• Title/Summary/Keyword: 2-dimensional liquid chromatography

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Comparative Proteomic Analyses of the Yeast Saccharomyces cerevisiae KNU5377 Strain Against Menadione-Induced Oxidative Stress

  • Kim, Il-Sup;Yun, Hae-Sun;Jin, In-Gnyol
    • Journal of Microbiology and Biotechnology
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    • v.17 no.2
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    • pp.207-217
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    • 2007
  • The Saccharomyces0 cerevisiae KNU5377 strain, which was isolated from spoilage in nature, has the ability to convert biomass to alcohol at high temperatures and it can resist against various stresses [18, 19]. In order to understand the defense mechanisms of the KNU5377 strain under menadione (MD) as oxidative stress, we used several techniques for study: peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) followed by two-dimensional (2D) gel electrophoresis, liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), and surface-enhanced laser desorption ionization-time of flight (SELDI-TOF) technology. Among the 35 proteins identified by MALDI-TOF MS, 19 proteins including Sod1p, Sod2p, Tsa1p, and Ahp1p were induced under stress condition, while 16 proteins were augmented under normal condition. In particular, five proteins, Sod1p, Sod2p, Ahp1p, Rib3p, Yaf9p, and Mnt1p, were induced in only stressed cells. By LC-ESI-MS/MS analysis, 37 proteins were identified in normal cells and 49 proteins were confirmed in the stressed cells. Among the identified proteins, 32 proteins were found in both cells. Five proteins including Yel047cp and Met6p were only upregulated in the normal cells, whereas 17 proteins including Abp1P and Sam1p were elevated in the stressed cells. It was interesting that highly hypothetical proteins such as Ynl281wp, Ygr279cp, Ypl273wp, Ykl133cp, and Ykr074wp were only expressed in the stressed cells. SELDI-TOF analysis using the SAX2 and WCX2 chips showed that highly multiple-specific protein patterns were reproducibly detected in ranges from 2.9 to 27.0 kDa both under normal and stress conditions. Therefore, induction of antioxidant proteins, hypothetical proteins, and low molecular weight proteins were revealed by different proteomic techniques. These results suggest that comparative analyses using proteomics might contribute to elucidate the defense mechanisms of KNU5377 under MD stress.

PAHs Formation Characteristics and Fullerenes $(C_{60},\;C_{70})$ Synthesis in a Low-Pressure $C_6H_6/Ar/O_2$ Flame (저압 $C_6H_6/Ar/O_2$ 화염에서 PAHs 생성 특성 및 플러렌$(C_{60},\;C_{70})$ 합성에 대한 연구)

  • Lee, G.W.;Kim, Y.W.;Hwang, J.;Jrung, J.;Choi, M.
    • Journal of the Korean Society of Combustion
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    • v.7 no.4
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    • pp.36-44
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    • 2002
  • Carbon molecules with closed-cage structures are called fullerenes $(C_{60},\;C_{70})$, whose applications include super-conductors, sensors, catalysts, optical and electronic device, polymer composites, and biological and medical materials. The synthesis of fullerenes has been recently studied with low-pressure benzene/argon/oxygen flames. The formation of fullerene is known as molecular weight growth processes of PAHs (polycyclic aromatic hydrocarbon). This study presents results of PAHs and fullerene measurements performed in a low-pressure benzene/argon/oxygen normal co-flow laminar diffusion flame. Through the central tube of the burner, benzene vapors carried by argon are injected. The benzene vapors are made in a temperature-controlled bubbler. The burner is located in a chamber, equipped with a sampling system for direct collection of condensable species from the flame, and exhausted to a vacuum pump. Samples of the condensable are analyzed by HPLC (High Performance Liquid Chromatography) to determine the yields of PAHs and fullerene. Also, we computed mole fraction of fullerene and PAHs in a nearly sooting low pressure premixed, one-dimensional benzene/argon/oxygen flame (equivalence ratio ${\Phi}=2.4$, pressure=5.33kPa). The object of computation was to investigate the formation mechanism of fullerenes and PAHs. The computations were performed with CHEMKIN/PREMIX. As a result of this study, fullerenes were synthesized in a low pressure (20torr) $C_6H_6/Ar/O_2$ flames and the highest concentration of fullerene was detected just above the visible surface of a flame.

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Physicochemical Characterization and NMR Assignments of Ginsenosides Rb1, Rb2, Rc, and Rd Isolated from Panax ginseng

  • Cho, Jin-Gyeong;Lee, Min-Kyung;Lee, Jae-Woong;Park, Hee-Jung;Lee, Dae-Young;Lee, Youn-Hyung;Yang, Deok-Chun;Baek, Nam-In
    • Journal of Ginseng Research
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    • v.34 no.2
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    • pp.113-121
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    • 2010
  • The fresh ginseng roots were extracted with aqueous methanol, and the obtained extracts were partitioned using ethyl acetate, n-butanol, and water, successively. The repeated silica gel and octadecyl silica gel column chromatogaraphy for n-butanol fraction afforded four diol ginseng saponins, ginsenosides $Rb_1$, $Rb_2$, $R_c$, and Rd. The physicochemical, spectroscopic, and chromatographic characteristics of these ginsenosides were measured and compared with those reported in the literature. Some of the peak assignments in previously published $^1H$- and $^{13}C$-nuclear magnetic resonance (NMR) spectra were inaccurate. This study employed two-dimensional NMR experiments, including $^1H-^1H$ correlation spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond connectivity, to determine exact peak assignments.

A Chemical Study of the Saponins and Flavonoids of Dwarf Ginseng (Panax trifolius L.) and Its Comparison to Related Species in the Araliaceae (왜생삼 (Panax trifolius L.)의 사포닌과 프라보노이드의 화학적 연구 및 오가과에 속하는 유연종과의 성분 비교연구)

  • Lee Taikwang M.;Marderosian Ara Der
    • Proceedings of the Ginseng society Conference
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    • 1988.08a
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    • pp.141-146
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    • 1988
  • Dwarf ginseng (Panax trifolius L.) is a member of the ginseng family (Araliaceae). which is indigenous to North America and is distributed from Southern Canada to the Northern United States. In total. nine compounds were isolated from the leaves of Dwarf gineng. Of these. four were identified as flavonoids and five were found to be ginsenosides. Two of the flavonoids were identified to be kaempferol-3. 7-dirhamnoside and kaempferol-3-gluco-7-rhamnoside. Four of the ginsenosides were identified as notoginsenoside-Fe. ginsenoside-Rd. ginsenoside-Rc and $ginsenoside-Rb_1$ The common aglycone of these ginsenosides was shown to be (20S)-protopanaxadiol. The identification of flavonoids and ginsenosides from the root. stem. leaf. flower and fruit of Dwarf ginseng was detected by Two-Dimensional Thin-Layer Chromatography (2D-TLC) and High Performance Liquid Chromatography (HPLC). The quantitation of flavonoids and ginsenosides from the root. stem. leaf. flower and fruit of Dwarf ginseng and related species such as Korean gineng (Panax ginseng C.A. Meyer) and American ginseng (Panax quinquefolium L.) was analyzed by HPLC only. Three flavonoids (Kaempferol derivatives) labelled compound 1 $(10.8\%)$, compound 3 ($2.8\%$), and compound 4 ($8.4\%)$ were found in the root of Dwarf ginseng but not found in the roots of Korean ginseng and American ginseng. This is the first time that flavonoids have been found and identified in roots of the ginseng family (Araliaceae).

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Identification of Cisplatin-Resistance Associated Genes through Proteomic Analysis of Human Ovarian Cancer Cells and a Cisplatin-resistant Subline

  • Zhou, Jing;Wei, Yue-Hua;Liao, Mei-Yan;Xiong, Yan;Li, Jie-Lan;Cai, Hong-Bing
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.12
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    • pp.6435-6439
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    • 2012
  • Chemoresistance to cancer therapy is a major obstacle to the effective treatment of human cancers with cisplatin (DDP), but the mechanisms of cisplatin-resistance are not clear. In this study, we established a cisplatin-resistant human ovarian cancer cell line (COC1/DDP) and identified differentially expressed proteins related to cisplatin resistance. The proteomic expression profiles in COC1 before and after DDP treatment were examined using 2-dimensional electrophoresis technology. Differentially expressed proteins were identified using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and high performance liquid chromatography-electrospray tandem MS (NanoUPLC-ESI-MS/MS). 5 protein spots, for cytokeratin 9, keratin 1, deoxyuridine triphosphatase (dUTPase), aarF domain containing kinase 4 (ADCK 4) and cofilin1, were identified to be significantly changed in COC1/DDP compared with its parental cells. The expression of these five proteins was further validated by quantitative PCR and Western blotting, confirming the results of proteomic analysis. Further research on these proteins may help to identify novel resistant biomarkers or reveal the mechanism of cisplatin-resistance in human ovarian cancers.

Adenyl Cyclase Activity in Cold-acclimatized Animals (한냉적응이 Adenyl Cyclase Activity에 미치는 영향)

  • Kang, Bok-Soon;Lee, Sang-Ho;Kang, Doo-Hee
    • The Korean Journal of Physiology
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    • v.8 no.2
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    • pp.67-74
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    • 1974
  • The object of this research is aimed to determine the activity of adenyl cyclase in both skeletal muscle sarcolemma and fat cell ghost of epididymal adipose tissue isolated from rats exposed to cold for various length of time in an attempt to evaluate whether the tissue sensitivity to catecholamine is increased when rats are exposed to cold for long periods of time Methods: a)Animals: Albino rats ranging in weight from 150 to 200 gm were used throughout this study. For experimental purposes, the rats are divided into two groups: experimental animals were place4 in a cold room at $4^{\circ}C$, controls being kept at $25^{\circ}C$. At the end of 2, 4, 6, 12, and 16 weeks. exposure to cold the rats were used to measure the adenyl cyclase activity. b) Isolation of plasma membrane from skeletal muscle and adipose tissue: The Plasma membrane of skeletal muscle from hind limbs of rats are prepared by the method employed by Rosenthal et at. and fat cell ghost of epididymal adipose tissue of rats by the method employed by Rodbell. c) Adenyl cyclase assay: Adenyl cyclase activity were measured by the method employed by Marinetti et al. Briefly, plasma membrane was incubated with $3^H-ATP$, various amount of noradrenaline and other incubation mixture at $37^{\circ}C$ for 20 minutes. After stopping the enzyme reaction by immersion in boiling water, carrier 3',5'-AMP was added to the system as a marker and $100\;{\mu}1$ aliquots of incubation mixture were pipetted on $20{\time}20$ Whatman No. 3 MM filter paper for one dimensional chromatography. The cyclic AMP spots were cut off and placed in counting vials containing 10ml of Bray's scintillation cocktail. Radioactivity was determined with a Packard Tri-Carb liquid scintillation counter. The enzyme activity is expressed as nanomoles of cyclic AMP produced per mg of membrane per hour. Result: 1. Average adenyl cyclase activity in the plasma membrane of skeletal muscle before and after noradrenaline administration was significantly higher in the cold-exposed rats as compared to the control. Continuous exposure to cold Produced an increased adenyl cyclase activity before and after noradrenaline administration. Adenyl cyclase activity reached peak levels at the 6 weeks exposure to told and level of adenyl cyclase activity remained high. Noradrenaline administration to the incubation medium induced a significant increase in adenyl cyclase activity and the degree of stimulation were proportional to the hormonal concentration But the rate of inclement in adenyl cyclase activity by noradreasline was the same in both groups. 2. Adenyl cyclase activity in fat cell ghost between cold exposed and control rats showed no significant differences before and after noradreualine administration. In summary, it can be concluded that cold adaptation give rise an increased activity of adenyl cyclase in plasma membrane of skeletal muscle in rats.

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Factors Influencing Frequency of Abnormal Peak in the Measurement of HbA1c by HPLC (HPLC법을 이용한 HbA1c 측정시 Abnormal Peak의 빈도와 원인)

  • Kim, Sun-Kyung;Bae, Ae-Young;Choi, Dae-Yong;Kim, Myung-Soo;Yoo, Kwang-Hyun;Ki, Chang-Seok
    • Korean Journal of Clinical Laboratory Science
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    • v.37 no.2
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    • pp.71-77
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    • 2005
  • We experienced the specimen that contains a hemoglobin variant known as interference from HbAS in October 2003. It was the first case of Hb variants since Samsung Medical Center began conducting glycohemoglobin College of American Pathologists surveys in 1997. The purpose of this study is to share our experience with the specimen and promote the understanding of Hb variants & derivatives. We've performed cross checks to examine HbA1c by using two pieces of equipment; the TOSHOH G7 and BIO-RAD VARIANT-T(turbo), and Automatic High Performance Liquid Chromatography(HPLC) as an analytic measurement method. HPLC provides different fractional information of hemoglobin with a two-dimensional graph as well as numeric results. We have been performing a "Systematic Checking Process". Three specimen suspicious of Hb variants & derivatives were found through this process. College of American Pathologists notified that it is important for users to be aware of the limitation of their glycohemoglobin method to avoid reporting incorrect results due to interference from hemoglobin variants or hemoglobin adducts. Therefore, laboratory findings of Hb variants & derivatives are very important. The experience of qualified technicians with professional knowledge in Hb variants is the most important aspect in finding Hb variants. Korea is homogeneous in race and is not in an area with a higher finding rate of Hb variants. While 1,024 cases of Hb variants have been found in Japan, we do not have specific data on how many cases of Hb variants have been found in Korea. Considering Hb variant cases in Japan, which is geographically close to us, it is presumed that there must be various Hb variant cases in Korea. If domestic laboratories set a systemic protocol and build a network to share our experience in Hb variants, I expect the Korean Hb variants could also be listed on the world's Hb variant list.

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