| Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring. | |
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MedLine Citation:
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PMID: 19730443 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. A novel method for monitoring caspase activity in living Xenopus oocytes and early embryos is described here. The approach, using microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we show that brief caspase activity (10 min) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing the inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that the eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggest that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators. |
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Authors:
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C E Johnson; C D Freel; S Kornbluth |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Cell death and differentiation Volume: 17 ISSN: 1476-5403 ISO Abbreviation: Cell Death Differ. Publication Date: 2010 Jan |
Date Detail:
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Created Date: 2009-12-16 Completed Date: 2010-02-24 Revised Date: 2011-09-26 |
Medline Journal Info:
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Nlm Unique ID: 9437445 Medline TA: Cell Death Differ Country: England |
Other Details:
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Languages: eng Pagination: 170-9 Citation Subset: IM |
Affiliation:
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Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Animals Apoptosis* Caspases, Effector / metabolism Cytochromes c / metabolism Embryo, Nonmammalian / cytology, enzymology* Fluorescence Resonance Energy Transfer* Fluorescent Dyes / metabolism Indoles / administration & dosage Inhibitor of Apoptosis Proteins / metabolism Microinjections Mitochondrial Proteins / metabolism Oocytes / cytology, enzymology*, metabolism Spectroscopy, Near-Infrared Xenopus Proteins / metabolism Xenopus laevis |
| Grant Support | |
ID/Acronym/Agency:
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CA102707/CA/NCI NIH HHS; R01 GM08033/GM/NIGMS NIH HHS; R01 GM080333-01A1/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Fluorescent Dyes; 0/IRDye800; 0/Indoles; 0/Inhibitor of Apoptosis Proteins; 0/Mitochondrial Proteins; 0/Smac protein, Xenopus; 0/Xenopus Proteins; 9007-43-6/Cytochromes c; EC 3.4.22.-/Caspases, Effector |
| Comments/Corrections | |
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