• Title/Summary/Keyword: X-gal

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Polymerization of p-Chlorophenyl Propargyl Ether by Molybdenum- and Tungsten- Based Catalysts

  • Lee, Won-Chul;Seo, Jang-Hyuk;Gal, Yeong-Soon;Jin, Sung-Ho;Choi, Sam-Kwon
    • Bulletin of the Korean Chemical Society
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    • v.14 no.6
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    • pp.708-712
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    • 1993
  • The Polymerization of p-chlorophenyl propargyl ether (CPE) was carried out using various transition metal catalysts. The catalytic activity of $MoCl_{5}$-based catalysts was greater than that of $WCl_6$-based catalysts. $MoCl_5$ alone and $MoCl_{5}$-cocatalyst systems polymerized CPE very effectively to give a high yield of poly(CPE). In most cases, the polymer yield was quantitative and the average molecular weight $({\bar{M}}n)$ was in the range of 9,000 and 17,000. The NMR, IR, UV-visible spectra indicated that the present poly(CPE) has a linear conjugated polyene structure having p-chlorophenyl oxymethylene substituent. The poly(CPE) was mostly dark-brown colored powder and was completely soluble in various organic solvents such as chloroform, methylene chloride, THF, chlorobenzene, etc. The X-ray diffraction analysis indicated that the present poly(CPE) is amorphous.

Inhibitory Mechanism on NF-${\kappa}B$ Transactivation by Dexamethasone in Pulmonary Epithelial Cells (폐상피세포에서 Dexamethasone에 의한 NF-${\kappa}B$ Transactivation 억제기전에 관한 연구)

  • Lee, Kye-Young;Kim, Yoon-Seop;Ko, Mi-Hye;Park, Jae-Seok;Jee, Young-Koo;Kim, Keun-Youl;Kwak, Sahng-June
    • Tuberculosis and Respiratory Diseases
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    • v.48 no.5
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    • pp.682-698
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    • 2000
  • Glucocorticoid receptor (GR) functions as a suppressor of inflammation by inhibiting the expression of many cytokine genes activated by NF-${\kappa}B$. The goal of this study is to investigate the mechanism by which GR repress NF-${\kappa}B$ activation in lung epithelial cells. We used A549 and BEAS-2B lung epithelia! cell lines. Using Ig$G{\kappa}$-NF-${\kappa}B$ luciferase reporter gene construct, we found that dexamethasone significantly suppressed TNF-$\alpha$-induced NF-${\kappa}B$ activation and the overexpression of GR showed dose-dependent reduction of TNF-$\alpha$-induced NF-${\kappa}B$ activity in both cell lines. However, DNA binding of NF-${\kappa}B$ induced by TNF-$\alpha$ in electromobility shift assay was not inhibited by dexamethasone. Super shift assay with anti-p65 antibody demonstrated the existence of p65 in NF-${\kappa}B$ complex induced by $\alpha$ Western blot showed that $I{\kappa}B{\alpha}$ degradation induced by TNF-$\alpha$ was not affected by dexamethasone and $I{\kappa}B{\kappa}$ was not induced by dexamethasone, neither. To evaluate p65 specific transactivation, we adopted co-transfection study of Gal4-p65TA1 or TA2 fusion protein expression system together with 5xGal4-luciferase vector. Co-transfection of GR with Gal4-p65TA1 or TA2 repressed luciferase activity profoundly to the level of 10-20% of p65TA1- or TA2-induced transcriptional activity. And this transrepressional effect was abolished by co-transfection of CBP of SRC-1 expression vectors. These results suggest that GR-mediated transrepression of NF-${\kappa}B$ in lung epithelial cells is through competing for binding to limiting amounts of transcriptional coactivators, CBP or SRC-1.

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Study on the Relations to Estimate Instrumental Seismic Intensities for the Moderate Earthquakes in South Korea (국내 중규모 지진에 대한 계측진도 추정식 연구)

  • Yun, Kwan-Hee;Lee, Kang-Ryel
    • Journal of the Earthquake Engineering Society of Korea
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    • v.22 no.6
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    • pp.323-332
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    • 2018
  • Recent two moderate earthquakes (2016 $M_w=5.4$ Gyeongju and 2017 $M_w=5.5$ Pohang) in Korea provided the unique chance of developing a set of relations to estimate instrumental seismic intensity in Korea by augmenting the time-history data from MMI seismic intensity regions above V to the insufficient data previously accumulated from the MMI regions limited up to IV. The MMI intensity regions of V and VI was identified by delineating the epicentral distance from the reference intensity statistics in distance derived by using the integrated MMI data obtained by combining the intensity survey results of KMA (Korea Meteorological Administration) and 'DYFI (Did You Feel It)' MMIs of USGS. The time-histories of the seismic stations from the MMI intensity regions above V were then preprocessed by applying the previously developed site-correction filters to be converted to a site-equivalent condition in a manner consistent with the previous study. The average values of the ground-motion parameters for the three ground motion parameters of PGA, PGV and BSPGA (Bracketed Summation of PGA per second for 30 seconds) were calculated for the MMI=V and VI and used to generate the dataset of the average values of the ground-motion parameters for the individual MMIs from I to VI. Based on this dataset, the linear regression analysis resulted in the following relations with proposed valid ranges of MMI. $MMI=2.36{\times}log_{10}(PGA(gal))+1.44$ ($I{\leq}MMI$$MMI=2.44{\times}log_{10}(PGV(kine))+4.86$ ($I{\leq}MMI$$MMI=2.59{\times}log_{10}(BSPGA(gal{\cdot}sec))-1.02$ ($I{\leq}MMI$

Construction of a Lactococcal Shuttle/Expression Vector Containing a $\beta$-Galactosidase Gene as a Screening Marker (선별마커로써 $\beta$-Galactosidase 유전자를 포함한 Lactococcus용 셔틀/발현 벡터 제조)

  • Han Tae Un;Jeong Do-Won;Cho San Ho;Lee Jong-Hoon;Chung Dae Kyun;Lee Hyong Joo
    • Microbiology and Biotechnology Letters
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    • v.33 no.4
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    • pp.241-247
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    • 2005
  • A new lactococcal shuttle/expression vector for lactococci, pWgal13T, was constructed using a $\beta$-galactosi-dase gene (lacZ) from Lacfococcus lactis ssp. lactis ATCC 7962 as a screening marker. The pWgal 13T was introduced into Escherichia coli DH5a and L. lactis MG1363, and was easily detected by the formation of blue colonies on a medium containing X-gal without any false transformants. Also, the quantitatively lacZ activity of pWgal13T was measured in L. lactis ssp. cremoris MG1363, and was found to be four times higher than that of L. lactis ssp. lactis ATCC7962 grown on a medium containing glucose, which shows that the lacZ gene of pWgal13T can be used for the efficient screening of L. lactis on general media. The pWgal13T was equipped with a lactococcal replicon of pWV01 from L. lactis Wg2, the new promoter P13C from L. lactis ssp. cremoris LM0230, multiple cloning sites, and a terminator for the expression of a relevant gene. The vee-tor pWgal13T was used for the expression of the EGFP gene in E. coli and L. lactis. These results show that the lactococcal expression/shuttle vector constructed in the present study can be used for the production of foreign proteins in E. coli and L. lactis.

Characterization and Selection of Lactic Acid Bacteria Producing ${\beta}-Galactosidase$ (${\beta}-Galactosidase$ 생산 유산균 선별 및 특성 조사)

  • Lee, Young-Ki;Choi, Susanna;Park, Young-Il;Park, Chan-Sun;Yoon, Byung-Dae;Hwang, Yun-Sik;Kim, Hee-Sik
    • Korean Journal of Microbiology
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    • v.42 no.3
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    • pp.216-222
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    • 2006
  • This study was carried out to select the lactic acid bacteria producing ${\beta}-galactosidase$ (lactase) and investigate the properties of the ${\beta}-galactosidase$. About 100 strains of lactic acid bacteria showing blue colony on the MRS agar medium containing X-gal were isolated from several kinds of Kimchi. Among them, 2 strains were selected as potential ${\beta}-galactosidase$ producers. The selected strains, ET-1 and LA-12, were identified as Lactobacillus fermentum and L. acidophilus, respectively by the analysis of 16S rDNA sequences. They showed relatively high ${\beta}-galactosidase$ activity and cellular viability. Their ${\beta}-galactosidase$ showed the highest activity at $55^{\circ}C$. And the optimum pHs of the enzymes produced by ET-1 and LA-12 were pH 5.5 and pH 7.0, respectively. They were also highly resistant to artificial gastric juice and bile. Two selected strains showed little change of viable cell number for 3 hr incubation in artificial gastric juice, and maintained the viable cell number at $10^8CFU/ml$ for 24 hr in 0.3% oxgall after incubation for 2 hours in artificial gastric juice. Based on these results, ET-1 and LA-12 are expected to be applied in dairy industry.

Neuroanatomical studies on the mechanism of scalp acupuncture therapy using the pseudorabies virus (Pseudorabies virus(PRV)를 이용한 두침(頭針) 치료(治療) 기전(機轉)에 대한 신경해부학(神經解剖學的) 연구(硏究))

  • Lee, Tae-Young;Lee, Chang-Hyun;Lee, Sang-Ryoung;Yuk, Sang-Won;Lee, Kwang-Gyu;Yuk, Tae-Han
    • Journal of Acupuncture Research
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    • v.17 no.2
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    • pp.261-276
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    • 2000
  • 본 실험은 pseudorabies 바이러스 (PRV) 의 Bartha strain 을 안면신경의 측두지, 하지를 지배하는 신경 (좌골신경) 및 상지를 지배하는 신경 (요골, 척골, 정중신경) 에 주입한 후 4 일간의 생존시간이 경과한 후 척수와 뇌를 적출하여 동결절편을 제작한 후 면역조직화학적 염색기법과 X-gal 조직화학 염색법을 시행하여 염색된 신경세포체를 척수와 뇌에 투사된 공통영역을 관찰하고 두침의 영역중 하나인 운동구와 사지와의 관계에 대한 실험적 증거를 제시하고자 시행하였다. 위의 실험에서 얻어진 결과는 아래와 같다. 1. 안면신경의 측두지, 하지를 지배하는 신경 (좌골신경) 및 상지를 지배하는 신경 (요골, 측골, 정중신경) 에서 투사된 공통된 영역은 척수에서 경수의 층판 1-IV, 흉수의 intermediolateral nucleus(IML), dorsal nucleus(D) 및 층판 X, 요수의 층판 IV, V, 천수의 층판 IV, V, IX, X 등의 영역에서 관찰되었고, 뇌줄기에서는 caudoventrolateral reticular nucleus(CVL), nucleus solitary tract(Sol), rostroventrolateral nucleus(RVL), area postrema(AP), raphe nuclei(raphe pallidus, raphe obscurus, raphe magnus), inferior olivary nucleus 의 등쪽부분 (gigantocellular reticular nucleus, Gi), Kolliker-Fuse nucleus(KF), central gray(CG), dorsal raphe nucleus (DR) and A5 영역에 표지된다. 또한 paraventricular hypothalamic nucleus(PRV) 와 lateral hypothalamic reticular nucleus(LH)에서도 관찰되고 locus coeruleus(LC) 와 subcoeruleus nuc!eus(SubCA) 에서도 관찰된다.

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Determination of plasma C16-C24 globotriaosylceramide (Gb3) isoforms by tandem mass spectrometry for diagnosis of Fabry disease (패브리병(Fabry) 진단을 위한 혈장 중 Globotriaosylceramide (Gb3)의 탠덤매스 분석법 개발과 임상 응용)

  • Yoon, Hye-Ran;Cho, Kyung-Hee;Yoo, Han-Wook;Choi, Jin-Ho;Lee, Dong-Hwan;Zhang, Kate;Keutzer, Joan
    • Journal of Genetic Medicine
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    • v.4 no.1
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    • pp.45-52
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    • 2007
  • Purpose : A simple, rapid, and highly sensitive analytical method for Gb3 in plasma was developed without labor-ex tensive pre-treatment by electrospray ionization MS/ MS (ESI-MS/MS). Measurement of globotriaosy lceramide (Gb3, ceramide trihex oside) in plasma has clinical importance for monitoring after enzyme replacement therapy in Fabry disease patients. The disease is an X-linked lipid storage disorder that results from a deficiency of the enzyme ${\alpha}$-galactosidase A (${\alpha}$-Gal A). The lack of ${\alpha}$-Gal A causes an intracellular accumulation of glycosphingolipids, mainly Gb3. Methods : Only simple 50-fold dilution of plasma is necessary for the extraction and isolation of Gb3 in plasma. Gb3 in diluted plasma was dissolved in dioxane containing C17:0 Gb3 as an internal standard. After centrifugation it was directly injected and analyzed through guard column by in combination with multiple reaction monitoring mode of ESI-MS/MS. Results : Eight isoforms of Gb3 were completely resolved from plasma matrix. C16:0 Gb3 occupied 50% of total Gb3 as a major component in plasma. Linear relationship for Gb3 isoforms w as found in the range of 0.001-1.0 ${\mu}g$/mL. The limit of detection (S/N=3) was 0.001 ${\mu}g$/mL and limit of quantification was 0.01 ${\mu}g$/mL for C16:0 Gb3 with acceptable precision and accuracy. Correlation coefficient of calibration curves for 8 Gb3 isoforms ranged from 0.9678 to 0.9982. Conclusion : This quantitative method developed could be useful for rapid and sensitive 1st line Fabry disease screening, monitoring and/or diagnostic tool for Fabry disease.

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Engineering lacZ Reporter Gene into an ephA8 Bacterial Artificial Chromosome Using a Highly Efficient Bacterial Recombination System

  • Kim, Yu-Jin;Song, Eun-Sook;Choi, Soon-Young;Park, Soo-Chul
    • BMB Reports
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    • v.40 no.5
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    • pp.656-661
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    • 2007
  • In this report, we describe an optimized method for generation of ephA8 BAC transgenic mice expressing the lacZ reporter gene under ephA8 regulatory sequences. First, we constructed a targeting vector that carries a 1.2 kb ephA8 DNA upstream of its first exon, a lacZ expression cassette, a kanamycin cassette, and a 0.7 kb ephA8 DNA downstream of its first exon. Second, the targeting vector was electroporated into cells containing the ephA8 BAC and pKOBEGA, in which recombinases induce a homologous recombination between the ephA8 BAC DNA and the targeting vector. Third, the FLP plasmid expressing the Flipase was electroporated into these bacteria to eliminate a kanamycin cassette from the recombinant BAC DNA. The appropriate structures of the modified ephA8 BAC DNA were confirmed by Southern analysis. Finally, BAC transgenic mouse embryos were generated by pronuclear injection of the recombinant BAC DNA. Whole mount X-gal staining revealed that the lacZ reporter expression is restricted to the anterior region of the developing midbrain in each transgenic embryo. These results indicate that the ephA8 BAC DNA contains most, if not all, regulatory sequences to direct temporal and spatial expression of the lacZ gene in vivo.

Analysis of Two Promoters that Control the Expression of the GTP cyclohydrolase I Gene in Drosophila melanogaster

  • Byun, Jaegoo;Yoon, Jaeseung;Baek, Kwanghee
    • Molecules and Cells
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    • v.27 no.5
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    • pp.583-589
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    • 2009
  • GTP cyclohydrolase I (GTPCH) is a key enzyme in the de novo synthesis of tetrahydrobiopterin. Previously, the Drosophila melanogaster GTPCH gene has been shown to be expressed from two different promoters (P1 and P2). In our study, the 5'-flanking DNA regions required for P1 and P2 promoter activities were characterized using transient expression assay. The DNA regions between -98 and +31, and between -73 and +35 are required for efficient P1 and P2 promoter activities, respectively. The regions between -98 and -56 and between -73 and -41 may contain critical elements required for the expression of GTPCH in Drosophila. By aligning the nucleotide sequences in the P1 and P2 promoter regions of the Drosophila melanogaster and Drosophila virilrs GTPCH genes, several conserved elements including palindromic sequences in the regions critical for P1 and P2 promoter activities were identified. Western blot analysis of transgenic flies transformed using P1 or P2 promoter-lacZ fusion plasmids further revealed that P1 promoter expression is restricted to the late pupae and adult developmental stages but that the P2 promoter driven expression of GTPCH is constitutive throughout fly development. In addition, X-gal staining of the embryos and imaginal discs of transgenic flies suggests that the P2 promoter is active from stage 13 of embryo and is generally active in most regions of the imaginal discs at the larval stages.

Thermostable ${\beta}$-Glycosidase-CBD Fusion Protein for Biochemical Analysis of Cotton Scouring Efficiency

  • Ha, Jae-Seok;Lee, Young-Mi;Choi, Su-Lim;Song, Jae-Jun;Shin, Chul-Soo;Kim, Ju-Hea;Lee, Seung-Goo
    • Journal of Microbiology and Biotechnology
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    • v.18 no.3
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    • pp.443-448
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    • 2008
  • Multidomain proteins for the biochemical analysis of the scouring efficiency of cotton fabrics were constructed by the fusion of a reporter moiety in the N-terminal and the cellulose binding domain (CBD) in the C-terminal. Based on the specific binding of the CBD of Cellulomonas fimi exoglucanase (Cex) to crystalline cellulose (Avicel), the reporter protein is guided to the cellulose fibers that are increasingly exposed as the scouring process proceeds. Among the tested reporter proteins, a thermostable ${\beta}$-glycosidase (BglA) from Thermus caldophilus was found to be most appropriate, showing a higher applicability and stability than GFP, DsRed2, or a tetrameric ${\beta}$-glycosidase (GUS) from Escherichia coli, which were precipitated more seriously during the expression and purification steps. When cotton fabrics with different scouring levels were treated with the BglA-CBD and incubated with X-Gal as the chromogenic substrate, an indigo color became visible within 2 h, and the color depth changed according to the conditions and extent of the scouring.