• Parasites, Hosts and Diseases
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• v.56 no.5
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• pp.419-427
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• 2018

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, $FAM^{TM}$, $HEX^{TM}$, $Cy5^{TM}$, and CAL Fluor $Red^{(R)}$ 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was $2{\times}10$ copies for C. parvum and for C. cayetanensis, while it was $2{\times}10^3$ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.

• Korean Journal of Microbiology
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• v.33 no.4
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• pp.257-261
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• 1997

To scrutinize the bacterial community composition of Lake Soyang in winter, bacterial numbers belonging to Eubacteria, Proteobacteria and Cytophaga-Flavobacterium group were estimated by using 16S and 23S rRNA targeted oligonucleotide probes. Total bacterial numbers ranged from $0.7{\times}10^6$ to $1.1{\times}10^6cells{\cdot}ml^{-1}$, and vertical profile of total bacteria showed a peak at 5 m depth. The ratio of eubacteria to total bacteria were 34~90% and at 5 m and 10 m depths those were low exhibiting, 39 and 34%, respectively. The percentage of proteobacteria ${\alpha}$-group ranged 10.8~28.7%, ${\beta}$-group 4.5~53.5%, ${\gamma}$-group 4.9~35.5% and Cytophaga-Flavobacterium group 6.1~21.1%. The dominant groups were ${\beta}$-group at 0, 2 and 5 m, ${\gamma}$-group at 10 m, ${\alpha}$-group at 30 m and Cytophaga-Flavobacterium group at 50 m depth. In winter season, Lake Soyang can be divided into three layer, 0~2 m, 5~10 m and 30~50 m, by the bacteria community composition. By this method, new informations about aquatic ecosystem were developed.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.2
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• pp.93-99
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• 2004

Pathogens pose a significant threat to humans, animals, and plants. Consequently, a considerable effort has been devoted to developing rapid, convenient, and accurate assays for the detection of these unfavorable organisms. Recently, DNA-microarray based technology is receiving much attention as a powerful tool for pathogen detection. After the target gene is first selected for the unique identification of microorganisms, species-specific probes are designed through bioinformatic analysis of the sequences, which uses the info rmation present in the databases. DNA samples, which were obtained from reference and/or clinical isolates, are properly processed and hybridized with species-specific probes that are immobilized on the surface of the microarray for fluorescent detection. In this study, we review the methods and strategies for the development of DNA microarray for pathogen detection, with the focus on probe design.

• Bulletin of the Korean Chemical Society
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• v.33 no.9
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• pp.3055-3060
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• 2012

A simple, rapid and sensitive spectrofluorometric method was developed for the determination of folic acid (FA), based on its quenching effect on the fluorescence intensity of enoxacin (ENX)-europium ($Eu^{3+}$) complex as a fluorescent probe. Fluorometric interaction between ENX-$Eu^{3+}$ complex and FA was studied using UV-visible and fluorescence spectroscopy. The quenched fluorescence intensity at an emission wavelength of 614 nm was proportional to the concentration of FA. Optimum conditions for the determination of FA were investigated. Under optimal conditions, the reduced fluorescence intensity at 614 nm was responded linearly with the concentration of FA. The linearity was maintained in the range of $1.25{\times}10^{-9}$ to $1.50{\times}10^{-7}$ M (R = 0.9986) with the limit of detection ($3S_b/m$) (where $S_b$ is the standard deviation of blank and m is the slop of linear calibration curve) of $6.94{\times}10^{-10}$ M. The relative standard deviation (RSD) for 9 repeated measurements of $1.0{\times}10^{-9}$ M FA was 1.42%. This method was simple, cost effective, and relatively free of interference from coexisting substances. Successful determinations of FA in pharmaceutical formulation and biological samples with the developed method were demonstrated.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.5
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• pp.409-415
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• 2000

Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to evaluate the effects of dopamine HCl on the range of the rotatioanl mobility of bulk bilayer structure of the synaptosomal plasma membrane vesicles (SPMV) isolated from whole bovine brain. In a dose-dependent manner, dopamine decreased the anisotropy $({\gamma}),$ limiting anisotropy $({\gamma}{infty})$ and order parameter (S) of DPH in the membranes. These indicate that dopamine increased the rotational mobility of the probe in the neuronal membranes. Cationic 1-[4-(trimethylammonio)-phenyl]-6-phenylhexa-1,3,5-hexatriene (TMA-DPH) and anionic 3-[p-(6-phenyl)-1,3,5-hexatrienyl]-phenylpropionic acid (PRO-DPH) were utilized to examine the range of transbilayer asymmetric rotational mobility of the neuronal membranes. Dopamine had a greater increasing effect on the mobility of the inner monolayer as compared to the outer monolayer of the neuronal membranes. It has been proven that dopamine exhibits a selective rather than nonselective fluidizing effect within the transbilayer domains of the SPMV.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.17
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• pp.7997-8002
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• 2015

Gene amplification is an important mechanism in the development and progression of cancer. Currently, gene amplification status is generally determined by in situ hybridization (ISH). Multiplex ligation-dependent probe amplification (MLPA) is a PCR-based method that allows copy number detection of up to 50 nucleic acid sequences in one reaction. The aim of the present study was to compare results for HER2, CCND1, MYC and ESR1 gene amplification detected by MLPA with fluorescent in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) as clinically approved methods. Tissue samples of 170 invasive breast cancers were collected. All were ER positive. Tissue samples had previously been tested for HER2 using immunohistochemistry. Amplification of the selected genes were assessed using MLPA, FISH and CISH and results were compared. HER2 MLPA and ISH results were also compared with HER2 immunohistochemistry (IHC) which detects protein overexpression. Amplification of HER2, CCND1, MYC and ESR1 by MLPA were found in 9%, 19%, 20% and 2% of samples, respectively. Amplification of HER2, CCND1, MYC and ESR1 by FISH was noted in 7%, 16%, 16% and 1% of samples, respectively. A high level of concordance was found between MLPA/FISH (HER2: 88%, CCND1: 88%, MYC: 86%, ESR1: 92%) and MLPA/CISH (HER2: 84%). Of all IHC 3+ cases, 91% were amplified by MLPA. In IHC 2+ group, 31% were MLPA amplified. In IHC 1+ group, 2% were MLPA amplified. None of the IHC 0 cases were amplified by MLPA. Our results indicate that there is a good correlation between MLPA, IHC and ISH results. Therefore, MLPA can serve as an alternative to ISH for detection of gene amplification.

• Korean Journal of Microbiology
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• v.35 no.3
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• pp.242-247
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• 1999

This sludy was conducted to investigate the change of bacterial co~munities with season and depth in Lake Soyang. Korea, using fluorescence in situ hybridization (FISH). The oligonucleotide probes used in this study were EUB338, ALF I b. GAM42a, and CF, The percentage of h e Proleobacteria a $\alpha$-group ranged from 0.70 to 33% the $\beta$-group from 1.0 to 26% they -group from 2.4 Lo 37% and Cytophagn and Flavobactefin groups from 4.7 to 24% duing the study period (April Lo November, 1998). They $\gamma$-group was dominant in spring when Asterionella was dominant. and a $\alpha$-group was dominant in summer when the organic content was low and Dinobryon was dominant. However, a specific group was not dominant in ？dl when cyanobacteria group was dominant and the ratio of eubacleria to total bacteria was very low. Therefore, the bacterial communities in Lake Soyang changed with season and depth, which seems to be associated with the telnporal succession of phytoplanlaons.

• Journal of Nutrition and Health
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• v.35 no.10
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• pp.1053-1059
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• 2002

In post-genome period, the technique for identifying gene expression has been changed to high throughput screening. In the field of molecular nutrition, the need for this technique to clarify molecular function of the specific nutrient is essential. In this study, we have tested the zinc-regulated gene expression in zinc-deficient U937 cells, using cDNA microarray which is the cutting-edge technique to screen large numbers of gene expression simultaneously. The study result can be used for the preliminary gene screening data for clarifying, using monocyte U937 cell line, molecular Zn aspect in atherosclerosis. U937 cells were cultured in Zn-adequate (control, 12 $\mu$M Zn) or Zn-deficient (experimental, 0 $\mu$M Zn) ESMI media during 2 days, respectively. Cells were harvested and RNA was extracted. Total RNA was reverse-transcriptinized and synthesized cDNA probe labeled with Cy-3. fluorescent labeled cDNA probe was applied to microarray slide for hybridization slide, and after then, the slide was scanned using fluorescence scanner. ‘Highly expressed genes’ in Zn-deficient U937 cells, comparing to Zn-adequate group, are mainly about the genes for motility protein, immune system protein, oncogene and tumor suppressor and ‘Less highly expressed genes’ are about the genes for transcription, apoptosis associated protein, cell cycle, and several basic transcription factors. The results of this preliminary study imply the effectiveness of cDNA microarray for expression profiling of a singly nutrient deficiency, specially Zn. Furthur study, using tailored-cDNA array and capillary endothelial cell lines, would be beneficial to clarify molecular Zn function, more in detail.

• Archives of Pharmacal Research
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• v.15 no.3
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• pp.196-203
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• 1992

Intramolecular excimer formation of 1, 3-di(1-pyrenyl)propane (Py-3-Py) and fluorescence polarization of 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) were used to investigate the effects of barbiturates on the fluidity of model membranes of phosphatidycholine (SPMVPC), phosphatidylserine (SPMVPS), and phosphatidylinositol (SPMVPI) fractions of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex. In a dose-dependent manner, barbiturates decreased the excimer to monomer fluorescence intensity ratio (I'/I) of Py-3-Py and increased the anisotropy(r), rotational relaxation time (P), limiting anisotropy $(r_infty)$, and order parameter (S) of DPH in SPMVPC, SPMVPS and SPMVPI. This indicates that barbiturates decreased both the lateral and rotational diffusion of the probes in SPMVPC, SPMVPS and SPMVPI. The relative potencies of barbiturates in ordering the membranes were in the order: pentobarbital > hexobarbital > amobarbital > phenobarbital. This order correlates well with the anesthetic potencies of barbiturates and the potencies for enhancement of $\gamma$-aminobutyric acid-stimulated chloride uptake. Thus, it is strongly suggested that a close relationship might exist between the membrane ordering effects of barbiturates and the chloride fluxes across SPMV.

• Journal of Microbiology and Biotechnology
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• v.28 no.11
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• pp.1782-1790
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• 2018

Assessment of microorganism viability is useful in many industrial fields. A large number of methods associated with the use of fluorescent probes have been developed, including fluorimetry, fluorescence microscopy, and cytometry. In this study, a microvolume spectrofluorometer was used to measure the membrane potential variations of Escherichia coli. In order to estimate the sensitivity of the device, the membrane potential of E. coli was artificially disrupted using an ionophore agent: carbonyl cyanide 3-chlorophenylhydrazone. The membrane potential was evaluated using two ratiometric methods: a Rhodamine 123/4',6-diamidino-2-phenylindole combination and a JC-10 ratiometric probe. These methods were used to study the impact of freezing on E. coli, and were compared with the conventional enumeration method. The results showed that it was beneficial to use this compact, easy-to-use, and inexpensive spectrofluorometer to assess the viability of bacterial cells via their membrane potential.