A hypothesis is proposed for premature death in Barred Plymouth Rock (PBR) and White Leghorn (WL) chicken melanocytes. BPR melanocytes are genetically sensitive due to a defect in their SOD and GSH levels caused by the barring gene and die prematurely from oxygen radical toxicity. WL chickens carry the dominant white gene in addition to the barring gene and have a further reduction of SOD and their melanocytes die much earlier than the BPR melanocytes. Some forms of human vitiligo may be caused by a similar mechanism.
The specific aims are focused to further test this hypothesis and to elucidate at the molecular gene level why SOD and GSH levels are low in the mutant birds and to determine the role that the keratinocytes may play in these vitiliginous avian models. The in vivo experiments are as follows: (1) Isolation of Cytosolic (CT) Cu/Zn bird using a competitive reference standard and RT-PCR. Sequence and compare the CT Cu/Zn SOD of the WLH with the BPR and JF. Comparisons will also include other species. Compare and assess differences in CT SOD between the 3 bird types by expression of the enzyme under a single promoter. (2) Analysis of three CT SOD between the 3 bird types by expression of the enzyme under a single promoter. (2) Analysis of three important enzymes in glutathione metabolism will be performed in JF, BPR and WL feature tissue with the JF serving as a control. If the down regulation of GSH is determined to be caused by one or more of the enzymes involved, then a molecular characterization of the mutant gene will be performed in the same manner as that for the SOD gene. (3) A thorough light and electron microscope study of the JF, BPR and WL feature melanocytes and keratinocytes will be performed to determine if any structural aberrations like those described in human vitiligo melanocytes occur in these mutant line cells. Enzymes associated with melanogenesis and cell death will be analyzed cytochemically in the melanocytes. In all cases, the JF will serve as a control. The in vitro studies consist of: (1) establishing the primary melanocyte cultures of the JF, BPR MSH to the media or by not changing the media. (2) Thorough electron microscope studies will be done on these three genotypes of melanocytes in normal and premature death conditions to see if the morphology is similar to that of the in vivo normal and dying melanocytes. (3) Since keratinocytes may play a large role in the survival and differentiation of the melanocytes, SOD, GSH, catalase,. GSH-peroxidase under normal and oxygen radical stress conditions. The measurements of the above parameters of the feather tissue, which predominately includes the keratinocytes and few melanocytes, has already been performed.
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