• Food Science of Animal Resources
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• v.33 no.5
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• pp.610-616
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• 2013

Loop-mediated isothermal amplification (LAMP) is an emerging detection technology for the amplification of DNA under isothermal conditions. The aim of this study was to develop a rapid and reliable LAMP technique for the detection of Cronobacter sakazakii in milk powder. In order to enhance the sensitivity and specificity, LAMP primers targeting outer membrane protein A (ompA) gene of C. sakazakii were designed using Explorer V4 software. Thirty seven C. sakazakii strains and 13 pathogenic microorganisms were used for comparative detection of C. sakazakii using polymerase chain reaction (PCR), real-time PCR, and LAMP. LAMP developed in this study could specifically detect C. sakazakii strains without cross-reactivity with other foodborne pathogens. LAMP products amplified from ompA gene of C. sakazakii were digested with with HhaI and NruI enzyme. The specificity of LAMP was confirmed by restriction fragment length polymorphism (RFLP) analysis. LAMP could detect C. sakazakii within 1 h without bacterial culture and its detection limit was as low as 1 CFU/mL C. sakazakii in milk. In the comparison of the sensitivity, LAMP showed 10,000- and 100-times higher detection limit than PCR or real-time PCR, respectively. Therefore, this study can conclude that LAMP is a rapid and reliable detection technique for C. sakazakii contaminated in powdered milk.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.4
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• pp.1041-1054
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• 2004

Electroacupuncture (EA) has been reported to increase pain threshold, and to enhance the NK cell activity by up-regulation of IFN-γ and endogenous β-endolphin. For the purpose of understanding the molecular mechanism of EA stimulation, we analyzed the gene expression profile of rat hypothalamus, treated on Zusanli (ST36) with EA, in comparison with control group by oligonucleotide chip microarray (Affymetrix GeneChip Rat Neurobiology U34 Array) and real-time RT-PCR. Sprague-Dawley (S-D) male rats were stimulated at the Zusanli (ST36) acupoint in restriction holder. Simultaneously the control group was given only holder stress without EA stimulation. In order to prove the appropriateness of EA treatment, we measured spleen NK cell activity with standard 51Cr release assay. NK cell activity of EA group was significantly increased comparing to control group. The microarray and PCR results show that EA treatment up-regulates expression of genes associated with 1) nerve growth such as NGF induced factor A and VGF, 2) signal transduction such as 5HT3 receptor subunit, AMPA receptor binding protein and Na-dependent neurotransmitter transporter, and 3) anti-oxidation such as superoxide dismutase and glutathione S-transferase. In addition, the activity of the anti-oxidative enzyme, SOD of hypothalamus, liver and RBC was enhanced compared to that of control. The list of differentially expressed genes may implicate further insight on the mechanism of acupuncture effects.

• Journal of the Korean Society of Physical Medicine
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• v.9 no.1
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• pp.45-53
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• 2014

PURPOSE: In this study, we tried to find out what kind of exercise was more effective in sleep disorder by comparing melatonin in blood after applying low intensity with high intensity exercise to sleep disordered rats induced by experiment. METHODS: We used male Sprague-Dawley rats which were 8weeks old and weighted 300g. They were supplied with water and food without any restriction. We kept the room temperature at $25^{\circ}C$ and controld the length of day and night in 12 hours blocks, respectively. We divided the rats 60 into 2 groups. To one group we applied low intensity exercise, and to the other we applied high intensity exercise for 15minutes per day over a period of 4 weeks. We extracted the blood from abdominal aorta before, after exercise, moved into EDTA tube, performed centrifugation. We decanted the serum $200{\mu}l$ from the blood into microcentrifuge tube by samples and moved into polypropylene culture tubes with micro pipette. We split enzyme solution $50{\mu}l$ into the tubes with melatonin direct kits and make them react at $37^{\circ}C$ for 2 hours. We split assay buffer $50{\mu}l$ into each tube and mixed melatonin tracer $50{\mu}l$ and melatonin antiserum $50{\mu}l$, respectively. After we made them react in room temperature, we decanted the superficial layer with a centrifuge and measured the activity for 1 minute by competitive method with ${\gamma}$-counter equipment. We draw a standard curve through logit-log graph with CPM(counts per minute) and counted the melatonin by B/B0. We conducted independent t-test to examine the homogeneous of melatonin value of before low-intensity and high-intensity exercise. We performed paired t-test to compare before and after low-intensity and high-intensity exercise, respectively. We carried out independent t-test to compare melatonin value after low-intensity and high-intensity exercise. Significance level was .05. RESULTS: The results were as follows; firstly melatonin was more increased in the group who was exposed to high intensity exercise when we compared before to after high and low intensity exercise, respectively. Secondly, high intensity exercise was more effective than low intensity exercise when we compared the two. CONCLUSION: In conclusion, secretion of melatonin which is the material of sleep improvement could be promoted by high intensity exercise. Low intensity exercise acted as a stress rather than improving sleep and had a negative effect on the secretion of melatonin because the melatonin was affected by stress.

• Korean Journal of Microbiology
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• v.46 no.1
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• pp.80-85
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• 2010

Two different ${\alpha}$-amylase isozymes (AMY1 and AMY2) found in barley malt share up to 80% of amino acid sequence identity with each other, but their enzymatic properties differ remarkably. AMY1 shows the highest activity at low concentration of calcium ion, while AMY2 is highly active at high calcium concentration. Meanwhile, BASI (Barley ${\alpha}$-Amylase/Subtilisin Inhibitor) protein specifically inhibits only AMY2. In the present study, three separate regions in AMY genes (I, II, and III) were assigned on the basis of restriction enzyme sites and four kinds of chimeric amylases have been obtained by swapping a part of regions with each other. Each chimera gene was successfully over-expressed in Pichia pastoris. From the results of enzymatic characterization, both AMY211 and AMY122 showed the mixed or intermediate type of calcium-dependent activity between AMY1 and 2. Meanwhile, only AMY221 chimera could be significantly inhibited by BASI protein. As a result, it can be proposed that some amino acid residues in the region I and II, except region III, of barley ${\alpha}$-amylases play very important roles in calcium-dependency and interaction with BASI.

• Korean Journal of Microbiology
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• v.49 no.1
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• pp.1-7
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• 2013

During meiosis, genetic recombinants are formed by homologous recombination accompanying with the programmed double-strand breaks (DSBs) and strand exchanges between homologous chromosomes. The mechanism is generated by recombination intermediates such as single-end invasions (SEIs) and double-Holliday junctions (dHJs), and followed by crossover (CO) or non-crossover (NCO) products. Our study was focused on the analysis of meiotic recombination intermediates (DSBs, SEIs, and dHJs) and final recombination products (CO and NCO). We identified these meiotic recombination intermediates using DNA physical analysis under HIS4LEU2 "hot spot" system in budding yeast, Saccharomyces cerevisiae. For DNA physical analysis, when the hot spot locus is recognized by restriction enzyme from synchronous meiotic cells, the fragmented DNA that are forming recombination intermediates can be detected and quantified through Southern hybridization analysis. Our study suggests that this system can analyze the structural change of recombination intermediates during DSB-SEI transition, double-Holiday junctions and crossover/non-crossover products in meiosis.

• Microbiology and Biotechnology Letters
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• v.32 no.1
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• pp.91-95
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• 2004

For the detection of the oil-degrading bacterium, Nocardia sp. Hl7-1, inoculated during the bioremediation of oil-contaminated soil, a species-specific primer was constructed based on the 16S rDNA sequence of this strain. Two forward primers and two reverse primers were designed and tested against both closely and distantly related bacterial strains. All the primers designed were specific to the Nocardia sp. H17-1. Particularly, primer sets NH169F-NH972R and NH575F-NH972R could be used to detect 50 fg of template DNA and TEX>$1.2${\times}$10^4$ CFU/g of sandy soil. These two PCR primer sets successfully detected the H 17-1 strain in the oil-con-laminated soil samples containing heterogeneous DNA. We also conformed the primer specificity by restriction-enzyme cleavage of the PCR products and denaturing gradient gel electrophoresis.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.3
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• pp.215-222
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• 2002

Objective : Previous studies have suggested that hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR C677T) mutations are associated with increased risk of recurrent spontaneous abortion (RSA). Recently, a second site polymorphism in MTHFR, 1298A-->C, which changes a glutamic acid into an alanine residue, was shown to be associated with a decreased enzyme activity. We tested whether the variant alleles of MTHFR C677T and A1298C are risk factor (biomarker) for RSA. Materials and Methods: We analyzed DNA from a case-control study in the Korean DNA was extracted from blood samples of 118 patients with RSA and 123 healthy fertile patients as the controls. MTHFR variant alleles were determined by a PCR-restriction fragment length polymorphism assay. Results: We found no evidence for an association between 677TT genotype and risk of RSA (OR=1.95, 95% CI=$0.84{\sim}4.50$, p=0.12). However, the MTHFR 1298AC (OR=0.36, 95% CI=$0.20{\sim}0.63$, p=0.0004) and 1298AC+CC (OR=0.35, 95% CI=$0.20{\sim}0.61$, p=0.0002) genotypes were lower among 118 RSA cases compared with 123 controls, conferring a 2.8-fold decrease in risk of RSA, respectively. Moreover, the combined genotypes of MTHFR 677CC/1298AC (OR=0.30, 95% CI=$0.10{\sim}0.88$, p=0.029) and 677CT/1298AC (OR=0.77, 95% CI=$0.60{\sim}0.99$, p=0.043) also showed significantly lower risk than those with MTHFR 677CC/1298AA type. Conclusion: MTHFR 1298AC, MTHFR 677CC/1298AC and 677CT/1298AC genotypes may represent genetic markers for the protection of RSA at least in Korean women.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.627-632
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• 2007

FoxO1, FoxO3a and FoxO4 belong to the FoxO gene family, which play important roles in the PI3K/PKB pathway. In this study, we cloned the porcine FoxO1, FoxO3a and FoxO4 sequences and assigned them to SSC11p11-15, SSC1p13 and SSC xq13 using somatic cell hybrid panel (SCHP) and radiation hybrid panel (IMpRH). RT-PCR results showed that these three genes are expressed in multiple tissues. Sequencing of PCR products from different breeds identified a synonymous T/C polymorphism in exon 2 of FoxO3a. This FoxO3a single nucleotide polymorphism (SNP) can be detected by AvaII restriction enzyme. The allele frequencies of this SNP were investigated in Dahuabai, Meishan, Tongcheng, Yushan, Large White, and Duroc pigs. Association of the genotypes with growth and carcass traits showed that different genotypes of FoxO3a were associated with carcass length and backfat thickness between 6th and 7th ribs (BTR) and drip loss (p<0.05).

• The Korean Journal of Mycology
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• v.26 no.2 s.85
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• pp.173-181
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• 1998

To identify genetic difference of 13 strains in three Pleurotus species, analyses of rDNA, AP-PCR and RFLP were carried out. IGRI and $ITSI{\sim}II$ regions of rDNA amplified by PCR were about 0.9 and 0.7 kb, respectively. These PCR products were digested with six restriction enzymes to analyse polymorphism. Especially, treatment of HaeIII enzyme on $ITSI{\sim}II$ regions showed specific bands in three Pleurotus sajor-caju strains. Genetic differences among three species were classified by similarity analyses based on rDNA polymorphism. Various band patterns of $2,500{\sim}150\;bp$ were showed by AP-PCR. Identification of species and varieties in 13 Pleurotus strains was possible according to primers used in AP-PCR. In order to develop genetic markers, RFLPs using IGRI and $ITSI{\sim}II$ probes derived from ASI 2180 and 2070 were carried out on eight Pleurotus varieties. RFLP patterns using IGRI probe were more various than that of $ITSI{\sim}II$ probe.

• Journal of Aquaculture
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• v.8 no.3
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• pp.209-220
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• 1995

The nuclear matrix was isolated from Misgumus mizolepis liver nuclei by low salt extraction and restriction enzyme treatment. The structure was digested with proteinase K. After centrifugation, matrix attachment regions (MARs) were obtained by RNase treatment and phenol-chloroform extraction. The result leads to the appearance of smeared bands in the range of about 0.3-15 kb. pURY19 vector was constructed by inserting 2.13 kb Eco47 III fragment of the yeast uracil 3 gene into the unique Ssp I site of pUC19 plasmid vector as a selection marker. This vector is unable to be maintained in Sacrharomyces cerevisiae by itself since it cannot replicate as an extrachromosomal element. Using this system, we attempted cloning the ARS (autonomously replicating sequence) from M. mizelepis to develop an efficient expression vector for the transgenic fish. pURY19N_{l-62}$ were constructed by inserting MARs in pURY19 plasmid vector and transformation of E. coli $DH5\alpha$. Replication origins (ARS) of M. mizolepis were isolated, which enabled the vector to replicate autonomously in S. cerevisiae. The cloned DNA fragments were sequenced by Sanger's dideoxy-chain termination method. All clones were AT-rich. $pURY19N_6$, one of the clones, expecially contained ARS consensus sequence, Topoisomerase II consensus, near A-box and T-box.