Document Type

Thesis - University Access Only

Award Date

1997

Degree Name

Master of Science (MS)

Department / School

Biology

Abstract

Alleles of the agouti locus regulate pigmentation in follicular melanocytes of the mouse (Silvers, 1979). Agouti protein (AP) expression in the wild-type agouti mouse (AWJ / AWJ) correlates with a switch from black pigment (eumelanin) to yellow pigment (phaeomelanin). Ectopic overexpression of AP in the lethal yellow mouse (AY / a) leads to an intense yellow coat as well as the Lethal Yellow Syndrome (L YS). The agouti locus has dramatic effects on a number of enzymes of rnelanogenesis, but the causal relationships between agouti expression and enzyme activity remain unknown. Agouti protein may modulate pigmentation by influencing thiol concentrations within the hair follicle. Thiol concentrations appear to control the conversion of dopaquinone to phaeomelanin in hair follicle melanocytes. Previous studies have demonstrated that AP expression correlates with an increase in cysteine and a decrease in GSH levels within the hair follicle. In addition, preliminary studies indicate that there may be similar thiol imbalances in other tissues of the AY I a mouse. Because the liver is the primary site of CSH production, an agouti-mediated modulation of GSH metabolism within this organ may have widespread negative effects which could possibly be manifested as symptoms of the LYS. The purpose of this study was: 1.) to assess the effects of allelic substitutions at the agouti locus on gamma-glutamyltranspeptidase (GGT) activity, 2.) to correlate any genotype-specific differences in GGT activity within regenerating hair follicles to cysteine levels previously measured, and 3.) to determine the relative abundance of cysteine and glutathione (GSH) in liver tissue at different developmental stages. This study hypothesizes that 1.) the agouti gene regulates thiol concentrations within the hair follicle (and therefore the type of pigment made) and possibly other tissues by modulating the activity of gamma-glutamyltranspeptidase (GGT), an enzyme which initiates the breakdown of GSH to cysteine, 2.) the agouti locus may cause a long-term chronic shift from GSH to cysteine in extrafollicular tissues of the Ay / a mouse. Hair fo11icle, spleen, and pancreas samples were taken from mice during periods of hair growth and GGT activity was measured using a modification of the colorimetric assay of Jacobs (1971). Cysteine and GSH levels in liver and spleen tissue were also measured using the reverse-phase HPLC method described by Fahey and Newton (1987). Results of this study failed to show any differences in GGT activity between genotypes on day 9 when results were expressed on a per protein basis. When activity was expressed as units activity/ mg sample, h0wever, GGT activity in the agouti (LSMean = 2.42) was seen to be elevated over that of the yellow and black mice (LSMeans = 1.27 & 1.52 respectively) and may reflect a short, intense demand for cysteine during the relatively brief period of yellow pigment production in the agouti mouse. In addition, GGT activity was seen to increase slightly in both yellow and agouti mice between days 6 and 12, confirming the findings of others that GGT activity is a marker of hair growth induction. Results of HPLC analysis of liver tissue failed to show a developmental shift from GSH to cysteine which could be correlated with the onset of the L YS. Plucking-induced hair growth was shown to dramatically increase liver cysteine levels in Ay I a, AwJ / AwJ, and a/ a mice over that of telogen phase littermates, implicating the liver as a source of the increased cysteine seen within regenerating hair follicles. The results of this study do not lend strong support to the hypothesis that agouti protein influences thiol metabolism in the hair follicle by directly modulating the activity of the enzyme GGT, or that agouti-induced misregulations of thiol metabolism in extrafollicular tissues are an underlying cause of the L YS. Study funded by SDSU-AES-HD089, Eagles' Ehrmann Cancer Fund, and NIH (AR42757).

Library of Congress Subject Headings

Mice -- Genetics

Lethal mutation

Thiols

Hair follicles

Format

application/pdf

Number of Pages

127

Publisher

South Dakota State University

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