Roles of reactive oxygen species in UVA‐induced oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid‐melanin as studied by differential spectrophotometric method

Eumelanin photoprotects pigmented tissues from ultraviolet (UV) damage. However, UVA‐induced tanning seems to result from the photooxidation of preexisting melanin and does not contribute to photoprotection. We investigated the mechanism of UVA‐induced degradation of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA)‐melanin taking advantage of its solubility in a neutral buffer and using a differential spectrophotometric method to detect subtle changes in its structure. Our methodology is suitable for examining the effects of various agents that interact with reactive oxygen species (ROS) to determine how ROS is involved in the UVA‐induced oxidative modifications. The results show that UVA radiation induces the oxidation of DHICA to indole‐5,6‐quinone‐2‐carboxylic acid in eumelanin, which is then cleaved to form a photodegraded, pyrrolic moiety and finally to form free pyrrole‐2,3,5‐tricarboxylic acid. The possible involvement of superoxide radical and singlet oxygen in the oxidation was suggested. The generation and quenching of singlet oxygen by DHICA‐melanin was confirmed by direct measurements of singlet oxygen phosphorescence.     

Differences in the Induction of Defence Responses in Resistant and Susceptible Muskmelon Plants Infected with Colletotrichum lagenarium

Defence reactions occurring in resistant (cv. Gankezaomi) and susceptible (cv. Ganmibao) muskmelon leaves were investigated after inoculating with Colletotrichum lagenarium. Lesion restriction in resistant cultivars was associated with the accumulation of hydrogen peroxide (H₂O₂). The activity of antioxidants catalase (CAT) and peroxidase (POD) significantly increased in both cultivars after inoculation, while levels of both CAT and POD activity were significantly higher in the resistant cultivar. Ascorbate peroxidase (APX) increased in both cultivars after inoculation, and level of APX activity was significantly higher in the resistant cultivar. Glutathione reductase (GR) activity significantly increased in both cultivars following inoculation, but was higher in the resistant cultivar, resulting in higher levels of ascorbic acid (AsA) and reduced glutathione (GSH). Phenylalanine ammonia lyase (PAL) significantly increased in inoculated leaves of both cultivars, resulting in higher levels of total phenolic compounds and flavonoids. The pathogenesis‐related proteins chitinase (CHT) and β‐1, 3‐glucanase (GLU) significantly increased following inoculation with higher activity in the resistant cultivar. These findings show that resistance of muskmelon plants against C. lagenarium is associated with the rapid accumulation of H₂O₂, resulting in altered cellular redox status, accumulation of pathogenesis‐related proteins, activation of phenylpropanoid pathway to accumulation of phenolic compounds and flavonoids.     

Epidermal fatty acid‐binding protein protects nerve growth factor‐differentiated PC12 cells from lipotoxic injury

Epidermal fatty acid‐binding protein (E‐FABP/FABP5/DA11) binds and transport long‐chain fatty acids in the cytoplasm and may play a protecting role during neuronal injury. We examined whether E‐FABP protects nerve growth factor‐differentiated PC12 cells (NGFDPC12 cells) from lipotoxic injury observed after palmitic acid (C16:0; PAM) overload. NGFDPC12 cells cultures treated with PAM/bovine serum albumin at 0.3 mM/0.15 mM show PAM‐induced lipotoxicity (PAM‐LTx) and apoptosis. The apoptosis was preceded by a cellular accumulation of reactive oxygen species (ROS) and higher levels of E‐FABP. Antioxidants MCI‐186 and N‐acetyl cysteine prevented E‐FABP's induction in expression by PAM‐LTx, while tert‐butyl hydroperoxide increased ROS and E‐FABP expression. Non‐metabolized methyl ester of PAM, methyl palmitic acid (mPAM), failed to increase cellular ROS, E‐FABP gene expression, or trigger apoptosis. Treatment of NGFDPC12 cultures with siE‐FABP showed reduced E‐FABP levels correlating with higher accumulation of ROS and cell death after exposure to PAM. In contrast, increasing E‐FABP cellular levels by pre‐loading the cells with recombinant E‐FABP diminished the PAM‐induced ROS and cell death. Finally, agonists for PPARβ (GW0742) or PPARγ (GW1929) increased E‐FABP expression and enhanced the resistance of NGFDPC12 cells to PAM‐LTx. We conclude that E‐FABP protects NGFDPC12 cells from lipotoxic injury through mechanisms that involve reduction of ROS.

     

The use of in vivo microscopy to image the cnidarian stress response in three octocoral species

Energy‐converting symbionts and organelles may be central to the cnidarian stress response. Stress may damage photochemistry in the endosymbiotic dinoflagellate Symbiodinium spp., leaving photosystems I and II relatively reduced and increasing reactive oxygen species (ROS). Alternatively, ROS may emanate from mitochondria of either the host or the symbiont, or both. These sources of ROS can be distinguished by using microscopy to examine the effects of light on stressed cnidarians incubated in the dark with a fluorescent, ROS‐detecting probe. Experiments were carried out with three species of alcyonacean octocoral, Phenganax parrini, Sarcothelia sp., and Sympodium sp. After incubation of colonies for 1 h at elevated temperature, imaging and illumination (excitation 450–490 nm, emission 515–565 nm) were begun simultaneously. Formation of ROS largely corresponded to the onset of illumination. On the other hand, chlorophyll fluorescence (excitation 530–580 nm, emission 620–690 nm) did not conform to this pattern. This difference is consistent with the expected rates of reaction. Remarkably, treatment with the inhibitor 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU) resulted in dramatically higher levels of light‐induced ROS. Chlorophyll fluorescence was higher in the DCMU treatment but not significantly so. By controlling for variation between individual symbionts, however, DCMU produced significantly greater levels of chlorophyll fluorescence, indicating the expected greater reduction of photosystem II. A brief exposure to light and thermal stress produced a similar effect in all three species. In addition to ROS being initiated by light, these results indicate that: (1) a brief period of stress shifts photosystem redox state toward reduction, (2) photosystem II can donate electrons to oxygen when blocked with DCMU, and (3) chlorophyll fluorescence is highly variable among individuals of Symbiodinium. Imaging of individual symbionts in hospite thus provides a powerful method for understanding the initial steps of the cnidarian stress response.     

Redox signaling

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have recently been shown to be involved in a multiplicity of physiological responses through modulation of signaling pathways. Some of the specific signaling components altered by reactive oxygen and nitrogen species (RONS) have begun to be identified. We will discuss RONS signaling by detailing the chemistry of signaling, the roles of antioxidant enzymes as signaling components, thiol chemistry in the specificity of RONS signaling, NO-heme interactions, and some do's and don'ts of redox signal research. The principal points raised are that: (1) as with classic signaling pathways, signaling by RONS is regulated; (2) antioxidant enzymes are essential 'turn-off' components in signaling; (3) spatial relationships are probably more important in RONS signaling than the overall 'redox state' of the cell; (4) deprotonation of cysteines to form the thiolate, which can react with RONS, occurs in specific protein sites providing specificity in signaling; (5) although multiple chemical mechanisms exist for producing nitrosothiols, their formation in vivo remains unclear; and (6) caution should be taken in the use of 'antioxidants' in signal transduction.     

The path from mitochondrial ROS to aging runs through the mitochondrial permeability transition pore

Excessive production of mitochondrial reactive oxygen species (mROS) is strongly associated with mitochondrial and cellular oxidative damage, aging, and degenerative diseases. However, mROS also induces pathways of protection of mitochondria that slow aging, inhibit cell death, and increase lifespan. Recent studies show that the activation of the mitochondrial permeability transition pore (mPTP), which is triggered by mROS and mitochondrial calcium overloading, is enhanced in aged animals and humans and in aging‐related degenerative diseases. mPTP opening initiates further production and release of mROS that damage both mitochondrial and nuclear DNA, proteins, and phospholipids, and also releases matrix NAD that is hydrolyzed in the intermembrane space, thus contributing to the depletion of cellular NAD that accelerates aging. Oxidative damage to calcium transporters leads to calcium overload and more frequent opening of mPTP. Because aging enhances the opening of the mPTP and mPTP opening accelerates aging, we suggest that mPTP opening drives the progression of aging. Activation of the mPTP is regulated, directly and indirectly, not only by the mitochondrial protection pathways that are induced by mROS, but also by pro‐apoptotic signals that are induced by DNA damage. We suggest that the integration of these contrasting signals by the mPTP largely determines the rate of cell aging and the initiation of cell death, and thus animal lifespan. The suggestion that the control of mPTP activation is critical for the progression of aging can explain the conflicting and confusing evidence regarding the beneficial and deleterious effects of mROS on health and lifespan.     

Reactive Oxygen Species Activity and Antioxidant Properties of Fusarium Infected Bananas

Fusarium infection of bananas is a global problem that threatens the production of bananas. This study looks at the effects of the infection upon the reactive oxygen species (ROS) system, as well as the induced antioxidant properties in the roots, stems, leaves and fruits. Results show that there is a greater amount of damage in infected tissue samples as opposed to non‐infected. The damage was observed to be higher in the root samples. ROS assays were divided into two classes: ROS assays and ROS‐scavenging assays. Of the ROS assays, lipoxygenase was observed to be higher in the infected samples, while peroxidase (POD) and polyphenol oxidase (PPO) were significantly higher in infected stem, leaf and fruit samples. Among root samples, there was no significant difference in POD activity and PPO was lower in infected samples. Induction of ROS is important for the hypersensitive response (HR) to function properly. The ROS‐scavenging enzymes, namely ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase, exhibited higher levels in the infected tissue. This is most likely to counter the build‐up of the ROS enzymes and to prevent further cell death. The increase in ROS‐scavenging assays also correlates with higher antioxidant properties as antioxidants play a critical role in regulating the HR free radicals.     

The exceptional longevity of the naked mole‐rat may be explained by mitochondrial antioxidant defenses

Naked mole‐rats (NMRs) are mouse‐sized mammals that exhibit an exceptionally long lifespan (>30 vs. <4 years for mice), and resist aging‐related pathologies such as cardiovascular and pulmonary diseases, cancer, and neurodegeneration. However, the mechanisms underlying this exceptional longevity and disease resistance remain poorly understood. The oxidative stress theory of aging posits that (a) senescence results from the accumulation of oxidative damage inflicted by reactive oxygen species (ROS) of mitochondrial origin, and (b) mitochondria of long‐lived species produce less ROS than do mitochondria of short‐lived species. However, comparative studies over the past 28 years have produced equivocal results supporting this latter prediction. We hypothesized that, rather than differences in ROS generation, the capacity of mitochondria to consume ROS might distinguish long‐lived species from short‐lived species. To test this hypothesis, we compared mitochondrial production and consumption of hydrogen peroxide (H₂O₂; as a proxy of overall ROS metabolism) between NMR and mouse skeletal muscle and heart. We found that the two species had comparable rates of mitochondrial H₂O₂ generation in both tissues; however, the capacity of mitochondria to consume ROS was markedly greater in NMRs. Specifically, maximal observed consumption rates were approximately two and fivefold greater in NMRs than in mice, for skeletal muscle and heart, respectively. Our results indicate that differences in matrix ROS detoxification capacity between species may contribute to their divergence in lifespan.     

A recent advance in the intracellular and extracellular redox post‐translational modification of proteins in plants

Plants, as sessile organisms, have acquired through evolution sophisticated regulatory signal pathways to overcome external variable factors during each stage of the life cycle. Among these regulatory signals, two pathways in particular, reactive oxygen species and reactive nitrogen species, have become of significant interest in several aspects of plant biology, underpinning these molecules as critical regulators during development, cellular differentiation, and plant‐pathogen interaction. Recently, redox posttranslational modifications (PTM), such as S‐nitrosylation on cysteine residues and tyrosine nitration, have shed light on multiple protein targets, as they are associated with signal networks/downstream metabolic pathways, capable of transducing the imbalance of redox hemostasis and consequently redirecting the biochemical status under stress conditions. However, most of the redox PTM have been studied only in the intracellular compartment, providing limited information concerning redox PTM in the extracellular matrix of plant cells. Nevertheless, recent studies have indicated the plausibility of redox PTM in extracellular proteins, including cell wall associated proteins. Accordingly, in this review, we endeavor to examine evidence of redox PTM supported by mass spectrometry data in the intracellular and extracellular space in plant cells. As a further example, we focus the last section of this review on illustrating, using molecular dynamics simulation, the effect of S‐nitrosylation on the structural conformation of well‐known cell wall‐associated proteins including pectin methylesterase and xyloglucan endo‐transglycosylases.     

A Strobilurin Fungicide Relieves Bipolaris oryzae‐Induced Oxidative Stress in Rice

Although strobilurins are one of the most effective and broad spectrum classes of systemic fungicides, they may also increase plant stress tolerance by modulating the activity of antioxidant enzymes. To address this issue, the effect of azoxystrobin (Az) on the activity of antioxidant enzymes and on the concentrations of antioxidant metabolites and oxidative stress‐related compounds was studied in rice plants (cv. Metica‐1) either inoculated or not with Bipolaris oryzae, the causal agent of brown spot (BS). The Az minimally affected the enzyme activities, but consistently increased the glutathione reduced (GSH) concentrations in the noninoculated plants. The activities of superoxide dismutase, peroxidase, ascorbate peroxidase, glutathione peroxidase, glutathione reductase and glutathione‐S‐transferase were increased upon B. oryzae infection, but such increases were greatly limited in the Az‐sprayed plants. Catalase activity decreased in the inoculated plants compared to the noninoculated plants regardless of fungicide treatment. The GSH concentration increased in response to the B. oryzae infection, and the Az‐sprayed plants sustained higher levels of GSH at advanced stages of fungal infection than did the nonsprayed plants. The inoculated plants exhibited an extensive oxidative stress as evidenced by higher concentrations of hydrogen peroxide and malondialdehyde compared to the noninoculated plants, but lower and later increases were recorded in the Az‐sprayed plants than in the nonsprayed plants. Therefore, Az greatly reduces B. oryzae‐induced oxidative stress by limiting BS development rather than by activating antioxidant enzymes. The GSH, however, seems to be Az‐modulated, and this may partially explain the constrained oxidative stress observed in the Az‐sprayed plants.