One of our major publications in FY2006 related specifically to this project was a detailed structurefunction study of chicken leukocyte RNases A1 and A2 (Nitto et al., JBC 2006). Of pragmatic importance, we demonstrated that chicken leukocyte RNase A2 had both bactericidal and angiogenic activity. Furthermore, we determined that the divergent domains II (amino acids 7176) and III (amino acids 89104) of RNase A2 are both important for bactericidal activity, which led to the provisional patent Bactericidal peptides from avian leukocyte ribonucleases to support the development of bactericidal RNase A2 and encoded peptides as novel antibacterial agents (Nitto et al., J Biol Chem. 2006) During FY2007 we explored a larger range of bactericidal activities of RNase A2 and peptides derived therefrom, which includes important pathogens, such as Salmonella sps. We have also carried out preliminary experiments that will permit us to begin testing RNase A2 and A2derived peptides in bacterial pathogenesis and treatment studies in vivo (Rosenberg et al., unpublished results) and have submitted a proposal for lab expansion funds to permit us to initiate in vivo testing in mouse models of bacterial infection ? ? Given our findings on the crucial role of GATA transcription factors in developing eosinophils in vivo and in vitro (see AI00094104) we have initiated a project designed to evaluate the role of GATA1 and GATA2 in the transcription of the two human eosinophil RNase A ribonucleases, the eosinophilderived neurotoxin (EDNRNase 2) and eosinophil cationic protein (ECPRNase 3). To explore the impact of the distal 5 regions on the differential transcription of the genes encoding EDN and ECP, 0.5, 0.8 and 1.4 kb of fragments including sequence directly 5 to the introns within the genes encoding EDN and ECP were subcloned into pGL3 reporter vector. Regulatory activity was assessed in the butyricacid (BA) differentiated eosinophil promyelocyte clone 15 cell line. The MatInspector program was employed to determine consensus transcription factor binding sites, followed by verification through mutagenesis studies and EMSAs. Quantitative PCR and western blotting were used to determine quantitative changes in expression of transcription factors as well as EDN and ECP in response to differentiationinducing stimuli. Luciferase activity of the 1.4 kb EDN 5 promoter fragment construct was about 2fold greater than that of 0.5 and 0.8 kb promoter fragments. Given the 92% nucleotide sequence identity between the 5 promoter regions of EDN and ECP, we were not surprised to find similar levels of luciferase activity when comparing the different size fragments. Mutational analysis revealed functional GATA1 and GATA2 consensus binding sites in the region between 500 and 1140 in the EDN promoter. In contrast, although consensus GATA1 and GATA2 binding sites were present in the ECP 5 promoter, analogous to those of EDN, mutational analysis indicated that these sites had no impact on transcription of ECP. EMSAs demonstrated specific binding of GATA1 and GATA2 to the identified consensus elements in the 5 promoter region of EDN. Furthermore, eosinophilic promyelocyte clone 15 cells respond to BA with increased transcription and translation of GATA1 and GATA2 as well as EDN. These results support the proposal that both GATA1 and GATA2 binding sites coordinately contribute to transcription of EDN, with each deletion resulting in a decrease in promoter function (Qiu et al., manuscript in preparation). ? ? Finally, I have been invited to submit two reviews on RNase A ribonuclease biology, the first, Rosenberg HF and Siegel SJ. RNase A Ribonucleases as Biomarkers of Disease, in The Open Clinical Chemistry Journal, Bentham Publishers, to be submitted in September 2007, and the second, Rosenberg HF. 2007. RNase A Ribonucleases and Host Defense, in Contributions to Microbiology, Karger AG, Basel, Switzerland to be submitted in October 2007.
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