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The 1918 influenza pandemic caused over 40 million deaths worldwide, with 675,000 deaths in the United States alone. Studies in several experimental animal models showed that 1918 influenza virus infection resulted in severe lung pathology associated with dysregulated immune and cell death responses. To determine if reactive oxygen species produced by host inflammatory responses play a central role in promoting severity of lung pathology, we treated 1918 influenza virus-infected mice with the catalytic catalase/superoxide dismutase mimetic, salen-manganese complex EUK-207 beginning 3 days postinfection. Postexposure treatment of mice infected with a lethal dose of the 1918 influenza virus with EUK-207 resulted in significantly increased survival and reduced lung pathology without a reduction in viral titers. In vitro studies also showed that EUK-207 treatment did not affect 1918 influenza viral replication. Immunohistochemical analysis showed a reduction in the detection of the apoptosis marker cleaved caspase-3 and the oxidative stress marker 8-oxo-2'-deoxyguanosine in lungs of EUK-207-treated animals compared to vehicle controls. High-throughput sequencing and RNA expression microarray analysis revealed that treatment resulted in decreased expression of inflammatory response genes and increased lung metabolic and repair responses. These results directly demonstrate that 1918 influenza virus infection leads to an immunopathogenic immune response with excessive inflammatory and cell death responses that can be limited by treatment with the catalytic antioxidant EUK-207.

Several interventions increase lifespan in model organisms, including reduced insulin/insulin-like growth factor-like signaling (IIS), FOXO transcription factor activation, dietary restriction, and superoxide dismutase (SOD) over-expression. One question is whether these manipulations function through different mechanisms, or whether they intersect on common processes affecting aging.A doxycycline-regulated system was used to over-express manganese-SOD (MnSOD) in adult Drosophila, yielding increases in mean and maximal lifespan of 20%. Increased lifespan resulted from lowered initial mortality rate and required MnSOD over-expression in the adult. Transcriptional profiling indicated that the expression of specific genes was altered by MnSOD in a manner opposite to their pattern during normal aging, revealing a set of candidate biomarkers of aging enriched for carbohydrate metabolism and electron transport genes and suggesting a true delay in physiological aging, rather than a novel phenotype. Strikingly, cross-dataset comparisons indicated that the pattern of gene expression caused by MnSOD was similar to that observed in long-lived Caenorhabditis elegans insulin-like signaling mutants and to the xenobiotic stress response, thus exposing potential conserved longevity promoting genes and implicating detoxification in Drosophila longevity.The data suggest that MnSOD up-regulation and a retrograde signal of reactive oxygen species from the mitochondria normally function as an intermediate step in the extension of lifespan caused by reduced insulin-like signaling in various species. The results implicate a species-conserved net of coordinated genes that affect the rate of senescence by modulating energetic efficiency, purine biosynthesis, apoptotic pathways, endocrine signals, and the detoxification and excretion of metabolites.

Methionine residues in protein can be oxidized by reactive oxygen or nitrogen species to generate methionine sulfoxide. This covalent modification has been implicated in processes ranging from normal cell signaling to neurodegenerative diseases. A general method for detecting methionine sulfoxide in proteins would be of great value in studying these processes, but development of a chemical or immunochemical technique has been elusive. Recently, an antiserum raised against an oxidized corn protein, DZS18, was reported to be specific for methionine sulfoxide in proteins (Arch. Biochem. Biophys. 485:35–40; 2009). However, data included in that report indicate that the antiserum is not specific. Utilizing well-characterized native and methionine-oxidized glutamine synthetase and aprotinin, we confirm that the antiserum does not possess specificity for methionine sulfoxide.

Group B Streptococcus is the most common cause of bacterial infection in the newborn. Our aim was to purify and identify molecules produced by the bacterium, which cause pulmonary hypertension. Guided by bioassays performed in neonatal lambs, we utilized standard biochemical techniques for the purification of these bioactive compounds. The compounds were identified by mass spectrometry. Fully synthetic compounds were then tested using the bioassay to confirm their ability to induce pulmonary hypertension. The purified bacterial components causing pulmonary hypertension were the phospholipids cardiolipin and phosphatidylglycerol. Synthetic cardiolipin or phosphatidylglycerol also induced pulmonary hypertension in lambs. Bacterial phospholipids are capable of causing pulmonary hypertension. This finding opens new avenues for therapeutic intervention in persistent pulmonary hypertension of the newborn and generates hypotheses regarding the etiology of respiratory distress in the newborn and the possible effect of antibiotic therapy.

It is well established that many amino acid residues of proteins are susceptible to oxidation by various forms of reactive oxygen species (ROS), and that oxidatively modified proteins accumulate during aging, oxidative stress, and in a number of age-related diseases. Methionine residues and cysteine residues of proteins are particularly sensitive to oxidation by ROS. However, unlike oxidation of other amino acid residues, the oxidation of these sulfur amino acids is reversible. Oxidation of methionine residues leads to the formation of both R- and S-stereoisomers of methionine sulfoxide (MetO) and most cells contain stereospecific methionine sulfoxide reductases (Msr's) that catalyze the thioredoxin-dependent reduction of MetO residues back to methionine residues. We summarize here results of studies, by many workers, showing that the MetO content of proteins increases with age in a number of different aging models, including replicative senescence and erythrocyte aging, but not in mouse tissues during aging. The change in levels of MetO may reflect alterations in any one or more of many different mechanisms, including (i) an increase in the rate of ROS generation; (ii) a decrease in the antioxidant capacity; (iii) a decrease in proteolytic activities that preferentially degrade oxidized proteins; or (iv) a decrease in the ability to convert MetO residues back to Met residues, due either to a direct loss of Msr enzyme levels or indirectly to a loss in the availability of the reducing equivalents (thioredoxin, thioredoxin reductase, NADPH generation) involved. The importance of Msr activity is highlighted by the fact that aging is associated with a loss of Msr activities in a number of animal tissues, and mutations in mice leading to a decrease in the Msr levels lead to a decrease in the maximum life span, whereas overexpression of Msr leads to a dramatic increase in the maximum life span.

Carbonic anhydrase III is a cytosolic protein which is particularly abundant in skeletal muscle, adipocytes, and liver. The specific activity of this isozyme is quite low, suggesting that its physiological function is not that of hydrating carbon dioxide. To understand the cellular roles of carbonic anhydrase III, we inactivated the Car3 gene. Mice lacking carbonic anhydrase III were viable and fertile and had normal life spans. Carbonic anhydrase III has also been implicated in the response to oxidative stress. We found that mice lacking the protein had the same response to a hyperoxic challenge as did their wild-type siblings. No anatomic alterations were noted in the mice lacking carbonic anhydrase III. They had normal amounts and distribution of fat, despite the fact that carbonic anhydrase III constitutes about 30% of the soluble protein in adipocytes. We conclude that carbonic anhydrase III is dispensable for mice living under standard laboratory husbandry conditions.

Group B Streptococcus is the most common cause of bacterial infection in the newborn. Infection in many cases causes persistent pulmonary hypertension, which impairs gas exchange in the lung. We purified the bacterial components causing pulmonary hypertension and identified them as cardiolipin and phosphatidylglycerol. Synthetic cardiolipin or phosphatidylglycerol also induced pulmonary hypertension in lambs. The recognition that bacterial phospholipids may cause pulmonary hypertension in newborns with Group B streptococcal infection opens new avenues for therapeutic intervention.

Iron regulatory protein 2 coordinates cellular regulation of iron metabolism by binding to iron responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. This iron-dependent degradation requires the presence of a 73-residue degradation domain, but its functions have not yet been established. We now show that the domain can act as an iron sensor, mediating its own covalent modification. The domain forms an iron-binding site with three cysteine residues located in the middle of the domain. It then reacts with molecular oxygen to generate a reactive oxidizing species at the iron-binding site. One cysteine residue is oxidized to dehydrocysteine and other products. This covalent modification may thus mark the protein molecule for degradation by the proteasome system.

Patterns of antioxidant gene expression and of oxidative damage were measured throughout the adult life span of a selected long-lived strain (La) of Drosophila melanogaster and compared to that of their normal-lived progenitor strain (Ra). Extended longevity in the La strain is correlated with enhanced antioxidant defense system gene expression, accumulation of CuZnSOD protein, and an increase in ADS enzyme activities. Extended longevity is strongly associated with a significantly increased resistance to oxidative stress. Reverse-selecting this long-lived strain for shortened longevity (RevLa strain) yields a significant decrease in longevity accompanied by reversion to normal levels of its antioxidant defense system gene expression patterns and antioxidant enzyme patterns. The significant effects of forward and reverse selection in these strains seem limited to the ADS enzymes; 11 other enzymes with primarily metabolic functions show no obvious effect of selection on their activity levels whereas six other enzymes postulated to play a role in flux control may actually be involved in NADPH reoxidation and thus support the enhanced activities of the ADS enzymes. Thus, alterations in the longevity of these Drosophila strains are directly correlated with corresponding alterations in; 1) the mRNA levels of certain antioxidant defense system genes; 2) the protein level of at least one antioxidant defense system gene; 3) the activity levels of the corresponding antioxidant defense system enzymes, and 4) the ability of the organism to resist the biological damage arising from oxidative stress.

The ability of iron to catalyze formation of reactive oxygen species significantly contributes to its toxicity in cells and animals. Iron uptake and distribution is regulated tightly in mammalian cells, in part by iron regulatory protein 2 (IRP2), a protein that is degraded efficiently by the proteasome in iron-replete cells. Here, we demonstrate that IRP2 is oxidized and ubiquitinated in cells before degradation. Moreover, iron-dependent oxidation converts IRP2 into a substrate for ubiquitination in vitro . A regulatory pathway is described in which excess iron is sensed by its ability to catalyze site-specific oxidations in IRP2, oxidized IRP2 is ubiquitinated, and ubiquitinated IRP2 subsequently is degraded by the proteasome. Selective targeting and removal of oxidatively modified proteins may contribute to the turnover of many proteins that are degraded by the proteasome.

Protein carbonylation is the most commonly used measure of oxidative modification of proteins. It is most often measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent 2,4 dinitrophenylhydrazine (DNPH). We present protocols for the derivatization and quantification of protein carbonylation with these two methods, including a newly described dot blot with greatly increased sensitivity.

In our technical note entitled “Wanted and Wanting: Antibody Against Methionine Sulfoxide” [1], we pointed out several errors of fact and unsupportable interpretations of experimental observations that were included in a paper reporting attempts to raise a rabbit polyclonal antiserum specific for methionine sulfoxide [2]. The immunogen employed was a corn protein rich in methionine that had been exposed to hydrogen peroxide to convert many of the methionines to methionine sulfoxide. While we do not know the epitopes recognized by the antiserum, we certainly established that there is no specificity for methionine sulfoxide itself. Dr. Moskovitz, the senior author of that paper, now states in his Letter to the Editor that our conclusion of the lack of specificity “has no merit”. He cites several studies that employed the antiserum and found differences between control and experimental samples. He argues that these studies establish specificity. In fact, the cited studies do not report investigations of specificity; they assume that it is specific for methionine sulfoxide based on the claim made in Moskovitz and colleague’s original report [2]. As pointed out in our note, multiple investigators have failed to produce an antiserum or antibody specific for methionine sulfoxide. Both Dr. Moskovitz and we are members of that group of investigators.

Protein carbonylation is the most commonly used measure of oxidative modification of proteins. It is most often measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent 2,4 dinitrophenylhydrazine (DNPH). We present protocols for the derivatization and quantification of protein carbonylation with these two methods, including a newly described dot blot with greatly increased sensitivity.

Protein carbonylation is the most commonly used measure of oxidative modification of proteins. It is frequently measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent, 2,4-dinitrophenylhydrazine. We developed an immunochemical dot blot method for quantitation of protein carbonylation in homogenates or purified proteins. Dimethyl sulfoxide was employed as the solvent because it very efficiently extracts proteins from tissues and keeps them soluble. It also readily dissolves 2,4-dinitrophenylhydrazine and wets polyvinylidene difluoride (PVDF) membranes. The detection limit is 0.19 ± 0.04 pmol of carbonyl, and 60 ng of protein is sufficient to measure protein carbonyl content. This level of sensitivity allowed measurement of protein carbonylation in individual Drosophila.

The oxidative modification of proteins by reactive species, especially reactive oxygen species, is implicated in the etiology or progression of a panoply of disorders and diseases. For the most part, oxidatively modified proteins are not repaired and must be removed by proteolytic degradation. The level of these modified molecules can be quantitated by measurement of the protein carbonyl content, which has been shown to increase in a variety of diseases and processes, most notably during aging. However, these studies have required invasive techniques to obtain cells for analysis. We examined the possibility that desquamating skin cells (corneocytes) would also show an age-related increase in protein carbonyl content, thus providing a noninvasive method for assessing biological age. This was not the case, as we found no age-dependent relationship in the protein carbonyl content of skin cells from volunteers aged 20 to 79 years.

For Deinococcus radiodurans and other bacteria which are extremely resistant to ionizing radiation, ultraviolet radiation, and desiccation, a mechanistic link exists between resistance, manganese accumulation, and protein protection. We show that ultrafiltered, protein-free preparations of D. radiodurans cell extracts prevent protein oxidation at massive doses of ionizing radiation. In contrast, ultrafiltrates from ionizing radiation-sensitive bacteria were not protective. The D. radiodurans ultrafiltrate was enriched in Mn, phosphate, nucleosides and bases, and peptides. When reconstituted in vitro at concentrations approximating those in the D. radiodurans cytosol, peptides interacted synergistically with Mn(2+) and orthophosphate, and preserved the activity of large, multimeric enzymes exposed to 50,000 Gy, conditions which obliterated DNA. When applied ex vivo, the D. radiodurans ultrafiltrate protected Escherichia coli cells and human Jurkat T cells from extreme cellular insults caused by ionizing radiation. By establishing that Mn(2+)-metabolite complexes of D. radiodurans specifically protect proteins against indirect damage caused by gamma-rays delivered in vast doses, our findings provide the basis for a new approach to radioprotection and insight into how surplus Mn budgets in cells combat reactive oxygen species.

Protein carbonyl content is widely used as both a marker for oxidative stress and a measure of oxidative damage. Widely used methods for determination of protein carbonylation utilize the reaction of carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) to form protein-bound 2,4-dinitrophenylhydrazones. Hydrazones can be quantitated spectrophotometrically or, for greater sensitivity, detected immunochemically with anti-dinitrophenyl antibodies. Attention to methodology is important to avoid artifactual elevation in protein carbonyl measurements. We studied extracts of Escherichia coli to identify and eliminate such effects. Nucleic acid contamination caused serious artifactual increases in the protein carbonyl content determined by spectrophotometric techniques. Both in vitro synthesized DNA oligonucleotides and purified chromosomal DNA reacted strongly with 2,4-DNPH. Treatment of cell extracts with DNase+RNase or with streptomycin sulfate to precipitate nucleic acids dramatically reduced the apparent carbonyl, while exposure to proteinase K did not. The commercial kit for immunochemical detection of protein carbonylation (OxyBlot from Chemicon/Millipore) recommends a high concentration of thiol in the homogenizing buffer. We found this recommendation leads to an artifactual doubling of the protein carbonyl, perhaps due to a thiol-stimulated Fenton reaction. Avoiding oxidizing conditions, removal of nucleic acids, and prompt assay of samples can prevent artifactual effects on protein carbonyl measurements.

Coomassie blue staining of gels and blots is commonly employed for detection and quantitation of proteins by densitometry. We found that Coomassie blue or Fast Green FCF bound to protein fluoresces in the near infrared. We took advantage of this property to develop a rapid and sensitive method for detection and quantitation of proteins in gels and on blots. The fluorescence response is quantitative for protein content between 10 ng and 20 microg per band or spot. Staining and destaining require only 30 min, and the method is compatible with subsequent immunodetection.

Iron regulatory protein 2 coordinates cellular regulation of iron metabolism by binding to iron responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. This iron-dependent degradation requires the presence of a 73-residue degradation domain, but its functions have not yet been established. We now show that the domain can act as an iron sensor, mediating its own covalent modification. The domain forms an iron-binding site with three cysteine residues located in the middle of the domain. It then reacts with molecular oxygen to generate a reactive oxidizing species at the iron-binding site. One cysteine residue is oxidized to dehydrocysteine and other products. This covalent modification may thus mark the protein molecule for degradation by the proteasome system.

The mechanisms that underlie iron toxicity in cells and organisms are poorly understood. Previous studies of regulation of the cytosolic iron sensor, iron-regulatory protein 2 (IRP2), indicate that iron-dependent oxidation triggers ubiquitination and proteasomal degradation of IRP2. To determine if oxidization by iron is involved in degradation of other proteins, we have used a carbonyl assay to identify oxidized proteins in lysates from RD4 cells treated with either an iron source or iron chelator. Protein lysates from iron-loaded or iron-depleted cells were resolved on two-dimensional gels and these iron manipulations were also repeated in the presence of proteasomal inhibitors. Eleven abundant proteins were identified as prone to iron-dependent oxidation and subsequent proteasomal degradation. These proteins included two putative iron-binding proteins, hNFU1 and calreticulin; two proteins involved in metabolism of hydrogen peroxide, peroxiredoxin 2 and superoxide dismutase 1; and several proteins identified in inclusions in neurodegenerative diseases, including HSP27, UCHL1, actin and tropomyosin. Our results indicate that cells can recognize and selectively eliminate iron-dependently oxidized proteins, but unlike IRP2, levels of these proteins do not significantly decrease in iron-treated cells. As iron overload is a feature of many human neurological diseases, further characterization of the process of degradation of iron-dependently oxidized proteins may yield insights into mechanisms of human disease.

Reactive oxygen species are thought to play a role in a variety of physiologic and pathophysiological processes. One possible mediator of oxidant effects at the molecular level is a subset of proteins containing reactive cysteine thiols that can be readily oxidized. The transient incorporation of glutathione into cellular proteins is an established response to oxidant stress and could provide a mechanism for reversible covalent modification in response to reactive oxygen species. To better understand the function of protein S-glutathiolation in vivo, a biotinylated membrane-permeant analogue of glutathione, biotinylated glutathione ethyl ester, was developed and used to detect proteins into which glutathione is incorporated under oxidant stress. Oxidant stress from exogenous hydrogen peroxide or generated in response to TNF-alpha was found to increase incorporation of biotinylated glutathione ethyl ester into several HeLa cell proteins. The identity of two of these proteins was determined by peptide sequencing and mass spectrometric peptide mapping. A 23 kDa S-glutathiolated protein was identified as thioredoxin peroxidase II, a member of the peroxiredoxin family of peroxidases known to play a role in redox-dependent growth factor and cytokine signal transduction. A second, 36 kDa, protein was identified as annexin II. Further investigation revealed a single reactive cysteine in the annexin II tail domain. Deletion of the identified cysteine was found to abolish S-glutathiolation of annexin II. These findings demonstrate a specific posttranslational modification associated with an endogenously generated oxidant stress and suggest a mechanism by which TNF-alpha might selectively regulate protein function in a redox-dependent fashion.

Hydrogen peroxide is a component of cigarette smoke known to be essential for inactivation of alpha(1)-antitrypsin, the primary inhibitor of neutrophil elastase. To establish the molecular basis of the inactivation of alpha(1)-antitrypsin, we determined the sites oxidized by hydrogen peroxide. Two of the nine methionines were particularly susceptible to oxidation. One was methionine 358, whose oxidation was known to cause loss of anti-elastase activity. The other, methionine 351, was as susceptible to oxidation as methionine 358. Its oxidation also resulted in loss of anti-elastase activity, an effect not previously recognized. The equal susceptibility of methionine 358 and methionine 351 to oxidation was confirmed by mass spectrometry. To verify this finding, we produced recombinant alpha(1)-antitrypsins in which one or both of the susceptible methionines were mutated to valine. M351V and M358V were not as rapidly inactivated as wild-type alpha1-antitrypsin, but only the double mutant M351V/M358V was markedly resistant to oxidative inactivation. We suggest that inactivation of alpha(1)-antitrypsin by oxidation of either methionine 351 or 358 provides a mechanism for regulation of its activity at sites of inflammation.

Transcriptional profiling of MnSOD-mediated life-span extension in Drosophila identifies a set of candidate biomarkers of aging, consisting primarily of carbohydrate metabolism and electron transport genes., Background Several interventions increase lifespan in model organisms, including reduced insulin/insulin-like growth factor-like signaling (IIS), FOXO transcription factor activation, dietary restriction, and superoxide dismutase (SOD) over-expression. One question is whether these manipulations function through different mechanisms, or whether they intersect on common processes affecting aging. Results A doxycycline-regulated system was used to over-express manganese-SOD (MnSOD) in adult Drosophila, yielding increases in mean and maximal lifespan of 20%. Increased lifespan resulted from lowered initial mortality rate and required MnSOD over-expression in the adult. Transcriptional profiling indicated that the expression of specific genes was altered by MnSOD in a manner opposite to their pattern during normal aging, revealing a set of candidate biomarkers of aging enriched for carbohydrate metabolism and electron transport genes and suggesting a true delay in physiological aging, rather than a novel phenotype. Strikingly, cross-dataset comparisons indicated that the pattern of gene expression caused by MnSOD was similar to that observed in long-lived Caenorhabditis elegans insulin-like signaling mutants and to the xenobiotic stress response, thus exposing potential conserved longevity promoting genes and implicating detoxification in Drosophila longevity. Conclusion The data suggest that MnSOD up-regulation and a retrograde signal of reactive oxygen species from the mitochondria normally function as an intermediate step in the extension of lifespan caused by reduced insulin-like signaling in various species. The results implicate a species-conserved net of coordinated genes that affect the rate of senescence by modulating energetic efficiency, purine biosynthesis, apoptotic pathways, endocrine signals, and the detoxification and excretion of metabolites.