When cells are exposed to external H 2 O 2 , the H 2 O 2 rapidly diffuses inside and oxidizes ferrous iron, thereby forming hydroxyl radicals that damage DNA. Thus the process of oxidative DNA damage requires only H 2 O 2 , free iron, and an as-yet unidentified electron donor that reduces ferric iron to the ferrous state. Previous work showed that H 2 O 2 kills Escherichia coli especially rapidly when respiration is inhibited either by cyanide or by genetic defects in respiratory enzymes. In this study we established that these respiratory blocks accelerate the rate of DNA damage. The respiratory blocks did not substantially affect the amounts of intracellular free iron or H 2 O 2 , indicating that that they accelerated damage because they increased the availability of the electron donor. The goal of this work was to identify that donor. As expected, the respiratory inhibitors caused a large increase in the amount of intracellular NADH. However, NADH itself was a poor reductant of free iron in vitro. This suggests that in non-respiring cells electrons are transferred from NADH to another carrier that directly reduces the iron. Genetic manipulations of the amounts of intracellular glutathione, NADPH, ␣-ketoacids, ferredoxin, and thioredoxin indicated that none of these was the direct electron donor. However, cells were protected from cyanide-stimulated DNA damage if they lacked flavin reductase, an enzyme that transfers electrons from NADH to free FAD. The K m value of this enzyme for NADH is much higher than the usual intracellular NADH concentration, which explains why its flux increased when NADH levels rose during respiratory inhibition. Flavins that were reduced by purified flavin reductase rapidly transferred electrons to free iron and drove a DNA-damaging Fenton system in vitro. Thus the rate of oxidative DNA damage can be limited by the rate at which electron donors reduce free iron, and reduced flavins become the predominant donors in E. coli when respiration is blocked. It remains unclear whether flavins or other reductants drive Fenton chemistry in respiring cells.

West Nile virus (WNV) is an emerging flavivirus that has caused frequent epidemics since 1996. Besides natural transmission by mosquitoes, WNV can also be transmitted through blood transfusion and organ transplantation, thus heightening the urgency of development of a specific and rapid serologic assay of WNV infection. The current immunoassays lack specificity because they are based on detection of antibodies against WNV structural proteins and immune responses to structural proteins among flaviviruses cross-react to each other. Here, we describe microsphere immunoassays that detect antibodies to nonstructural proteins 3 and 5 (NS3 and NS5). In contrast to immunoassays based on viral envelope and NS3 proteins, the NS5-based assay (i) reliably discriminates between WNV infections and dengue virus or St. Louis encephalitis virus infections, (ii) differentiates between flavivirus vaccination and natural WNV infection, and (iii) indicates recent infections. These unique features of the NS5-based immunoassay will be very useful for both clinical and veterinary diagnosis of WNV infection.West Nile virus (WNV) is a member of the genus Flavivirus, which includes many significant human pathogens of global epidemiological importance, including four serotypes of dengue (DEN) virus, yellow fever (YF) virus, Japanese encephalitis (JE) virus, St. Louis encephalitis (SLE) virus, and tickborne encephalitis (TBE) virus, as well as WNV (3). Among them, DEN virus, YF virus, TBE virus, JE virus, and WNV are listed by the National Institutes of Health as potential biodefense pathogens. Since its introduction into the United States in 1999, WNV has resulted in more than 4,156 known human cases, with 284 deaths (for updates, see http://www.cdc.gov /ncidod/dvbid/westnile/surv&controlCaseCount03.htm). Recent studies have shown that, besides natural transmission by mosquitoes, WNV can also be transmitted through blood transfusion, organ transplantation (9), breast feeding (8), intrauterine exposure (6), and laboratory-acquired infection (7). These findings have underlined the importance of developing an accurate serologic assay for diagnosis of WNV infection.Flavivirus genomic RNA contains a single open reading frame encoding 10 viral proteins: three structural and seven nonstructural (NS) proteins (Fig. 1A). Viral envelope protein (E protein) (14), NS1 (18,25,26), and NS3 (24) are the most immunogenic proteins during flavivirus infection (15). The current serologic diagnosis of WNV infection is based on detection of antibodies against viral structural proteins, mainly the E protein (12, 21). Unfortunately, the high cross-reactivity of the E protein among flaviviruses limits the specificity of the assay. Positive sera or spinal fluids identified by the current assay must be verified by cross-species plaque reduction neutralization tests (PRNT) to exclude the possibility of infection with cross-reactive viruses such as SLE and DEN. These confirmatory tests have to be performed in level 3 biocontainment for many flaviviruses and substanti...

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SummaryThe presence of nitric oxide (NO) greatly accelerates the rate at which hydrogen peroxide (H 2 O 2 ) kills Escherichia coli . Workers have suggested that this effect may be important in the process of bacteriocide by phagocytes. The goal of this study was to determine the mechanism of this synergism. The filamentation of the dead cells, and their protection by cell-permeable iron chelators, indicated that NO/H 2 O 2 killed cells by damaging their DNA through the Fenton reaction. Indeed, the number of DNA lesions was far greater when NO was present during H 2 O 2 exposure. In the Fenton reaction, free intracellular iron transfers electrons from adventitious donors to H 2 O 2 , producing hydroxyl radicals. Although NO damaged the [Fe-S] clusters of dehydratases, this did not increase the amount of free iron and was therefore not the reason for acceleration of Fenton chemistry. However, NO also blocked respiration, an event that previous studies have shown can stimulate oxidative DNA damage. The resultant accumulation of NADH accelerates the reduction of free flavins by flavin reductase, and these reduced flavins drive Fenton chemistry by transferring electrons to free iron. Indeed, mutants lacking the respiratory quinol oxidases were sensitive to H 2 O 2 , and NO did not have any further effect. Further, mutants that lack flavin reductase were resistant to NO/H 2 O 2 , and overproducing strains were hypersensitive. We discuss the possibility that H 2 O 2 and NO synergize when macrophages attack captive bacteria.

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