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Abstract: The higher resistance of stationary-phase Saccharomyces cerevisiae to H2O2 when compared with exponential phase is well characterized, but the molecular mechanisms underlying it remain mostly unknown. By applying the steady-state H2O2-delivery model, we show that (a) cellular permeability to H2O2 is five times lower in stationary – than in exponential phase; (b) cell survival to H2O2 correlates with H2O2 cellular gradients for a variety of cells; and, (c) cells in stationary phase are predicted to be more susceptible to intracellular H2O2 than in exponential phase. In conclusion, limiting H2O2 diffusion into cells is a key protective mechanism against extracellular H2O2. [Copyright &y& Elsevier]

The aim of this study was to evaluate the involvement of oxidative stress in glutamate-evoked transglutaminase (TGase) upregulation in astrocyte cultures (14 DIV). A 24 h exposure to glutamate caused a dose-dependent depletion of glutathione intracellular content and increased the ROS production in cell cultures. These effects were receptor-mediated, as demonstrated by inhibition with GYKI 52466. The pre-incubation with glutathione ethyl ester or cysteamine recovered oxidative status and was effective in significantly reducing glutamate-increased tissue TGase. These data suggest that tissue TGase upregulation may be part of a biochemical response to oxidative stress induced by a prolonged exposure of astrocyte cultures to glutamate. [Copyright &y& Elsevier]

Abstract: Repression of activation of c-Jun N-terminal kinase (JNK) participates in the anti-apoptotic effect of nuclear factor-κB (NF-κB) in TNFα-treated Ewing sarcoma cells. As oxidative stress is one of the most prominent activators of JNK, we investigated the relationship between TNFα-induced NF-κB activation and the control of oxidative stress. Inhibition of NF-κB activation resulted in an increase in TNFα-induced ROS production, lipid peroxidation and protein oxidation. Those ROS and lipid peroxides were both involved in TNFα-induced apoptosis, whereas only ROS elevation triggered sustained JNK activation. TNFα increased the level of two antioxidant enzymes, thioredoxin and manganese superoxide dismutase by an NF-κB-dependent mechanism. Inhibition of expression or activity of these enzymes sensitized cells to TNFα-induced apoptosis, indicating their functional role in protection from cell death. Thus, agents that inhibit activities of these enzymes may prove helpful in the treatment of Ewing tumors. [Copyright &y& Elsevier]

The redox regulator thioredoxin-1 (Trx-1) is required for the redox potential of the cell and exerts important functions in cell growth and apoptosis. Severe oxidative stress has been implicated in the oxidation of proteins and cell death. However, the role of low doses of reactive oxygen species (ROS) is poorly understood. Here, we show that 10 and 50 μM H2O2 and short-term exposure to shear stress significantly increased Trx-1 mRNA and protein levels in endothelial cells. Since it is known that Trx-1 exerts anti-apoptotic functions, we next investigated whether low doses of ROS can inhibit basal and serum-depletion induced endothelial cell apoptosis. Indeed, treatment of endothelial cells with 10 and 50 μM H2O2 significantly reduced apoptosis induction. Reduction of Trx-1 expression using an antisense oligonucleotide approach resulted in the induction of apoptosis and abolished the inhibitory effect of low doses of H2O2. Taken together, our results demonstrate that low doses of ROS act as signaling molecules and exert anti-apoptotic functions in endothelial cells via upregulation of the redox-regulator Trx-1. [Copyright &y& Elsevier]

Mutations in mitochondrial DNA (mtDNA) cause excessive production of mitochondrial reactive oxygen species (ROS) and shorten animal life span. We examined the mechanisms responsible for removal of mitochondria with deleterious mtDNA mutations by autophagy. Incubation of primary cells and cell lines in the absence of serum promotes autophagy of mitochondria with deleterious mtDNA mutations but spares their normal counterparts. The effect of serum withdrawal on the autophagy of dysfunctional mitochondria is prevented by the addition of IGF-1. As a result of the elimination of mitochondria with deleterious mutations, excessive ROS production, characteristic of dysfunctional mitochondria, is greatly reduced. Mitochondrial autophagy shares a common mechanism with mitochondrial-induced cell apoptosis, including mitochondrial transition pore formation and increased ROS production. [Copyright &y& Elsevier]

Identification of mechanisms of modulation of the TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis is important for its potential use in anticancer therapy. Ethanol can induce cell death in vitro and in vivo by different signalling pathways. Its effect in combination with death ligands is unknown. We investigated how ethanol modulates the effects of TRAIL in colon cancer cells. After combined TRAIL and ethanol treatment, a potentiation of caspase-8, -9, -3 activation, a proapoptotic Bid protein cleavage, a decrease of mitochondrial membrane potential, a complete poly(ADP)ribose polymerase cleavage, and disappearance of antiapoptotic Mcl-1 protein were demonstrated. Ethanol acts as a potent agent sensitizing colon cancer cells to TRAIL-induced apoptosis. [Copyright &y& Elsevier]

The physiological role of the prion protein is largely unknown. Here, clustering of prion at the surface of GT1-7 cells was observed upon anti-prion antibody treatments. This clustering was associated with a rapid and transient phosphorylation of the mitogen activated protein kinases (MAPKs) extracellular receptor kinases 1 and 2 (ERK1/2), and also of the microtubule-destabilizing protein stathmin at serine 16. The specificity of this antibody-mediated activation was ascertained by its inhibition by prion small interfering RNA. The phosphorylation of ERK1/2 but not that of stathmin was abolished by the MAPK/ERK kinase 1 inhibitor U0126, whereas both signaling pathways were blocked by the specific inhibitor of the epidermal growth factor receptor AG1478, suggesting the likely recruitment of this receptor upon prion clustering. [Copyright &y& Elsevier]

The purpose of this study was to investigate whether, after oxidative injury in vivo, liver mitochondrial phospholipids suffered from structural defects similar to those we have previously observed after either chemical oxidation or respiration state IV incubation of isolated mitochondria in vitro. Oxidative injury of the liver was simulated by endogastric administration of CCl4 to rats in variable amounts for different times, under various conditions. Measurements of the phospholipid bilayer packing order were carried out by electron paramagnetic resonance (EPR) spectrometry of oriented planar samples of phospholipids extracted from liver mitochondria, spin labeled with 5-doxylstearoyl-lecithin. Disordering of the bilayer was revealed by the anisotropy loss of EPR spectra and reached a maximum value 4.5 h after CCl4 administration, vanishing thereafter. The observed disorder also increased with the amount of CCl4 administered, showing distinct dose-dependence, while administration of resveratrol soon after carbon tetrachloride decreased bilayer disordering by 50%. On the contrary, the order parameter S of spin labeled lecithin in isolated mitochondrial membranes from intoxicated rats revealed no change in membrane fluidity after oxidative stress. It is concluded that the phospholipid damage leading to disturbed bilayer geometry after oxidative attack already observed in model membranes and in isolated mitochondria in vitro also occurs in a simulated pathological state in vivo, indicating its possible occurrence also in real oxidative stress-linked pathologies as a contribution to the onset/sustaining of related diseases. [Copyright &y& Elsevier]

Serum withdrawal rapidly induces apoptosis in rat 423-cells, while addition of bFGF results in cell survival. However, surviving cells initially display morphological changes characteristic for apoptotic cells and even process caspases. Active caspase-3 was detected at the single-cell level in those finally bFGF-rescued cells, while mitochondrial integrity was maintained. Generation of cleavage products of caspase targets was confirmed in surviving cells. Proteome analysis indicated multi-faceted survival activities of bFGF including upregulation of inhibitor-of-apoptosis and heat shock protein family members directly interfering with caspases. Our data suggest that the “point-of-no-return” in death-induced cells has to be moved downstream of activated caspase-3. [Copyright &y& Elsevier]

Point mutations and deletions in mitochondrial DNA (mtDNA) accumulate as a result of oxidative stress, including ionizing radiation. As a result, dysfunctional mitochondria suffer from a decline in oxidative phosphorylation and increased release of superoxides and other reactive oxygen species (ROS). Through this mechanism, mitochondria have been implicated in a host of degenerative diseases. Associated with this type of damage, and serving as a marker of total mtDNA mutations and deletions, the accumulation of a specific 4977-bp deletion, known as the common deletion (Δ-mtDNA4977), takes place. The Δ-mtDNA4977 has been reported to increase with age and during the progression of mitochondrial degeneration. The purpose of this study was to investigate whether ionizing radiation induces the formation of the common deletion in a variety of human cell lines and to determine if it is associated with cellular radiosensitivity. Cell lines used included eight normal human skin fibroblast lines, a radiosensitive non-transformed and an SV40 transformed ataxia telangiectasia (AT) homozygous fibroblast line, a Kearns Sayre Syndrome (KSS) line known to contain mitochondrial deletions, and five human tumor lines. The Δ-mtDNA4977 was assessed by polymerase chain reaction (PCR). Significant levels of Δ-mtDNA4977 accumulated 72 h after irradiation doses of 2, 5, 10 or 20 Gy in all of the normal lines with lower response in tumor cell lines, but the absolute amounts of the induced deletion were variable. There was no consistent dose–response relationship. SV40 transformed and non-transformed AT cell lines both showed significant induction of the deletion. However, the five tumor cell lines showed only a modest induction of the deletion, including the one line that was deficient in DNA damage repair. No relationship was found between sensitivity to radiation-induced deletions and sensitivity to cell killing by radiation. [Copyright &y& Elsevier]

The mitochondria-targeted antioxidant MitoQ comprises a ubiquinol moiety covalently attached through an aliphatic carbon chain to the lipophilic triphenylphosphonium cation. This cation drives the membrane potential-dependent accumulation of MitoQ into mitochondria, enabling the ubiquinol antioxidant to prevent mitochondrial oxidative damage far more effectively than untargeted antioxidants. We sought to fine-tune the hydrophobicity of MitoQ so as to control the extent of its membrane binding and penetration into the phospholipid bilayer, and thereby regulate its partitioning between the membrane and aqueous phases within mitochondria and cells. To do this, MitoQ variants with 3, 5, 10 and 15 carbon aliphatic chains were synthesised. These molecules had a wide range of hydrophobicities with octan-1-ol/phosphate buffered saline partition coefficients from 2.8 to 20 000. All MitoQ variants were accumulated into mitochondria driven by the membrane potential, but their binding to phospholipid bilayers varied from negligible for MitoQ3 to essentially total for MitoQ15. Despite the span of hydrophobicites, all MitoQ variants were effective antioxidants. Therefore, it is possible to fine-tune the degree of membrane association of MitoQ and other mitochondria targeted compounds, without losing antioxidant efficacy. This indicates how the uptake and distribution of mitochondria-targeted compounds within mitochondria and cells can be controlled, thereby facilitating investigations of mitochondrial oxidative damage. [Copyright &y& Elsevier]

Chemotherapeutic drugs that inhibit the synthesis of DNA precursor thymidine triphosphate cause apoptosis, although the mechanism underlying this process remains rather unknown. Here, we describe thymineless death of human myeloid leukemia U937 cells treated with the thymidylate-synthase inhibitor 5′-fluoro-2′-deoxyuridine (FUdR). This apoptotic process was shown to be independent of p53, reactive oxygen species generation and CD95 activation. Caspases were activated downstream of cytochrome c but upstream of mitochondrial depolarization. Furthermore, FUdR-induced apoptosis required the presence of glucose in the culture medium at a step upstream of the release of cytochrome c from mitochondria. [Copyright &y& Elsevier]

In the present study, we characterized hydrogen peroxide (H2O2)-induced cell apoptosis and related cell signaling pathways in cultured embryonic neural stem/progenitor cells (NS/PCs). Our data indicated that H2O2 induced acute cell apoptosis in NS/PC in concentration- and time-dependent manners and selectively, it transiently increased PI3K-Akt and Mek-Erk1/2 in a dose-dependent manner. Inhibition of PI3K-Akt with wortmannin, a PI3-K inhibitor, was found to significantly increase H2O2-induced acute apoptosis and dramatically decrease basal pGSK3β levels. The level of pGSK3β remained unchanged with H2O2 exposure. We conclude that the transient activation of PI3K-Akt signaling delays the H2O2-induced acute apoptosis in cultured NS/PCs in part through maintaining the basal pGSK3β level and activating other downstream effectors. [Copyright &y& Elsevier]

We studied the regulation of glucose-6-phosphate dehydrogenase (G6PD) gene expression by chronic hypoxia. G6PD mRNA level and activity were increased in PC12 cells by hypoxia in a dose- and time-dependent manner. Cobalt chloride and dimethyloxalylglycine, which can mimic hypoxia, also activated G6PD gene expression. Interestingly, hypoxia-induced G6PD expression followed a time course much slower than that of phosphoglycerate kinase 1 (PGK1), a hypoxia-inducible factor (HIF)-dependent glycolytic enzyme. Hypoxic-G6PD induction was almost negligible in non-excitable Buffalo rat liver cells, although in these cells PGK1 was strongly upregulated by low PO2. Furthermore, G6PD but not PGK1 induction was blocked by the antioxidants glutathione and N-acetylcysteine. These results suggest the dependence of G6PD gene expression on HIF and intracellular redox status and the differential hypoxic regulation of glucose-metabolizing enzymes. [Copyright &y& Elsevier]

Oxidants are known to modulate cell proliferation and apoptosis, and induce synthesis of growth factors that play an important role in tumor growth and invasion. Antioxidant enzymes and thiol proteins regulating cellular redox state constitute the major cellular protection against oxidants. Consequently, they are also associated both with carcinogenesis and tumor progression. Superoxide dismutases, glutamate cysteine ligase, catalase, thioredoxins and peroxiredoxins, which are the most important of these enzymes, are expressed in lung malignancies, and especially in pleural mesothelioma. This has consequences not only for tumor behavior but also for resistance of tumor cells to cytotoxic drugs and radiation. [Copyright &y& Elsevier]

Stimulation of cells with tumor necrosis factor-α (TNF-α) results in the increase in generation of H2O2 in mitochondria that leads to apoptosis. The effect of H2O2 produced by TNF-α on the redox status of selenocysteine (SeCys) residue essential for mitochondrial thioredoxin reductase (TrxR2) was investigated in HeLa cells. TNF-α caused accumulation of oxidized TrxR2 with a thioselenide bond. The conditional induction of SeCys-deficient TrxR2 resulted in the increased production of H2O2 and apoptosis. These results suggest that the SeCys residue of TrxR2 plays a critical role in cell survival by serving as an electron donor for Trx-II and subsequent peroxiredoxin-III, which is a primary line of defense against H2O2 in mitochondria. [Copyright &y& Elsevier]

4-Hydroxyhexenal (HHE) is known to affect redox balance during aging, included are vascular dysfunctions. To better understand vascular abnormality through the molecular alterations resulting from HHE accumulation in aging processes, we set out to determine whether up-regulation of mitogen-activated protein kinase (MAPK) by HHE is mediated through nuclear factor kappa B (NF-κB) activation in endothelial cells. HHE induced NF-κB activation by inhibitor of κB (IκB) phosphorylation via the IκB kinase (IKK)/NF-κB inducing kinase (NIK) pathway. HHE increased the activity of p38 MAPK and extracellular signal regulated kinase (ERK), but not c-jun NH2-terminal kinase, indicating that p38 MAPK and ERK are closely involved in HHE-induced NF-κB transactivation. Pretreatment with ERK inhibitor PD98059, and p38 MAPK inhibitor SB203580, attenuated the induction of p65 translocation, IκB phosphorylation, and NF-κB luciferase activity. These findings strongly suggest that HHE induces NF-κB activation through IKK/NIK pathway and/or p38 MAPK and ERK activation associated with oxidative stress in endothelial cells. [Copyright &y& Elsevier]

Bax-induced lethality in yeast is accompanied by morphological changes in mitochondria, giving rise to a reduced number of swollen tubules. Although these changes are completely abolished upon coexpression of the Bax inhibitor, Bcl-2, coexpression of Bax with Bax inhibiting-glutathione S-transferase (BI-GST) leads to aggregation, but not fusion of the mitochondria. In addition, Bax affects the integrity of yeast vacuoles, resulting in the disintegration and eventual loss of the organelles, and the disruption of intracellular protein traffic. While Bcl-2 coexpression only partially corrects this phenotype, coexpression of BI-GST fully restores the organelles, indicating a different mode of protection exerted by Bcl-2 and BI-GST. [Copyright &y& Elsevier]

Three mitogen activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2, MK2) interacting proteins were identified using a yeast two-hybrid approach. ShcA, a signaling phospho-protein, human polyhomeotic 2 (HPH2), a transcriptional regulator, and highly similar to smoothelin (HSTS), which is related to the cytoskeletal associated protein smoothelin, interact specifically with MK2. The interaction of MK2 with the 66 kDa isoform of ShcA, p66ShcA, and HPH2 was confirmed using co-immunoprecipitation. MK2 is activated with p66ShcA co-expression and p66ShcA is an in vitro substrate for MK2, further demonstrating their association and suggesting a biological role for p66Shc in MK2 activation. [Copyright &y& Elsevier]

In contrast to other oxidative modifications of amino acids, methionine sulfoxide can be enzymatically reduced back to methionine in proteins by the peptide methionine sulfoxide reductase system, composed of MsrA and MsrB. The expression of MsrA and one member of the MsrB family, hCBS-1, was analyzed during replicative senescence of WI-38 human fibroblasts. Gene expression decreased for both enzymes in senescent cells compared to young cells, and this decline was associated with an alteration in catalytic activity and the accumulation of oxidized proteins during senescence. These results suggest that downregulation of MsrA and hCBS-1 can alter the ability of senescent cells to cope with oxidative stress, hence contributing to the age-related accumulation of oxidative damage. [Copyright &y& Elsevier]

We studied the effect of ageing on the mRNA levels of mitochondria-encoded polypeptides in human platelets. We used quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to investigate the expression of selected cytochrome c oxidase (COX) genes (subunits I and III) and Complex I genes (subunits reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ND)1 and ND5) in platelets from young and aged healthy subjects. Northern blot analysis confirmed the PCR results. COX I expression is higher than that of COX III in both young and aged platelets. A significant increase of transcripts for Complex I was found during ageing. On the contrary, the mRNA levels of the two COX subunits did not significantly vary during ageing. [Copyright &y& Elsevier]

Glutathione is the most abundant non-protein thiol and a major source of reducing equivalents in eukaryotes. We examined the role of glutathione in Candida albicans by the disruption of γ-glutamylcysteine synthetase (GCS1), an essential enzyme in glutathione biosynthesis. The gcs1/gcs1 null mutants exhibited glutathione auxotrophy, which could be rescued by supplementing with reduced and oxidized glutathione and γ-glutamylcysteine. When the mutants were depleted of glutathione, they showed typical markers of apoptosis. These results suggest that glutathione itself is an essential metabolite and C. albicans lacking GCS1 undergoes apoptosis. [Copyright &y& Elsevier]

Redox factor-1 (Ref-1) is a ubiquitously expressed protein with proven roles as a modulator of redox-sensitive transcription, and as an endonuclease in the base excision repair pathway of oxidatively damaged DNA. Although Ref-1 is induced by a variety of oxidative stress and protects cells against oxidative stress, the function of Ref-1 in regulating nitric oxide (NO) synthesis has not been elucidated to date. We investigated the role of Ref-1 in regulating NO synthesis and NO-mediated apoptosis employing adenoviral-mediated overexpression of Ref-1 in lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells. LPS treatment produced NO synthesis and NO-mediated apoptosis. Forced overexpression of Ref-1 suppressed LPS-stimulated NO synthesis. In parallel with this, Ref-1 also mitigated alteration of inducible NO synthase expression and NO-mediated apoptosis. Our findings suggest that Ref-1 is implicated in protection against cell death resulting from oxidative stimuli containing NO. [Copyright &y& Elsevier]

The impact of intensity of glycolysis and oxidative phosphorylation on death of photosensitized murine hepatoma MH22 cells in vitro has been investigated. Cells photosensitized with meso-tetra(4-sulfonatophenyl)-porphine localized to lysosomes died mostly by necrosis, and the mode of cell death did not depend on the energy metabolism. Photosensitization with 5-aminolevulinic acid-stimulated endogenous porphyrins localized mainly in mitochondria or 5,10,15,20-tetrakis(m-hydroxyphenyl)-chlorine localized to cell membranes, including mitochondria, led to cell death mostly by apoptosis. In this case, the mode of cell death depended on the medium: under conditions unfavorable to glycolysis the ratio apoptosis/necrosis decreased significantly. [Copyright &y& Elsevier]

Preadipocytes are present and can proliferate to increase fat mass throughout adult life. The importance of mitochondria in these cells has never been investigated, although we recently reported that mitochondrial oxidative metabolism is non-negligible in white preadipocytes. Mitochondrial reactive oxygen species generation is intimately associated with respiratory chain function. An increasing number of reports support their role as signalling molecules. The aim of this work was to study the effects of mitochondrial reactive oxygen species on proliferation of white preadipocytes. Rotenone and oligomycin, inhibitors of complex I and of ATP synthase respectively, increased H2O2 and inhibited cell growth of preadipocytes (without inducing necrosis or apoptosis). These effects were partly prevented by addition of radical scavengers. A chemical uncoupler had opposite effects on reactive oxygen species generation and cell growth. Propofol, which inhibits complex I but also scavenges free radicals, had effects similar to those of the uncoupler on both parameters. Thus, mitochondrial reactive oxygen species can influence development of adipose tissue by affecting the size of the white preadipocyte pool. [Copyright &y& Elsevier]

Mitochondria are known to play a fundamental role in apoptosis by releasing apoptogenic molecules such as cytochrome c into the cytoplasm, thereby sequentially activating initiator caspase-9. However, the mechanisms of cytochrome c release or caspase-9 activation in response to hypoxia are unclear. In this report, we show that caspase-9 is activated by reactive oxygen species (ROS) without involvement of cytochrome c release in hypoxic injury. In addition, activated caspase-9 induces permeability transition (PT)-independent cytochrome c release, suggesting that caspase-9 may disrupt mitochondrial diffusion limit of cytochrome c and serve to amplify further release of cytochrome c. [Copyright &y& Elsevier]

Neopterin is synthesized by human monocyte-derived macrophages primarily upon stimulation with the cytokine interferon-γ. We studied the influence of neopterin on the generation of reactive oxygen species (ROS) in human peripheral blood neutrophils. Radical formation was measured using a biochemiluminometer. Neutrophils were isolated from peripheral blood of healthy donors. The generation of ROS by neutrophils suspended in Earl’s solution (pH=7.4) at 37°C was investigated by monitoring of chemiluminescence using luminol and lucigenin as light emitters. Neopterin induced chemiluminescence in suspensions of neutrophils in the presence of luminol, but not of lucigenin. Neopterin affected only adhesive cells. Addition of neopterin into the suspension of the cells involving D-mannitol, L-histidine and diazabicyclo[2.2.2]octane (DABCO) decreased luminol-dependent chemiluminescence (LDCL) of the neutrophils. The action of superoxide dismutase (SOD) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) reduced neopterin-induced LDCL of neutrophils. Data suggest that neutrophils respond on exposure to neopterin with additional generation of singlet oxygen, hydroxyl radical and nitric oxide by nicotinamide adenine dinucleotide phosphate (NADPH)-independent pathways. [Copyright &y& Elsevier]

In parasitism, Plasmodium falciparum is exposed to toxic reactive oxygen species and reactive nitrogen species (RNS). Peroxiredoxins (Prx) are ubiquitously distributed antioxidant enzymes. In bacteria and yeast, Prx have also been implicated in detoxifying RNS. Here, we used a gene targeting strategy to investigate the physiological role of 2-Cys Prx of P. falciparum, PfTPx-1, in living parasite cells. The PfTPx-1-null parasite line was more sensitive to paraquat (a superoxide donor) and sodium nitroprusside (a nitric oxide donor), than wildtype. These findings suggest that PfTPx-1 protects the parasite cells from oxidative and nitrosative stresses. [Copyright &y& Elsevier]

Exposure of PC12 cells to 100 μM peroxynitrite promotes phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) sensitive to PD98059 or U0126. At higher concentrations, however, ERK1/2 phosphorylation was prevented by U0126 and increased by PD98059 via a U0126-sensitive mechanism. PD98059, unlike U0126, enhanced the peroxynitrite-dependent formation of reactive oxygen species (ROS). These results, along with others obtained using respiratory chain inhibitors and respiration-deficient cells, lead to the conclusion that PD98059, while effectively inhibiting the peroxynitrite-induced Raf/MEK signaling leading to ERK1/2 phosphorylation, promotes an enforced mitochondrial formation of ROS inducing ERK1/2 phosphorylation via a Raf-1-independent/MEK-dependent mechanism. [Copyright &y& Elsevier]

Post-hypoxic injuries in plants are primarily caused by bursts of reactive oxygen species and acetaldehyde. In agreement with previous studies, we found accumulations of acetaldehyde in rice during re-aeration following submergence. During re-aeration, acetaldehyde-oxidizing aldehyde dehydrogenase (ALDH) activity increased, thereby causing the acetaldehyde content to decrease in rice. Interestingly, re-aerated rice plants showed an intense mitochondrial ALDH2a protein induction, even though ALDH2a mRNA was submergence induced and declined upon re-aeration. This suggests that rice ALDH2a mRNA is accumulated in order to quickly metabolize acetaldehyde that is produced upon re-aeration. [Copyright &y& Elsevier]

Although initially viewed as housekeeping enzymes, research over the last 15 years has revealed that the protein tyrosine phosphatases (PTPs) are critical regulators of tyrosine phosphorylation-dependent signaling events and may represent novel targets for therapeutic intervention in a variety of human diseases. In this review I will describe some of the key advances in the characterization of the structure, regulation and function of the prototypic PTP, PTP1B, and illustrate how our understanding of the properties of this enzyme has revealed principles that apply to the PTP family as a whole. [Copyright &y& Elsevier]

Human peroxiredoxin 5 is a recently discovered mitochondrial, peroxisomal and cytosolic thioredoxin peroxidase able to reduce hydrogen peroxide and alkyl hydroperoxides. To gain insight into peroxiredoxin 5 antioxidant role in cell protection, we investigated the resistance of yeast cells expressing human peroxiredoxin 5 in mitochondria or in the cytosol against oxidative stress induced by paraquat. The herbicide paraquat is a redox active drug known to generate superoxide anions in mitochondria and the cytosol of yeast and mammalian cells leading to the formation of several reactive oxygen species. Here, we report that mitochondrial and cytosolic human peroxiredoxin 5 protect yeast cells from cytotoxicity and lipid peroxidation induced by paraquat. [Copyright &y& Elsevier]

We have studied changes in hepatic mitochondrial efficiency induced by 24-h fasting or acclimation at 29°C, two conditions of reduced thermogenesis. Basal and palmitate-induced proton leak, which contribute to mitochondrial efficiency, are not affected after 24-h fasting, when serum free triiodothyronine decreases significantly and serum free fatty acids increase significantly. In rats at 29°C, in which serum free triiodothyronine and fatty acids decrease significantly, basal proton leak increases significantly, while no variation is found in palmitate-induced proton leak. The present results indicate that mitochondrial efficiency in the liver is not related to a physiological decrease in whole body thermogenesis. [Copyright &y& Elsevier]

In this study we show that micromolar Ca2+ concentrations (>10 μM) strongly stimulate the release of reactive oxygen species (ROS) in rotenone-treated isolated rat forebrain mitochondria. Ca2+-stimulated mitochondrial ROS release was associated with membrane lipid peroxidation and was directly correlated with the degree of complex I inhibition by rotenone. On the other hand, Ca2+ did not increase mitochondrial ROS release in the presence of the complex I inhibitor 1-methyl-4-phenylpyridinium. Cyclosporin A had no effect on Ca2+-stimulated mitochondrial ROS release in the presence of rotenone, indicating that mitochondrial permeability transition is not involved in this process. We hypothesized that Ca2+-induced mitochondrial oxidative stress associated with partial inhibition of complex I may be an important factor in neuronal cell death observed in the neurodegenerative disorder Parkinson’s disease. [Copyright &y& Elsevier]

Thyroid hormone (TH/T3) exerts many of its effects on energy metabolism by affecting gene transcription. However, although this is an important target for T3, only a limited number of T3-responsive genes have been identified and studied. Among these, the genes for uncoupling proteins (UCPs) have attracted the interest of scientists. Although the role of UCP1 seems quite well established, uncertainty surrounds the physiological function of the recently discovered UCP1 analogs, UCP2 and UCP3. The literature suggests that T3 affects both the expression and the activity of each of these UCPs but further studies are needed to establish whether the mechanisms activated by the hormone are the same. Recently, because of their larger range of expression, much attention has been devoted to UCP2 and UCP3. Most detailed studies on the involvement of these proteins as mediators of the effects of T3 on metabolism have focused on UCP3 because of its expression in skeletal muscle. T3 seems to be unique in having the ability to stimulate the expression and activity of UCP3 and this may be related to the capacity of T3 to activate the integrated biochemical processes linked to UCP activity, such as those related to fatty acids, coenzyme Q and free radicals. [Copyright &y& Elsevier]

Employing a spin trapping agent combined with electron spin resonance spectroscopy, we were able to capture reactive oxygen species (ROS) in living hepatoma cells and first found that the trapped ROS was superoxide anion (O2⋅−). O2⋅− suppressed by treatment with diphenylene iodonium, a flavoprotein inhibitor, was generated by the flavoprotein-containing NADPH-oxidase complex. Applying endogenous/exogenous pro-oxidant or antioxidant causes different redox states in hepatoma cells. Akt activity and cell growth were significantly stimulated by treating hepatoma cells with low concentration of ROS, which could be abolished by adding antioxidants. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (0.15 μM) inhibited Akt phosphorylation induced by ROS. Our results indicate that hepatoma cell growth is ROS-dependent, and fluctuation of the intracellular redox state may regulate hepatoma cell growth through Akt phosphorylation and the PI3K/Akt pathway, resulting in a broad array of responses from cellular proliferation to apoptosis. [Copyright &y& Elsevier]

A tridecapeptide (MITLAIPVNKPGR) that stimulates phagocytosis of human neutrophils was isolated from a trypsin digest of soybean proteins. This peptide is derived from the soybean β-conglycinin α′ subunit and was named soymetide-13. The N-terminal methionine residue of soymetide-13 is essential for its activity, and removal of C-terminal residues revealed that soymetide-4 (MITL) is the minimal structure required for phagocytosis stimulation. Although they are not formylated at their N-termini, soymetides have a weak affinity for the N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor and their phagocytosis-stimulating activity is inhibited by the fMLP antagonist Boc-MLP. Interestingly, soymetide-4 promotes tumor necrosis factor α production at a higher level than soymetide-13 following oral administration in mice. [Copyright &y& Elsevier]

Wheat gliadin and other cereal prolamins have been said to be involved in the pathogenic damage of the small intestine in celiac disease via the apoptosis of epithelial cells. In the present work we investigated the mechanisms underlying the pro-apoptotic activity exerted by gliadin-derived peptides in Caco-2 intestinal cells, a cell line which retains many morphological and enzymatic features typical of normal human enterocytes. We found that digested peptides from wheat gliadins (i) induce apoptosis by the CD95/Fas apoptotic pathway, (ii) induce increased Fas and FasL mRNA levels, (iii) determine increased FasL release in the medium, and (iv) that gliadin digest-induced apoptosis can be blocked by Fas cascade blocking agents, i.e. targeted neutralizing antibodies. This favors the hypothesis that gliadin could activate an autocrine/paracrine Fas-mediated cell death pathway. Finally, we found that (v) a small peptide (1157 Da) from durum wheat, previously proposed for clinical practice, exerted a powerful protective activity against gliadin digest cytotoxicity. [Copyright &y& Elsevier]

In this study, we show that ultraviolet B radiation (UVB)-induced apoptosis of human keratinocytes involves mainly cytosolic signals with mitochondria playing a central role. Overexpression of Bcl-2 inhibited UVB-induced apoptosis by blocking the early generation of reactive oxygen species, mitochondrial cardiolipin degradation and cytochrome c release, without affecting Fas ligand (FasL)-induced cell death. It also prevented the subsequent activation of procaspase-3 and -8 as well as Bid cleavage in UVB-treated cells. Comparative analysis of UVB and FasL death pathways revealed a differential role and mechanism of caspase activation, with the UVB-induced activation of procaspase-8 only being a bystander cytosolic event rather than a major initiator mechanism, as is the case for the FasL-induced cell death. Our results suggest that Bcl-2 overexpression, by preventing reactive oxygen species production, helps indirectly to maintain the integrity of lysosomal membranes, and therefore inhibits the release of cathepsins, which contribute to the cytosolic activation of procaspase-8 in UVB-irradiated keratinocytes. [Copyright &y& Elsevier]

Cell culture studies have given much valuable information about mechanisms of metabolism and signal transduction and of regulation of gene expression, proliferation, senescence, and death. However, cells in culture may behave differently from cells in vivo in many ways. One of these is that cell culture imposes a state of oxidative stress on cells. I argue that cells that survive and grow in culture might use ROS-dependent signal transduction pathways that rarely or never operate in vivo. A further problem is that cell culture media can catalyse the oxidation of compounds added to them, resulting in apparent cellular effects that are in fact due to oxidation products such as ROS. Such artefacts may have affected many studies on the effects of ascorbate, thiols, flavonoids and other polyphenolic compounds on cells in culture. [Copyright &y& Elsevier]

On the sperm surface, glutathione S-transferases (GSTs) exist as oocyte binding proteins but their detoxification function in this unique cell type is not known. Using H2O2- and 4-hydroxynonenal-induced sperm dysfunction models, this study demonstrates that the sperm surface GSTs are able to use extracellular reduced glutathione to inhibit the loss of functional competence of goat spermatozoa; however, in the presence of GST inhibitors, they are unable to do so. In the context of susceptibility of spermatozoa to oxidative stress, this finding that strategically located sperm surface GSTs are important for maintaining the functional competence of sperm is relevant to studies on male infertility. [Copyright &y& Elsevier]

Mitochondrial ATP-sensitive K+ channel (mitoKATP) opening was shown previously to slightly increase respiration and decrease the membrane potential by stimulating K+ cycling across the inner membrane. Here we show that mitoKATP opening reduces reactive oxygen species generation in heart, liver and brain mitochondria. Decreased H2O2 release is observed when mitoKATP is active both with respiration stimulated by oxidative phosphorylation and when ATP synthesis is inhibited. In addition, decreased H2O2 release is observed when mitochondrial ΔpH is enhanced, an effect expected to occur when mitoKATP is open. We conclude that mitoKATP is an effective pathway to trigger mild uncoupling, preventing reactive oxygen species release. [Copyright &y& Elsevier]

The predominance of oxidative stress in many tumour cell environments provides a means to selectively target these cells via protein oxidation. The zinc fingers of transcription factors utilise cysteine thiols for structural zinc coordination. Redox control of DNA binding regulates transcription and therefore the overall rates of proliferation, apoptosis and necrosis in the carcinoma. We report here the adverse effects of glutathione peroxidase (GPx) mimics towards zinc finger motifs and PC12 cell survival. Nanomolar catalyst concentrations facilitated H2O2-induced oxidation of an Sp1 transcription factor fragment. In PC12 cells GPx catalysis triggered a significant increase in cell death, correlating with severity of oxidative stress. As a consequence, we conclude that GPx mimics are potential chemotherapeutic agents. [Copyright &y& Elsevier]

In this study we show that mitochondria of Dictyostelium discoideum contain both alternative oxidase (AOX) and uncoupling protein (UCP). AOX was stimulated by purine mononucleoside and was monomeric. UCP was stimulated by free fatty acids and was poorly sensitive to GTP. Both proteins collaborated in energy dissipation when activated together. AOX expression in free-living ameboid cells decreased strongly from exponential to stationary phase of growth but much less during starvation-induced aggregation. In contrast, UCP expression was constant in all conditions indicating permanent need. Our results suggest that AOX could play a role in cell differentiation, mainly by protecting prespore cells from programmed cell death. [Copyright &y& Elsevier]

We previously reported that paclitaxel acted directly on mitochondria isolated from human neuroblastoma SK-N-SH cells. Here, we demonstrate that the direct mitochondrial effect of paclitaxel observed in vitro is relevant in intact SK-N-SH cells. After a 2 h incubation with 1 μM paclitaxel, the mitochondria were less condensed. Paclitaxel (1 μM, 1–4 h) also induced a 20% increase in respiration rate and a caspase-independent production of reactive oxygen species by mitochondria. The paclitaxel-induced release of cytochrome c was detected only after 24 h of incubation, was caspase-independent and permeability transition pore-dependent. Thus, paclitaxel targets mitochondria upstream of caspase activation, early during the apoptotic process in intact human neuroblastoma cells. [Copyright &y& Elsevier]

Reactive oxygen species (ROS) are implicated in the pathogenesis of osteoarthritis (OA). However, little is known about the antioxidant defence system in articular cartilage. We investigated the expression and regulation of peroxiredoxin 5 (PRDX5), a newly discovered thioredoxin peroxidase, in human normal and osteoarthritic cartilage. Our results show that human cartilage constitutively expresses PRDX5. Moreover, the expression is up-regulated in OA. Inflammatory cytokines tumour necrosis factor α and interleukin 1 β contribute to this up-regulation by increasing intracellular ROS production. The present study suggests that PRDX5 may play a protective role against oxidative stress in human cartilage. [Copyright &y& Elsevier]

Recent data on the cellular ceramide functions and its involvement in the apoptotic/necrotic cell death as well as its anticarcinogenic properties are presented. The emphasis is on the connections between the ceramide and caspase signaling pathways during the apoptotic cell death process. Notably, the experimental strategies and pharmacological tools used for establishment of the role of ceramide in triggering cell death are described. Moreover, the importance of a compartmentation of endogenous ceramide within the plasma membrane microdomains, lysosomes and mitochondria is discussed. Information on the deregulated functions of ceramide and caspase signaling pathways in several metastatic cancer types is also presented. [Copyright &y& Elsevier]

During recent years, several pieces of indirect evidence of a programmed death in yeast have been published. Among them there are observations that some mammalian pro- or anti-apoptotic proteins induce or prevent the death of yeast; some toxic compounds kill yeast at lower concentrations if protein synthesis is operative; this death, as well as the death due to certain mutations, shows some apoptotic markers. In April 2002, the yeast programmed death concept received direct support. Madeo et al. [Madeo et al., Mol. Cell 9 (2002) 911–917] disclosed a caspase which is activated by H2O2 or aging and is required for the protein-synthesis-dependent death of yeast. Thus, a specific apoptosis-mediating protein was identified for the first time in Saccharomyces cerevisiae. Independently, Severin and Hyman [Severin, F.F., Hyman, A.A., Curr. Biol. 12 (2002) R233–R235] discovered that death of yeast, induced by a high level of a pheromone, is programmed. In particular, the death was found to be prevented by cycloheximide and cyclosporin A. It required mitochondrial DNA, cytochrome c and the pheromone-initiated protein kinase cascade. When haploids of opposite mating types were mixed, some cells died, the inhibitory pattern being the same as in the case of the killing by pheromone. Inhibition of mating proved to be favorable for death. Thus, pheromone not only activates mating but also eliminates yeast cells failing to mate. Such an effect should (i) stimulate switch of the yeast population from vegetative to sexual reproduction, and (ii) shorten the life span and, hence, accelerate changing of generations. As a result, the probability of appearance of new traits could be enhanced when ambient conditions turned for the worse. [Copyright &y& Elsevier]

Exposure to methoxyacetic acid (MAA), a major byproduct of the paint industry, causes testicular atrophy in multiple species. This study demonstrates DNA breakdown in rat germ cells after exposure to MAA in vivo within 12 h, leading to 40% germ cell death by 24 h. Within 4 h of treatment, cytochrome c is released from the mitochondria into the cytosol without the involvement of mitochondrial potential loss, reactive oxygen species generation or lipid peroxidation events. Peak activation of caspase-9 and caspase-3 is detectable post treatment at 4 and 8 h respectively. There is a decrease in germ cell glutathione levels within 2 h of MAA treatment. Replenishment of glutathione by pretreatment of the animals with the antioxidant N-acetylcysteine prior to MAA treatment could prevent the release of cytochrome c, DNA fragmentation and cell death. [Copyright &y& Elsevier]

Nuclear factor (NF)-κB pathways are influenced by the intracellular reduction–oxidation (redox) balance. While NF-κB is activated through inhibitor (I)-κB degradation by oxidative stress, its DNA binding is accelerated in the reduced state. We found that N,N′-diacetyl-L-cystine dimethylester (DACDM) suppressed the UVB-induced NF-κB binding activity at a much lower concentration (50–100 μM) than N-acetyl-L-cysteine (NAC, 10–30 mM). While NAC suppressed the I-κB degradation but not the DNA binding, DACDM prevented the activated NF-κB from binding DNA, without influencing the I-κB degradation. These properties of DACDM make it possible to effectively regulate the intracellular redox balance. [Copyright &y& Elsevier]