The ability of protein tyrosine kinases to phosphorylate a synthetic peptide was inhibited 51% by peroxynitritemediated nitration of tyrosine. Exposure of endothelial cells to peroxynitrite decreased the intensity of tyrosine phosphorylated proteins and increased the intensity of nitrotyrosine-containing proteins. Peroxynitrite-modified BSA was degraded by human red blood cell lysates. However, human plasma in a concentration-, time-, and temperature-dependent manner, removed the protein nitrotyrosine epitope. These results suggest that tyrosine nitration interferes with phosphorylation and targets proteins for degradation. Specific enzymatic process(es) for removing nitrotyrosine may be present in vivo.
The present study identifies proteins modified by nitration in the plasma of patients with ongoing acute respiratory distress syndrome (ARDS). The proteins modified by nitration in ARDS were revealed by microsequencing and specific antibody detection to be ceruloplasmin, transferrin, alpha(1)-protease inhibitor, alpha(1)-antichymotrypsin, and beta-chain fibrinogen. Exposure to nitrating agents did not deter the chymotrypsin-inhibiting activity of alpha(1)-antichymotrypsin. However, the ferroxidase activity of ceruloplasmin and the elastase-inhibiting activity of alpha(1)-protease inhibitor were reduced to 50.3 +/- 1.6 and 60.3 +/- 5.3% of control after exposure to the nitrating agent. In contrast, the rate of interaction of fibrinogen with thrombin was increased to 193.4 +/- 8.5% of the control value after exposure of fibrinogen to nitration. Ferroxidase activity of ceruloplasmin and elastase-inhibiting activity of the alpha(1)-protease inhibitor in the ARDS patients were significantly reduced (by 81 and 44%, respectively), whereas alpha(1)-antichymotrypsin activity was not significantly altered. Posttranslational modifications of plasma proteins mediated by nitrating agents may offer a biochemical explanation for the reported diminished ferroxidase activity, elevated levels of elastase, and fibrin deposits detected in patients with ongoing ARDS.
Recent reports of fatal transfusion-associated Yersinia enterocolitica sepsis prompted a study of the feasibility of adding a question to the routine donor health history as a method of reducing this risk. In three American Red Cross blood centers, 11,323 donors were asked one of two questions about gastrointestinal symptoms during their health history screenings. Affirmative responses were obtained from 0.6 or 4.0 percent of the donors, depending on how the question was asked. In one center, more than 6 percent of donors gave affirmative answers. The efficacy of asking a relatively simple question about gastrointestinal symptoms as a way of preventing Y. enterocolitica should be evaluated further, because relatively large numbers of donors may respond affirmatively. Other methods of reducing the risk of transfusion-associated Y. enterocolitica infection should be pursued.
Enviroxime and related analogs are potent inhibitors of rhinoviruses and enteroviruses in cell culture. Previous analyses of resistant mutants implicated the viral nonstructural protein 3A(B) as the likely target of drug activity. In this study, we used site-directed mutagenesis and selection of spontaneous rhinovirus 14 mutants with several enviroxime analogs to confirm the link between domains in rhinovirus 14 3A(B) and the function blocked by enviroxime. We also produced recombinant 3A and 3AB proteins for biochemical analyses. Despite extensive efforts, however, we were unable to demonstrate direct binding between enviroxime and any of several viral proteins, nor could we demonstrate binding of enviroxime to a host protein. In addition, enviroxime did not disrupt 3AB's ability to bind RNA or 3D polypeptide, the association of 3AB with membranes, or the cleavage of 3AB by 3C protease. Finally, we identified an enviroxime-resistant mutant with an increased level of resistance which apparently has mutations in multiple proteins or RNA sequences. Taken together, these results suggest that enviroxime targets a complex of proteins and/or cellular factors. Such a complex mechanism of inhibition might explain the low levels of viral resistance to these inhibitors as compared with other picornaviral inhibitors.
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