• Title/Summary/Keyword: affinity analysis

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Characterization of Laccase Purified from Korean Pycnoporus cinnabarinus SCH-3 (한국산 주걱송편버섯(Pycnoporus cinnabarinus) SCH-3로부터 정제 된 Laccase의 특성)

  • Park, Eun-Hye;Yoon, Kyung-Ha
    • The Korean Journal of Mycology
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    • v.31 no.2
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    • pp.59-66
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    • 2003
  • Laccase produced by Pycnoporus cinnabarinus SCH-3 isolated from Korea was partially purified using ultrafiltration, anion exchange chromatography and affinity chromatography, The laccase was produced as the predominant extracellular phenoloxidase during primary metabolism. Neither lignin peroxidase nor manganese-dependent peroxidase were detected in the culture fluid. In order to examine the effect of inducers in laccase production, 2,5-xylidine was added in the culture of Pycnoporus cinnabarinus SCH-3. Addition of 2,5-xylidine enhanced 25-fold laccase production. Purified laccase was a single polypeptide having a molecular mass of approximately 66 kDa, as determined by SDS-polyacrylamide gel electrophoresis, and carbohydrate content of 9%. $K_{m}\;and\;V_{max}$ values for laccase with ABTS [2,2-azinobis (3-ethylbenzthiazoline 6-sulfonic acid)] as a substrate (Lineweaver-Burk plot) was determined to be $44.4{\mu}M\;and\;56.0{\mu}mole$, respectively. The optimal pH for laccase activity was found to be 3.0. The enzyme was very stable for 1 hour at $60{\circ}C$. Half-life ($t_{1/2}$) of the enzyme was about 10 min at $80{\circ}C$. Spectroscopic analysis of purified enzyme indicated that the enzyme was typical of copper-containing protein. Substrate specificity and inhibitor studies for laccase also indicated to be a typical fungal laccase. The N-terminal amino acid sequence of the P. cinnabarinus SCH-3 laccase showed 94% of homology to the N-terminal sequences of laccases from P. cinnabarinus PB and P. coccineus.

Influence of Phenobarbital on the Circadian Rhythm of Opiate Receptor in Rat Brain (백서의 뇌내 Opiate 수용체의 일중 변동에 미치는 Phenobarbital의 영향)

  • Park, Yeoung-Gul;Kim, Kee-Won;Cho, Kyu-Park
    • The Korean Journal of Pharmacology
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    • v.21 no.2
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    • pp.128-141
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    • 1985
  • To investigate the influence of phenobarbital sodium on the action of morphine and on the diurnal rhythms of both opiate receptor binding and ${\beta}-endorphin$ contents, the amount of specifically bound $(^3H)$-morphine and immunoreactive ${\beta}-endorphin$ were measured in the midbrain of phenobarbital-treated rats at 4h intervals in a day. Rats were housed and adapted to a controlled cycle of either 12 h light-12 h dark or 24 h constant dark. After 3 weeks of adaptation, 0.5 ml of physiological saline or phenobarbital sodium (20mg/kg/day, i.p.) were administered twice a day for 2 weeks. Highly significant diurnal rhythms of opiate receptor binding and ${\beta}-endorphin$ were present in rat midbrain. In control group, the peak of maximum $(^3H)$-morphine binding was observed at 22:00 h, whereas the peak of ${\beta}-endorphin$ content was found at 06:00 h. Even in the absence of time cues these diurnal rhythms persisted, but they were highly modified with respect to the wave form as well as differences in the timing of peak and nadir. In the phenobarbital-treated group, these diurnal rhythms were also modified in shape, phase and amplitude, as well as in timing of peak and nadir. In this group, 24 h mean of opiate receptor binding was significantly decreased, while the 24 h mean level of ${\beta}-endorphin$ content was highly increased. However, Kd values in all experimental groups did not change. This indicates that differences in binding were not due to changes in the affinity, but in the number of binding sites. Statistical analysis of regression line indicates that changes of receptor binding were closely correlated with the changes of ${\beta}-endorphin$ content. These results suggest that phenobarbital may influence the action of morphine by changing the number of opiate receptors and that the modification of diurnal rhythm of opiate receptor by the agent is possibly due to changes of ${\beta}-endorphin$ content.

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Destabilization of TNF-α mRNA by Rapamycin

  • Park, Jong-Woo;Jeon, Ye-Ji;Lee, Jae-Cheol;Ahn, So-Ra;Ha, Shin-Won;Bang, So-Young;Park, Eun-Kyung;Yi, Sang-Ah;Lee, Min-Gyu;Han, Jeung-Whan
    • Biomolecules & Therapeutics
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    • v.20 no.1
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    • pp.43-49
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    • 2012
  • Stimulation of mast cells through the high affinity IgE receptor (Fc${\varepsilon}$RI) induces degranulation, lipid mediator release, and cytokine secretion leading to allergic reactions. Although various signaling pathways have been characterized to be involved in the Fc${\varepsilon}$RI-mediated responses, little is known about the precious mechanism for the expression of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) in mast cells. Here, we report that rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR), reduces the expression of TNF-${\alpha}$ in rat basophilic leukemia (RBL-2H3) cells. IgE or specific antigen stimulation of RBL-2H3 cells increases the expression of TNF-${\alpha}$ and activates various signaling molecules including S6K1, Akt and p38 MAPK. Rapamycin specifically inhibits antigeninduced TNF-${\alpha}$ mRNA level, while other kinase inhibitors have no effect on TNF-${\alpha}$ mRNA level. These data indicate that mTOR signaling pathway is the main regulation mechanism for antigen-induced TNF-${\alpha}$ expression. TNF-${\alpha}$ mRNA stability analysis using reporter construct containing TNF-${\alpha}$ adenylate/uridylate-rich elements (AREs) shows that rapamycin destabilizes TNF-${\alpha}$ mRNA via regulating the AU-rich element of TNF-${\alpha}$ mRNA. The antigen-induced activation of S6K1 is inhibited by specific kinase inhibitors including mTOR, PI3K, PKC and $Ca^{2+}$chelator inhibitor, while TNF-${\alpha}$ mRNA level is reduced only by rapamycin treatment. These data suggest that the effects of rapamycin on the expression of TNF-${\alpha}$ mRNA are not mediated by S6K1 but regulated by mTOR. Taken together, our results reveal that mTOR signaling pathway is a novel regulation mechanism for antigen-induced TNF-${\alpha}$ expression in RBL-2H3 cells.

Development of the feedback resistant pheAFBR from E. coli and studies on its biochemical characteristics (E. coli 유래 pheA 유전자의 되먹임제어 저항성 돌연변이의 구축과 그 단백질의 생화학적 특성 연구)

  • Cao, Thinh-Phat;Lee, Sang-Hyun;Hong, KwangWon;Lee, Sung Haeng
    • Korean Journal of Microbiology
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    • v.52 no.3
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    • pp.278-285
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    • 2016
  • The bifunctional PheA protein, having chorismate mutase and prephenate dehydratase (CMPD) activities, is one of the key regulatory enzymes in the aromatic amino acid biosynthesis in Escherichia coli, and is negatively regulated by an end-product, phenyalanine. Therefore, PheA protein has been thought as useful for protein engineering to utilize mass production of essential amino acid phenylalanine. To obtain feedback resistant PheA protein against phenylalanine, we mutated by using random mutagenesis, extensively screened, and obtained $pheA^{FBR}$ gene encoding a feedback resistant PheA protein. The mutant PheA protein contains substitution of Leu to Phe at the position of 118, displaying that higher affinity (about $290{\mu}M$) for prephenate in comparison with that (about $850{\mu}M$) of wild type PheA protein. Kinetic analysis showed that the saturation curve of $PheA^{FBR}$ against phenyalanine is hyperbolic rather than that of $PheA^{WT}$, which is sigmoidal, indicating that the L118F mutant enzyme has no cooperative effects in prephenate binding in the presence of phenylalanine. In vitro enzymatic assay showed that the mutant protein exhibited increased activity by above 3.5 folds compared to the wild type enzyme. Moreover, L118F mutant protein appeared insensitive to feedback inhibition with keeping 40% of enzymatic activity even in the presence of 10 mM phenylalanine at which the activity of wild type $PheA^{WT}$ was not observed. The substitution of Leu to Phe in CMPD may induce significant conformational change for this enzyme to acquire feedback resistance to end-product of the pathway by modulating kinetic properties.

Seismic study of the Ulleung Basin crust and its implications for the opening of the East Sea (탄성파 탐사를 통해 본 울릉분지의 지각특성과 동해형성에 있어서의 의미)

  • Kim, Han Jun
    • Journal of the Korean Geophysical Society
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    • v.2 no.1
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    • pp.9-26
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    • 1999
  • The Ulleung Basin (Tsushima Basin) in the southwestern East Sea (Japan Sea) is floored by a crust whose affinity is not known whether oceanic or thinned continental. This ambiguity resulted in unconstrained mechanisms of basin evolution. The present work attempts to define the nature of the crust of the Ulleung Basin and its tectonic evolution using seismic wide-angle reflection and refraction data recorded on ocean bottom seismometers (OBSs). Although the thickness of (10 km) of the crust is greater than typical oceanic crust, tau-p analysis of OBS data and forward modeling by 2-D ray tracing suggest that it is oceanic in character: (1) the crust consists of laterally consistent upper and lower layers that are typical of oceanic layers 2 and 3 in seismic velocity and gradient distribution and (2) layer 2C, the transition between layer 2 and layer 3 in oceanic crust, is manifested by a continuous velocity increase from 5.7 to 6.3 km/s over the thickness interval of about 1 km between the upper and lower layers. Therefore it is not likely that the Ulleung Basin was formed by the crustal extension of the southwestern Japan Arc where crustal structure is typically continental. Instead, the thickness of the crust and its velocity structure suggest that the Ulleung Basin was formed by seafloor spreading in a region of hotter than normal mantle surrounding a distant mantle plume, not directly above the core of the plume. It seems that the mantle plume was located in northeast China. This suggestion is consistent with geochemical data that indicate the influence of a mantle plume on the production of volcanic rocks in and around the Ulleung Basin. Thus we propose that the opening models of the southwestern East Sea should incorporate seafloor spreading and the influence of a mantle plume rather than the extension of the crust of the Japan Arc.

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Analysis of Environmental Factors and Change of Vascular Plant Species along an Elevational Gradients in Baekdansa, Mt. Taebaeksan National Park (태백산국립공원 백단사코스의 고도별 관속식물상 변화와 환경요인 분석)

  • An, Ji-Hong;Park, Hwan-Joon;Lee, Sae-rom;Seo, In-Soon;Nam, Gi-Heum;Kim, Jung-Hyun
    • Korean Journal of Environment and Ecology
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    • v.33 no.4
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    • pp.378-401
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    • 2019
  • This study generated a list of plants in eight sections from the Baekdansa ticket office (874m) to Cheonjedan (1,560m) divided in the interval of 100m above sea level to examine the species diversity patterns and distribution changes of the vascular plants at different altitudes in Taebaeksan National Park. Four site surveys found a total of 385 taxa: 89 families, 240 genera, 345 species, 5 subspecies, 34 varieties, and 1 form. A result of analyzing the change of species diversity along elevational gradients showed that it decreased with increasing elevation and then increased from a certain section. A result of analyzing habitat affinity types showed that the proportion of forest species increased with increasing elevation. On the other hand, the ruderal species appeared at a high rate in the artificial interference section. A result of comparing the proportion of woody and herb plants showed that the woody plants gradually increased with elevation and rapidly decreased in the artificial interference section. On the other hand, the herb plants showed the opposite trend. A result of analyzing the change of distribution of species according to altitude with the DCA technique showed that the vascular plants were divided into three groups according to the elevation in order on the I axis with the boundaries at 900m and 1,300m above sea level. The arrangement of each stand from right to left along the altitude on the I axis with a significant correlation with warmth index (WI) confirmed that the temperature change along the altitude could affect the distribution of vascular plants, composition, and diversity. Therefore, the continuous monitoring is necessary to confirm ecological and environmental characteristics of vegetation, distribution ranges, changes of habitat. We expect that the results of this study will be used as the basic data for establishing the measurement measures related to the preservation of biodiversity and climate change.

SHRIMP Zircon U-Pb Age and Geochemistry of Igneous Rocks in the Ssangyong and Yongchu Valleys and Mungyeong Saejae Geosites, Mungyeong Geopark (문경지질공원 쌍룡계곡, 용추계곡, 문경새재 지질명소 화성암류의 SHRIMP 저어콘 U-Pb 연령과 지구화학)

  • Wonseok Cheong;Yoonsup Kim;Giun Han;Taehwan Kim
    • Korean Journal of Mineralogy and Petrology
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    • v.36 no.1
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    • pp.73-94
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    • 2023
  • We carried out the sensitive high resolution ion microprobe (SHRIMP) zircon U-Pb age dating and whole-rock geochemical analysis of granitoids and felsic porphyries in the Ssangyong Valley, Yongchu Valley, and Mungyeong Saejae geosites in the Mungyeong Geopark. The igneous rocks crop out in the western, northwestern and central parts of the Mungyeong city area, respectively, and intruded (meta)sedimentary successions of the Ogcheon Metamorphic Belt, Cambro-Ordovician Mungyeong Group and Jurrasic Daedong Group. The U-Pb isotopic compositions of zircon from two felsic porphyries and one granite samples in the Ssanyeong Valley yielded the Cretaceous intrusion ages of 93.9±3.3 Ma (tσ), 95.1±4.0 Ma (tσ) and 94.4±2.0 Ma (tσ), respectively. On the other hand, a felsic dike sample and a granite in the Yongchu Valley and a porphyritic granite in the Mungyeong Saejae had intrusion ages of 90.2±2.0 Ma (tσ), 91.0±3.0 Ma (tσ) and 88.6±1.5 Ma (tσ), respectively. Based on the average standard error calculated in combination with results of previous studies in this area (Lee et al., 2010; Yi et al., 2014; Aum et al., 2019), the geochronological results show that spatial variation in intrusion age of ~5 Myr between the Ssangyong (94.5±0.2 Ma) and Yongchu Valleys (89.7±0.4 Ma) is apparent. The geochemical compositions of major and trace elements in the samples showed an affinity of typical post-orogenic granite, indicating their petrogenesis during the late stage of Early Cretaceous magmatic activity possibly in association with subduction events of the Izanagi Plate.