• Journal of Food Hygiene and Safety
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• v.29 no.4
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• pp.312-315
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• 2014

The present study demonstrated the development and validation of the method for the quantification of phenol in food using gas chromatography coupled with mass spectrometry (GC-MS). After spiking of internal standard (Phenol-$d_5$) to food, those samples were extracted with organic solvent mixture (acetone : dichloromethane = 1 : 1, v/v) using ultra sonic extractor and cleaned by gel permeation chromatography (GPC) technique. The amount of phenol was determined by GC/MS. To validate the developed method, we evaluated parameters were the selectivity, linearity, accuracy, precision, and recovery. To demonstrate the selectivity of the method, blank samples of rice, corn, and fish(mackerel) were prepared and subjected to GC-MS analysis. To verify the linearity of the method, six different standard concentrations of phenol at 0.01, 0.05, 0.1, 0.5, 1 and 2.5 mg/kg were evaluated. The correlation coefficient ($r^2$) of calibration curve was 0.9999. The recovery rate for phenol standard calculated by internal standard method were 82.2~101.5% for samples fortified with 0.25, 0.50, and 1.0 mg/kg, respectively. Also the repeatability and reproducibility for validation of precision were 0.2~5.5%. According to the result of the validation, this established method was suitable for AOAC guideline. The limit of detection (LOD) for phenol analysis were 0.03~0.1 mg/kg, and the limit of quantification (LOQ) were 0.1~0.3 mg/kg. Therefore, we established the optimal analysis method for determination of phenol in food using GPC and GC/MS.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.67 no.4
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• pp.211-221
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• 2022

Recently, the demand for the cultivation of upland soil has been increasing, and the rate of conversion of paddy soil into upland soil is also increasing. Theincrease in uneven precipitation due to climate change has resulted in dramatic effects of waterlogging stress on upland crops. Therefore, the present study was conducted to investigate the changes in growth characteristics and the expression patterns of proteins at the two-leaf stage of adzuki beans. The domestic cultivar, Arari (Miryang No. 8), was used to test waterlogging stress. At the two-leaf stage of adzuki beans, plant height slightly decreased androot fresh weight showed significant changes after 3 days of waterlogging treatment. Chlorophyll content was also significantly different after 3 days of waterlogging treatment compared to its content in control plants. Using two-dimensional gel electrophoresis, more than 400 protein spots were identified. Twenty-one differentially expressed proteins from the two-leaf stage were analyzed using linear trap quadrupole-Fourier transform-ion cyclotron resonance mass spectrometry. Of these 21 proteins, 9 were up-regulated and 12 were down-regulated under waterlogging treatment. Protein information resource (https://pir.georgetown.edu/) categories were assigned to all 49 proteins according to their molecular function, cellular component localization, and biological processes. Most of the proteins were found to be involved in the biological process, carbohydrate metabolism and were localized in chloroplasts.

• Korean journal of applied entomology
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• v.24 no.1 s.62
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• pp.45-50
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• 1985

The use of a modified procedure for the isolation of extrachromosomal DNA of low to high molecular weight, followed by agarose gel electrophoresis of the crude lysates, provided a simple screening procedure for detecting plasmids ranging in molecular weights from approximately 1 to more than 135 megadaltons from serovars of Bacillus thuringiensis. The procedure provides for a relatively large-volume stable lysate for isolation of plasmids for restriction endonuclease mapping and cloning procedures. The method was used for screening of plasm ids in 6 differenentially effective serovars of B. thuringiensis toxic to dipteran and lepidopteran insects. Relatively large plasmid DNAs of masses above 50 megadaltons (Mdal) were isolated from all of the serovars examined using this technique. The number of extrachromosomal DNAs detected in serovars of B. thuringiensis was 8 for israelensis, 10 for kurstaki, 13 for aizawai, 2 for dendrolimus, 1 for finitimus, and 6 for yunnanensis. Smaller plasmid DNAs were isolated in four of the six serovars that ranged in mass down to approximately 2 Mdal.

• Korean Journal of Environmental Biology
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• v.39 no.4
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• pp.445-451
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• 2021

In this study, we described the production of an antibody to living modified organisms (LMOs) containing the gene encoding for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium tumefaciens strain CP4 EPSPS provides resistance to the herbicide glyphosate (N- (phosphonomethyl) glycine). These LMOs were approved and have recently been used in the feed, food production, and processing industries in South Korea. Highly efficient monoclonal antibody (mAb) production is crucial for developing assays that enable the proper detection and quantification of the CP4 EPSPS protein in LMOs. This study describes the purification and characterization of recombinant CP4 EPSPS protein in E. coli BL21 (DE3) based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrixassisted laser desorption/ionization time-of-flight mass spectrometry. The production of mAbs was undertaken based on the standard operating procedure of Abclon, Inc.(South Korea), and the purity of the mAbs was assessed using SDS-PAGE. The following five mAb clones were produced: 2F2, 4B9, 6C11, 10A9, and 10G9. To verify the efficiency and specificity of the five developed mAbs, we performed Western blotting analysis using the LM (living modified) cotton crude extracts. All mAbs could detect the CP4 EPSPS protein in the LM cotton traits MON1445 and MON88913 with high specificity, but not in any other LM cottons or non-LM cottons. These data indicate that these five mAbs to CP4 EPSPS could be successfully used for the further development of antibody-based detection methods to target CP4 EPSPS protein in LMOs.

• Molecules and Cells
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• v.46 no.9
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• pp.545-557
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• 2023

Sphingomyelinase (SMase) catalyzes ceramide production from sphingomyelin. Ceramides are critical in cellular responses such as apoptosis. They enhance mitochondrial outer membrane permeabilization (MOMP) through self-assembly in the mitochondrial outer membrane to form channels that release cytochrome c from intermembrane space (IMS) into the cytosol, triggering caspase-9 activation. However, the SMase involved in MOMP is yet to be identified. Here, we identified a mitochondrial Mg²⁺-independent SMase (mt-iSMase) from rat brain, which was purified 6,130-fold using a Percoll gradient, pulled down with biotinylated sphingomyelin, and subjected to Mono Q anion exchange. A single peak of mt-iSMase activity was eluted at a molecular mass of approximately 65 kDa using Superose 6 gel filtration. The purified enzyme showed optimal activity at pH of 6.5 and was inhibited by dithiothreitol and Mg²⁺, Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Fe²⁺, and Fe³⁺ ions. It was also inhibited by GW4869, which is a non-competitive inhibitor of Mg²⁺-dependent neutral SMase 2 (encoded by SMPD3), that protects against cytochrome c release-mediated cell death. Subfractionation experiments showed that mt-iSMase localizes in the IMS of the mitochondria, implying that mt-iSMase may play a critical role in generating ceramides for MOMP, cytochrome c release, and apoptosis. These data suggest that the purified enzyme in this study is a novel SMase.

• Journal of Life Science
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• v.31 no.1
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• pp.90-99
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• 2021

In this study, to reduce the production cost of poly(3-hydroxyalkanoates) (PHA), optimal cell growth and PHA biosynthesis conditions of the isolated strain Pseudomonas sp. EML8 were established using waste frying oil (WFO) as the cheap carbon source. Gas chromatography (GC) and GC mass spectrometry analysis of the medium-chain-length PHA (mcl-PHAWFO) obtained by Pseudomonas sp. EML8 of WFO indicated that it was composed of 7.28 mol% 3-hydrxoyhexanoate, 39.04 mol% 3-hydroxyoctanoate, 37.11 mol% 3-hydroxydecanoate, and 16.58 mol% 3-hydroxvdodecanoate monomers. When Pseudomonas sp. EML8 were culture in flask, the maximum dry cell weight (DCW) and the mcl-PHAWFO yield (g/l) were showed under WFO (20 g/l), (NH4)2SO4 (0.5 g/l), pH 7, and 25℃ culture conditions. Based on this, the highest DCW, mcl-PHAWFO content, and mcl-PHAWFO yield from 3-l-jar fermentation was obtained after 48 hr. Similar results were obtained using 20 g/l of fresh frying oil (FFO) as a control carbon source. In this case, the DCW, the mcl-PHAFFO content, and the mcl-PHAFFO yields were 2.7 g/l, 62 wt%, and 1.6 g/l, respectively. Gel permeation chromatography analysis confirmed the average molecular weight of the mcl-PHAWFO and mcl-PHAFFO to be between 165-175 kDa. Thermogravimetric analysis showed decomposition temperature values of 260℃ and 274.7℃ for mcl-PHAWFO and mcl-PHAFFO, respectively. In conclusion, Pseudomonas sp. EML8 and WFO could be suggested as a new candidate and substrate for the industrial production of PHA.

• The Korean Journal of Nuclear Medicine Technology
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• v.14 no.2
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• pp.45-49
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• 2010

Purpose: Several radiopharmaceuticals were used for cisternography. But recently, due to more short acquisition time, high resolution than other radiopharmaceuticals like In-111 DTPA, we were using Tc-99m DTPA in cisternography. Using of Tc-99m DTPA for intrathecal, was not officially recognised by the FDA. And there are matters of aseptic meningitis, muscular tetany, seizures by inappropriate radiopharmaceuticals handling. So, it is necessary to prevent adverse reactions while handling the radiopharmaceuticals using in cisternography. Therefore, this study aims to evaluation of usefulness and procedures for safety of radiopharmaceuticals in cisternography. Materials and Methods: Subjects were 12radioactive tracer vials using in cisternography in 2008 Dec. 16 - 2009 Dec. 30. (1) Radioactive tracer Vial test - We were measured NaPertechnetate radiation dose and volume, normal saline volume for dilution, source volume and dose activity for patient injection. And then, calculated mass of pure DTPA. (2) Bacterial endotoxin test - We performed pyrogen test using by negative/positive control vials which was added normal saline 0.2 mL and added normal saline 0.1 mL, Tc-99m DTPA 0.1 mL in test control vial. And then, reacted by digital hot plate in $37.5^{\circ}C$ for 1 hour and compared of gel-clot in each control vials. (3) Compliance safety procedure - We were checked safety issues and wrote out a safety procedure exam sheet. Results: (1) Radioactive tracer Vial test - Mass of DTPA per dose for patient injection (mg) was 0.88 (mg) on average, and Mass of DTPA per volume for patient injection (mg) was 0.74 (mg) on average. (2) Bacterial endotoxin test - All control test vials showed negative reactions. (3) Compliance safety procedure - We were checked safety issues and wrote out a safety exam sheet in all the exams. So, there were no adverse reactions. Conclusion: We could examine easier to safety tests using by Techscan - DTPA (Mallinckrodt): CaNa3. Each test results were passed the safety tests and there are no adverse reactions. The use of Tc-99m DTPA for cisternography, always has been become an issue. Since it has occur adverse reaction while examine the cisternography using by Tc-99m DTPA, it needs to set up the 'Standard Operating procedures'.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.58 no.2
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• pp.149-160
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• 2013

The objective of present study was to simultaneously isolate of isoflavone and soyasaponin compounds from the germ of soybean seeds. Soy germ flours were defatted with hexane for 48h at room temperature, and methanolic extracts were prepared using reflux apparatus at $90^{\circ}C$ for 6h, two times. After extraction, extracts were separated with preparative RP-$C_{18}$ packing column ($125{\AA}$, $55-105{\mu}m$, $40{\times}150mm$), and collected 52 fractions were identified with TLC plate (Kieselgel 60 F-254) and HPLC, respectively. Among the identified isoflavone and soyasaponin fractions, isoflavone fractions were re-separated using a recycling HPLC with gel permeation column (Jaigel-W252, $20{\times}500mm$). Final fractions were air-dried, and the purified compounds of two isoflavones (ISF-1-1, ISF-1-2) and four soyasaponins (SAP-1, SAP-2, SAP-3, SAP-4) were obtained. Two isoflavone compounds (ISF-1-1, ISF-1-2) were acid-hydrolyzed for the identification of their aglycones, and confirmed by comparing with 12 types of isoflavone isomers. While the four kinds of soyasaponins were identified by using a micro Q-TOF mass spectrometer in the ESI positive mode with capillary voltage of 4.5kV, and dry temperature of $200^{\circ}C$. Base on the obtained results, it was conclude that ISF-1-1 is the mixture isomers of daidzin (43.4%), glycitin (47.0%), and genistin (9.6%), but ISF-1-2 is the single compound of genistin (99.8% <). On the other hand, soyasaponin SAP-1 is the mixture compounds of soyasaponin A-group (Aa, Ab, Ac, Ae, Af); SAP-2 is soyasaponin B-group (Ba, Bb, Bc) and E-group (Bd, Be); SAP-3 is soyasaponin B-group (Ba, Bb, Bc), E-group (Bd, Be), and DDMP-group (${\beta}g$); SAP-4 is soyasaponin B-group (Ba, Bb, Bc), E-group (Bd, Be), and DDMP-group (${\beta}g$, ${\beta}a$), respectively.

• The Korean Journal of Mycology
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• v.31 no.2
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• pp.59-66
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• 2003

Laccase produced by Pycnoporus cinnabarinus SCH-3 isolated from Korea was partially purified using ultrafiltration, anion exchange chromatography and affinity chromatography, The laccase was produced as the predominant extracellular phenoloxidase during primary metabolism. Neither lignin peroxidase nor manganese-dependent peroxidase were detected in the culture fluid. In order to examine the effect of inducers in laccase production, 2,5-xylidine was added in the culture of Pycnoporus cinnabarinus SCH-3. Addition of 2,5-xylidine enhanced 25-fold laccase production. Purified laccase was a single polypeptide having a molecular mass of approximately 66 kDa, as determined by SDS-polyacrylamide gel electrophoresis, and carbohydrate content of 9%. $K_{m}\;and\;V_{max}$ values for laccase with ABTS [2,2-azinobis (3-ethylbenzthiazoline 6-sulfonic acid)] as a substrate (Lineweaver-Burk plot) was determined to be $44.4{\mu}M\;and\;56.0{\mu}mole$, respectively. The optimal pH for laccase activity was found to be 3.0. The enzyme was very stable for 1 hour at $60{\circ}C$. Half-life ($t_{1/2}$) of the enzyme was about 10 min at $80{\circ}C$. Spectroscopic analysis of purified enzyme indicated that the enzyme was typical of copper-containing protein. Substrate specificity and inhibitor studies for laccase also indicated to be a typical fungal laccase. The N-terminal amino acid sequence of the P. cinnabarinus SCH-3 laccase showed 94% of homology to the N-terminal sequences of laccases from P. cinnabarinus PB and P. coccineus.

• Restorative Dentistry and Endodontics
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• v.25 no.4
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• pp.561-574
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• 2000

Poly-P has been used to prevent decomposition of foods and has been shown to have inhibitory effect on the growth of gram positive bacteria. The purpose of this study was to evaluate the effect of poly-P on the growth of Porphyromonas endodontalis, a gram negative obligate anaerobic rod, endodontopathic bacterium. P. endodontalis ATCC 35406 was in BHI broth containing hemin and vitamin K with or without poly-P. Inhibitory effect of each poly-P which was added at the beginning(lag phase) or during(exponential phase) the culture, MIC(minimum inhibitory concentration) was determined by measuring the optical density of the bacterial cell at 540nm. Viable cell counts were measured to determined whether poly-P has a bactericidal effect. Leakage of intracellular nucleotides from P. endodontalis was determined at 260nm and morphological change of P. endodontalis was observed under the TEM(transmission electron microscope). Binding of 32P-labeled poly-P to P. endodontalis was examined. SDS-polyacrylamide gel electrophoresis and zymography were performed to observe the changes in protein and enzyme profiles of P. endodontalis, respectively. The results from this study were as follows : 1. The minimal inhibitory concentration(MIC) of poly-P to P. endodontalis appeared to be 0.04~0.05%. 2. Poly-P added to the P. endodontalis culture during the exponential phase of P. endodontalis was as much effective as poly-P added at the begining of the culture, suggesting that the antibacterial effect of poly-P is not much dependent on the initial inoculum size of P. endodontalis. 3. Poly-P are bactericidal to P. endodontalis, demonstrating the decrease of the viable cell counts. 4. Intracellular nucleotide release from the P. endodontalis, was not increased in the presence of poly-P and was not reversed by the addition of divalent cations like $Ca^{2+}$ and $Mg^{2-}$. 5. Under the TEM, it was observed that fine electro-dense materials were prominent in the poly-P grown P. endodontalis, appearing locally in the cell, and the materials were more abundant and more dispersed in the cell as the incubation time with poly-P increased. In addition, highly electron dense granules accumulated in many poly-P grown cells, most of which were atypical in their shape. 6. Binding of 32P-labeled poly-P to P. endodontalis appeared to be 32.8 and 45.5 and 53.4% at 30 minutes, 1 hours and 2 hours, respectively. 7. In the presence of poly-P. the synthesis of proteins with apparent molecular masses of 25, 27, 35, 45 was lost or drastically decreased whereas expression of a protein with an apparent molecular mass of 75 was elevated. 8. Proteolytic activity of P. endodontalis was decreased by poly-P. The overall results suggest that use of poly-P may affect the growth of P. endodontalis, and the anti-bacterial activity of poly-P seems largely bactericidal. Changes in shape, protein expression, and proteolytic activity of P. endodontalis by poly-P may be directly and indirectly attributed to the antibacterial effect of poly-P. Further studies will be needed to confirm the effect of poly-P.