Functional and structural analysis of the mouse ATPase II gene.
Item
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Title
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Functional and structural analysis of the mouse ATPase II gene.
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Identifier
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AAI3213256
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identifier
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3213256
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Creator
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Jayman, Farah A.
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Contributor
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Adviser: Probal Banerjee
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Date
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2006
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Biology, Molecular
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Abstract
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Normally, PS is located in the inner leaflet of the plasma membrane, however during apoptosis externalization of PS to the outer leaflet of apoptotic cells induces macrophages containing surface PS receptors to engulf these cells. In healthy cells a bifunctional enzyme, which is a Mg2+-ATPase, as well as an aminophospholipid translocase (APTL), is responsible for the translocation of PS from the outer leaflet of the plasma membrane to the inner. Phospholipid asymmetry is relatively stable and is believed to be maintained by the enzymes APTL, Scramblase (which bidirectionally translocates all phospholipids) and floppase (which slowly translocates phospholipids from the inner leaflet to the outer). ATPase II is a Mg2+-ATPase II that is also a good candidate for the enzyme aminophospholipid translocase (APTL). The main objective of this project is to provide evidence for the hypothesis that ATPase II is indeed an APTL. In doing so, we have shown that the overexpression of the mouse ATPase II cDNA in mouse neuroblastoma cells causes increased PS translocation, inhibition of caspase-3, and also activation of Erk 1/2, which regulates gene expression and apoptosis. We also expressed antisense, full-length ATPase II cDNA, which showed externalization of PS and the inhibition of its APTL activity in healthy cells. This observation shows us that ATPase II could indeed have an APTL like activity or could be involved in regulating the APTL activity. Additionally, our results indicate that ATPase II could also have signaling activity. Previous studies have shown that the expression of ATPase II mRNA is tissue-specific, with the highest level of expression seen in brain and skeletal muscle. In order to study the mechanism of cell type-specific expression of ATPase II, the 5' upstream sequence of the mouse ATPase II gene has been isolated. A 1.5-kb 5' flanking sequence of this gene bears considerable homology to and some significant differences from the human ATPase II promoter that has been isolated and analyzed. The transcription start site and regulatory elements have been analyzed using 5'-RACE and deletion analysis of the putative mouse ATPase II promoter. Results from RACE and promoter activity studies show that there is some cell type specific activity of this ATPase promoter. It has been shown by many groups including ours that macrophages (microglia in the brain) recognize phosphatidylserine (PS) when it is exposed on the cell surface. Therefore, a strategy of down regulating ATPase II could be used to trigger the engulfment of unwanted cells via recognition of externalized PS by the PS receptors present on scavenger cells.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
