• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.928-933
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• 2001

Approximately 0.1 mg/ml of polyvinyl alcohol (PVA) was degraded by the growing cell, Brevibacillus laterospours, for 30 h, and 0.2 mg/ml of PVA was degraded by the cell-free extract that was isolated from Brevibacillus laterosporus. Approximately $0.29{\mu}g$/ml of acetic acid was produced from PVA by using the cell-free extract as a catalyst for 40 min. $V_{max}\;and\;K_m$ value of purified PAV-degradation enzyme was 3.75g/l and 2.75 g/l/min in reaction with EDTA and 3.99 g/l and 2.98 g/l/min in reaction without EDTA, respectively. Molecular weight of the purified enzyme determined by SDS-PAGE was 63,000 Da. Alcohol dehydrogenase and aldehyde dehydrogenase activities were qualitatively detected on a native acrylamide gel by an active staining method, indicating the existence of the metabolic pathway to use PVA as a substrate.

• Korean Journal of Human Ecology
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• v.14 no.3
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• pp.485-489
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• 2005

The purpose of this study is to investigate changes of isoflavone and nutrients during germination of soybean varieties and to determine the optimal germination condition for soybean processing. After 5 days' germination, water content of the soybeans increased in the order of Poongsannamoolkong> Sinpaldal#2> Black#1> Jinpumkong. Ash content was higher in the order of Poongsannamoolkong> Sinpaldal#2> Jinpumkong> black#1 soybean. All varieties of the soybean samples increased in ash content during germination. Crude fat content was higher in the order of Poongsannamoolkong> Black#1> Jinpumkong> Shinpaldalkong. Crude protein content was higher in the order of Shinpaldalkong> Black#1> Jinpumkong> Poongsannamoolkong. Polypeptide subunits separated from the SDS-PAGE of the entire proteins were showing comparatively high level in the range of 2-16KD in Jinpumkong and 2-21KD in Poongsannamoolkong till the first day of germination, 2-25KD in Sinpaldal#2 till the second day, and 6-27KD in Black#l till the third day. Changes of total isoflavone contents during germination showed different patterns in soybean varieties: Jinpumkong and Black#1 had the highest total isoflavone content on the fourth day of germination, Poongsannamoolkong and Sinpaldal#2 on the first and the second day of germination, respectively.

• 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.

• Korean Journal of Microbiology
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• v.44 no.2
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• pp.122-129
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• 2008

The purpose of this work was to investigate the synergically bactericidal effects and cellular responses of green tea polyphenols (TPPs) and nalidixic acid (NA) on nalidixic acid-resistant (NAR) Salmonella typhimurium. The bactericidal activities of $>3,500{\mu}g/ml$ TPPs and $<256{\mu}g/ml$ NA were investigated for S. typhimurium of which initial cell number was approximately adjusted to 107 cell/ml. Complete elimination of NAS-S. typhimurium was achieved within 6 hr of incubation at the concentrations of $3,500{\mu}g/ml$ TPP or $256{\mu}g/ml$ NA, whereas only partial bactericidal effect was achieved under the same conditions. However, the combinations of $3,000{\mu}g/ml$ TPPs and $32{\mu}g/ml$ NA against NAS-S. typhimurium and $3,500{\mu}g/ml$ TPPs and $64{\mu}g/ml$ nalidixic acid against NAR-S. typhimurium showed complete removal within 5 hr of incubation. The stress shock proteins (SSPs) were induced at different concentrations of TPP o rNA used as stressors against cell culture of S. typhimurium. The proteins were identified as 70-kDa DnaK and 60-kDa GroEL by SDS-PAGE and Western blot. SSPs induced by the stressors were found to increase in proportion to the TPPs or NA. Scanning electron microscopy analyses revealed the presence of perforations and irregular rod shape with wrinkled surfaces for cells treated with TPPs or NA.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.9
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• pp.1296-1302
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• 2004

This study was conducted to evaluate the possible application of 2 D-SDS-PAGE (2 DE)-based proteome analysis techniques to the assessment of extreme proteolysis in postmortem skeletal muscle. Eight Hanwoo longissimus muscles were incubated immediately after slaughter for 24 h at 5$^{\circ}C$, 15$^{\circ}C$ or 36$^{\circ}C$. Warner Bratzler (WB)-shear force and ultrastructural configuration were determined at 24 h, and rate of proteolysis to 24 h was determined by 1 D-SDS-PAGE (1 DE) and 2 DE. In addition, tentative protein identification was performed from peptide mass fingerprints of MALDI-ToF analysis of major protein groups on 2 DE profiles. The result showed that although ultrastructural configuration was similar between the 5$^{\circ}C$ and 36$^{\circ}C$ treatments, meat at 5$^{\circ}C$ had higher WBshear force (approximately 5 kg greater). A higher rate of protein degradation at 36$^{\circ}C$ was observed based on Troponin-T degradation, 1 DE, and 2 DE analysis. This indicates that proteolysis during the early postmortem period was a significant determinant of shear force at 24 h. Little difference in proteolysis between 5$^{\circ}C$ and 15$^{\circ}C$ treatments was found based on classic 1 DE profile assessment. Meanwhile, considerable differences in the 2 DE profiles between the two treatments were revealed, with substantially higher rate of proteolysis at 15$^{\circ}C$ compared to 5$^{\circ}C$. Nuclease treatment improved 2 DE profile resolution. 400 ${\mu}$g and 600 ${\mu}$g of sample loading appeared to be appropriate for 24 cm pH 3-10 and pH 5-7 IPG strips, respectively. Protein detection and quantification of the 5$^{\circ}C$, 15$^{\circ}C$ and 36$^{\circ}C$ 2 DE profiles revealed 78, 163 and 232 protein spots respectively that were differentially modified in terms of their electrophoretic properties between approximately pI 5.3-7.7 with the molecular weight range of approximately 71-12 kDa. The current results demonstrated that 2 DE was a superior tool to 1 DE for characterising proteolysis in postmortem skeletal muscle.

• Journal of Life Science
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• v.8 no.5
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• pp.497-503
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• 1998

Cyclodextrinase from B. stearothemophilus KJ16 that can produce both cyclodextrin(CD) glucanotransferase and cyclodextrinase was purified 87.6-fold with 7% yield by ammonium sulfate precipitation, DEAE-cellulose chromatog-raphy, Sephadex G-100 chromatography, and FPLC. The molecular weight of the purified enzyme was about 68,000 dalton by SDS-PAGE. The optimal pH and temperature were 6.0 and 55$^{\circ}C$, respectively. The enzyme was stable at 5$0^{\circ}C$ for 2 hr in the pH range of 5.5 and 8.5. The enzyme activity was inhibited strongly by mercaptoethanol, di-thiothreitol, p-chloromercuribenzoate, N-bromosuccinimide, $Cu^{+2}$and $Hg^{+2}$. The purified enzyme hydrolyzed CDs with$\gamma$-CD>$\beta$-CD>$\alpha$-CD. The enzyme also hydrolyzed linear maltodextrins and polysaccharides, but the rates of hyd-rolysis for such substrates were slow as compared to that for $\gamma$-CD. The final degradation products with all substrates were maltose and glucose. Maltose was not further hydrolyzed.

• Korean Journal of Microbiology
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• v.42 no.4
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• pp.294-298
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• 2006

An immunoaffinity chromatography for the specific binding of Streptomyces somaliensis phospholipase D (PLD) that is considered as an industrially potential enzyme was developed. By using the protein structure prediction programs and the X-ray crystal structure of a Streptomyces PLD, 5 different epitopes with high antigenicity that are predicted to locate on the surface of the S. somaliensis PLD were selected and then synthesized for the preparation of antipeptide antibodies. Each purified rabbit IgG was coupled with NHS-activated Sepharose to prepare the immunoaffinity resins. After one-step purification of the culture concentrate on the antipeptide IgG-coupled Sepharose column, SDS-PAGE and the Western blot analysis of the purified samples showed that purification of PLD on the affinity columns was satisfactory, indicating that the peptide design using the structural information of Streptomyces PLDs was rational. However, the purified PLD in the solution aggregated rapidly, which resulted in poor specific activity and low purification yield.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.30 no.3
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• pp.186-192
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• 2004

Purpose: Human tooth proteins are highly heterogeneous, comprising diverse proteins derived from a number of genes. The attempts to identify protein for activity of tooth matrix proteins have been defied by several factors. First, the amount of proteins within teeth is very small relative to many extracellular matrix proteins of other tissues. Second, the bioassay system is tedious and needed for long time. Therefore we tried to find easy techniques, which increase the product rate, and an assay of small proteins, with which amino acid sequence is possible without additional procedures. Materials and Methods: Total protein were extracted from 300 g enamel removed teeth and 600 g teeth with 4 mol/L guanidine HCl and purified by gel chromatography. Aliquot of proteins was implanted into muscle pouches in Sprague-Dawley rats for bioassay. By SDS-PAGE and membrane blotting, molecular weight of each protein was estimated and a partial amino acid sequence was obtained. Each fraction blotted on the membrane was cut out and inserted in rat ectopic model. Results: In dissociative method, total tooth proteins were obtained 1mg/ml from enamel removed teeth and 3.5 mg/ml from teeth. In SDS-PAGE, four clear bands at the sites corresponding to 66, 40, 20 and 18 kD. Especially The 66 kD band was clearly exhibited. Amino acid sequencing from tooth could be possible using PVDF membrane blotting technique. In amino acid sequencing, 66 kD protein was identified as albumin. Conclusion: Compared with conventional method for extraction of teeth protein and bioassay of proteins, the methods in this study were easy, time-saving and more productive technique. The matured tooth proteins omitting additional procedure of mechanical removal of enamel were simply analyzed using blotted PVDF membrane. This method seems to make a contribution as a technique for bioassay and amino acid sequencing of protein.

• The Journal of Pediatrics of Korean Medicine
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• v.13 no.1
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• pp.253-275
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• 1999

The effects of Sagunjatang and Samultang on the immunosuppression induced by methotrexate(MTX) in rats were investigated in this study. The multiple parameters of immunity assessed in each rats included leukocyte count, lymphocyte rate, the number of lymphocyte in tibial bone marrow, contact hypersensitivity to DNFB, morphological change of thymocyte and IgG antibody on SDS-PAGE. Sprague-Dawley male rats were used and divided into five groups at random. Group A was normal control. Group B, the MTX treatment control, was injected i.v. with 2mg/kg of on days 9, 11 after sensitization with SRBC on 5th day. Group C, the experimental control, was treated Sagunjatang for 18days and MTX. Group D was treated Samultang for 18days and MTX. Group E was treated Sagunjatang-Samultang for 18days and MTX. The dosage of Sagunjatang and Samultang was $1m{\ell}/day$ respectively. In the case of Group E, rats Were fed Sagunjatang $1m{\ell}$ in the morning and Samultang $1m{\ell}$ in the afternoon. The results are summarized as follows: 1. Leukocyte count in rats induced by intravenous sensitization with SRBC was decreased significantly in Group E. 2. Leukocyte counts of 2weeks later after being treated MTX were increased significantly in Groups C and D. 3. Lymphocyte rate in rats induced by intravenous sensitization with SRBC wasn't changed significantly in all the experimental groups. 4. Lymphocyte rate of 2weeks. later after being treated MTX was increased significantly in Group D. 5. The number of lymphocyte in tibial bone marrow was incereased significantly in Group C. 6. Contact hypersensitivity wasn't changed significantly in all the experimental groups. 7. Morphological finding of thymocyte in group C was similar to normal group as compared with control group. 8. Purified IgG of all the experimental groups showed two bands of 50,000 and 25,000 on SDS-PAGE. But there was no difference among experimental groups.

• Restorative Dentistry and Endodontics
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• v.20 no.2
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• pp.549-576
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• 1995

Bacteria are one of the most important causative agents of the pulpal and periapical diseases. Streptococci are one of the most frequently isolated facultative anarerobic bacteria in the infected root canals. Bacterial cell wall components have a direct effect in the pathogenesis of the pulpal and periapical infections. Hyaluronidase produced by bacteria has been implicated in dissemination of the diseases. The purpose of this study was to evaluate the effect of cell wall extract of streptococci on the L929 cells using inverted microscope and the transmission electron microscopy (TEM). Hyaluronidase production of streptococcal strains were investigated to determine the correlation between the severity of cell damage and the activity of enzymes. Bacterial cell wall extracts of S. sanguis, S. mitis and S. uberis isolated from infected root canals and ATCC type strains of S. mutans (ATCC 10449) and E. faecalis (ATCC 19433) were prepared by sonication and confirmed with SDS-PAGE. Silver stain of SDS-PAGE of sonic extract was efficient at $100{\mu}g$/ml concentration of cell wall protein, while Coomasie blue stain was efficient at $100{\mu}g$/ml concentration. Inverted microscope showed that sonic extract-treated L929 cells were round and detached from the substratum while others lost their fibroblastic shapes. Transmission electron microscopic examination revealed that streptococcal extracts induced death of L929 cells. Sonic extracts of streptococci had variable effect on microstructure of L929 cells. significant chromatin condensation was observed in the nucleus of the cells. Disappearance of cell surface microvilli and nuclear fragments with dense chromatin were observed. The cell nucleus had an irregular shape and numerous large vacuoles were seen in the cytoplasm and some breaks of the cell membrane could be seen. Cell organelles were in various stages of destruction and cristae of mitochondria were disoriented or disappeared. Eighteen strains of streptococci did not produce hyaluronidase.