• Korean Journal of Microbiology
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• v.38 no.2
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• pp.86-95
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• 2002

As a result of genome projects, the research to elucidate the function of a protein of interest has recently been well-recognized. In order to facilitate functional genomics, a useful mammalian gene expression vector is required. Using an infectious CDNA clone of BVDV pNADLclns-, we have developed a mammalian gene expression vector. In this study, a replication-competent full-length infectious CDNA clone containing puremycin acetyltransferase (pac) gene (pNADLclns-/pac) was successfully generated. The viral RNA replication and viral protein NS3 synthesis were examined by detecting metabollically $^{32}P$-labelled genomic viral RNA and immunoblotting with a mouse anti-NS3 antibody. To generate viral replicon as an expression vector, we examine if the viral structural genes (C, E0, El, E2) are required for viral replication by deletion analysis. As a result, all of the structural proteins are dispensable for viral replication per se, but essential for infectious viral particle formation. Based on our deletion analysis, we have generated a replication-competent BVDV viral replicon (pNADLclns-/pac/${\Delta}S$), whose structural genes are all deleted. In addition to NADLclns- /pac/${\Delta}S$, NADLclns-/ luc/${\Delta}S$ viral replicon containing luciferase gene as a reporter was constructed and fecund to be replication-compotent in HeLa and BHK cells as well as MDBK cells. Therefore, BVDV viral replicon developed in our study will be a useful tool to express a protein of interest in various mammalian cells.

• Clinical and Experimental Pediatrics
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• v.53 no.3
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• pp.373-379
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• 2010

Purpose : This study was performed to investigate the epidemiologic and clinical features of 13 respiratory viruses in children with acute lower respiratory tract infections (ALRIs). Methods : Nasopharyngeal aspirates were prospectively obtained from 325 children aged 15 years or less from May 2008 to April 2009 and were tested for the presence of 13 respiratory viruses by multiplex real-time-polymerase chain reaction (RT-PCR). Results : Viruses were identified in 270 children (83.1%). Co-infections with ${\geq}2$ viruses were observed in 71 patients (26.3 %). Respiratory syncytial virus (RSV) was the most common virus detected (33.2%), followed by human rhinovirus (hRV) (19.1%), influenza virus (Flu A) (16.9%), human metapneumovirus (hMPV) (15.4%), parainfluenza viruses (PIVs) (8.3%), human bocavirus (hBoV) (8.0%), adenovirus (ADV) (5.8%), and human coronavirus (hCoV) (2.2%). Clinical diagnoses of viral ALRIs were bronchiolitis (37.5%), pneumonia (34.5%), asthma exacerbation (20.9%), and croup (7.1%). Clinical diagnoses of viral bronchiolitis and pneumonia were frequently demonstrated in patients who tested positive for RSV, hRV, hMPV, or Flu A. Flu A and hRV were most commonly identified in children older than 3 years and were the 2 leading causes of asthma exacerbation. hRV C was detected in 14 (4.3%) children, who were significantly older than those infected with hRV A ($mean{\pm}SD$, $4.1{\pm}3.5$ years vs. $1.7{\pm}2.3$ years; P =0.009). hBoV was usually detected in young children ($2.3{\pm}3.4$ years) with bronchiolitis and pneumonia. Conclusion : This study described the features of ALRI associated with 13 respiratory viruses in Korean children. Additional investigations are required to define the roles of newly identified viruses in children with ALRIs.

• Applied Biological Chemistry
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• v.50 no.2
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• pp.95-100
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• 2007

The cellulase gene of Bacillus licheniformis K11 which has plant growth-promoting activity by auxin and antagonistic ability by siderophore was cloned in pUC18 using PCR employing heterologous primers. The 1.6kb PCR fragment contained the full sequence of the cellulase gene, denoted celW which has been reported to encode a 499 amino acid protein. Similarity search in protein data base revealed that the cellulase from B. licheniformis K11 was more than 97% identical in amino acid sequence to those of various Bacillus spp. The cellulase protein from B. licheniformis K11, overproduced in E. coli DH5${\alpha}$ by the lac promoter on the vector, had apparent molecular weight of 55 kDa upon CMC-SDS-PAGE analysis. The protein not only had enzymatic activity toward carboxymethyl-cellulose (CMC), but also was able to degrade insoluble cellulose, such as Avicel and filter paper (Whatman$^{\circledR}$ No. 1). In addition, the cellulase could degrade a fungal cell wall of Phytophthora capsici. Consequently B. licheniformis K11 was able to suppress the peperblight causing P. capsici by its cellulase. Biochemical analysis showed that the enzyme had a maximum activity at 60$^{\circ}C$ and pH 6.0. Also, the enzyme activity was activated by Co$^{2+}$ of Mn$^{2+}$ but inhibited by Fe$^{3+}$ or Hg$^{2+}$. Moreover, enzyme activity was not inhibited by SDS or sodium azide.

• Restorative Dentistry and Endodontics
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• v.31 no.6
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• pp.437-444
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• 2006

This study was aimed to elucidate the biological function of OD314 (Apin protein), which is related to ameloblast differentiation and amelogenesis. Apin protein, calcifying epithelial odontogenic (pindborg) tumors (CEOTs)-associated amyloid, were isolated from CEOTs, and has similar nucleotide sequences to OD314. We examined expression of the OD314 mRNA using in-situ hybridization during tooth development in mice. Expression of OD314 and several enamel matrix proteins were examined in the cultured ameloblast cell line up to 28 days by reverse transcription-polymerase chain reaction (RT-PCR) amplification. After inactivation and over-expression of the OD314 gene in ameloblast cell lines using U6 vectordriven RNA interference and CMV-OD314 construct, RT-PCR were performed to evaluate the effect of the OD314 during amelogenesis. The results were as follows: 1. In in-situ hybridization, OD314 mRNAs were more strongly expressed in ameloblast than odontoblast. 2. When ameloblast cells were cultured in the diffcrentiation and mineralization medium for 28 days, the tuftelin mRNA expression was maintained from the beginning to day 14, and then gradually decreased to day 28. The expressions of amelogenin and enamelin were gradually decreased according to the ameloblast differentiation. 3. Inactivation of OD314 by U6-OD314 siRNA construct down-regulated the expression of OD314, MMP-20, and tuftelin, whereas over-expression of OD314 by CMV-OD314 construct up-regulated the expression of OD314 and MMP-20 without change in tuftelin. These results suggest that OD314 is considered as an ameloblast-enriched gene and may play the important roles in ameloblast differentiation and mineralization.

• Journal of the Korean Society of Food Science and Nutrition
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• v.37 no.1
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• pp.8-15
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• 2008

Saussurea lappa (SL) has been used to reduce abdominal pain and tenesmus in traditional oriental medicine. SL and major compounds of SL, sesquiterpene lactones, have been suggested to possess various biological effects, including anti-tumor, anti-ulcer, anti-inflammatory, anti-viral and cardiotonic activities. Recently, it has been reported that ethanol extracts from roots of SL have antiproliferative effects on gastric cancer cells. To explore the possibility that SL has chemopreventive effects on prostate cancer, we examined whether the hexane extract of SL (HESL) inhibits the growth of LNCaP human prostate cancer cells. Cells were incubated with various concentrations ($0{\sim}4$ mg/L) of HESL in DMEM/F12 containing 5% charcoal stripped fetal bovine serum. HESL substantially decreased viable cell numbers and induced apoptosis of LNCaP cells in dose-dependent manners. HESL increased the levels of cleaved caspase-8, -9, -7 and -3, and poly (ADP-ribose) polymerase. HESL increased the levels of the pro-apoptotic Bak and truncated-Bid proteins whereas it had no effect on the anti-apoptotic Bcl-2, Bcl-xL, or Mcl-1. The present results indicate that HESL inhibits the growth of human prostate cancer cells by inducing apoptosis, which involves the activation of the caspase cascades.

• Journal of the Korean Society of Food Science and Nutrition
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• v.32 no.1
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• pp.35-41
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• 2003

The study was conducted to develope a rapid method for the detection of Yersinia enterocolitica in spring water and vegetables via multiplex polymerase chain reaction (PCR) technique using ail, yst, uirF and subgenus-specific Y16S primers. Specificity and sensitivity of multiplex PCR and application of best primers for the detection of Y. enterocolitica from spring water and vegetables were investigeted. Y. enterocolitica ATCC 27729 strains gave 356 bP and 200 bp (Y16S) and 134 bp (yst) bands. but Y. enterocolitica ATCC 9610 and ATCC 23715 strains gave 200 bp and 134 bp bands.In the meanwhile, non-pathogenic Yersinia species, such as Y. frederikseni, Y. inter-media, Y. kristenseni and Y. pseudotuberculosis gave only single 200 bp band, and other bacteria including Escherichia coli O157:H7 ATCC 25392, Shigella dysenteri. Staphylococcu aureus ATCC 25923 and Listeria mo-nocytogenes ATCC 19111 did not show any bands. Among primers, yst and Y16S primer showed the best sensitivity. Seven CFU/mL Y. enterocolitica cells could be detected with yst and Y16S primers and the sensitivity was significantly improved by the further 2nd PCR after 38 cycles of first PCR amplication. Spring water, cabbage and mushroom were inoculated with Y. enterocolitica to determine the sensitivity of multiplex-PCR for the rapid detection of Y. enterocolitica. Multiplex-PCR assay could detect 7 or 70 cells in spring water and vegetables using whole cell lysate with repeating PCR amplication.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.5
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• pp.562-571
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• 2017

In this study, we investigated the anti-oxidant activities [electron-donating ability (EDA), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging ability, and reactive oxygen species (ROS) inhibitory activity], anti-wrinkle activities [collagenase inhibitory activity, suppression and/or phosphorylation of matrix metalloproteinases (MMPs), and mitogen-activated protein (MAP) kinase activity], and mRNA expression levels using reverse transcription-polymerase chain reaction (RT-PCR) assay in ultraviolet (UV) B ray ($50mJ/cm^2$)-irradiated human keratinocyte HaCaT cells. Josaengheugchal, Sinneungheugchal (SE), Shintoheug rice, Heugjinju rice, and Heugseol (HE) among colored rice varieties were reported to have excellent antioxidant properties. In the EDA and ABTS radical scavenging assays, extracts of the five colored rice varieties had scavenging activities of 72% at concentrations higher $50{\mu}g/mL$. In the collagenase inhibition assay, ethanol extracts of the five colored rice varieties showed high inhibitory effects of about 60% at concentrations higher $25{\mu}g/mL$. In the ROS inhibition assay, ethanol extracts of HE and SE showed very excellent inhibition efficacies at all concentrations. We determined molecular biological mechanisms of MMPs (MMP-1, -3, -8, and -13) and mitogen-activated protein kinase (MAPK) with HE, and the results show that HE suppressed expression of MMPs and phosphorylation of MAPK and increased expression of pro-collagen type I in UVB-irradiated cells. It was also confirmed by RT-PCR that HE reduced expression of MMPs mRNA. Therefore, these results suggest that HE has anti-wrinkle and collagen production effects and may be used as a material in the development of functional food and cosmetic industries.

• Journal of Gastric Cancer
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• v.7 no.1
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• pp.9-15
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• 2007

Purpose: DNA methylation is an important epigenetic factor in tumorigenesis. We hypothesized that polymorphism of the promoter of the DNA methyltransferase 3b (DNMT3b) genes, which are responsible for regulating the methylation status of tumor suppressor genes, are associated with increased risk of gastric cancer. Materials and Methods: In this hospital-based case-control study, to determine the role of this polymorphism of the promoter of DNA methyltransferase 3b (DNMT3b) genes in gastric cancer, we genotyped 176 cases and 70 control subjects. To determine the genotype, we used a polymerase chain reaction restriction fragment length polymorphism assay. We compared alleles and genotypes between the two groups and revealed an association of DNMT3b promoter polymorphism with increased risk of gastric cancer in the Korean population. Results: Genotype frequencies were 14.8% (Cytosine-Cytosine), 71.6% (Cytosine-Thymine), and 13.6% (Thymine- Thymine) in the case patients and 40.0% (Cytosine-Cytosine), 42.9% (Cytosine-Thymine), and 17.1% (Thymine-Thymine) in the control subjects, respectively. Compared with CC homozygotes, CT heterozygotes had a 4.523-fold increased risk (OR, 2.13; 95% CI, 2.324~8.803), and the TT homozygotes had a 2.154-fold elevated risk (OR, 1.42; 95% CI, 0.899~85.165). For the T variant genotype (CT+TT), there was a 3.846-fold increased risk (OR, 1.88; 95% CI, 2.040~7.251). However, no significance was observed in the genotype distributions of both polymorphisms according to histopathology, stage of stomach cancer. The Ssame results were observed with Helicobacter infection. Conclusion: DNMT3b promoter polymorphism, especially the T variant genotype, is associated significantly with thean increased risk of gastric cancer.

• Tuberculosis and Respiratory Diseases
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• v.49 no.5
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• pp.546-557
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• 2000

Background : Oligonucleotide chip technology has proven to be a very useful tool in the rapid diagnosis of infectious disease. Rifampin resistance is considered as a useful marker of multidrug-resistance in tuberculosis. Mutations in the rpoB gene coding $\beta$ subunit of RNA polymerase represent the main mechanism of rifampin resistance. The purpose of this study was to develop a diagnosis kit using oligonucleotide chip for the rapid and accurate detection of rifampin-resistance in Mycobacterium tuberculosis. Method : The sequence specific probes for mutations in the rpoB gene were designed and spotted onto the glass slide, oligonucleotide chip. 38 clinical isolates of Mycobacterium were tested. A part of rpoB was amplified, labelled, and hybridized on the oligonucleotide chip with probes. Results were analyzed with a laser scanner. Direct sequencing was done to verify the results. Result : The low-density oligonucleotide chip design어 to determine the specific mutations in the rpoB gene of M. tuberculosis accurately detected rifampin resistance associated with mutations in 28 clinical isolates. Mutations at codons 531, 526, and 513 were confirmed by direct sequencing analysis. Conclusion : Mutant detection using oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of multidrug-resistance in M. tuberculosis.

• Tuberculosis and Respiratory Diseases
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• v.43 no.6
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• pp.842-851
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• 1996

Background : Rifampicin(RFP) is a key component of the antituberculous shon-course chemotherapy and the RFP-resistance is a marker of multi-drug resistant(MDR) M. tuberculosis. rpoB gene encodes the ${\beta}$-subunit of RNA polymerase of M. tuberculosis which is the target of RFP. Recent reports show that rpoB gene mutations are the cause of RFP resistance of M. tuberculosis and the main mechanism of rpoB gene mutation is point mutation. And PCR-SSCP is a rapid and easy method for detecting point mutations. So we performed PCR-SSCP of rpoB gene of M. tuberculosis and compared the result with traditional RFP sensitivity test. Method : The 27 RFP sensitive M. tuberculosis culture isolates and 25 RFP resistant isolates were evaluated. The RFP sensitivity test was done at the Korean Tuberculosis istitute. The DNA was extracted by bead beater method and was amplified with primers TR-8 and TR-9 in a 20ul PCR reaction containing 0.1ul(luCi) [${\alpha}-^{32}P$] - dCTP. After amplification, SSCP was done using non-denaturaring polyacrylamide gel electrophoresis. Then direct sequencing was done in cases of different eletrophoretic mobility compared with that of H37Rv. In 19 cases, we compared PCR-SSCP results with patient's clinical course and the results of traditional RFP sensitivity test. Results : 1) All 27 RFP sensitive M. tuberculosis isolates showed the same electrophoretic mobility compared with that of H37Rv. And all 25 RFP resistant M. tuberculosis isolates showed different electrophoretic mobility. 2) The mechanism of rpoB gene mutation of M. tuberculosis is mainly point mutation. 3) The PCR-SSCP results correlate well with traditional RFP sensitivity and patient's clinical response to antituberculous treatment. Conclusion: The PCR-SSCP of rpoB gene is a very sensitive and rapid mehod in detecting RFP- resistant M. tuberculosis.