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Observation of amyloid precursor protein cleavage and A beta generation in living cells by using multiphoton laser scanning microscopy / 神经科学通报·英文版
Neuroscience Bulletin ; (6): 256-262, 2007.
Article in English | WPRIM | ID: wpr-264716
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the proteolytic mechanism of amyloid precursor protein (APP) and to explore amyloid-beta (A beta) generation in living neurons.</p><p><b>METHODS</b>DNA fragments were amplified by PCR or synthesized. The four fragments, CFP, 54bp, YFP and C99 were ligated into pcDNA3.0 vector to construct the recombinant plasmids pcDNA3.0-CFP-54bp-YFP and pcDNA3.0-CFP-54bp-YFP-C99. The SH-SY5Y cells were transiently transfected with pcDNA3.0-CFP-54bp-YFP or pcDNA3.0-CFP-54bp-YFP-C99. The expression of fusion gene was examined under a multiphoton laser scanning microscope. Fluorescence resonance energy transfer (FRET) was used to measure the beta cleavage and gamma cleavage of APP. A beta generation was confirmed by immunocytochemistry and multiphoton laser scanning microscopy. Cell viability was tested by MTT assay at different time points.</p><p><b>RESULTS</b>(1) The double restriction endonuclease digestion and sequencing analysis confirmed the authenticity of the recombinant plasmids pcDNA3.0-CFP-54bp-YFP and pcDNA3.0-CFP-54bp-YFP-C99. (2) Blue and yellow fluorescences were detected in the transfected cells. (3) FRET occurred in pcDNA3.0-CFP-54bp-YFP-transfected cells but not in pcDNA3.0-CFP-54bp-YFP-C99-transfected cells. (4) A beta was produced in the pcDNA3.0-CFP-54bp-YFP-C99 transfected cells. (5) A beta-deposition was widespread in the cell. (6) Cell viability decreased along with the intracellular A beta deposition.</p><p><b>CONCLUSION</b>C99 is important for the APP beta cleavage. A beta may be generated and deposited in cells at the early stage of Alzheimeros disease. Intracellular A beta accumulation brings deleterious effects on cells.</p>
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Full text: Available Index: WPRIM (Western Pacific) Main subject: Peptide Fragments / Plasmids / Immunohistochemistry / Transfection / Polymerase Chain Reaction / Amyloid beta-Peptides / Amyloid beta-Protein Precursor / Microscopy, Confocal / Fluorescence Resonance Energy Transfer / Cell Line, Tumor Limits: Humans Language: English Journal: Neuroscience Bulletin Year: 2007 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Peptide Fragments / Plasmids / Immunohistochemistry / Transfection / Polymerase Chain Reaction / Amyloid beta-Peptides / Amyloid beta-Protein Precursor / Microscopy, Confocal / Fluorescence Resonance Energy Transfer / Cell Line, Tumor Limits: Humans Language: English Journal: Neuroscience Bulletin Year: 2007 Type: Article