• Bulletin of the Korean Chemical Society
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• v.28 no.12
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• pp.2303-2309
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• 2007

Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its Cterminal fragment (EPSPSC, residues Met237-238CYS-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing ~1 mM of EPSPSN-thioester with ~2 mM of EPSPSCCYS (residues 238CYS-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was ~85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and ~115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins.

• Korean Journal of Soil Science and Fertilizer
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• v.8 no.3
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• pp.161-170
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• 1975

Status of agriculture in Jeju province were investigated to find out problems and solutions in view of weather, soils and crop productions. Two hundred harmful windy days per year force to build strong wind-break forest near to farm. Somewhat severe drought situation in croping could be completely avoided by good irrigation system using abundant underground water resources. Poor fertility of soils should be improved by using much compost, lime, fused phosphate, and high dose fertilizers. Optimum fertilizer level for each crop should be determined with special consideration on characteristics of volcanic ash soil.

• The Journal of Korean Academy of Prosthodontics
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• v.23 no.1
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• pp.145-154
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• 1985

To study the effect of the vent and dense surface of the phophate-bonded investment on the castability of a non-precious alloy used in PFM restoration, one-ended polypropylene straw patterns of 0.15mm thick, 5mm radius. 10mm-high were cast with Vera Bond. The following results were obtained. 1. Thin patterns could be cast completely with a non-precious alloy. 2. No difference in the castability was found among the patterns with vent, chill vent and with out vent. 3. Dense surface of the phosphate-bonded investment had no effect on the cast ability of a nonprecious alloy.

• YAKHAK HOEJI
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• v.43 no.4
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• pp.429-436
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• 1999

A simple, accurate and reproducible Capillary electrophoresis (CE) assay has been developed for the determination of baicalin, baicalein, wogonin and chrysin in Scutellaria baicalensis. Successful separation of these compounds has been obtained in 35 mM phosphate butter (pH 7.0) using a untreated fused silica capillary ($57{\;}cm{\times}75{\;}{\mutextrm{m}}$ i.d.) at $25^{\circ}C$ with the electric field of 19kV. Baicalin, baicalein wogonin and chrysin was separated and detected at 280 nm 13 min. The detection limits of CE were acceptable compared to HPLC. Reproducibilities of migration time and peak area were 0.66~1.11% (within-run), 2.18~3.38% (between-run) and 3.50~4.55% (within-run), 3.97~4.82%(between-run) at CE. The results indicate that CE could be a promising technique for quality and quantity control analysis of Scutellaria baicalensis as a validation method.

• Bulletin of the Korean Chemical Society
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• v.15 no.11
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• pp.945-949
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• 1994

Mixtures of cyano complexes of palladium(II) and platinum(II) were separated by capillary electrophoresis using a fused silica capillary as a separation column and 30 mM phosphate buffer (pH 7) containing 15 wt. % acetonitrile as a running buffer. By virtue of the high ionic mobilities of the negatively charged cyano complexes of Pd(II) and Pt(II), they were separated using a cathodic injection and anodic detection scheme. The metal complexes eluted through the capillary were detected by direct UV absorption at 214 nm. A linear relationship between peak area and concentration was obtained for both ions and the detection limit was lower than $10^{-14}$ mole. The proposed method was applied to real sample, e.g., anode slime obtained from an electrolytic copper refinary, as a method for the simultaneous determination of palladium and platinum.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.51-61
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• 2001

This study was conducted to determine developmental ability of reconstructed embryos by nuclear transfer using somatic cell of Korean bull with high genetic value. Fibroblast cells obtained from ear biopsy of the bull were cultured in Dulbecco's Modified Eagle's medium (DMEM) at 37$^{\circ}C$ in air containing 5% $CO_2$. The cummulus-oocyte complexes were collected from slaughterhouse and were matured in vitro for 20 h in TCM 199 culture medium and the oocytes were then enucleated in modified phosphate buffered saline with cytochalasin B. Matured bovine oocytes were enucleated by aspirating the first polar body and metaphase chromatin using a beveled pipette in modified phosphate buffered saline. The ear fibroblast cells were fused into enucleated oocyte by electrical stimulation. The reconstructed oocytes were activated with ionomycine and 6-dimethylaminopurine, and then cultured in CR1aa medium for 7.5 days. Out of 524 bovine eggs reconstructed by nuclear transfer 65.6%(277/422) embryos were cleaved, and 30.7% (85/277) cleaved embryos were developed to the morula to blastocysts. There was no difference of developmental ability in vitro of reconstructed embryos regardless of donor cell passages. In order to determine fate of foreign mitochondria of donor nucleus, the Mito Tracker stained cells were fused into enucleated oocytes. The donor mitochondria were detected early stage of embryos, but disappeared rapidly. The developmental ability of reconstructed embryos was not impaired by Mito Tracker treatments. The results indicate that viable reconstructed embryos can be producted by nuclear transfer using somatic cell of Korean bulls.bulls.

• Applied Biological Chemistry
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• v.46 no.4
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• pp.380-384
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• 2003

Optimal extraction, separation and capillary rinsing conditions for capillary electrophoresis (CE) were established to discriminate the geographical origin of ligusticum root (Ligusticum wallichii) using 113 samples (domestic sample n = 62, foreign sample n = 51). Ligusticum root was extracted with 30% ethanol and separated on a uncoated fused-silica $(50\;{\mu}m{\times}27\;cm)$ capillary. Conditions for optimal analysis include: temperature, $40^{\circ}C$; voltage, 10 kV; and pressure injection time, domestic and foreign samples were 5 sec and 2 sec, respectively. The optimal separation buffer was 0.1 M phosphate buffer (pH 2.5) containing 15 mM iminodiacetic acid with 40% methanol. Under the optimal conditions established for CE, the ratio of specific peak area (peak LW-1) to other peak area (peak LW-5) was effective in discrimination geographical origin of ligusticum root. The mean accuracy for correct discrimination of geographical origin of domestic and foreign ligusticum roots were 65% and 63%, respectively.

• Korean Journal of Food Science and Technology
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• v.37 no.4
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• pp.513-518
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• 2005

Capillary electrophoresis (CE) method was developed to determine (+)-catechin and (-)-epicatechin contents in grape seed ethanol extract. CE separation was achieved using 100 mM phosphate and borate buffer at pH 6.0 as background electrolyte and fused silica capillary with 50 microns x 375 microns O.D. (effective length 20.0cm) maintained at $25^{\circ}C$. The applied voltage was 10kV, and detection was performed by DAD at 210 nm, Two catechins were well separated within 6 min with repeatability of <0.8% RSD for migration time and <2.0% RSD for peak area, and correlation coefficients higher than 0.994 were obtained from 58.0 to 174.0 mg/L with detection limit of 0.035 mg/L. Separated compounds were successfully determined. CE method was easy to handle and showed good reproducibility. CE method was compared with conventional coloring and HPLC methods, and main advantages of CE method were low amount of sample required, simple pre-sample treatment, good recovery rate, and short analysis time.

• Korean Journal of Soil Science and Fertilizer
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• v.18 no.4
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• pp.378-385
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• 1985

In order to investigate the application effects of soil amendment on wetland rice soil field and laboratory experiment were conducted on ill drained paddy field on which rice straw, Compost, Lime and Silicate materlials such as wallostonite and fused phosphate were applied. 1. As compared with check plot, rice yield was increased by application of soil amendments significantly in all plots but rice straw one. 2. The effects of soil amendment on rice production were in the order of fused phosphate and N.K, addition > silicate material and N.K, addition=Compost > rice straw and lime plot. 3. The significant correlation was showed among the rice yield and the numbers of panicle and grain. 4. The application of the rice straw restrained the rice growth because of nitrogen defficiency in early stage but increased rice yield due to the number of panicle due to supplying nitrogens late stage. 5. The application of rice straw in wetland soil promoted to occur organic acids such as $PCO_2$ and $HCO_3$, and decreased tillering due to these organic acids occuring in early growth stage.

• Korean Journal of Soil Science and Fertilizer
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• v.15 no.3
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• pp.172-177
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• 1982

This experiment was conducted to evaluate the broadcast and band applications of phosphate fertilizer and the application amounts of fertilizer during the soybean-barley cropping periods from 1976 to 1979 on the Sam-gag sand loam of a newly reclaimed hilly land. 1. The broadcast application was superior to band in view of yields for both soybean and barley. The yield was the highest in the broadcast application with the amount 38 kg $P_2O_5$ per 10a of fused phosphate which was calculated from soil testing at the first crop period and thereafter additional 6 kg at every crop period. 2. According to the quadratic relationship between yield and the available phosphate in soil, the maximum soybean Yield, 322 kg per 10a obtained at 220 ppm of available phosphate under broadcast and 259 kg per 10a at 200ppm under band. 3. By broadcast application of phosphorus, root weight was higher and horizontal root expansion was much better than by band application.