• Archives of Pharmacal Research
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• 제29권5호
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• pp.418-423
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• 2006

Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-lle-Asp-Lys-Val-Arg. The protein was activated by $Cu^{2+}\;and\;Zn^{2+}$, and the optimal conditions were found to be at pH $3\sim6\;and\;40\sim70^{\circ}C$. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.

• 자원환경지질
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• 제32권1호
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• pp.73-82
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• 1999

The objectives of this study are to investigate soil contamination in the vicinity of abandoned Au-Ag mine and to apply a remediation technique of liming to tailings. In the study area of the Imcheon Au-Ag mine, soils were sampled in and around the mine the analyzed by Atomic Absorption Spectrometry extracted by both 0.1N HCl and aqua regia. Elevated levels of Cd, Cu, Pb and Zn concentrations extracted by 0.1N HCl were found in soils taken from tailings site. These high contents directly influenced metal concentrations in soils taken in the vicinity of the site. This is mainly due to clastic movement by wind and effluent of mine waste water. In addition, relatively enriched concentrations of the metals were found in soils extrated by aqua regia due to strong decomposition of the samples compared with 0.1N HCl extration. According to the statistical approach, metal concentrations in soils by 0.1N HCl had a positive correlation with those by aqua regia extraction. Mine waste waters and stream waters were also sampled around the mine in spring and summer and analyzed by AAS for Cd, Cu, Pb and Zn, and by Ion Chromatography for anions. Like soils developed over tailings, significant levels of metals and sulphates were found in the mine waste waters ranging of 0.2~0.3, 0.5~2.0, 0.2~2.8, 30~50 and 1,240~4,700 mg/l of Cd, Cu, Pb, Zn and $SO_4^{2-}$, respectively. These elevated levels influenced in the stream waters in the vicinity of the tailings site. In seasonal variation of metal and anion contents, relatively high levels were found in waters sampled on summer due to leaching the metals and anions from tailings by rain. This study also examined the possibility of lime treatment for remediation of acid mine tailings and assumed to be 46 tones of pulverized lime for neutralization of the tailings.

• 한국수산과학회지
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• 제38권1호
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• pp.6-11
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• 2005

Vasopressin (VP)-related peptide was purified from the brain extract of conger eel (Conger myriaster) by reverse-phase, ion-exchange high performance liquid chromatography (HPLC). This peptide with a molecular weight of 1,051.2 Da was determined as $H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NH_2$, whose Cys residues made an intramolecular disulfide bridge by the automated amino acid sequence analysis, MALDI- TOF mass spectrometry. It's sequence was confirmed by identity of the elution position with the synthetic peptide in HPLC system. As a result of homology investigation, the primary structure of this peptide was the same as that of VP-superfamily member, $[Arg^8]-vasotocin$. The synthetic peptide showed a contractile activity at a minimal effective concentration of $10^{-10}\;M$ on the intestinal smooth muscle of goldfish.

• 한국대기환경학회지
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• 제21권4호
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• pp.439-458
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• 2005

An automated analysis system for water soluble constituents in $PM_{2.5}$ has been developed. The system consists of a high capacity multi tube diffusion scrubber (MTDS), a low temperature particle impactor (LTPI), and two ion (anion and cation) chromatography (IC) systems. Atmospheric particles have been collected by passing sample air through a thermostated MTDS followed by a LTPI. This system allows simultaneous measurements of soluble ions in $PM_{2.5}$ at 30 minutes interval. At the air sampling flow rate of 1.0L/min, the detection limits of the overall system are in the order of tens of $ng/m^3$. This system has been successfully used for the measurement of particulate components of Seoul air from April 2003 to January 2004. $SO_4^{2-},\;NO_3^-,\;NH_4^+,\;NO_2^-,\;Cl^-,\;Na^+,\;K^+,\;Ca^{2+},\;and\;Mg^{2+}$ are the major ionic species for $PM_{2.5}$ at Seoul. Among them, $SO_4^{2-},\;NO_3^-\;and\;NH_4^+$ are the most abundant ions, contributed up to $86\%$ of the total and the concentrations were higher than those in any other urban sites in the world except for Chinese cities. There are high pollutant episodes which contribute about $15\~20\%$ of annual average values of the major ions. During the episode, the all parcels were transported from the asian continent and $PM_{2.5}$ were significantly neutralized. This suggests that aged and long range transported pollutants caused the high pollutant episodes. They showed a distinct daily and seasonal variations:they showed a peak in the early morning caused by the night-time accumulation of particulate matters. Atmospheric reactions including gas-to-particle reactions and inter-particle reactions and meteorological parameters including relative humidity and ambient temperature were described with related to the $PM_{2.5}$ 5 concentrations. All of the ionic species showed higher concentrations during the spring than those for summer and winter.

• 한국잠사곤충학회지
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• 제45권2호
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• pp.90-95
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• 2003

최근 천연색소의 다양한 생리 활성이 보고됨에 따라 오디에 함유된 C3G 색소에 대해서도 관심이 높아지고 있다. 이에 오디로부터 C3G를 분리$.$정제하고 정제된 C3G와 표준물질의 분자량을 비교ㆍ동정하였다. 또한 유전자원으로 보존되고 있는 뽕 품종 및 계통에 대한 C3G 함량을 분석하여 고함유 계통을 선발하였다. 1. Amberlite IRC-50 ion exchange resin이 충진된 유리 column을 산성화시킨 후 이온교환수로 비흡착색소, 수용성 당류, 유기산 및 아미노산 등을 제거하였으며, column에 흡착된 색소를 용출 및 감압농축하여 분말상태로 정 제된 색소물질 C3G(cyanidin-3-glucoside)를 얻었다. 2. Waters 996 Photodiods Array Detector를 이용하여 정제된 C3G(cyanidin-3-glucoside)의 흡수 spectrum을 조사한 결과, 각각 516nm과 280nm에서 최대흡수 spectrum을 보임으로써 anthocyanin계 색소의 특이적 흡수 파장 영역인 500-550nm(Band I)과 280nm전후(Band II)와 일치하여 open column에 의해 분리된 색소가 anthocyanin 계열의 색소임을 확인하였다. 3. LC-Mass(Waters 2690 alliance Separation Module)를 사용하여 분자량을 확인한 결과, 449로서 C3G 표준물질의 분자량과 일치하였다. 4. 유전자원으로 보존되고 있는 뽕 품종 및 계통에 대한 C3G 함량을 분석한 결과, 공시한 35계통의 C3G 평균 함량은 0.9%였으며 계통간 함량의 차이가 있는 것으로 나타났다. 5. '수성뽕'. '강선' 및 '절곡조생(충북)'은 C3G함량은 물론 수량, 과중 및 당도에 대한 과실적 특성조사에서 양호한 성적을 나타내어 오디생산용 유망 계통으로 선발하였다.

• 대한미생물학회지
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• 제21권1호
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• pp.151-161
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• 1986

In order to develop sensitive and sepcific assay methods for E. coli heat labile enterotoxin(LT) hybridoma cell lines secreting LT specific monoclonal antibody were obtained. LT was purified from cell lysate of E. coli O15H11. The steps included disruption of bacteria by French pressure, DEAE Sephacel ion exchange chromatography, Sephadex G200 gel filtration, and second DEAE Sephacel ion exchange chromatography, successively. Spleen cells from Balb/c mice immunized with the purified LT and $HGPRT^{(-)}$ plasmacytomas, $P3{\times}63Ag8.V653$ were mixed and fused by 50% (w/v) PEG. Hybrid cells were grown in 308 wells out of 360 wells, and 13 wells out of them secreted antibodies reacting to LT. Among these hybridoma cell 1G8-1D1 cell line was selected since it had produced high-titered monoclonal antibody continuously. By using culture supernatant and ascites from 1G8-1D1 cells the monoclonal antibody was characterized, and an assay system for detecting enterotoxigenic E. coli was established by double sandwich enzyme-linked immunosorbent assay (ELISA). The following results were obtained. 1. Antibody titers of culture supernatant and ascites from 1G8-1D1 hybridoma cells were 512, and 102, 400, respectively by GM1-ELISA and its immunoglobulin class was IgM. 2. The maximum absorption ratio of 1G8-1D1 cell culture supernatant to LT was 90% at $300\;{\mu}g/ml$ of LT concentration. LT concentration shown at 50% absorption ratio was $103.45{\mu}g$ and the absorption ratio was decreased with tile reduction of LT concentration. This result suggests that monoclonal antibody from 1G8-1D1 hybridoma cell bound with LT specifically. 3. The reactivities of 1G8-1D1 cell culture supernatant to LT and V. cholerae enterotoxin(CT) were 0.886 and 0.142(O.D. at 492nm) measured by the GM1-ELISA, indicating 1G8-1D1 monoclonal antibody reacted specifically with LT but not with CT. 4. The addition of 0.1ml of ascites to 0.6mg and 0.12mg of LT decreased the vascular permeability factor to 41% and 44% respectively, but it did not completely neutralize LT. 5. By double sandwich ELISA using monoclonal antibody, as little as 75ng of the purified LT per ml could be detected. 6. The results by assay of detecting LT in culture supernatants of 14 wild strains E. coli isolated from diarrhea patients by the double sandwich ELISA were almost the same level as those by reverse passive latex agglutination.

• 한국응용과학기술학회지
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• 제18권1호
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• pp.67-77
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• 2001

In search for several fatty acid with unusual structure in vegetable oils, we have found that unknown peaks were shown on GLC in the analysis of fatty acids of the lipids from the pulp of ripened jujube (Zizypus jujuba var. inermis) fruits. These fatty acids were identified as a series of cis-monoenoic acids with ${\omega}-5$ double bond system such as $C_{14:1{\omega}5}$, $C_{16:1{\omega}5}$ and $C_{18:1{\omega}5}$, including ${\omega}-7$ fatty acid as $C_{16:1{\omega}7}$ and $C_{18:1{\omega}7}$, by GLC, solid-phase extraction silver ion-column chromatographic, GLC-mass spectrometric and IR techniques. First of all, total fatty acid methyl esters were resolved into saturated and branched fatty acid, monoenoic acid, dienoic acid, and trienoic acid fraction, respectively, with 100% dichloromethane (DCM), DCM/acetone (9:1, v/v) 100% acetone, and acetone/ acetonitrile (97:3, v/v) solvent system. Unknown fatty acids were included in the monoenoic fraction and were confirmed to have cis-configuration by IR. Picolinyl esters of monoenoic fatty acids gave distinct molecular ion peak and dominant diagnostic peaks, for example, m/z 317, 220 and 260 fragment for $cis-C_{14:1{\omega}5}$, m/z 345, m/z 248 and 288 fragment for $cis-C_{16:1{\omega}5}$ and m/z 373, m/z 276 and 316 fragment for $cis-C_{18:1{\omega}5}$. In this way the occurrence of $cis-C_{16:1{\omega}7}$ and $cis-C_{18:1{\omega}7}$ could be deduced from the appearance of prominent fragments as m/z 345, 220 and 260, and m/z 373, 248 and 280. Level of total ${\omega}-5$ fatty acids amounted to about 30% in the fatty acid composition with the predominance of $C_{16:1{\omega}5}$ $ (18.7{\sim}25.0%)$, in the semi-ripened and/or ripened samples collected in September 14 ($C_{16:1{\omega}5}$ ; 18.7%, $C_{14:1{\omega}5}$ ; 3.6% and $C_{18:1{\omega}5}$ ; 3.0%), September 22 ($C_{16:1{\omega}5}$ ; 25.0%, $C_{14:1{\omega}5}$ ; 1.4% and $C_{18:1{\omega}5}$ ; 2.6%), and October $7 (C_{16:1{\omega}5}$ ; 24.7%, $C_{14:1{\omega}5}$ ; 7.7% and $C_{18:1{\omega}5}$ ; 2.5%). However, the lipids extracted from unripened jujube in July and August contain these unusual fatty acids as low as negligible. It could be observed that the level of ${\omega}-5$ fatty acids in the pulps increased sharply with an elapse of ripening time of jujube fruits. Other monoenoic fatty acids with ${\omega}-7$ series, $C_{16:1{\omega}7}$ (palmitoleic acid) and $C_{18:1{\omega}7}$ (cis-vaccenic acid) could be detected. And in the lipids of the kernel and leaf of jujube, none of ${\omega}-5$ fatty acids could be detected.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1583-1590
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• 2006

T4 endonuclease V (T4 endo V) [EC 3. 1. 25. 1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a cyclobutane pyrimidine dimer DNA glycosylase (CPD glycosylase) and an apyrimidic/apurinic endonuclease (AP lyase). T4 denV (414 bp cDNA) encoding T4 en do V (138 amino acid) was synthesized and expressed using either an expression vector, pTriEx-4, in E. coli or a baculovirus AcNPV vector, pBacPAK8, in insect cells. The recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed from bacteria was purified using one-step affinity chromatography with a HiTrap Chelating HP column and used to make rabbit anti-His-Tag/T4 endo V polyclonal antibody for detection of recombinant T4 endo V (rT4 endo V) expressed in insect cells. In the meantime, the recombinant baculovirus was obtained by cotransfection of BacPAK6 viral DNA and pBP/T4 endo V in Spodoptera frugiperda (Sf21) insect cells, and used to infect Sf21 cells to overexpress T4 endo V protein. The level of rT4 endo V protein expressed in Sf21 cells was optimized by varying the virus titers and time course of infection. The optimal expression condition was set as follows; infection of the cells at a MOI of 10 and harvest at 96 h post-infection. Under these conditions, we estimated the amount of rT4 endo V produced in the baculovirus expression vector system to be 125 mg/l. The rT4 endo V was purified to homogeneity by a rapid procedure, consisting of ion-exchange, affinity, and reversed phase chromatographies, based on FPLC. The rT4 endo V positively reacted to an antiserum made against rHis-Tag/T4 endo V and showed a residual nicking activity against CPD-containing DNA caused by UV. This is the first report to have T4 endo V expressed in an insect system to exclude the toxic effect of a bacterial expression system, retaining enzymatic activity.

• 한국대기환경학회지
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• 제34권3호
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• pp.457-468
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• 2018

To improve understanding of the physico-chemical characteristics of aerosols in the national park and comparing the air pollution between national park and the urban area nearby national park, the aerosol characterization study was conducted in Bukhansan National Park, Seoul, from July through September 2017. Semi-continuous measurements of $PM_{2.5}$ using PILS (Particle Into Liquid System) coupled with IC (Ion Chromatography) and TOC (Total Organic Carbon) analyzer allowed quantification of concentrations of major ionic species($Cl^-$, $SO_4{^{2-}}$, $NO_3{^-}$, $Na^+$, $NH_4{^+}$, $K^+$, $Mg{^{2+}}$ and $Ca{^{2+}}$) and water soluble organic carbon (WSOC) with 30-minute time resolution. The total mass concentration of $PM_{2.5}$ was measured by T640 (Teledyne) with 5-minute time resolution. The black carbon (BC) and ozone were measured with a minute time resolution. The timeline of aerosol chemical compositions reveals a strong influence from urban area (Seoul) at the site in Bukhansan National Park. Inorganic aerosol composition was observed to be dominated by ammoniated sulfate at most times with ranging from $0.1{\sim}32.6{\mu}g/m^3$ (6.5~76.1% of total mass of $PM_{2.5}$). The concentration of ammonium nitrate, a potential indicator of the presence of local source, ranged from below detection limits to $20{\mu}g/m^3$ and was observed to be highest during times of maximum local urban (Seoul) impact. The total mass of $PM_{2.5}$ in Bukhansan National Park was observed to be 10~23% lower than the total mass of $PM_{2.5}$ in urban area (Gireum-dong and Bulgwang-dong, Seoul). In general, ozone concentration in Bukhansan National Park was observed to be similar or higher than urban sites in Seoul, suggesting additional biogenic VOCs with $NO_x$ from vehicle emission were to be precursors for ozone formation in Bukhansan National Park.

• 대한한의학방제학회지
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• 제31권1호
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• pp.1-10
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• 2023

Objectives : The main aim of this study was to examine the LC-MS/MS used to identify phenolic compounds of CRE(Coptidis Rhizoma 70% EtOH Extract). Also, we investigated antioxidative activities and Anti-inflammatory activities. Methods : LC-MS/MS Analysis HPLC and LC-MS/MS were performed on a 1260 series HPLC system (Agilent Technologies, Inc., California, USA) and 3200 QTrap tandem mass system (Sciex LLC) operated in positive ion mode (spray voltage set at -4.5 kV). The solvent used was DW and Acetonitrile containing 0.1% formic acid, a gradient system was used at a flow rate of 0.5 mL/min for analysis, and a Prontosil C18 column (length, 250 mm; inner diameter, 4.6 mm; particle size, 5 ㎛; Phenomenex Co., Ltd., California, USA, Biochoff Chromatography) was used. The solvent conditions used in the mobile phases were 0-10 min at 10-15% B, 10-20 min at 20% B, 20-30 min at 25%, 30-40 min at 40%, 40-50 min at 70%, 50-60 min at 95%, and 60-70 min at 95%. The analysis was performed at a wavelength of 284 nm and a temperature of 35℃. The cell viability was measured using a 3-(4,5-dimethyethiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. We examined the effects of CRE on the lipopolysaccharide (LPS)-induced production of nitric oxide (NO) in a RAW 264.7 cells Results : The chemical analysis CRE by Liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that Rosmarinic acid, Ferrulic acid, 3-O-feruloylquinic acid, and 5-O-feruloylquinic acid as phenolic components. DPPH radical scavenging activity was the inhibitory activity of CRE showed at 200 ㎍/mL a statistically significant level. MTT assay demonstrated that the CRE did not have a cytotoxic effect in RAW 264.7 and LPS-induced RAW264.7 cells. Also, CRE reduced NO production in RAW 264.7 cells stimulated with LPS. Conclusions : Based on these findings, The chemical analysis 4 major components CRE such as Rosmarinic acid, Ferrulic acid, 3-O-feruloylquinic acid, and 5-O-feruloylquinic acid. Moreover, we confirmed that CRE has effects antioxidant and anti-inflammatory. The results demonstrate that CRE can be used as an antioxidant and a powerful chemopreventive ingredient against inflammatory diseases.