CURRENT PROJECTS

1. ETHANOL RESPONSIVE ELEMENTS IN THE MITOCHONDRIAL ASPARTATE AMINOTRANSFERASE PROMOTER
When exposed to ethanol, hepatocytes increase their expression of mitochondrial aspartate aminotransferase. This leads to an increase in the activity of this enzyme in serum and is the biological basis for the characteristic rise in AST seen in alcoholic liver disease. However, this enzyme has been found to be identical to plasma membrane fatty acid binding protein, the first protein identified as promoting facilitated uptake of long-chain free fatty acids into cells. The increase in expression may also lead to hepatic steatosis seen in alcoholic liver disease. This dual role makes this gene of extreme interest in the study of alcoholic liver disease. Analysis of the promoter is underway, to locate and define transcription factor binding sites that may be involved in the response to ethanol. Reporter constructs with various portions of the promoter regulating expression of secreted alkaline phosphatase (SEAP) are employed, and transfected into human hepatoma cells. After selection, cells are exposed to medium with 0 or 40 mM ethanol for 24 hours and medium is assayed for SEAP activity using a fluorescent assay. Regions responsive to ethanol will be analyzed for transcription factor binding sites using bioinformatics, DNA Footprinting, EMSA, and various newer assay formats becoming available to determine what factors alter promoter function. Comparisons may be made with promoters of other genes known to be responsive to ethanol.

2. DELINEATION OF THE FATTY ACID BINDING SITE OF MITOCHONDRIAL ASPARTATE AMINOTRANSFERASE
Molecular modeling of mitochondrial aspartate aminotransferase has identified a specific region in which a large number of hydrophobic residues face a cleft in the surface. The volume of the cleft is suitable for binding a long-chain fatty acid. Also, an arginine residue (R201) is present at one end, similar to the placement of arginine or lysine residues in the fatty acid binding sites of albumin. A rat cDNA clone has been mutated to alter specific residues to their cognate forms found in the cytoplasmic isozyme, which has no significant fatty acid binding capacity. Although the cytoplasmic and mitochondrial forms catalyze the same reaction, they are only ~50% identical at the amino acid sequence level. There are numerous residues that are conserved in either form, where all mammals have a specific amino acid at one position in the mitochondrial form, while all cytoplasmic forms share a different residue. In the cleft region defined by just over 100 residues, 23 of these conserved substitutions are present. Preliminary findings indicate that the substitutions R201T and A219P decrease fatty acid binding and/or uptake substantially. A construct with a complete replacement of the binding site region with cytoplasmic sequence is also under investigation, as well as double mutants and a mutation, R201K, which retains a basic residue at the key site. These structure-function studies will help us determine how this single protein participates in two disparate cellular processes.

3. HISTOLOGICAL ANALYSIS OF THE MOUSE GERM CELL LINEAGE
This project is based upon previous observations made while studying serial sections of developing mouse fetuses and adult gonads. A by-product of a staining reaction gave a specific staining pattern to mouse germ cells. This pattern was seen in all stages from day 11 of fetal development onwards, and was present in germ cells of all stages in adult gonads. These observations will be extended to include the earlier stages of fetal and embryonic development, to determine when it appears, or if it is maintained throughout development, which would argue for a mode of continuity of the germ plasm. Also, another method employed in paraffin sections appeared to delineate the acrosomal cap as it forms in spermatogenesis, something not generally visible except in electron microscopy. This method will be investigated for its compatibility with other staining methods suitable for analysis spermatogenesis and may be useful in the study of defects in that process.