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Volumn 279, Issue 5347, 1998, Pages 102-105

Ultraviolet-induced cell death blocked by a selenoprotein from a human dermatotropic poxvirus

Author keywords

[No Author keywords available]

Indexed keywords

ANTIOXIDANT; HYDROGEN PEROXIDE; MESSENGER RNA; SELENIUM; SELENOPROTEIN;

EID: 0032472279     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5347.102     Document Type: Article
Times cited : (136)

References (32)
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    • The BS-C-1 cells were infected with vTF7-3 and transfected as described [T. R. Fuerst, E. G. Niles, F. W. Studier, B. Moss, Proc. Natl. Acad. Sci. U.S.A. 83, 8122 (1986)] except for the use of lipofectin (Life Technologies). After 16 hours, the cells were lysed and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to a polyvinylidene difluoride membrane (Millipore) and incubated with mAb 12CA5 (BABCO, Richmond, CA) followed by an alkaline phosphatase-conjugated antibody to murine immunoglobulin G (Promega), Membranes were developed using a stabilized substrate for alkaline phosphatase (Promega).
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    • Fuerst, T.R.1    Niles, E.G.2    Studier, F.W.3    Moss, B.4
  • 16
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    • The DNA segment corresponding to nucleotides 661 to 932 or 661 to 797 (Fig. 1) as subcloned downstream of the rat type 1 deiodinase coding region in the mammalian expression vector pUHD10 [M. Gossen and H. Bujard, Proc. Natl. Acad. Sci. U.S.A. 89, 5547 (1992)]. Plasmids containing the wild-type deiodinase SECIS element or a nonfunctional mutant of this element downstream of the deiodinase coding region served as positive and negative controls, respectively. The plasmids with deiodinase sequences were transfected, together with a control plasmid that expresses human growth hormone, into human embryonic kidney 293 cells. Deiodinase activity, in counts per minute of iodine released per microliter of transfected cell sonicate, was normalized to units of human growth hormone in 100 μl of medium, as described [G. W. Martin III, J. W. Harney, M. J. Berry, RNA 2, 171 (1996)].
    • (1992) Proc. Natl. Acad. Sci. U.S.A. , vol.89 , pp. 5547
    • Gossen, M.1    Bujard, H.2
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    • 0029832601 scopus 로고    scopus 로고
    • The DNA segment corresponding to nucleotides 661 to 932 or 661 to 797 (Fig. 1) as subcloned downstream of the rat type 1 deiodinase coding region in the mammalian expression vector pUHD10 [M. Gossen and H. Bujard, Proc. Natl. Acad. Sci. U.S.A. 89, 5547 (1992)]. Plasmids containing the wild-type deiodinase SECIS element or a nonfunctional mutant of this element downstream of the deiodinase coding region served as positive and negative controls, respectively. The plasmids with deiodinase sequences were transfected, together with a control plasmid that expresses human growth hormone, into human embryonic kidney 293 cells. Deiodinase activity, in counts per minute of iodine released per microliter of transfected cell sonicate, was normalized to units of human growth hormone in 100 μl of medium, as described [G. W. Martin III, J. W. Harney, M. J. Berry, RNA 2, 171 (1996)].
    • (1996) RNA , vol.2 , pp. 171
    • Martin III, G.W.1    Harney, J.W.2    Berry, M.J.3
  • 18
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    • note
    • Transfection procedures: HeLa cells were co-transfected with 1 μg of cytomegalovirus (CMV)-β-Gal (Stratagene) and 5 μg of pCl vector (Promega), pCl-P35 (18), or pCl-MC066L-A mixed with lipofectin. Thirty hours after transfection, the cells were treated with UV (312 nm) light for 15 s at room temperature or continuously with 100 μM hydrogen peroxide or cycloheximide (1 μg/ml; Sigma) and TNF-α (10 ng/ml; Boehringer Mannheim) or cycloheximide and anti-Fas (1.25 μg/ml; CH-11 antibody. Kamiya Biomedical, Seattle, WA). Twelve hours later, cells were fixed, stained with X-Gal (Promega), and examined microscopically. The control wells contained 100 to 200 flat, blue cells per microscopic field. The number of flat, blue cells in six fields of the untreated well of each transfectant was compared to the number of flat, blue cells in six fields of the treated well, and the percent viability was determined. The HaCaT cells were co-transfected with 0.33 μg of CMV-β-Gal and 1.67 μg of pCl, pCl-P35, or pCl-MC066L-A mixed with lipofectamine reagent (Life Technologies). Thirty hours after transfection, the cells were treated for 30 s with UV (312 nm) light or 10 μM hydrogen peroxide. Cells were fixed, stained, and scored for viability as described for HeLa cells.
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  • 27
    • 0030970013 scopus 로고    scopus 로고
    • M. Thome et al., Nature 386, 517 (1997); S. Hu, C. Vincenz, M. Buller, V. M. Dixit, J. Biol. Chem. 272, 9621 (1997).
    • (1997) Nature , vol.386 , pp. 517
    • Thome, M.1
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    • R. M. L. Buller, J. B. W. Chen, J. Kreider, Virology 213, 655 (1995); K. H. Fife et al., ibid. 226, 95 (1996).
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    • Fife, K.H.1
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    • note
    • We thank D. Hatfield for suggestions regarding selenium labeling of proteins and J. Bertin and J. Cohen for critically reading the manuscript, providing pCl-P35, and offering advice regarding transfections and viability assays. Supported in part by NIH grant DK47320 (M.J.B.).


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