Oxidation states of the manganese cluster during the flash-induced S-state cycle of the photosynthetic oxygen-evolving complex.

The Mn K-edge x-ray absorption spectra for the pure S states of the tetranuclear Mn cluster of the oxygen-evolving complex of photosystem II during flash-induced S-state cycling have been determined. The relative S-state populations in samples given 0, 1, 2, 3, 4, or 5 flashes were determined from fitting the flash-induced electron paramagnetic resonance (EPR) multiline signal oscillation pattern to the Kok model. The edge spectra of samples given 0, 1, 2, or 3 flashes were combined with EPR information to calculate the pure S-state edge spectra. The edge positions (defined as the zero-crossing of the second derivatives) are 6550.1, 6551.7, 6553.5, and 6553.8 eV for S0, S1, S2, and S3, respectively. In addition to the shift in edge position, the S0--> S1 and S1--> S2 transitions are accompanied by characteristic changes in the shape of the edge, both indicative of Mn oxidation. The edge position shifts very little (0.3 eV) for the S2--> S3 transition, and the edge shape shows only subtle changes. We conclude that probably no direct Mn oxidation is involved in this transition. The proposed Mn oxidation state assignments are as follows: S0 (II, III, IV, IV) or (III, III, III, IV), S1 (III, III, IV, IV), S2 (III, IV, IV, IV), S3 (III, IV, IV, IV).

Thyroid hormone (T3) inhibits ciprofibrate-induced transcription of genes encoding beta-oxidation enzymes: cross talk between peroxisome proliferator and T3 signaling pathways.

Peroxisome proliferators cause rapid and coordinated transcriptional activation of genes encoding peroxisomal beta-oxidation system enzymes by activating peroxisome proliferator-activated receptor (PPAR) isoform(s). Since the thyroid hormone (T3; 3,3',5-triiodothyronine) receptor (TR), another member of the nuclear hormone receptor superfamily, regulates a subset of fatty acid metabolism genes shared with PPAR, we examined the possibility of interplay between peroxisome proliferator and T3 signaling pathways. T3 inhibited ciprofibrate-induced luciferase activity as well as the endogenous peroxisomal beta-oxidation enzymes in transgenic mice carrying a 3.2-kb 5'-flanking region of the rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene fused to the coding region of luciferase. Transfection assays in hepatoma H4-II-E-C3 and CV-1 cells indicated that this inhibition is mediated by TR in a ligand-dependent fashion. Gel shift assays revealed that modulation of PPAR action by TR occurs through titration of limiting amounts of retinoid X receptor (RXR) required for PPAR activation. Increasing amounts of RXR partially reversed the inhibition in a reciprocal manner; PPAR also inhibited TR activation. Results with heterodimerization-deficient TR and PPAR mutants further confirmed that interaction between PPAR and TR signaling systems is indirect. These results suggest that a convergence of the peroxisome proliferator and T3 signaling pathways occurs through their common interaction with the heterodimeric partner RXR.

Yeast flavin-containing monooxygenase is induced by the unfolded protein response

Flavin-containing monooxygenase from yeast (yFMO) carries out the O2- and NADPH-dependent oxidation of biological thiols, including oxidizing glutathione to glutathione disulfide. FMO provides a large fraction of the oxidizing necessary for proper folding of disulfide bond-containing proteins; deletion of the enzyme reduces proper folding of endogenous carboxypeptidase Y by about 40%. The enzyme is not essential to cell viability because other enzymes can generate a significant fraction of the oxidizing equivalents required by the cell. However, yFMO is vital to the yeast response to reductive stress. FMO1 deletion mutants grow poorly under reductive stress, and carboxypeptidase Y activity is less than 10% of that in a stressed wild type. The FMO1 gene appears to be under control of an unfolded protein response element and is inducible by factors, such as reductive stress, that elicit the unfolded protein response. Reductive stress can increase yFMO activity at least 6-fold. This increased activity allows the cell to process endogenous disulfide bond-containing proteins and also to allow correct folding of disulfide-bonded proteins expressed from multicopy plasmids. The unfolded protein response is mediated by the Hac1p transcription factor that mediates virtually all of the induction of yFMO triggered by exogenous reducing agents.

Age-Induced Protein Modifications and Increased Proteolysis in Potato Seed-Tubers1

Long-term aging of potato (Solanum tuberosum) seed-tubers resulted in a loss of patatin (40 kD) and a cysteine-proteinase inhibitor, potato multicystatin (PMC), as well as an increase in the activities of 84-, 95-, and 125-kD proteinases. Highly active, additional proteinases (75, 90, and 100 kD) appeared in the oldest tubers. Over 90% of the total proteolytic activity in aged tubers was sensitive to trans-epoxysuccinyl-l-leucylamido (4-guanidino) butane or leupeptin, whereas pepstatin was the most effective inhibitor of proteinases in young tubers. Proteinases in aged tubers were also inhibited by crude extracts or purified PMC from young tubers, suggesting that the loss of PMC was responsible for the age-induced increase in proteinase activity. Nonenzymatic oxidation, glycation, and deamidation of proteins were enhanced by aging. Aged tubers developed “daughter” tubers that contained 3-fold more protein than “mother” tubers, with a polypeptide profile consistent with that of young tubers. Although PMC and patatin were absent from the older mother tubers, both proteins were expressed in the daughter tubers, indicating that aging did not compromise the efficacy of genes encoding PMC and patatin. Unlike the mother tubers, proteinase activity in daughter tubers was undetectable. Our results indicate that tuber aging nonenzymatically modifies proteins, which enhances their susceptibility to breakdown; we also identify a role for PMC in regulating protein turnover in potato tubers.

Pathogen-Induced Changes in the Antioxidant Status of the Apoplast in Barley Leaves

Leaves of two barley (Hordeum vulgare L.) isolines, Alg-R, which has the dominant Mla1 allele conferring hypersensitive race-specific resistance to avirulent races of Blumeria graminis, and Alg-S, which has the recessive mla1 allele for susceptibility to attack, were inoculated with B. graminis f. sp. hordei. Total leaf and apoplastic antioxidants were measured 24 h after inoculation when maximum numbers of attacked cells showed hypersensitive death in Alg-R. Cytoplasmic contamination of the apoplastic extracts, judged by the marker enzyme glucose-6-phosphate dehydrogenase, was very low (less than 2%) even in inoculated plants. Dehydroascorbate, glutathione, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were present in the apoplast. Inoculation had no effect on the total foliar ascorbate pool size or the redox state. The glutathione content of Alg-S leaves and apoplast decreased, whereas that of Alg-R leaves and apoplast increased after pathogen attack, but the redox state was unchanged in both cases. Large increases in foliar catalase activity were observed in Alg-S but not in Alg-R leaves. Pathogen-induced increases in the apoplastic antioxidant enzyme activities were observed. We conclude that sustained oxidation does not occur and that differential strategies of antioxidant response in Alg-S and Alg-R may contribute to pathogen sensitivity.

Repair of hydantoins, one electron oxidation product of 8-oxoguanine, by DNA glycosylases of Escherichia coli

8-Oxoguanine (8-oxoG), induced by reactive oxygen species and arguably one of the most important mutagenic DNA lesions, is prone to further oxidation. Its one-electron oxidation products include potentially mutagenic guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) because of their mispairing with A or G. All three oxidized base-specific DNA glycosylases of Escherichia coli, namely endonuclease III (Nth), 8-oxoG-DNA glycosylase (MutM) and endonuclease VIII (Nei), excise Gh and Sp, when paired with C or G in DNA, although Nth is less active than the other two. MutM prefers Sp and Gh paired with C (kcat/Km of 0.24–0.26 min–1 nM–1), while Nei prefers G over C as the complementary base (kcat/Km – 0.15–0.17 min–1 nM–1). However, only Nei efficiently excises these paired with A. MutY, a 8-oxoG·A(G)-specific A(G)-DNA glycosylase, is inactive with Gh(Sp)·A/G-containing duplex oligonucleotide, in spite of specific affinity. It inhibits excision of lesions by MutM from the Gh·G or Sp·G pair, but not from Gh·C and Sp·C pairs. In contrast, MutY does not significantly inhibit Nei for any Gh(Sp) base pair. These results suggest a protective function for MutY in preventing mutation as a result of A (G) incorporation opposite Gh(Sp) during DNA replication.

Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae.

The cause for death after lethal heat shock is not well understood. A shift from low to intermediate temperature causes the induction of heat-shock proteins in most organisms. However, except for HSP104, a convincing involvement of heat-shock proteins in the development of stress resistance has not been established in Saccharomyces cerevisiae. This paper shows that oxidative stress and antioxidant enzymes play a major role in heat-induced cell death in yeast. Mutants deleted for the antioxidant genes catalase, superoxide dismutase, and cytochrome c peroxidase were more sensitive to the lethal effect of heat than isogenic wild-type cells. Overexpression of catalase and superoxide dismutase genes caused an increase in thermotolerance. Anaerobic conditions caused a 500- to 20,000-fold increase in thermotolerance. The thermotolerance of cells in anaerobic conditions was immediately abolished upon oxygen exposure. HSP104 is not responsible for the increased resistance of anaerobically grown cells. The thermotolerance of anaerobically grown cells is not due to expression of heat-shock proteins. By using an oxidation-dependent fluorescent molecular probe a 2- to 3-fold increase in fluorescence was found upon heating. Thus, we conclude that oxidative stress is involved in heat-induced cell death.

Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity.

Tumor necrosis factor (TNF) is selectively cytotoxic to some types of tumor cells in vitro and exerts antitumor activity in vivo. Reactive oxygen intermediates (ROIs) have been implicated in the direct cytotoxic activity of TNF. By using confocal microscopy, flow cytometry, and the ROI-specific probe dihydrorhodamine 123, we directly demonstrate that intracellular ROIs are formed after TNF stimulation. These ROIs are observed exclusively under conditions where cells are sensitive to the cytotoxic activity of TNF, suggesting a direct link between both phenomena. ROI scavengers, such as butylated hydroxyanisole, effectively blocked the formation of free radicals and arrested the cytotoxic response, confirming that the observed ROIs are cytocidal. The mitochondrial glutathione system scavenges the major part of the produced ROIs, an activity that could be blocked by diethyl maleate; under these conditions, TNF-induced ROIs detectable by dihydrorhodamine 123 oxidation were 5- to 20-fold higher.

The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the Fc epsilon RII/CD23 surface antigen.

Serum IgE concentrations and the expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) are increased in cutaneous leishmaniasis or after immune challenge with Leishmania antigens. In vitro, the ligation of CD23 by IgE-anti-IgE immune complexes (IgE-IC) or by anti-CD23 monoclonal antibody (mAb) induces nitric oxide (NO) synthase and the generation of various cytokines by human monocytes/macrophages. The present study shows that IgE-IC, via CD23 binding, induce intracellular killing of Leishmania major in human monocyte-derived macrophages through the induction of the L-arginine:NO pathway. This was demonstrated by increased generation of nitrite (NO2-), the stable oxidation product of NO, and by the ability of NG-monomethyl-L-arginine to block both NO generation and parasite killing. A similar NO-dependent effect was observed with interferon gamma-treated cells. Tumor necrosis factor alpha is involved in this process, since both the induction of NO synthase and the killing of parasites caused by anti-CD23 mAb were inhibited by an anti-tumor necrosis factor alpha mAb. Treatment of noninfected CD23+ macrophages with IgE-IC provided protection against subsequent in vitro infection of these cells by Leishmania major promastigotes. Thus, IgE-IC promote killing of L. major by inducing NO synthase in human macrophages.