Synthesis and characterization of a novel organic nitrate NDHP: Role of xanthine oxidoreductase-mediated nitric oxide formation

In this report, we describe the synthesis and characterization of 1,3-bis(hexyloxy)propan-2-yl nitrate (NDHP), a novel organic mono nitrate. Using purified xanthine oxidoreductase (XOR), chemiluminescence and electron paramagnetic resonance (EPR) spectroscopy, we found that XOR catalyzes nitric oxide (NO) generation from NDHP under anaerobic conditions, and that thiols are not involved or required in this process. Further mechanistic studies revealed that NDHP could be reduced to NO at both the FAD and the molybdenum sites of XOR, but that the FAD site required an unoccupied molybdenum site. Conversely, the molybdenum site was able to reduce NDHP independently of an active FAD site. Moreover, using isolated vessels in a myograph, we demonstrate that NDHP dilates pre-constricted mesenteric arteries from rats and mice. These effects were diminished when XOR was blocked using the selective inhibitor febuxostat. Finally, we demonstrate that NDHP, in contrast to glyceryl trinitrate (GTN), is not subject to development of tolerance in isolated mesenteric arteries.</p

Improved diagnostic performance of a commercial Anaplasma

The current study tested the hypothesis that removal of maltose binding protein (MBP) from recombinant antigen used for plate coating would improve the specificity of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay (cELISA). The number of 358 sera with significant MBP antibody binding (≥30%I) in Anaplasma-negative herds was 139 (38.8%) when tested using the recombinant major surface protein 5 (rMSP5)-MBP cELISA without MBP adsorption. All but 8 of the MBP binders were rendered negative (<30%I) using the commercial rMSP5-MBP cELISA with MBP adsorption, resulting in 97.8% specificity. This specificity was higher than some previous reports, so to improve the specificity of the commercial cELISA, a new recombinant antigen designated rMSP5-glutathione S-transferase (GST) was developed, eliminating MBP from the antigen and obviating the need for MBP adsorption. Using the rMSP5-GST cELISA, only 1 of 358 Anaplasma-negative sera, which included the 139 sera with significant (≥30%I) MBP binding in the rMSP5-MBP cELISA without MBP adsorption, was positive. This resulted in an improved diagnostic specificity of 99.7%. The rMSP5-GST cELISA without MBP adsorption had comparable analytical sensitivity to the rMSP5-MBP cELISA with MBP adsorption and had 100% diagnostic sensitivity when tested with 135 positive sera defined by nested polymerase chain reaction. Further, the rMSP5-GST cELISA resolved 103 false-positive reactions from selected sera with possible false-positive reactions obtained using the rMSP5-MBP cELISA with MBP adsorption and improved the resolution of 29 of 31 other sera. In summary, the rMSP5-GST cELISA was a faster and simpler assay with higher specificity, comparable sensitivity, and improved resolution in comparison with the rMSP5-MBP cELISA with MBP adsorption