• BMB Reports
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• 제31권4호
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• pp.350-354
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• 1998

To understand the role of protein kinase C (PKC) in the regulation of chondrogenesis, we examined proteins which are phosphorylated by PKC. Stage 23/24 chick embryo wing mesenchymes were micromass-cultured to induce chondrogenesis and cell extracts were phosphorylated in a condition that activates PKC. Several proteins including 63 and 66 kDa proteins were phosphorylated. The 66 kDa protein was phosphorylated only in the presence of phorbol 12-myristate 13-acetate (PMA) and phosphatidylserine CPS), and the phosphorylation was almost completely diminished by bisindolylmaleimide, a PKC inhibitor. In addition, partially purified PKC increased the phosphorylation of the 66 kDa protein. Treatment of cultures with lysophosphatidylcholine (LPC) promoted chondrogenesis and phosphorylation of 66 kDa protein, while PMA and thymeleatoxin inhibited both of the two events. Our results suggest that the 66 kDa protein is a putative substrate of PKC, and phosphorylation of the 66 kDa protein, probably by $PKC\alpha$ is required for chondrogenesis.

• 한국임상수의학회지
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• 제28권1호
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• pp.75-80
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• 2011

Proteomics is a useful approach to know protein expression, post-translational modification and protein function. We investigated the protein expression pattern and identity in pigs fed with the tissue culture medium waste after harvest of Korean wild ginseng (TCM-KWG) (Panax ginseng). Two groups (n = 30/group) of pigs were administered with 0 (control) and 16 ml/L (treatment) TCM-KWG through drinking water. After 4 weeks, we examined the protein expression pattern of longissimus dorsi muscle by Two-dimensional electrophoresis analysis. TCM-KWG treatment significantly increased two spot's density, and markedly reduced one spot's density in the muscles. We identified 3 proteins (heat shock protein 90-alpha, myosin binding protein and cofilin 2) by the ESI-MS/MS (Q-TOF2, Micromass). These results demonstrate that TCM-KWG treatment may play a protection role against physiological stress in pigs, like as increased heat shock protein 90-alpha.

• 한국동물학회지
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• 제38권2호
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• pp.286-293
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• 1995

The present studies were undertaken to examine the activitites of PKC isoforms in cultures of chick limb mesenchvme. Micromass cultures were prepared using wing buds of stage 23/24 (Hamburger and Hamilton, 19511 chick embryo. The cells were homogenized and DEAE-cellulose column chromatography was performed to get fraction containing protein kinase C (PKC) activity. PKC isoforms were resolved with hvdroxyapatitie column chromatography. Profile of PKC isoforms of cultures were compared with that of rat brain. Activity of $PKC-\beta$ isoform was appeared at the early stage of chondrogenesis. On 3 daw of culture, activities of both PKC a and $\beta$ were observed with remarkable increase but no activity of y isoform was appeared. Treatment of phorbol-12-mvristate-13-acetate (PMA) (10-7 M) to the culture inhibited chondrosenesis and down-regulated a and $\beta$ isoforms. Staurosporine promoted chondro!genesis without any effect on PKC isioforms profile. These data indicate that PKC a and $\beta,$ especiallv $\beta$ isoform is related to chondrosenesis and the promoting effect of staurosporine on chondrogenesis is not related to PKC isoforms activities.

• 한국동물학회지
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• 제34권4호
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• pp.460-468
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• 1991

계배 limb bud간충직 연골원성 세포로부터 연골원세포로의 분화과정에서 Ca2+의 역할을 추구해 보기 위하여 Hamburger-Hamilton stage 23/24의 계배 limb bud간충직세포들을 미세배양법으로 배양하면서, Ca2+, calcium ionophore인 A23187, calcium channel blocker인 D600을 각각 농도 및 처리기간을 변화시켜 처리하면서 연골화의 정도를 검정하여, 연골세포 분화에 미치는 Ca2+의 작용양상을 분석하였다. 그 결과 Ca2+은 3 mM의 농도로 배양초기에 처리하였을 때 가장 효과적으로 연골화를 촉진하였으며, A23187(0.05 $\mu$ M) 처리는 세포내로 Ca2+유입을 증가시켜 연골화를 촉진시킨 반면, D6OO(30 $\mu$ M이하) 처리는 세포내로 Ca2+ 유입을 차단시킴으로서 연골화를 억제하는 것으로 나타났다. 이러한 결과에 따른 세포내로의 칼슘 유입 변화는 45Ca로 확인하였다. 그러므로 Ca2+에 의한 간충직세포의 연골세포로의 분화촉진 작용은 Ca2+이 연골화의 응집시기에 세포간의 접촉을 유도할 뿐만 아니라 배양 초기에 Ca2+이 세포 내로 들어감으로써 수반되는 일련의 기작에 의한 것임을 알았다.

• Animal cells and systems
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• 제4권4호
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• pp.361-366
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• 2000

In order to investigate the role of protein kinase C (PKC) in chondrogenic differentiation, we examined the localization of PKC isoforms in a limb bud micromass culture system. PKC$\alpha$ is specifically localized in the regions which would become cartilage nodules, while PKC$\lambda/l$ and $\zeta$ display widespread distribution in the whole culture. Distribution of PKC$\alpha$ change along with promotion or inhibition of chondrogenesis by lysophosphatidylcholine or phorbol 12-myristate 13-acetate. On the other hand, localization of PKC$\lambda/l$ or $\zeta$ a was not changed by the modulation of chondrogenesis. Peanut agglutinin binding protein which is associated with cell aggregation during chondrogenesis was present in the cell condensation regions and its expression in those regions was influenced by PKC activity. Expression of fibronectin and N-cadherin in the cell condensing area were also affected by modulation of PKC activity. These results suggest involvement of PKC$\alpha$ in the cell condensation, possibly through regulating expression of fibronectin and N-cadherin.

• Journal of Ginseng Research
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• 제40권3호
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• pp.220-228
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• 2016

Background: Among the various ginseng strains, Shizhu ginseng is endemic to China, mainly distributed in Kuandian Manchu Autonomous County (Liaoning Province, China); however, not much is known about the compounds (especially saponins) in Shizhu ginseng. Methods: A rapid, sensitive, and reliable ultra-high performance liquid chromatography coupled with MS/MS (UHPLC-MS/MS) method was developed to separate and identify saponins in Shizhu ginseng. Results: The separation was carried out on a Waters ACQUITY UPLC BEH $C_{18}$ column ($100mm{\times}2.1mm$, $1.7{\mu}m$) with acetonitrile and 0.1% formic acid aqueous solution as the mobile phase under a gradient elution at $40^{\circ}C$. The detection was performed on a Micromass Quattro Micro API mass spectrometer equipped with electrospray ionization source in both positive and negative modes. Under the optimized conditions, a total of 31 saponins were identified or tentatively characterized by comparing retention time and MS data with related literatures and reference substances. Conclusion: The developed UHPLC-MS/MS method was suitable for identifying and characterizing the chemical constituents in Shizhu ginseng, which provided a helpful chemical basis for further research on Shizhu ginseng.

• Journal of Applied Biological Chemistry
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• 제42권4호
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• pp.186-190
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• 1999

Isotope ratio ($^{15}N/^{14}N$) and nitrate-nitrogen concentration in groundwater were measured to investigate the effect of chemical fertilizer and livestock manure on temporal variations in nitrate-nitrogen concentration and to estimate the contribution of fertilizer and manure to groundwater contamination by nitrate. Four study wells from a rural area in Kyonggi province were selected. One well was located on an upper site from a livestock feedlot, and the others were situated at lower sites from the feedlot. The ${\delta}^{15}N$ values were analyzed by a stable isotope ratio mass spectrometer (Micromass, VG Optima IRMS). Reproducibility of the method and precision of the mass spectrometer were below 1.0 and 0.1‰, respectively Even though study wells were located at the same area, nitrate-nitrogen concentrations and ${\delta}^{15}N$ values differed and fluctuated during the sampling period. The ${\delta}^{15}N$ values of well located at upper site from the feedlot were extremely variable (-1.48~20.80‰). The ranges of ${\delta}^{15}N$ value of three wells situated at lower sites from the feedlot were 11.83~20.73 (ave. 16.11), 8.90~11.73 (ave.11.01), and 5.29~12.73‰ (ave. 8.21‰) with increasing distance from the feedlot. The average values of contribution proportion of nitrogen derived from livestock manure to nitrate-nitrogen in groundwater were 79% for the well closet to the feedlot, 44% for the well most distant from the feedlot, and 56% for the well in between the two wells.

• 대한한의학방제학회지
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• 제24권1호
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• pp.17-30
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• 2016

Objectives Rehmanniae Radix Preparata was prepared in the traditional Rehmanniae Radix Preparata preparation method set forth in the 『Treasured Mirror of Eastern Medicine(Donguibogam)』 with a view to measuring the contents of 5-Hydroxymethyl-2-furaldehyde(5-HMF) at individual stages of steaming and sundrying and identifying new chemical components.Methods Based on the traditional Rehmanniae Radix Preparata preparation method set forth in the 『Treasured Mirror of Eastern Medicine』, Rehmanniae Radix Preparata steamed and sundried once through nine times was prepared. Thereafter, 5-HMF contents were analyzed and new chemical components were identified in the Rehmanniae Radix Preparata using Waters HPLC e2695, 2640 detectors, a Waters Acquity UPLC system, and a Micromass Q-TOF Premier mass spectrometer.Results The Rehmanniae Radix Preparata preparation method set forth in the 『Treasured Mirror of Eastern Medicine』 is a unique preparation method in Republic of Korea different from that in China. In the first stage of the method, fresh Rehmanniae Radix Crudus was divided into high quality, medium quality, and low quality ones named Rehmanniae Radix Crudus (Caelum)(天黃), Rehmanniae Radix Crudus (Homo)(人黃), and Rehmanniae Radix Crudus (Terra)(地黃) respectively to use Rehmanniae Radix Crudus (Caelum) and Rehmanniae Radix Crudus (Homo) for preparation of juice while using Rehmanniae Radix Crudus (Terra) to make Rehmanniae Radix Preparata. In the second stage, Rehmanniae Radix Crudus (Caelum) and Rehmanniae Radix Crudus (Terra) were made into juice and Rehmanniae Radix Crudus (Terra) was soaked in the juice. In the third stage, among auxiliary materials, rice wine named Purum Vinum Oryzae(淸酒) brewed from sticky rice was sprinkled on Rehmanniae Radix Crudus (Terra) to the extent that Rehmanniae Radix Crudus (Terra) became wet. In the fourth stage, Rehmanniae Radix Preparata steamed in earthenware steamer was dried under natural sunlight. The contents of 5-HMF in Rehmanniae Radix Preparata steamed and sundried once through nine times were shown to be below 0.1% in all cases. Pomolic acid was identified as a new chemical component.Conclusions In conclusion, the Rehmanniae Radix Preparata preparation method set forth in the 『Treasured Mirror of Eastern Medicine』 is thought to be a unique preparation method in South Korea in which Rehmanniae Radix Preparata is completed through the first stage in which fresh Rehmanniae Radix Crudus collected from fields is divided into high, medium, and low quality ones and fresh Rehmanniae Radix Crudus juice is made, the second stage in which the high quality fresh Rehmanniae Radix Crudus is soaked in the fresh Rehmanniae Radix Crudus juice, the third stage in which the fresh Rehmanniae Radix Crudus is steamed, and the fourth stage in which the steamed Rehmanniae Radix Crudus is dried.

• 한국초지조사료학회지
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• 제25권1호
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• pp.1-6
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• 2005

With recent interest organic farming the use of legumes including vetch and clover to provide N to adjacent crops is increasing in Korea. In the present studies, we conducted a trial to investigate the effects of the application of N rate on nitrogen fixation and transfer from vetch to barley in mixed stands. The experiment was arranged in a randomized complete block design with three replications. Four different N rates(0, 75, 113 and 150/ha) was used and vetch+barley was broadcasted manually on 1.5 $\times$2 m plot in Oct. 2001. Half of urea and K$_{2}O, 200 Phosphate and 75 kg potash per ha were applied as basal dressing md half of N md 75 potash were used for topdressing to soil surface on MarctL 2002. The equivalent of 1kg ha$^{-1}$ at($^{15}$NH$_{4}$)$_{2}$SO$_{4}$ solution at 99.8 atom $\%$$^{15}$N excess was applied to the microplot in mid April. Forage was harvested from each plot at ground level and separated into barley and vetch. Total N content and It values of samples were determined using a continuous flow stable isotope ratio mass spectrometry(IsoPrime-EA. Micromass, UK.). The percentage of legume H fixed from atmospheric N2 were 95.0, 93.8, 94.4 and $84.8\%$ with increment of N levels. The percentage of N transfer from vetch to barley by N-difference method with increment of N fertilizer were from 58 to$49\%$ while 39 to $23\%$ in $^{15}$N-dilution method. The amount of transfer from vetch to barley were 87 to 68 kg/ ha with N level by N-difference moth여 and 58 to -56/ha with N application levels by $^{15}$N dilution method. The amount of nitrogen fixation per ha were from 150 kg / ha to 219 kg / ha by different method, but on the other side 49 to 105kg/ha by N$^{15}$-dilution.