Memorial University DAI: Antifreeze protein in winter flounder, Pleuronectes americanus, gill epithelial cells isolated and grown in culture

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Title	Antifreeze protein in winter flounder, Pleuronectes americanus, gill epithelial cells isolated and grown in culture
Author	Winsor, Stephen B., 1971-
Description	Thesis (M.Sc.)--Memorial University of Newfoundland, 2000. Medicine
Date	2000
Pagination	x, 79 leaves : ill.
Subject	Winter flounder--Physiology; Cryobiochemistry; Flounder--physiology; Antifreeze Proteins; Epithelial Cells
Degree	M.Sc.
Degree Grantor	Memorial University of Newfoundland. Faculty of Medicine
Discipline	Medicine
Language	eng
Notes	Bibliography: leaves 74-79
Abstract	Antifreeze proteins (AFPs) have been found in the blood of many teleost species and have the ability to bind to ice crystals and inhibit their growth. Type I AFP was later discovered in several body tissues of the winter flounder (Pleuronectes americanus) including the skin, scales and gills. In order to further our understanding of these proteins that are not produced in the liver, epithelial cells of the winter flounder gill were isolated and maintained in culture to look for the presence of skin type I AFP. The present study is the first that describes the isolation and culture of gill epithelial cells from winter flounder. The isolation procedure used was based in part on the gill cell isolation method developed by Part et al [Part, P., Norrgren, L., Bergstrom, E. & Sjoberg, P.(1993) J. Exp. Biol 175, 219 - 232]. The presence of type I AFP in the cells was determined using immuno-histochemistry. The results indicate that epithelial cells stained positive for winter flounder type I AFP antisera against liver, skin or a recombinant form of type I AFP whereas cells stained with sea raven type II AFP antiserum showed no reaction. The distribution pattern of AFP seen in these cells suggests the AFP is located intracellular^. The AFP produced within these cells is believed to be the skin type I AFP and is thought to react with the liver and recombinant antisera due to the close similarity between these AFPs. In short term culture, individual cells were of three predominate shapes. Round cells were 8.3 ± 2.6 , um in diameter, crescent cells were 19.8 ± 5.2 µm x 10.4 ± 2.9 pan and elongated cells were 21.5 ± 5.0 µm x 6.8 ± 1.7 /µm. Scanning electron microscopy (SEM) showed individual cells with an elevated nuclear region with a low and somewhat ruffled outer cytoplasmic area. Dishes that contained a relatively high number of isolated gill cells supported the formation of a confluent monolayer of cells. Scanning electron micrographs of confluent cells 8 days in culture show a highly flattened apical surface with no distinguishing characteristics. The cells attached to culture dishes and were capable of forming confluent monolayers of cells similar to pavement cells seen in other gill epithelial cultures. However, SEM revealed that they do not contain microridges typically seen in pavement cells isolated from the sea bass and rainbow trout. The use of the fluorescent stain DASPEI showed these cells to be mitochondria-rich, a characteristic of gill epithelial chloride cells. Preparation of cultures such as these provide a means of examining the mechanisms involved in skin type I AFP production, regulation and also how these proteins function in gill epithelia.
Type	Text
Resource Type	Electronic thesis or dissertation
Format	Image/jpeg; Application/pdf
Source	Paper copy kept in the Centre for Newfoundland Studies, Memorial University Libraries
Local Identifier	a1493339
Rights	The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission.
Collection	Electronic Theses and Dissertations
Scanning Status	Completed
PDF File	(8.91 MB) -- http://collections.mun.ca/PDFs/theses/Winsor_StephenB.pdf
CONTENTdm file name	3327.cpd