1998 Beckman Scholars at Carnegie MellonCarolyn Calisti, Carnegie Mellon University
(Advisor: Dr. Alan Koretsky)
Do Mice Expressing Creatine Kinase in Livers Have a Resistance to Endotoxic Shock?
Endotoxin, a molecule located in the outer membrane of gram negative bacteria, induces cell death. During bacterial infection, the endotoxin is released from the membrane and interacts with the immune system to produce an excess of inflammatory mediators. These mediators activate neutrophils producing lysosomal enzymes and oxygen-derived free radicals which injure adjacent hepatocyte cells, both necrotically and apoptotically. Evidence is accumulating that creatine kinase (CK), a highly conserved enzyme that plays a significant role in cellular energetics, can suppress cell death. CK catalyzes the synthesis of ATP from ADP and phosphocreatine during times when the cellular metabolism of ATP is greater than its production. There are three possible ways in which CK can affect endotoxic shock 1) by producing ATP to prevent necrotic cell death, 2) by producing ATP to prevent apoptotic cell death, and 3) by mitochondrial CK forming specific alterations in the mitochondrial membrane to block the release of cell death signals. To test whether CK can protect against endotoxic shock, transgenic mice overexpressing CK isoforms in liver, along with control mice, were fed CK substrate and injected with endotoxin (lipopolysaccharide, LPS) . After LPS injection, the glucose levels decreased in control mice and mice expressing mitochondrial-CK, signifying liver damage. The mice expressing mitochondrial CK demonstrated a higher tolerance to endotoxic shock than the control or brain-CK mice. The tolerance was independent of the presence of CK substrate. The results support the working hypothesis that mitochondrial CK plays a role in the make-up of the mitochondrial membrane, potentially inhibiting the cell death signal, cytochrome C, from passing to the cytoplasm.
David Fooksman, Carnegie Mellon University
(Advisor: Dr. Javier Lopez)
Alternative splicing of the Drosophila melanogaster Ultrabithorax gene: Mechanism of regulation by HRP48
Alternative splicing of RNAs from the homeotic gene Ultrabithorax (Ubx) of Drosophila melanogaster generates six mRNA isoforms that differ by the inclusion or exclusion of two internal microexons (mI and mII) and an element (B) that is defined by two 5' splice sites at the end of the 5' exon. Recent work in the Lopez laboratory has established that the hnRNP protein HRP48 is required for inclusion of exons mI and mII in Ubx mRNAs. Incorporation of mI is decreased by up to 50% in flies heterozygous for an hrp48 mutation that reduces but does not eliminate the transcription of this gene, indicating that HRP48 protein plays a dose-dependent role in the regulation of alternative splicing of Ubx pre-RNAs. To investigate the effect of complete absence of hrp48 function, candidates for null mutations in hrp48 were generated by imprecise excision of a transposable element (P-element) inserted within the upstream regulatory region of the hrp48 gene. These candidates are currently being characterized. Southern hybridization is being used to determine the extent of the deleted region, and coupled reverse transcription/amplification assays are being used to quantify the reduction of Ubx isoforms containing the mI exon in the mutant lines. The sequence of mI reveals a potential HRP48 binding site at the extreme 5' end, overlapping a 5' splice site regenerated at the junction with the upstream exon. To further understand the interaction between HRP48 and Ubx pre-RNAs, I will test whether HRP48 protein binds directly this site or to other flanking sequences using the yeast 3-hybrid RNA-binding assay. Work is currently underway to develop the RNA and protein constructs for the 3-hybrid assay.