Intracellular localization and functional implications of sterol carrier protein-2 (SCP-2) gene products and liver-fatty acid binding protein (L-FABP) in transfected L-cell fibroblasts /
Immunofluorescence double labeling and laser scanning confocal microscopy (LSCM) of transfected L-cells were used to determine sterol carrier protein-x (SCP-x) colocalization with catalase in and outside peroxisomes. The larger SCP-2 gene product, SCP-x, from which SCP-2 arises, when visualized in i...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2002.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=764789901&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Immunofluorescence double labeling and laser scanning confocal microscopy (LSCM) of transfected L-cells were used to determine sterol carrier protein-x (SCP-x) colocalization with catalase in and outside peroxisomes. The larger SCP-2 gene product, SCP-x, from which SCP-2 arises, when visualized in intact cells, suggests a role in cholesterol uptake and intracellular cycling. L-cell fibroblasts stably expressing SCP-2 showed SCP-2 localization in peroxisomes and endoplasmic reticulum, the latter indicating a role in phospholipid synthesis. SCP-2 in transfected L-cells overexpressing SCP-2 was colocalized with mitochondria, and absent from lysosomes. Both SCP-2 and its precursor, pro-SCP-2, were known to stimulate molecular sterol transfer from lysosomal to mitochondrial membranes, suggesting a potential mechanism for replenishing mitochondrial cholesterol pools depleted by cholesterol oxidation. LSCM, dot and Western blotting were used to demonstrate the peroxisomal targeting of pro-SCP-2, mediated by the C-terminal SKL sequence, and facilitated by the 20-amino acid presequence, which might alter SCP-2 structure. SCP-2 was colocalized in the highest level with the peroxisomal marker PMP70 in pro-SCP-2, and SCP-2 transfected L-cells, as well as McAR7777 hepatoma cells. Liver fatty acid binding protein (L-FABP) was localized in L-cells and embryonic stem (ES) clones expressing different levels of L-FABP. L-FABP expression altered ES cell morphology and expression of an embryonic marker, suggesting a role in differentiation control by acting in the nucleus as well as cytoplasm. Short-chain, medium-chain fatty acids, and LCFA were studied for their uptake and distribution in L-cell fibroblasts overexpressing L-FABP. L-FABP targeted LCFA to the nucleus of living cells. L-FABP overexpressing L-cell fibroblasts were analyzed for the presence of L-FABP, and the transcription factor that regulates its expression, peroxisome proliferator activator receptor (PPAR), by LSCM and Western blotting of cell and tissue homogenates, as well as nuclear extracts and membrane fractions. Markers for the inner membrane of the nuclear envelope and for the nucleoli were localized, and colocalized with PPAR in L-FABP overexpressing L-cell fibroblasts. L-FABP might participate in other functions than transport, such as the regulation of transcription and nuclear translation. |
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| Item Description: | In title numerals are used. Vita. "Major Subject: Veterinary Microbiology". |
| Physical Description: | ix, 219 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 177-218). |