• Korean Journal of Clinical Laboratory Science
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• v.41 no.4
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• pp.153-157
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• 2009

The production of extended-spectrum ${\beta}$-lactamases ($ESBL_S$) of the TEM or SHV type by bacterial pathogens is a major threat to the use of the clinically important expanded-spectrum cephalosporins. The characterization of the SHV ESBLs producing Klebsiella pneumoniae strains present in clinical isolates is time-consuming processes. We describe here in the development of a novel system, which consists of a real time PCR. We found 11 K. pneumoniae strains to be presumptive strains ESBLs producers by clinical and laboratory standards institute (CLSI) guidelines. The double disk synergy test showed 8 ESBL positive and conventional PCR showed 10 SHV ESBL positive, which were K. pneumoniae strains isolates. By real time PCR analysis, SHV gene in 11 of 11 strains were identified. When sequencing analysis was compared with real time PCR, both analysis were presented 99% similarity. In this study, we used a rapid, sensitive, and specific real-time PCR (RT-PCR) method for detection of the assay SHV ESBL producing K. pneumoniae strains in clinical isolates.

• Journal of Plant Biotechnology
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• v.42 no.2
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• pp.117-122
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• 2015

Canola is a crop globally used for production of oil and biofuel. Cultivation area and import volume of living modified (LM) canola have been increasing every year. As canola import dependence has reached 100% in Korea, efforts have been made for safety management of LM canola and ecological risk assessment. We developed a set of multiplex PCR method for simultaneous detection of 5 LM canola events (Topas 19/2, Rf3, Ms8, RT73 and T45) approved in Korea. The multiplex PCR assay developed allows amplification of estimated products of 5 LM canolas from event specific primer sets. Primer extension time was skipped for a time-consuming process and two annealing steps (20 cycles at $55^{\circ}C$ and 20 cycles at $60^{\circ}C$) were performed for yielding the best result which was sufficient to distinguish five LM canolas. Our results suggest that multiplex PCR method provides a cost and time-effective approach for LM canola detection.

• Tuberculosis and Respiratory Diseases
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• v.62 no.5
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• pp.406-416
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• 2007

Background: Single nucleotide polymorphisms (SNPs), which consist of a substitution of a single nucleotide pair, are the most abundant form of genetic variations occurring with a frequency of approximately 1 per 1000 base pairs. SNPs by themselves do not cause disease but can predispose humans to disease, modify the extent or severity of the disease or influence the drug response and treatment efficacy. Single nucleotide polymorphisms (SNPs), particularly those within the regulatory regions of the genes often influence the expression levels and can modify the disease. Studies examining the associations between SNP and the disease outcome have provided valuable insight into the disease etiology and potential therapeutic intervention. Traditionally, the genotyping of SNPs has been carried out using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP), which is a low throughput technique not amenable for use in large-scale SNP studies. Recently, TaqMan real-time PCR chemistry was adapted for use in allelic discrimination assays. This study validated the accuracy and utility of real-time PCR technology for SNPs genotyping Methods: The SNPs in promoter sequence (-37 and -524) of lung cancer suppressor gene, RRM1 (ribonucleotide reductase M1 subunit) with the genomic DNA samples of 89 subjects were genotyped using both real-time PCR and PCR-RFLP. Results: The discordance rates were 2.2% (2 mismatches) in -37 and 16.3% (15 mismatches) in -524. Auto-direct sequencing of all the mismatched samples(17 cases) were in accord with the genotypes read by real-time PCR. In addition, 138 genomic DNAs were genotyped using real-time PCR in a duplicate manner (two separated assays). Ninety-eight percent of the samples showed concordance between the two assays. Conclusion: Real-time PCR allelic discrimination assays are amenable to high-throughput genotyping and overcome many of the problematic features associated with PCR-RFLP.

• Journal of Animal Science and Technology
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• v.53 no.3
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• pp.269-274
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• 2011

DNA methyltransferases (DNMTs) are closely associated with the epigenetic change and the gene silencing through the regulation of methylation status in animal genome. But, the role of DNMTs in transgene silencing has remained unclear. So, we examined whether the knockdown of DNMT influences the reactivation of transgene expression in the transgenic quails. In this study, we investigated the expression of DNMT3a, and DNMT3b in blastoderm, quail embryonic fibroblasts (QEFs) and limited embryonic tissues such as gonad, kidney, heart and liver of E6 transgenic quails (TQ2) by RT-PCR. We further analyzed the expression of DNMT3a at different stages of whole embryos during early embryonic development by qRT-PCR. DNMT3a expression was detected in all test samples; however, it showed the highest expression in E6 whole embryo. Embryonic fibroblasts collected from TQ2 quails were treated with two DNMT3a-targeted siRNAs (siDNMT3a-51 and siDNMT3a-88) for RNA interference assay, and changes in expression were then analyzed by qRT-PCR. The siDNMT3a-51 and siDNMT3a-88 reduced 53.34% and 64.64% of DNMT3a expression in TQ2 QEFs, respectively. Subsequently the treatment of each siRNA reactivated enhanced green fluorescent protein (EGFP) expression in TQ2 (224% and 114%). Our results might provide a clue for understanding the DNA methylation mechanism responsible for transgenic animal production and stable transgene expression.

• International Journal of Oral Biology
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• v.35 no.2
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• pp.69-74
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• 2010

The mu opioid receptor (MOR) has been regarded as the main site of interaction with analgesics in major clinical use, particularly morphine. The repressor element-1 silencing transcription factor (REST) functions as a transcriptional repressor of neuronal genes in non-neuronal cells. However, it is expressed in certain mature neurons, suggesting that it may have complex and novel roles. In addition, the interactions between MOR and REST and their functions remain unclear. In this study, we examined the effects of morphine on the expression of REST mRNA and protein in human neuroblastoma NMB cells to investigate the roles of REST induced by MOR activation in neuronal cells. To determine the effects of morphine on REST expression, we performed RT-PCR, real-time quantitative RT-PCR, western blot analysis and radioligand binding assays in NMB cells. By RTPCR and real-time quantitative RT-PCR, the expression of REST was found to be unchanged by either the MOR agonist morphine or the MOR specific antagonist CTOP. By western blot, morphine was shown to significantly inhibit the expression of REST, but this suppression was completely blocked by treatment with CTOP. In the radioligand binding assay, the overexpression of REST led to an increased opioid ligand binding activity of endogenous MOR in the NMB cells. These results together suggest that morphine inhibits the expression of REST in human neuroblastoma cells through a post-transcriptional regulatory mechanism mediated through MOR.

• Journal of Preventive Medicine and Public Health
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• v.48 no.1
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• pp.53-61
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• 2015

Objectives: The seroprevalence of hepatitis E virus (HEV) among high-risk groups overseas is high, but studies in these groups are rare in South Korea. We conducted the present study from April to November 2012 to obtain data on the seroprevalence and associated risk factors for HEV among slaughterhouse workers in South Korea. Methods: Slaughterhouse workers from 80 workplaces nationwide were surveyed in South Korea in 2012. The subjects comprised 1848 cases: 1434 slaughter workers and 414 residual products handlers. By visiting 80 slaughterhouses, which were mixed with 75 of which also performed residual products handling, we conducted a questionnaire survey for risk factors and obtained blood samples in order to determine the seropositivity and seroprevalence of HEV. Anti-HEV IgG and IgM were measured using HEV IgG and IgM enzyme-linked immunospecific assay kits and HEV antigen was measured by reverse transcription polymerase chain reaction (RT-PCR). Results: The seropositivity of anti-HEV IgG was 33.5% (slaughter workers 32.8% and residual products handlers 36.2%), and among the seropositive individuals the seroprevalence of anti-HEV IgM was 0.5% (slaughter workers 0.5%, residual products handlers 0.7%). The response rate of HEV-antigen as measured by RT-PCR was 0.2%. Risk factors significantly related to anti-HEV IgG seropositivity were age, sex, and working duration (slaughter workers only). Conclusions: There were significant risk factors (sex, age, and working duration) for HEV identified in our study. All three positive cases for HEV-antigen by RT-PCR were related to pig slaughter but without statistical significance. To prevent HEV, an educational program and working guidelines may be needed for high risk groups.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.14 no.4
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• pp.1857-1862
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• 2013

Dermal fibroblasts produce the many components of the extracellular matrix (ECM) that are needed to maintain connective tissue integrity and repair tissue injuries. This study investigated the effects of Areca catechu extract (ACE) on dermal fibroblast cell activation. Cultured human dermal fibroblasts were treated with ACE, and then ECM production was determined by ELISA, Western blot and RT-PCR. ACE significantly accelerated the production of type 1 collagen, fibronectin, and transforming growth factor (TGF)-${\beta}1$ by ELISA and type 1 collagen by Western blot assay. ACE also increased the gene expression of COL1A1, TGF-${\beta}1$, keratinocyte growth factor (KGF) and insulin growth factor (IGF)-1. These results suggest that ACE has the potential to stimulate ECM production and that it might be suitable for maintaining skin texture.

• The Plant Pathology Journal
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• v.16 no.6
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• pp.297-305
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• 2000

Weeds are widely grown in the field and are infected by many viruses. A survey was conducted to identify viruses infecting weeds in Korea. Virus-infected weed samples including Rorippa indica (L.) Hiern, R. islandica (Oed.) Bord, Crepidiastrum denticulatum (Houtt.) Pak & Kawanno, Achyranthes japonica (Miq.) Nakai, and Chrysanthemum boreale (Makino) Makino were collected in Kyonggi Province. These weeds were grown in the greenhouse and were isolated on 10 test plants. Several virus isolates were isolated fron infected tissues and were further studied by host range assay, serological test, electron microscopy (EM), reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. Each isolated virus strain was mechanically transmitted to weeds and various hosts including Nicotiana spp., Brassica spp., Vigna unguiculata, Capsicum annuum, and Cucumis sativus and showed systemic mosaic, vein clearing, necrosis, mottle, malformation, chlorosis, and/or death of host plants in some cases. Each virus was then purified using infected leaves and observed by EM. From these results three viruses were isolated and identified as Turnip mosaic virus (TuMV), Broad bean wilt virus (BBWV), and Cucumber mosaic virus (CMV). RT-PCR using virus-specific oligonucleotide primers and the cloning were conducted to determine the nucleotide sequences of coat proteins of the three viruses their amino acid sequence were deduced. The amino acid sequence homologies were about 92.7 to 99.7%, 96.2 to 97.7%, and 93.9 to 98.6% to other reported TuMV, BBWV, and CMV strains, respectively. These results suggest that many weeds may serve as primary inoculum source of diseases caused by TuMV, BBWV, CMV and that the management of these viral diseases can be achieved through weed control.

• The Plant Pathology Journal
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• v.20 no.2
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• pp.127-130
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• 2004

Grapevine leaf roll-associated virus 3 (GLRaV-3) is one of the most important viral diseases of grapevine in the world. In this study, GLRaV-3 Ampelovirus was identi-fied from grapevines in Korea by analyzing viral coat protein size, nucleotide, and amino acid sequences. The molecular weight of viral coat protein from virus-infected in vitro plantlets was determined by western blot using a commercial GLRaV-3 polyclonal antibody. Western blot analysis showed a coat protein of about 43 kDa. RT-PCR product of about 942 bp which encoded the coat protein (CP) gene was amplified with specific primers. When the viruses existed at low titers in the host plant, the dsRNA had very specific template in RT- PCR amplification of fruit tree viruses. Especially, small-scale dsRNA extraction method was very reliable and rapid. Sequence analysis revealed that the CP of the GLRaV-3 Ko consisted of 942 bp nucleotide, which encoded 314 amino acid residues. The CP gene of GLRaV-3 Ko had 98.9% nucleotide sequence and 98.7% amino acid sequence identities with earlier reported GLRaV-3. This is the first report on molecular assay of GLRaV-3 Ampelovirus identified from Korea. The GLRaV-3 Ko CP clone would be very useful for breeding of virus resistant grapevines.

• Journal of Periodontal and Implant Science
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• v.35 no.2
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• pp.277-288
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• 2005

There is a potential role of collagenase-3 in alveolar bone loss and periodontal disease progression, we need to develope or find chemotherapeutic drugs or herbal agents which may regulate the expression of MMP-13. Ginseng saponin, one of the major components of Korea ginseng(panax ginseng) root, has many various biologic effects, such as cytotoxic effect, tumoricidal effects, cytokine regulations, and protein biosynthesis effect. The purpose of this study was to determine the effects of Korea red ginseng saponin on MMP-13 gene expression in osteoblasts. The experimental groups were cultured with ginseng saponin in concentration of 1.0, 10, 25, 50, 100, 250 and $500{\mu}g/ml$ for MTT assay. Primary rat calvarial cells were pre-treated for 1 hour with ginseng saponin(100 ${\mu}g/ml$) and then stimulated with $IL-1{\beta}(1.0ng/ml)$ and PTH(10 nM). MMP-13 gene expression was evaluated by RT-PCR. The results were as follows: Ginseng saponin was cytotoxic to osteoblast at concentration exceeding $250{\mu}g/ml$ for longer than 24 hours in tissue culture(p<0.01). In RT-PCR analysis, steady state MMP-13 mRNA levels were increased approximately 350% by $IL-1{\beta}$, and 400% by PTH when normalized to untreated control. $IL-1{\beta}-indued$ MMP-13 mRNA expression was reduced 50% by pretreatment with ginseng saponin. But ginseng saponin didn't inhibit MMP-13 expression from PTH stimulated cells. This results suggest that ginseng saponin Inhibit $IL-1{\beta}-indued$ MMP-13 mRNA expression.