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排序方式: 共有158条查询结果,搜索用时 15 毫秒
1.
Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells 总被引:19,自引:0,他引:19
The firefly enzyme luciferase catalyzes the luminescent reaction of luciferin with ATP and oxygen. The luciferase gene has recently been cloned and proposed as a reporter gene in procaryotic and eucaryotic cells. We present here a luciferase activity assay which relies on luminescence detection using a standard scintillation counter. This technique is simple, fast, inexpensive, and still very sensitive: as little as 0.02 pg (250,000 molecules) of enzyme is readily detected. The technique is optimized for the luciferase assay in mammalian cell lysates. Thus, the luciferase gene may become a very useful tool for gene regulation studies. 相似文献
2.
In an attempt to question the toxic effect of heat shock and related stress, we have studied the activity of reporter enzymes during stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase were synthesized in mouse and Drosophila cells after transfection of the corresponding genes. Both enzymes are rapidly inactivated during hyperthermia. The corresponding polypeptides are not degraded but become insoluble even in the presence of non-ionic detergents. The heat inactivation is more dramatic in vivo within the living cell than in vitro, in a detergent-free crude cell lysate. The extent of enzyme inactivation at a given temperature depends on the cell type in which the enzyme is expressed. Luciferase is inactivated at lower temperatures within Drosophila cells than within mouse cells, whereas beta-galactosidase is inactivated at higher temperatures in E. coli than in mouse cells. A "priming" heat shock confers a transient increased resistance (thermotolerance) of cells against a second "challenging" heat shock. Enzyme inactivation during heat shock or exposure of the cells to ethanol is attenuated in heat shock-primed cells. A comparable thermoprotection is raised by a priming heat shock for both luciferase activity and protein synthesis. Thus, the study of reporter enzyme inactivation is a promising tool for understanding the molecular basis of the toxicity of heat shock and related stress as well as the mechanisms leading to thermotolerance. 相似文献
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Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells. 总被引:22,自引:9,他引:13
In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells. 相似文献
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Paslaru L Davidson S Popescu I Morange M 《Journal of cellular and molecular medicine》2007,11(2):369-371
Cyclosporine A (CsA) is a powerful immunosuppressive drug which significantly improved the success of organ transplantation; however, the major limiting factors for the drug's clinical use are its long and short term adverse effects. The present study was conducted to examine, in a dose-dependent manner, in a model of cardiogenesis, the effect of CsA on cardiomyocytes differentiation. 相似文献
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Hsp and chaperone distribution during endochondral bone development in mouse embryo 总被引:1,自引:0,他引:1
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The process of endochondral bone formation was examined with regard to expression of seven heat shock proteins (Hsps): two small Hsps, the constitutive and the inducible forms of the 70 and the 90 Hsp families, the collagen chaperone Hsp47-and a cytosolic chaperone, TCP-1α, using immunohistochemistry. Around day 15.5 of embryo-genesis the calcification of the long endochondral bones occurs through progressive replacement of the cartilaginous scaffold (rich in type II collagen) with an ossified matrix (rich in type I collagen), and thus a longitudinal section of limb bone recapitulates all the steps of chondrogenesis and the early steps of osteogenesis. We observed that all these Hsps and chaperones are differentially expressed during bone development in a stage-specific pattern reaching very high levels at some specific stages. The involvement of chaperones during these important differentiation steps will be discussed. 相似文献
9.
Hugues Aschard Bjarni?J. Vilhjálmsson Nicolas Greliche Pierre-Emmanuel Morange David-Alexandre Trégou?t Peter Kraft 《American journal of human genetics》2014,94(5):662-676
Many human traits are highly correlated. This correlation can be leveraged to improve the power of genetic association tests to identify markers associated with one or more of the traits. Principal component analysis (PCA) is a useful tool that has been widely used for the multivariate analysis of correlated variables. PCA is usually applied as a dimension reduction method: the few top principal components (PCs) explaining most of total trait variance are tested for association with a predictor of interest, and the remaining components are not analyzed. In this study we review the theoretical basis of PCA and describe the behavior of PCA when testing for association between a SNP and correlated traits. We then use simulation to compare the power of various PCA-based strategies when analyzing up to 100 correlated traits. We show that contrary to widespread practice, testing only the top PCs often has low power, whereas combining signal across all PCs can have greater power. This power gain is primarily due to increased power to detect genetic variants with opposite effects on positively correlated traits and variants that are exclusively associated with a single trait. Relative to other methods, the combined-PC approach has close to optimal power in all scenarios considered while offering more flexibility and more robustness to potential confounders. Finally, we apply the proposed PCA strategy to the genome-wide association study of five correlated coagulation traits where we identify two candidate SNPs that were not found by the standard approach. 相似文献
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