Cyclooxygenases (COXs) are of important therapeutic value as they are the target site of aspirin-like drugs. Here I report nine new COX-1 splice variants in chapter 1, which I characterized with regard to heme-binding and other properties. Inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) are co-inducible in many tissues following mitogenic and proinflammatory stimulation. In chapter 2, I investigate the physical and enzymatic properties of human COX-2 and iNOS and demonstrate that, despite reports to the contrary by another laboratory, they do not interact. The only reported COX-1 splice variant to exhibit cyclooxygenase activity has been isolated from dog brain and is termed COX-3. It contains an in-frame insertion of intron 1. However the existence of human COX-3 remains questionable since intron 1 is out of frame. Two putative in-frame human COX-3 isozymes, COX-1b2 and COX-1b3, (herein designated as COX-3-72 and COX-3-50) have been reported in the literature, but only one of them, COX-3-72, has been characterized. In chapter 3, COX-3-50 and COX-3-72 are reported to be over-expressed and determined to be active cyclooxygenases. COX-3-72 and, to a greater extent, COX-3-50, were stimulated by rofecoxib at physiological concentrations. A similar rofecoxib-stimulated COX activity is observed in quiescent A549 cells. Immunoblot and immunoprecipitation analysis suggest that human platelet and potentially A549 cells, contain a COX-3-50 like protein. Lonicera japonica is used as an anti-inflammatory treatment in traditional Chinese medicine. Its working mechanism is not well known. In chapter 4, I report that extracts from this herb inhibit COX-2 by three mechanisms: direct inhibition, transcriptional and post-transcriptional down regulation. COX-1 and COX-2 are similar to each other in their crystallographic structures. One of the most striking differences is that there are eight amino acids immediately following the signal peptide in COX-1 which are not found in COX-2. The function of this sequence is unknown. In chapter 5, I found that deletion of these amino acids decreased COX-1 Vmax by approximately 4-fold, but had little effect on other properties of the enzyme. Selecting bacteria transformed with recombinant plasmids is a laborious step in gene cloning experiments. This selection process is even more tedious when large numbers of clones need to be screened. In appendix I, I describe an ultra fast plasmid screening method. This new method was frequently used in the experiments performed in chapters 2-6.



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Physical and Mathematical Sciences; Chemistry and Biochemistry



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cyclooxygenase-1, cyclooxygenase-2



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Chemistry Commons