• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.215-223
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• 2003

This study was to investigate the effects of fertilization time and culture medium of pig oocytes matured in-vitro by liquid boar sperm. The sperm rich fraction (30∼60 ml) was slowly cooled to room temperature (20∼23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min 800 ${\times}$ g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of the LEN diluent to provide 1.0${\times}$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$. The medium used for oocyte maturation was TCM-199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin B$_{12}$ , 25 mM HEPES, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 ${mu}ell$ mTBM fertilization media with 1.0${\times}$10$^{6}$ sperm/ml concentration, respectively. Thereafter, oocytes were transferred into 500 ${mu}ell$ NCSU-23, HEPES buffered NCSU-23, PZM-3 and PZM-4 culture media, respectively, for further culture of 6, 48 and 144 h. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times for 6 and 9 h than in those for 1 and 3 h. The rates of cleaved oocytes were higher in the fertilization times for 6 and 9 h (85.0 and 84.6%) than in those for 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time for 6 h (33.6%) than in that for 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9, 27.6, 26.3 and 24.4 in the fertilization times for 6, 9, 3 and 1 h, respectively. The rate of blastocyst from the cleaved oocytes and the number of cells per blastocyst were higher in HEPES buffered NCSU-23 culture medium than in NCSU-23, PZM-3 and PZM-4 culture media. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend the coincubation time of 6 h in 500 ${mu}ell$ TBM fertilization medium with 1${\times}$10$^{6}$ sperm/ml concentration and the HEPES buffered NCSU-23 culture medium for in-vitro fertilization of pig oocytes matured in-vitro.

• Bulletin of the Korean Chemical Society
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• v.27 no.3
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• pp.407-412
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• 2006

Bioluminescence single-site immunometric assay for methamphetamine (MA) using the native aequorin, a photoprotein, as a signal generator was developed for the first time. MA is a potent sympathomimetic amine with stimulant effects on the central nervous system. MA abuse induces hallucinations and, thus, may cause a serious social problem. The single-site immunometric MA assay was optimized and its dose-response behavior was examined. The dose-response curve shows that the detection limit is 1.1 ${\times}$ $10^{-10}$ M and a dynamic range is four orders of magnitude with 15 $\mu$g/mL BSA-MA conjugate and 1.0 ${\times}$ $10^{-8}$ M anti-MA antibody-biotin conjugate. In order to evaluate this assay, the structurally similar compounds, amphetamine, ephedrine, norephedrine, benzphetamine and N-4-(aminobutyl)methamphetamine were examined for their crossreactivity. None of these five compounds showed any cross-reactivity. Additionally, an artificial urine solution spiked with MA was analyzed by the MA assay, and the result of the analysis demonstrated the usefulness of the present assay for the determination of MA in urine.

• The Korean Journal of Nuclear Medicine
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• v.18 no.1
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• pp.55-62
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• 1984

For an effective solid-phase labelling of protein with $^{125}I$, studies on the immobilization of lactoperoxidase(LPO) on the inner wall of polystyrene tubes were carried out. Labelling of bovine serum albumin(BSA) and insulin was also practiced using the LPO immobilized tubes. The immobilized enzyme of about $2.5{\mu}g/tube$ was sufficient for small scale labelling since the results of radio-paper chromatography of the labelling mixture of insulin indicated that the yields were sufficiently high(80%) even in the reactions conducted at room temperature for 60 sec. The results of the Sephadex column chromatography indicated that the labelled products were not contaminated with $LPO-^{125}I$, and the radiochemical purity of the products was more than 90%. In considering the general trend that the $^{125}I$ labelled protein obtained by using LPO maintains its intactness better than those obtained by using chloramine-T, together with the tendency of yield enhancing with increase of reactants-concentration, the LPO immobilized tube method is estimated to be one of the simple methods of labelling. The product might be applicable without further purification.

• Bulletin of the Korean Chemical Society
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• v.32 no.4
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• pp.1315-1320
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• 2011

Whey protein (WP) is a mixture of proteins, and is of high nutritional values. WP has become an important source of functional ingredients in various health-promoting foods. In this study, size-exclusion chromatography (SEC) and asymmetrical flow field-flow fractionation (AsFlFFF) were used for separation and analysis of whey proteins. It was found that a lab-prepared WP from raw milk is mostly of ${\beta}$-lactoglobulin with small amount of higher molecular weight components, while a commercial whey protein isolate (WPI) powder contains relatively larger amount of components other than ${\beta}$-lactoglobulin, including IgG and protein aggregates. Results suggest that AsFlFFF provides higher resolution for the major whey proteins than SEC in their normal operation conditions. AsFlFFF could differentiate the BSA and Albumin, despite a small difference in their molecular weights, and also was able to separate much smaller amount of aggregates from monomers. It is noted that SEC was able to show the presence of low molecular weight components other than the major whey proteins in the WP samples, which AsFlFFF could not show, probably due to the partial loss of those low molecular weight species through the membrane.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.297-303
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• 1998

For the extraction and measurement of zearalenone in the corn, bean, wheat and barley contaminated with Fusarium graminearum, the zearalenone-oxime, zearalenone-oxime BSA and zearalenone monoclonal antibodies were studied to develop and apply the direct competitive enzyme linked immunosorbent assay (ELISA). The extraction range of zearalenone with the monoclonal antibodies produced in this experiment was 10ng to 500ng/g feed and the 50% inhibition value was 50ng/ml. The mean recoveries of zearalenone artificially spiked in the ground corn were 89%. The specificity of F-2 monoclonal antibody for the analogues was favorable for the direct competitive ELISA. The result of the experiment showed the zearalenone in the corn, bean, wheat and barely naturally contaminated with the mold would be suitable for extraction and measurement with the monoclonal antibodies.

• BMB Reports
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• v.29 no.5
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• pp.455-461
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• 1996

A serine proteinase was purified from Acanthamoeba culbertsoni by 41~80% ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography and gel filtration chromatography. The molecular weight of the purified enzyme was estimated to be 108.0 kDa by gel filtration chromatography and 54.0 kDa by SDS-PAGE. Therefore, the purified enzyme seemed to be a dimer. Isoelectric point was 4.5. The enzyme activity was highly inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate (OFP) and phenylmethyl sulfonylfluoride (PMSF). It had a narrow pH optimum of 6.5~7.5 with a maximum at pH 7.0. These data suggested that the purified enzyme was a neutral serine proteinase. Optimal temperature was $37^{\circ}C$. It was stable for at least 16 h at $4^{\circ}C$ and $37^{\circ}C$, but it was rapidly inactivated at $65^{\circ}C$ The activity of the purified enzyme was not influenced significantly by $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$ or $Ca^{2+}$. However, the enzyme activity was highly inhibited by $Hg^{2+}$ The enzyme degraded type I collagen and fibronectin, but not BSA, hemoglobin, lysozyme, immunoglobulin A or immunoglobulin G.

• Korean Journal of Pharmacognosy
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• v.46 no.4
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• pp.279-282
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• 2015

In order to quantify compound K(CK), anticancer component of Panax ginseng C. A. Meyer, high titer rabbit polyclonal antibodies (pAbs) were raised against a conjugate of CK and bovine serum albumin coupled by a periodate oxidation method. Coating antigen (CK-OVA) was also prepared by the same method with OVA. As a result of optimization of antiserum dilution (2,000 fold), coating antigen ($25{\mu}g/ml$) and other condition (incubation time, temperature and washing method), ELISA method for the determination of CK was established. The measuring range extended from 0.5 ng/ml to 25 ng/ml of CK. The antibodies exhibited minor or even no cross reactivities with protopanaxatriol (1.56%) and other tested ginsenosides, $GRb_1$ (0.11%), $GRg_1$ (0.07%) except protopanaxadiol (87.2%) from the structural similarity. And the antibody showed good correlation (r=0.987) between the assay values obtained by this ELISA method and HPLC. Therefore, the ELISA method could be very useful tools for the determination of CK in biological fluids because of their high sensitivity and specificity.

• Food Science and Biotechnology
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• v.18 no.1
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• pp.130-136
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• 2009

The present study was conducted to determine the protective ability of the ethanol extracts of Hypericum scabroides Robson & Poulter (HS) and Hypericum triquetrifolium Turra (HT) against the protein oxidation and DNA damage induced by Fenton system. The ability of HS and HT to prevent oxidative damage to bovine serum albumin (BSA) induced by $Fe^{3+}/H_2O_2$ and ascorbic acid was investigated. The ethanol extracts of HS and HT at different concentrations ($50-1,000{\mu}g/mL$) efficiently prevented protein oxidation induced by hydroxy radical as assayed by protein oxidation markers including protein carbonyl formation (PCO) and polyacrylamide gel electrophoresis. The effect of ethanol extracts of HS and HT on DNA cleavage induced by UV-photholysis of $H_2O_2$ using pBluescript M13+ plasmid DNA were investigated. These extracts significantly inhibited DNA damage induced by reactive oxygen species (ROS). Therefore, HS and HT extracts may be useful in the food industry as effective synthetic antioxidants.

• Journal of Food Hygiene and Safety
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• v.8 no.4
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• pp.205-214
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• 1993

The monoclonal antibody to chloramphenicol(CAP) was produced to develop an enzyme-linked immunosorbent assay(ELISA) for residual CAP. An immunogen(CAP-BSA) was prepared by immunogen, antibody titer was measured by indirect ELISA. Spleen cells form the immunized mouse were fused with SP2/OAg14 myeloma cells. Among hybridomas selected in HAT media, 6 clones shown high antibody titer to CAP were subjected to cloning by limit dilution, and all of the monoclonal antibodies(MCA1, 2, 3, 4, 5, 7 and 9) produced by each clone were identified as IgG1 by ELISA isotyping analysis. Competitive ELISA condition was established by using the purified monoclonal antibody MCA1 as primary antibody and CAP-HSA conjugate as coating antigen. Standard curve of CAP(n=28) showed that the lowest detection limit of CAP is 20ng/ml level. The cross-reactivities of the 6 monoclonal antibodies showed that CAP sodium succinate. CAP base, P-nitrophenol, and p-nitrobenzyl alcohol were 89∼178, 0.050∼2.237, 0.056∼0.794 and 0.013∼7.939%, respectively. No cross-reactivities were observed with phenylalanine, tyrosine, glutamine, thiamphenicol, neomycin, streptomycin, gentamicin, sulfamethazine, sulfathiazole, chlortetracycline and p-aminobenzoic acid.

• Archives of Pharmacal Research
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• v.25 no.4
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• pp.528-533
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• 2002

To evaluate the antithrombotic and antiallergic properties of rhaponticin extracted from Rhei Rhizoma, the in vitro and ex vivo inhibitory activities of rhaponticin and its metabolite, rhapontigenin, were measured. These compounds inhibited in vitro ADP- and collagen-induced platelet aggregation. Rhapontigenin was more potent, with $IC_{50}$ values of 4 and $70{\;}{\mu}g/ml$, respectively. In ex vivo ADP- and collagen-induced rat platelet aggregation, these compounds also exhibited a potent inhibitory effect. The antiplatelet aggregation effects of rhaponticin and rhapontigenin were more potent than those of aspirin. Rhapontigenin showed significant protection from death due to pulmonary thrombosis in mice. Rhapontigenin also showed the strongest inhibitory activity against $\beta-hexosaminidase$ release induced by DNP-BSA. These compounds inhibited PCA reaction in mice. Rhapontigenin intraperitoneally administered showed the strongest inhibitory activity and significantly inhibited PCA at doses of 25 and 50 mg/kg, with inhibitory activities of 48 and 85%, respectively. The inhibitory activity of orally administered rhaponticin was stronger than that of intraperitoneally administered rhaponticin. These results suggest that rhaponticin, in the rhizome of Rhei Rhizoma, is a prodrug that has extensive antiallergic and antithrombotic properties.