 2013

A variety of influenza A viruses from animal hosts are continuously prevalent throughout the world which cause human epidemics resulting millions of human infections and enormous industrial and economic damages. Thus, early diagnosis of such pathogen is of paramount importance for biomedical examination and public healthcare screening. To approach this issue, here we propose a fully integrated Rotary genetic analysis system, called Rotary Genetic Analyzer, for on-site detection of influenza A viruses with high speed. The Rotary Genetic Analyzer is made up of four parts including a disposable microchip, a servo motor for precise and high rate spinning of the chip, thermal blocks for temperature control, and a miniaturized optical fluorescence detector as shown Fig. 1. A thermal block made from duralumin is integrated with a film heater at the bottom and a resistance temperature detector (RTD) in the middle. For the efficient performance of RT-PCR, three thermal blocks are placed on the Rotary stage and the temperature of each block is corresponded to the thermal cycling, namely $95^{\circ}C$ (denature), $58^{\circ}C$ (annealing), and $72^{\circ}C$ (extension). Rotary RT-PCR was performed to amplify the target gene which was monitored by an optical fluorescent detector above the extension block. A disposable microdevice (10 cm diameter) consists of a solid-phase extraction based sample pretreatment unit, bead chamber, and 4 ${\mu}L$ of the PCR chamber as shown Fig. 2. The microchip is fabricated using a patterned polycarbonate (PC) sheet with 1 mm thickness and a PC film with 130 ${\mu}m$ thickness, which layers are thermally bonded at $138^{\circ}C$ using acetone vapour. Silicatreated microglass beads with 150~212 ${\mu}L$ diameter are introduced into the sample pretreatment chambers and held in place by weir structure for construction of solid-phase extraction system. Fig. 3 shows strobed images of sequential loading of three samples. Three samples were loaded into the reservoir simultaneously (Fig. 3A), then the influenza A H3N2 viral RNA sample was loaded at 5000 RPM for 10 sec (Fig. 3B). Washing buffer was followed at 5000 RPM for 5 min (Fig. 3C), and angular frequency was decreased to 100 RPM for siphon priming of PCR cocktail to the channel as shown in Figure 3D. Finally the PCR cocktail was loaded to the bead chamber at 2000 RPM for 10 sec, and then RPM was increased up to 5000 RPM for 1 min to obtain the as much as PCR cocktail containing the RNA template (Fig. 3E). In this system, the wastes from RNA samples and washing buffer were transported to the waste chamber, which is fully filled to the chamber with precise optimization. Then, the PCR cocktail was able to transport to the PCR chamber. Fig. 3F shows the final image of the sample pretreatment. PCR cocktail containing RNA template is successfully isolated from waste. To detect the influenza A H3N2 virus, the purified RNA with PCR cocktail in the PCR chamber was amplified by using performed the RNA capture on the proposed microdevice. The fluorescence images were described in Figure 4A at the 0, 40 cycles. The fluorescence signal (40 cycle) was drastically increased confirming the influenza A H3N2 virus. The real-time profiles were successfully obtained using the optical fluorescence detector as shown in Figure 4B. The Rotary PCR and off-chip PCR were compared with same amount of influenza A H3N2 virus. The Ct value of Rotary PCR was smaller than the off-chip PCR without contamination. The whole process of the sample pretreatment and RT-PCR could be accomplished in 30 min on the fully integrated Rotary Genetic Analyzer system. We have demonstrated a fully integrated and portable Rotary Genetic Analyzer for detection of the gene expression of influenza A virus, which has 'Sample-in-answer-out' capability including sample pretreatment, rotary amplification, and optical detection. Target gene amplification was real-time monitored using the integrated Rotary Genetic Analyzer system.

• Bulletin of the Korean Chemical Society
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• 제31권2호
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• pp.415-418
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• 2010

Highly ordered mesoporous silver material was successfully synthesized from a mesoporous silica template (KIT-6) with 3-D channel structure using the nano-replication method. The effects of $H_2$ or $O_2$ pretreatments on the catalytic performance of the mesoporous silver were investigated using a temperature programmed CO oxidation technique in a fixed bed reactor. The mesoporous silver material that was pretreated with $H_2$ exhibited an excellent catalytic activity compared to the as-prepared and $O_2$-pretreated catalysts. Moreover, this present mesoporous silver material showed good catalytic stability. For the CO oxidation, the apparent activation energy of the $H_2$-pretreated mesoporous silver catalyst was $61{\pm}0.5\;kJ\;mol^{-1}$, which was also much lower than the as-prepared ($132{\pm}1.5\;kJ\;mol^{-1}$) and $O_2$-pretreated ($124{\pm}1.4\;kJ\;mol^{-1}$) catalysts.

• 대한의생명과학회지
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• 제10권1호
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• pp.23-29
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• 2004

Nebulin is a (Mr 600∼900 kDa) large actin-binding protein specific to skeletal muscle and thought to act as a molecular template that regulates the length of thin filaments. Cardiac muscles of higher vertebrates have been shown earlier to lack nebulin. Recently, full-length nebulin mRNA transcripts have been detected in heart muscle, but at lower levels than in skeletal muscle. Nebulin expression also was detected in the kidney, eye, and otic canal, suggesting that nebulin isoforms may also be expressed in these organs. We have searched for nebulin isoforms in brain of human using PCR and Northern blot. Here, we provide evidence that nebulin mRNA transcripts are expressed in brain. Seven nebulin isoforms (B, C, D, E, F, G and H form) are obtained in human skeletal muscle and four isoforms (B, C, G and H form) in human brain cDNA library. We cloned the 1.3 kb of nebulin fragment from human adult brain library by PCR. The identity of the PCR product was confirmed by sequence analysis. The partial brain nebulin sequence was 99% identical to the skeletal muscle cDNA as determined by Blast alignment. It contains two simple-repeats HR1, HR2 and linker-repeats exon l35∼143 except exon 140. It was different from skeletal muscle B form, which contain HR1 and HR8. These data suggest that nebulin isoform diversity occurs even more extensively than previously known, likely contributing to the distinct thin filament architecture of different striated muscles.

• Plant Biotechnology Reports
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• 제4권3호
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• pp.201-211
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• 2010

We present a highly effective T-DNA inserted gene screening method as part of a reverse genetics model system using the Chinese cabbage (Brassica rapa L. spp. pekinensis). Three-step two-dimensional (2D) matrix strategies are potentially accurate and useful for the identification of specific T-DNA inserted mutants from a large population. To construct our Chinese cabbage model, we utilized a forward genetics screening approach for the abnormal phenotypes that were obtained from transgenic plants of Brassica rapa generated with Agrobacteria tumefaciens containing the pRCV2 vector. From one transgenic plant with an abnormal phenotype, we observed that the st1 gene (which is related to senescence-associated process proteins) contained a T-DNA fragment, and that its expression level was decreased. This T-DNA insert was then used as a control to construct an effective screening pool. As a result, the optimum template concentration was found to be 0.1-1 ng in our PCR strategy. For other conditions, positive changes to the Gibbs free energy prevented the formation of oligo dimers and hairpin loop structures, and autosegment extension gave better results for long fragment amplification. Using this effective reverse genetics screening method, only 23 PCR reactions were necessary to select a target gene from a pool of 100 individual DNAs. Finally, we also confirmed that the sequence we obtained from the above method was identical to the flanking sequence isolated by rescue cloning.

• 한국CDE학회논문집
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• 제19권2호
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• pp.138-147
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• 2014

In manufacturing companies, different types of production have been developed based on diverse production strategies and differentiated technologies. The production systems have become smart, factories are filled with unmanned manufacturing lines, and sustainable manufacturing technologies are under development. Nowadays, the digital manufacturing technology is being adopted and used in manufacturing industries. When this technology is applied, a lot of efforts, time and cost are required and training professionals in-house is limited. In this paper, we introduce e-FEED system (electronic based Front End Engineering and Design) that is the integrated design and analysis system for optimized manufacturing line development on virtual environment. This system provides the functions that can be designed easily using library and template based on standardized modules and analyzed automatically the logistic and capacity simulation by one-click and verified the result using visual reports. Also, we can review the factory layout using automatically created 3D virtual factory and increase the knowledge reuse by e-FEED system.

• 대한바이러스학회지
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• 제27권2호
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• pp.115-128
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• 1997

Three cDNA fragments located within NS5 region of HCV were synthesized by RT using viral RNA extracted from blood sample of Korean patient as a template. The cDNAs were amplified by PCR, cloned into the T-vector, and the nucleotide sequences were determined. Comparative analysis of the nucleotide and amino acid sequence of NS5 cDNAs showed that it is closely related with HCV type 1b. The cloned NS5 cDNA showed 91-94% homology at the nucleotide sequence level and 96-98% homology at the amino acid sequence level with several strains of the HCV type 1b. The NS5 cDNAs were subcloned into E. coli expression vectors to construct pRSETA5-1, pTHAN5-1, pRSETC5-2, pRSETBB1, pRESTCB1 and pRSETB-H3. Expression of the NS5 proteins was achieved by inducing the promoter with isopropyl-thio-${\beta}$-D-galactoside (IPTG) and confirmed by SDS-polyacrylamide gel electrophoresis. The NS5 proteins were immunoreactive against sera from Korean hepatitis C patients in Western blot analysis. Among the recombinant NS5 proteins, pRSETAS-1 plasmid derived protein, coded from aa2022 to aa2521 of HCV polyprotein, showed the strongest immunoreactivity against sera from Korean hepatitis C patients in immunoblot analysis. These results suggest that NS5 proteins would be useful as an antigen for detection of antibody against HCV in the blood samples.

• Asian-Australasian Journal of Animal Sciences
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• 제15권9호
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• pp.1237-1243
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• 2002

We have developed a reliable and noninvasive method for swine genotyping of single locus nuclear gene with aged single hair follicles delivered by general mail. The method is based on booster and nested PCR amplification with step-wise increase of primers and dNTPs concentrations followed by restriction endonuclease digestion. To establish this method, the ryanodine receptor (RYR 1) locus which is an economically important trait in swine industry was employed for genotyping experiment. The 3-step PCR amplication method is much less dependent on the quantity and quality of template DNA and produces enough amplification product for the detection on the ethidium bromide-stained gel such as RFLP analysis. A total of 120 pigs were subjected to the RYR 1 genotyping analysis using three-step PCR method which amplified enough quantity of PCR products from the aged single hair follicles for RFLP analysis and genotyping results were identical to the results of the corresponding ethanol-fixed skeletal muscle tissue. This approach will be a great help for porcine breeders and investigators in genotyping of swine. They can receive genotyping results later by simply plucking single hairs of their pigs at farm and sending them in general mail to the diagnostic laboratory which eliminates the inconveniences to collect ear tissue or blood cells from pigs, or the investigator's need for travel to farms in order to collect fresh hair samples.

• Archives of Pharmacal Research
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• 제29권1호
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• pp.50-58
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• 2006

Translationally controlled tumor protein (TCTP), also known as histamine releasing factor (HRF), is found abundantly in different eukaryotic cell types. The sequence homology of TCTP between different species is very high, belonging to the MSS4/DSS4 superfamily of proteins. TCTP is involved in both cell growth and human late allergy reaction, as well as having a calcium binding property; however, its primary biological functions remain to be clearly elucidated. In regard to many possible functions, the TCTP of Plasmodium falciparum (Pf) is known to bind with an antimalarial agent, artemisinin, which is activated by heme. It is assumed that the endoperoxide-bridge of artemisinin is opened up by heme to form a free radical, which then eventually alkylates, probably to the Cys14 of PfTCTP. Study of the docking of artemisinin with heme, and subsequently with PfTCTP, was carried out to verify the above hypothesis on the basis of structural interactions. The three dimensional (3D) structure of PfTCTP was built by homology modeling, using the NMR structure of the TCTP of Schizosaccharomyces pombe as a template. The quality of the model was examined based on its secondary structure and biological function, as well as with the use of structure evaluating programs. The interactions between artemisinin, heme and PfTCTP were then studied using the docking program, FlexiDock. The center of the peroxide bond of artemisinin and the Fe of heme were docked within a short distance of $2.6{\AA}$, implying the strong possibility of an interaction between the two molecules, as proposed. When the activated form of artemisinin was docked on the PfTCTP, the C4-radical of the drug faced towards the sulfur of Cys14 within a distance of $2.48{\AA}$, again suggesting the possibility of alkylation having occurred. These results confirm the proposed mechanism of the antimalarial effect of artemisinin, which will provide a reliable method for establishing the mechanism of its biological activity using a molecular modeling study.