• Title/Summary/Keyword: P-gal

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Protective effect of euonymus alatus extract on experimental liver injury in mice (Euonymus alatus 추출물의 실험적 간 손상 억제)

  • Shin, Sook-Jeong;Lee, Byung-Yong;Shin, Dong-Keun;Lee, Jeong-Ho
    • IMMUNE NETWORK
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    • v.1 no.3
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    • pp.213-220
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    • 2001
  • Background: A previous study has shown that Euonymus alatus (EA) has an antidotic activities against inflammation, suggesting possibility that EA can exert this beneficial effects to liver injury by an initial protection against drug-induced hepatocyte demage. The present study was undertaken to evaluate the protective effect of EA-extract on experimentally induced hepatitis in ICR mice and to investigate some mechanisms responsible for its action. Methods: Water EA extract was used in this experiments. The mice received i.p. a dose of 700 mg/kg galactosamine (GalN) together with $5{\mu}g/kg$ of endotoxin (LPS), or received i.v. 12 mg/kg of concanavalin A (Con A). EA (4 mg/mouse) was administrated on day -2, -1 and 0 before induction of liver injury. Liver injury was assessed by measurement of serum alanin amino-transferase (SGPT) levels on 9 hr after GaIN.LPS, or 8 hr after con A administration. Results: Treatment with either GaIN or LPS alone did not cause hepatitis. However, simultaneous administration of GalN and LPS to mice resulted in LPS-dose dependent fulminant hepatitis. GaLN/LPS-induced liver injury was reduced when mice were given EA for 3 days before induction. This preventive effect of Ea was more prominent when EA was given by intraperitoneal route rather then by oral route. Pretreatment of EA or dexamethasone inhibited significantly $TNF{\alpha}$ production in GalL/LPS-injured mice. However, EA-treatment did not influence $TNF{\alpha}$-induced hepatitis in GalN-sensitized mice, suggesting that $TNF{\alpha}$ is likely to act as one of final mediators of endotoxin action and the protective effect of EA might be manifested chiefly by inhibition of endotoxin-induced $TNF{\alpha}$ production, not by blocking the $TNF{\alpha}$-action. Injection of Con A into mice evoked remarkable liver injury in a dose dependent fashion. This liver damage was reduced by EA-pretreatment. Dexamethasone significantly reduced both GalL/LPS-induced and Con A-induced liver damages, showing synergism with EA. However, indomethacin reduced only GalN/ LPS-induced hepatitis, not for Con A-induced hepatitis. Conclusion: These results led to the conclusion that EA may be able to contribute at least in part to prevent the drug-induced hepatotoxicity, and that its anti-hepatitis effects might be manifested directly by modulation of endogenous mediators, such as leukotriese D4, $TNF{\alpha}$ and free radical, and indirectly by regulation of immune mediated responses. Also these results suggested that EA could be developed as a potential antidotic agent.

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Screening and Characterization of Drosophila Development Mutants Using Single P[en-lacZ] Element Mutagenesis (Drosophila single P[en-lacZ] element mutagenesis를 이용한 발생 관련 돌연변이체 작성)

  • Ha, Hye-Yeong;Lee, Heui-Jung;Park, Soon-Hee;Yoo, Mi-Ae;Lee, Won-Ho
    • Journal of Life Science
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    • v.7 no.1
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    • pp.49-58
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    • 1997
  • Single P[en-lacZ] element including 5.7 kb of engrailed upstream sequences and the E. coli lacZ fusion gene, localized on 48A in rxyho25 strain was transposed to different sites in the Drosophila genome by the jumpstart technique. From 3315 individual genetic crosses, 113 new insertion lines carrying P[en-lacZ] inserted at different sites were obtained. $\beta$-Galactosidase expression in larval tissues of 113 insertion lines were detected by X-gal staining. & among 113 lines have been indentified to be for recessive lethal mutations. Among 7 lines, the #1119 line being lethal during embryogenesis was examined about the ${\beta}$$-Galactosidase expression, nuclear behavior and cellularization pattern during embryogenesis. The P[en-lacZ] insertion lines obtained in this study could be utilized for studying structure and function of the Drosophila development-related genes.

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Transfer of Foreign Gene into Mud Loach, Misgurnus mizolepis I . Availability of the lacZ as a reporter gene for producing transgenic mud loach (미꾸라지, Misgurnus mizozepis에 외래 유전자 이식 I. lacZ의 reporter 유전자로서의 유용성 검토)

  • KIM Dong Soo;NAM Yoon Kwon
    • Journal of Aquaculture
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    • v.7 no.1
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    • pp.41-54
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    • 1994
  • In order to evaluate the availability of lacZ as a reporter gene for producing transgenic mud loach, foreign DNA, bacterial \beta-galactosidase$ gene (lacZ) was microinjected into mud loach eggs and its insertion and expression were examined. X-gal based histochemical assay, fluorimetric analysis of \beta-galactosidase$ with 4-methylumbelliferyl-$\beta$-D-galactoside (MUG) and molecular biological examination using polymerase chain reaction (PCR), dot blot, southern blot and sequence analysis of PCR products were carried out to analyze both microinjected group and non-injected controls. The results are disccussed in this paper.

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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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Promoter Polymorphisms of ST3GAL4 and ST6GAL1 Genes and Associations with Risk of Premalignant and Malignant Lesions of the Cervix

  • de los Angeles Rivera-Juarez, Maria;Rosas-Murrieta, Nora Hilda;Mendieta-Carmona, Victoriano;Hernandez-Pacheco, Raquel Esneidy;Zamora-Ginez, Irma;Rodea-Avila, Carlos;Apresa-Garcia, Teresa;Garay-Villar, Onix;Aguilar-Lemarroy, Adriana;Jave-Suarez, Luis Felipe;Diaz-Orea, Maria Alicia;Milflores-Flores, Lorena;Reyes-Salinas, Juan Salvador;Ceja-Utrera, Francisco Javier;Vazquez-Zamora, Victor Javier;Vargas-Maldonado, Tomas;Reyes-Carmona, Sandra;Sosa-Jurado, Francisca;Santos-Lopez, Gerardo;Reyes-Leyva, Julio;Vallejo-Ruiz, Veronica
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.3
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    • pp.1181-1186
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    • 2014
  • Sialyltransferase gene expression is altered in several cancers, including examples in the cervix. Transcriptional regulation of the responsible genes depends on different promoters. We aimed to determine the association of single-nucleotide polymorphisms in the B3 promoter of the ST3GAL4 gene and the P1 promoter of the ST6GAL1 gene with cervical premalignant lesions or cervical cancer. A blood sample and/or cervical scrapes were obtained from 104 women with normal cytology, 154 with premalignant lesions and 100 with cervical cancer. We also included 119 blood samples of random donors. The polymorphisms were identified by sequencing from PCR products. For the B3 promoter, a fragment of 506 bp (from nucleotide -408 to +98) was analyzed, and for the P1 promoter a 490 bp (-326 to +164) fragment. The polymorphism analysis showed that at SNP rs10893506, genotypes CC and CT of the ST3GAL4 B3 promoter were associated with the presence of premalignant lesions (OR=2.89; 95%CI 1.72-4.85) and cervical cancer (OR=2.23; 95%CI 1.27-3.91). We detected only one allele of each polymorphism in the ST6GAL1 P1 promoter. We did not detect any genetic variability in the P1 promoter region in our study population. Our results suggest that the rs10893506 polymorphism -22C/T may increase susceptibility to premalignant and malignant lesions of the cervix.

Inulooligosaccharide Production from Inulin by Saccharomyces cerevisiae Strain Displaying Cell-Surface Endoinulinase

  • Kim Hyun-Chul;Kim Hyun-Jin;Choi Woo-Bong;Nam Soo-Wan
    • Journal of Microbiology and Biotechnology
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    • v.16 no.3
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    • pp.360-367
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    • 2006
  • The endoinulinase gene (inu1) from Pseudomonas mucidolens was expressed on the cell surface of Saccharomyces cerevisiae by fusing with Aga2p linked to the membrane anchored protein, Aga1p. The inu1 gene of P. mucidolens was subcloned into the surface display vector, pCTcon (GAL1 promoter). The constructed plasmid, pCTENIU (8.5kb), was then introduced to S. cerevisiae EBY100 cells and the yeast transformants selected on synthetic defined media lacking uracil and inulin-containing media. The inu1 gene under the control of the GAL1 promoter was successfully expressed in the yeast transformants, and the surface display of endoinulinase confirmed by immunofluorescence microscopy, along with its enzymatic ability to form inulooligosaccharides (IOSs) from inulin. The total endoinulinase activity reached about 2.31 units/ml when the yeast transform ants were cultivated on a YPDG medium. To efficiently hydrolyze the inulin, various reaction conditions were examined, including the pH, temperature, and inulin source. The optimized conditions were then determined as follows: pH, 7.0; temperature, $50^{\circ}C$; inulin source, Jerusalem artichoke. Under the optimized condition and 46 units of endoinulinase per g of inulin, IOSs started to be produced after 10 min of enzymatic reaction. The highest yield, 71.2% of IOSs, was achieved after 30 h of reaction without any significant loss of the initial enzyme activity. As a result of the reaction with inulin, IOSs consisting of inulobiose (F2), inulotriose (F3), inulotetraose (F4), and inulopentaose (F5) were produced, and F4 was the major product.

Optimization for Production of Exo-β-1,3-glucanase (Laminarinase) from Aspergillus oryzae in Saccharomyces cerevisiae (Saccharomyces cerevisiae에서 Aspergillus oryzae 유래의 exo-β-1,3-glucanase (laminarinase)의 생산 최적화)

  • Kim, Min-Jung;Nam, Soo-Wan;Tamano, Koichi;Machida, Masayuki;Kim, Sung-Koo;Kim, Yeon-Hee
    • KSBB Journal
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    • v.26 no.5
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    • pp.427-432
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    • 2011
  • In this study, a EXGA gene code for exo-β-1,3-glucanase from Aspergillus oryzae was overexpressed and secretory produced in Saccharomyces cerevisiae. To overexpress the β-1,3-glucanase, pGInu-exgA and pAInu-exgA plasmids having GAL10 and ADH1 promoter, respectively, and exoinulinase signal sequence (Inu s.s) were constructed and introduced in S. cerevisiae SEY2102 and 2805. The recombinant β-1,3-glucanase was successfully expressed and secreted into the medium and the β--1,3-glucanase activity in 2102/pGInu-exgA and 2102/pAInu-exgA strain were 5.01 unit/mL and 4.09 unit/mL, respectively. In the 2805/pGInu-exgA and 2805/pAInu-exgA strain, the β-1,3-glucanase activity showed 3.23 unit/mL and 3.22 unit/mL, respectively. Secretory efficiency in each strain reached 95% to 98%. Subsequently, the recombinant β1,3-glucanase was used for ethanol production. Ethanol productivity in 2102/pAInu-exgA strain was 0.83 g/L when pre-treated Laminaria japonica which has initial reducing sugar of 1.4 g/L was used as substrate. It is assumed that the polysaccharides of Laminaria japonica was effectively saccharified by recombinant β-1,3-glucanase, resulting in increase of ethanol productivity. These results suggested that recombinant β-1,3-glucanase was efficiently overexpressed and secreted in S. cerevisiae SEY2102 as host strain by using ADH1 promoter-Inu s.s system.

Cell Surface Display of Cycloinulooligosaccharide Fructanotransferase Gene in Saccharomyces cerevisiae (Saccharomyces cerevisiae에서 Cycloinulooligosaccharide Fructanotransferase 유전자의 표층 발현)

  • Kim, Hyun-Jin;Lee, Jae-Hyung;Kim, Hyun-Chul;Kim, Yeon-Hee;Kwon, Hyun-Ju;Nam, Soo-Wan
    • Journal of Life Science
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    • v.17 no.2 s.82
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    • pp.241-247
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    • 2007
  • The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus macerans was subcloned into the surface display vector, pCTcon (GAL1 promoter). The constructed plasmid, pCTECFTN (9.0 kb) was introduced to S. cerevisiae EBY100 cell and then east transformants were selected on the synthetic defined medium lacking uracil and on the inulin containing medium. The surface display of CFTase was confirmed by immunofluorescence microscopy and its enzymatic ability to form cycloinulooligosaccharides(cyclofructans, CFs) from inulin. The total activity of the CFTase was reached about 5.52 unit/1 by cultivation of yeast transformant on YPDG medium. The optimized conditions determined were as follows; pH, 8.0; temperature, $50^{\circ}C$ ; substrate concentration, 5%; inulin source, Jerusalem artichoke. By the reaction with inulin, CFs consisting of cycloinulohexaose (CF6), cycloinuloheptaose (CF7), and cycloinulooctaose (CF8) were produced and CF6 was the major product.