Previous studies in our laboratory have demonstrated that consumption by mammals and birds of a low zinc diet is associated with a diminished output of gene products in response to either endogenous or exogenous estrogen challenge. A recent discovery of the highly conserved zinc finger (or twist) motif in the steroid receptor superfamily suggests that zinc may play a broad role in assuring swift and timely adaptive, regulatory, and developmental responses to this entire class of lipophilic substances. To determine the effect of zinc status at the molecular level, a zinc deficit was created in cell cultures and glucocorticoid regulated and constitutive gene expression monitored.
Using a cell impermeable zinc specific chelator, diethylenetriaminepentaacetic acid (DTPA), a zinc deficient state in HeLa cells was created. This deficit was quantified by a fluorescence assay using N-(6-methoxy-8-quinolyl 1)-p-toluenesulfonamide (TSQ) to measure exchangeable zinc and induced coupled plasma analysis to measure total zinc. The 83% reduction in total cellular zinc, corresponding to a 65% decrease in TSQ fluorescence was rapid, nearly maximal within 4 hours, and without significant loss of viability over 72 hours as tested by dye permeability. This was prevented by addition of zinc ions. This cellular system was used to study the effects of zinc deficits separately upon the individual steps in steroid-directed gene expression and constitutive protein synthesis. Differentiation between the effect of zinc supply upon hormone mediated and constitutive gene expression in HeLa cells was accomplished through transient transfections. By examining the expression of a glucocorticoid-responsive chloramphenicol acetyltransferase (CAT) reporter gene as compared to CAT activity derived from glucocorticoid-insensitive reporter plasmid, it was determined that glucocorticoid directed expression was reduced to a greater extent than constitutive (88% vs. 15%) when zinc concentrations were reduced 80%. Furthermore, neither the binding affinities of receptors for dexamethasone nor nuclear translocation was altered by zinc concentrations. However, glucocorticoid receptor concentrations were reduced by 50%. This reduction in receptor number is sufficient to produce decreases in gene induction of the magnitude observed. Based on these results in conjunction with previous studies in our laboratory we propose that the most sensitive need for zinc may be in permitting rapid induction of newly synthesized enzymes as seen during hormone responses, rather than serving as a cofactor of metalloenzymes involved in constitutive synthesis. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/38109 |
| Date | 06 June 2008 |
| Creators | Simon, Kathryn D. |
| Contributors | Biochemistry and Anaerobic Microbiology, Bunce, George E., Bender, Patrick K., Storrie, Brian, Falkinham, Joseph O. III, Hess, John L. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation, Text |
| Format | x, 147 leaves, BTD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 35205902, LD5655.V856_1996.S563.pdf |
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