Vascular pro-oxidant effects secondary to the autoxidation of gallic acid in rat aorta

Gallic acid autoxidation was monitored by absorption spectroscopy and H₂O₂ production; vascular effects related to the autoxidation process were studied on intact and rubbed aortic rings from WKY rats. Gallic acid autoxidation in an oxygenated physiological salt solution (37°C, pH=7.4) mostly occurred in a 2-h time period. Superoxide anions, H₂O₂ and gallic acid quinones were produced during gallic acid autoxidation. In rings partially precontracted with phenylephrine, 0.1–3 μM gallic acid induced marked and largely endothelium-dependent contractions, 10–30 μM gallic acid induced endothelium-independent contractions and 0.1–0.3 mM gallic acid induced complete, fast-developing, endothelium-independent relaxations. Superoxide dismutase (SOD) shifted the endothelium-dependent gallic acid contractions to the right, and N^G-nitro-l-arginine abolished them. Indomethacin suppressed the endothelium-independent gallic acid contractions, and catalase abolished the endothelium-independent contractions and relaxations. Gallic acid (30 μM) inhibited the relaxant effects of acetylcholine and sodium nitroprusside. In rings maximally precontracted with KCl, 0.1–100 μM gallic acid did not modify the tone, whereas 0.3 mM induced complete, slow-developing, endothelium-independent relaxations. Moreover, 0.3 mM gallic acid induced an irreversible impairment of ring reactivity and the release of lactate dehydrogenase. Catalase and N-acetyl cysteine suppressed the deleterious effects induced by gallic acid in the rings. In conclusion: (a) gallic acid is rapidly and nonenzymatically oxidized in physiological solutions, generating superoxide anions, H₂O₂ and quinones; (b) superoxide anions (by destroying NO) and low H₂O₂ levels (by activating cyclooxygenase) both increase vascular tone; (c) moderate H₂O₂ levels decrease vascular tone; (d) high H₂O₂ and quinone levels cause irreversible relaxations due to cellular damage.     

Hymenolepis diminuta: Chloride fluxes and membrane potentials associated with sodium-coupled glucose transport

Glucose transport by Hymenolepis diminuta was inhibited when Cl^? in the bathing medium was replaced with acetate (C₂H₃O₂^Post?), but was unaffected when Cl^? was replaced with SCN^?. The relative effectiveness of the anions to inhibit influx of 7.4 mM Cl^? in the presence of 1 mM glucose was SCN^? > Cl^? > C₂H₃O₂^Post?. Glucose stimulated the influxes of 120 mM Cl^? and SCN^?, but had little effect on 120 mM C₂H₃O₂^Post? influx. While the diffusion rates of the anions were C₂H₃O₂^Post? > SCN^? = Cl^?, the preference of the glucose transport system for the anions was SCN^? > Cl^? > C₂H₃O₂^Post?. Efflux of Cl^? was not affected by the rate of glucose influx. Finally, microelectrode recordings of worms anesthetized with 2 mM arecoline revealed a transmembrane potential (TMP) of ?45 ± 3.6 mV (inside negative). Three to four minutes after addition of glucose (5 mM) there was a progressive hyperpolarization of the TMP to ?58 mV. A revised model of the glucose transport system that is consistent with previous observations on this organism is proposed.     

Programmed cell death in plants: Protective effect of tetraphenylphosphonium and tetramethylrhodamine cations used as transmembrane quinone carriers

Tetraphenylphosphonium (TPP⁺) and tetramethylrhodamine ethyl ester (TMRE⁺) cations used as transmembrane carriers of ubiquinone (MitoQ) and plastoquinone (SkQ, SkQR) in mitochondria prevented at nanomolar concentrations the chitosanor H₂O₂-induced destruction of the nucleus in epidermal cells of epidermis isolated from pea leaves. The protective effect of the cations was potentiated by palmitate. Penetrating anions of tetraphenylboron (TB⁻) and phenyl dicarbaundecaborane also displayed protective effects at micromolar concentrations; the effect of TB⁻ was potentiated by NH₄Cl. It is proposed that the protective effect of the penetrating cations and anions against chitosan is due to suppression of the generation of reactive oxygen species in mitochondria as a result of the protonophoric effect of the cations plus fatty acids and the anions plus NH₄⁺. Phenol was suitable as the electron donor for H₂O₂ reduction catalyzed by horseradish peroxidase, preventing the destruction of cell nuclei. The penetrating cations and anions, SkQ1, and SkQR1 did not maintain the peroxidase or peroxidase/oxidase reactions measured by their suitability as electron donors for H₂O₂ reduction or by the oxidation of exogenous NADH.     

Superoxide anion and hydrogen peroxide production by chemically elicited peritoneal macrophages--induction by multiple nonphagocytic stimuli

The ability of a number of stimulants to activate an oxidative burst (OB) in oil-elicited guinea pig peritoneal exudate macrophages (MPs) was examined. The parameters of the OB were the generation and extracellular release of Superoxide anions (O₂^?) and hydrogen peroxide (H₂O₂). We found that: (1) The cocarcinogen and skin irritant phorbol myristate acetate (PMA) was the most potent OB activator—The weak cocarcinogen 4-O-methyl PMA was a proportionally less effective OB activator; (2) The lectins concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean, Lotus, and pokeweed lectins, were also quite effective OB activators—The ability of Con A to stimulate O₂^? production was abolished by succinylation and could be prevented by the presence of α-methyl-D-mannoside; (3) Other stimulators of an OB in MPs were: N-formyl-methionyl peptides, opsonized zymosan, the Ca²⁺ ionophore A23187, phospholipase C, NaF, antimacrophage antibody, microtubule-disrupting drugs, and sodium nitroprusside—O₂^? generation induced by A23187 (but not that stimulated by PMA) was dependent on extracellular Ca²⁺; (4) The amount of O₂^? produced per cell was higher at low cell densities; (5) The addition of Superoxide dismutase (SOD) to the medium totally prevented the detection of O₂^? and augmented twice the amount of H₂O₂ recovered; (6) Pretreatment of MPs with the SOD inhibitor sodium diethyldithiocarbamate had no effect on the release of O₂^? but blocked H₂O₂ release in a dose-dependent manner. These data were interpreted as indicating that the bulk of H₂O₂ was derived by enzymatic dismutation of O₂^?; (7) The common mechanism by which such a variety of stimuli provoke an OB in MPs was not elucidated. No evidence was found to suggest a role for a cyclic nucleotide messenger.     

Hemoglobin–Albumin Cluster Incorporating a Pt Nanoparticle: Artificial O2 Carrier with Antioxidant Activities

A covalent core–shell structured protein cluster composed of hemoglobin (Hb) at the center and human serum albumins (HSA) at the periphery, Hb-HSA_m, is an artificial O₂ carrier that can function as a red blood cell substitute. Here we described the preparation of a novel Hb-HSA₃ cluster with antioxidant activities and its O₂ complex stable in aqueous H₂O₂ solution. We used an approach of incorporating a Pt nanoparticle (PtNP) into the exterior HSA unit of the cluster. A citrate reduced PtNP (1.8 nm diameter) was bound tightly within the cleft of free HSA with a binding constant (K) of 1.1×10⁷ M⁻¹, generating a stable HSA-PtNP complex. This platinated protein showed high catalytic activities for dismutations of superoxide radical anions (O₂ ^•–) and hydrogen peroxide (H₂O₂), i.e., superoxide dismutase and catalase activities. Also, Hb-HSA₃ captured PtNP into the external albumin unit (K = 1.1×10⁷ M⁻¹), yielding an Hb-HSA₃(PtNP) cluster. The association of PtNP caused no alteration of the protein surface net charge and O₂ binding affinity. The peripheral HSA-PtNP shell prevents oxidation of the core Hb, which enables the formation of an extremely stable O₂ complex, even in H₂O₂ solution.     

Distribution of hydrogen peroxide during adventitious roots initiation and development in mung bean hypocotyls cuttings

Previous studies have shown that hydrogen peroxide (H₂O₂) may mediate the auxin response during the formation of adventitious roots (AR). However, the mechanism and distribution of H₂O₂ during AR formation remains unclear. In this study, we investigate the spatiotemporal changes and role of H₂O₂ in AR initiation and development. Application of 5?C100 mM H₂O₂ to Mung bean (Phaseolus radiatus L.) hypocotyl cuttings induced AR formation in a dose-dependent manner. The effect was blocked by ascorbic acid (AA), an important reducing substrate for H₂O₂ reduction. Depletion of endogenous H₂O₂ by AA resulted in the significant reduction of AR emergence, suggesting a physiological role for H₂O₂ in the regulation of AR formation. Determination of H₂O₂ content showed that the level of H₂O₂ increased gradually and reached the highest value 60 h after induction of AR. Further detection of endogenous H₂O₂ by the specific fluorescent probe dichlorofluorescein diacetate (H₂DCF-DA) and 3,3??-diaminobenzidine (DAB) staining in transverse sections of the basal region of cuttings revealed that obvious H₂O₂ signals were observed in the pericycle cells between the vascular bundles 24 h after the primary roots were removed. With the development of root primordia, H₂O₂ signals increased gradually and were mainly distributed in the root meristem. AA significant inhibited the H₂O₂-dependent fluorescence and the formation of AR, suggesting an essential role of H₂O₂ generation during AR initiation and development. Furthermore, the involvement of Ca²⁺ during H₂O₂-mediated AR formation was evaluated. Ca²⁺ channel inhibitors LaCl₃ and ruthenium red (RR) and Ca²⁺ chelator ethylene glycol-bis(2-aminoethylether)-N,N,N??,N??-tetraacetic acid (EGTA) prevent H₂O₂-induced AR formation, which indicate that the hypocotyl cuttings response to H₂O₂ depends on the availability of both intracellular and extracellular Ca²⁺ pools, and Ca²⁺ is a downstream messenger in the signaling pathway triggered by H₂O₂ to promote adventitious root formation.     

Dicarboxylatodirhodium derivatives of polyoxotungstates

Salts of the new complexes [PW₁₁O₃₉{Rh₂(O₂CR)₂}]⁵⁻; R = Prⁿ (1), CH₂Cl (2), CH₂OH (3), o-C₆H₄OH (4), p-C₆H₄OH (5), and [XW₁₁O₃₉{Rh₂(p-O₂CC₆H₄OH)₂}]⁶⁻; X = Si (6), Ge (7) have been prepared in good yield and characterized by elemental analysis, multinuclear NMR spectroscopy, and structural crystallography of the cesium salts of anions 1 and 5 with chloride ions in axial positions of the dirhodium moiety. The incorporation of the dirhodium group into the Keggin structure significantly increases the hydrolytic kinetic stability of the polyoxotungstates at pH 7–8.5, a result that has implications for the use of such complexes for imaging and for phase determination in structural studies of large biomolecules. Based on NMR studies, the axial sites of the dirhodium moiety of the polytungstate anions are accessible to ligation by molecules such as cysteine, methionine, and isonicotinic acid.     

A Mechanism of Resistance to Hydrogen Peroxide in Vibrio rumoiensis S-1

A possible mechanism of resistance to hydrogen peroxide (H₂O₂) in Vibrio rumoiensis, isolated from the H₂O₂-rich drain pool of a fish processing plant, was examined. When V. rumoiensis cells were inoculated into medium containing either 5 mM or no H₂O₂, they grew in similar manners. A spontaneous mutant strain, S-4, derived from V. rumoiensis and lacking catalase activity did not grow at all in the presence of 5 mM H₂O₂. These results suggest that catalase is inevitably involved in the resistance and survival of V. rumoiensis in the presence of H₂O₂. Catalase activity was constitutively present in V. rumoiensis cells grown in the absence of H₂O₂, and its occurrence was dependent on the age of the cells, a characteristic which is observed for the HP II-type catalase of Escherichia coli. The presence of the HP II-type catalase in V. rumoiensis cells was evidenced by partial sequencing of the gene encoding the HP II-type catalase from this organism. A notable difference between V. rumoiensis and E. coli is that catalase is accumulated at very high levels (~2% of the total soluble proteins) in V. rumoiensis, in contrast to the case for E. coli. When V. rumoiensis cells which had been exposed to 5 mM H₂O₂ were centrifuged, most intracellular proteins, including catalase, were recovered in the medium. On the other hand, when V. rumoiensis cells were grown on plates containing various concentrations of H₂O₂, individual cells had a colony-forming ability inferior to those of E. coli, Bacillus subtilis, and Vibrio parahaemolyticus. Thus, it is suggested that when V. rumoiensis cells are exposed to high concentrations of H₂O₂, most cells will immediately be broken by H₂O₂. In addition, the cells which have had little or no damage will start to grow in a medium where almost all H₂O₂ has been decomposed by the catalase released from broken cells.     

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H₂O₂) through the plasma membrane decreases during adaptation to H₂O₂ by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H₂O₂ the anisotropy of the plasma membrane increases. Adaptation to H₂O₂ was studied at several times (15min up to 90min) by applying the steady-state H₂O₂ delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H₂O₂ was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H₂O₂ adaptation. H₂O₂ diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H₂O₂ permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H₂O₂ is mediated by modulation of the biophysical properties of the plasma membrane.     

Individuals with higher metabolic rates have lower levels of reactive oxygen species in vivo

There is increasing interest in the effect of energy metabolism on oxidative stress, but much ambiguity over the relationship between the rate of oxygen consumption and the generation of reactive oxygen species (ROS). Production of ROS (such as hydrogen peroxide, H₂O₂) in the mitochondria is primarily inferred indirectly from measurements in vitro, which may not reflect actual ROS production in living animals. Here, we measured in vivo H₂O₂ content using the recently developed MitoB probe that becomes concentrated in the mitochondria of living organisms, where it is converted by H₂O₂ into an alternative form termed MitoP; the ratio of MitoP/MitoB indicates the level of mitochondrial H₂O₂ in vivo. Using the brown trout Salmo trutta, we tested whether this measurement of in vivo H₂O₂ content over a 24 h-period was related to interindividual variation in standard metabolic rate (SMR). We showed that the H₂O₂ content varied up to 26-fold among fish of the same age and under identical environmental conditions and nutritional states. Interindividual variation in H₂O₂ content was unrelated to mitochondrial density but was significantly associated with SMR: fish with a higher mass-independent SMR had a lower level of H₂O₂. The mechanism underlying this observed relationship between SMR and in vivo H₂O₂ content requires further investigation, but may implicate mitochondrial uncoupling which can simultaneously increase SMR but reduce ROS production. To our knowledge, this is the first study in living organisms to show that individuals with higher oxygen consumption rates can actually have lower levels of H₂O₂.     

Reduction of Hydrogen Peroxide Accumulation and Toxicity by a Catalase from Mycoplasma iowae

Mycoplasma iowae is a well-established avian pathogen that can infect and damage many sites throughout the body. One potential mediator of cellular damage by mycoplasmas is the production of H₂O₂ via a glycerol catabolic pathway whose genes are widespread amongst many mycoplasma species. Previous sequencing of M. iowae serovar I strain 695 revealed the presence of not only genes for H₂O₂ production through glycerol catabolism but also the first documented mycoplasma gene for catalase, which degrades H₂O₂. To test the activity of M. iowae catalase in degrading H₂O₂, we studied catalase activity and H₂O₂ accumulation by both M. iowae serovar K strain DK-CPA, whose genome we sequenced, and strains of the H₂O₂-producing species Mycoplasma gallisepticum engineered to produce M. iowae catalase by transformation with the M. iowae putative catalase gene, katE. H₂O₂-mediated virulence by M. iowae serovar K and catalase-producing M. gallisepticum transformants were also analyzed using a Caenorhabditis elegans toxicity assay, which has never previously been used in conjunction with mycoplasmas. We found that M. iowae katE encodes an active catalase that, when expressed in M. gallisepticum, reduces both the amount of H₂O₂ produced and the amount of damage to C. elegans in the presence of glycerol. Therefore, the correlation between the presence of glycerol catabolism genes and the use of H₂O₂ as a virulence factor by mycoplasmas might not be absolute.     

Interaction between HY1 and H2O2 in auxin-induced lateral root formation in Arabidopsis

Haem oxygenase-1 (HO-1) and hydrogen peroxide (H₂O₂) are two key downstream signals of auxin, a well-known phytohormone regulating plant growth and development. However, the inter-relationship between HO-1 and H₂O₂ in auxin-mediated lateral root (LR) formation is poorly understood. Herein, we revealed that exogenous auxin, 1-naphthylacetic acid (NAA), could simultaneously stimulate Arabidopsis HO-1 (HY1) gene expression and H₂O₂ generation. Subsequently, LR formation was induced. NAA-induced HY1 expression is dependent on H₂O₂. This conclusion was supported by analyzing the removal of H₂O₂ with ascorbic acid (AsA) and dimethylthiourea (DMTU), both of which could block NAA-induced HY1 expression and LR formation. H₂O₂-induced LR formation was inhibited by an HO-1 inhibitor zinc protoporphyrin IX (Znpp) in wild-type and severely impaired in HY1 mutant hy1-100. Simultaneously, HY1 is required for NAA-mediated H₂O₂ generation, since Znpp inhibition of HY1 blocked the NAA-induced H₂O₂ production and LR formation. Genetic data demonstrated that hy1-100 was significantly impaired in H₂O₂ production and LR formation in response to NAA, compared with wild-type plants. The addition of carbon monoxide-releasing molecule-2 (CORM-2), the carbon monoxide (CO) donor, induced H₂O₂ production and LR formation, both of which were decreased by DMTU. Moreover, H₂O₂ and CORM-2 mimicked the NAA responses in the regulation of cell cycle genes expression, all of which were blocked by Znpp or DMTU, respectively, confirming that both H₂O₂ and CO were important in the early LR initiation. In summary, our pharmacological, genetic and molecular evidence demonstrated a close inter-relationship between HY1 and H₂O₂ existing in auxin-induced LR formation in Arabidopsis.     

Diurnal changes in the xanthophyll cycle pigments of freshwater algae correlate with the environmental hydrogen peroxide concentration rather than non-photochemical quenching

Background and Aims In photosynthetic organisms exposure to high light induces the production of reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), which in part is prevented by non-photochemical quenching (NPQ). As one of the most stable and longest-lived ROS, H₂O₂ is involved in key signalling pathways in development and stress responses, although in excess it can induce damage. A ubiquitous response to high light is the induction of the xanthophyll cycle, but its role in algae is unclear as it is not always associated with NPQ induction. The aim of this study was to reveal how diurnal changes in the level of H₂O₂ are regulated in a freshwater algal community.Methods A natural freshwater community of algae in a temporary rainwater pool was studied, comprising photosynthetic Euglena species, benthic Navicula diatoms, Chlamydomonas and Chlorella species. Diurnal measurements were made of photosynthetic performance, concentrations of photosynthetic pigments and H₂O₂. The frequently studied model organisms Chlamydomonas and Chlorella species were isolated to study photosynthesis-related H₂O₂ responses to high light.Key Results NPQ was shown to prevent H₂O₂ release in Chlamydomonas and Chlorella species under high light; in addition, dissolved organic carbon excited by UV-B radiation was probably responsible for a part of the H₂O₂ produced in the water column. Concentrations of H₂O₂ peaked at 2 µm at midday and algae rapidly scavenged H₂O₂ rather than releasing it. A vertical H₂O₂ gradient was observed that was lowest next to diatom-rich benthic algal mats. The diurnal changes in photosynthetic pigments included the violaxanthin and diadinoxanthin cycles; the former was induced prior to the latter, but neither was strictly correlated with NPQ.Conclusions The diurnal cycling of H₂O₂ was apparently modulated by the organisms in this freshwater algal community. Although the community showed flexibility in its levels of NPQ, the diurnal changes in xanthophylls correlated with H₂O₂ concentrations. Alternative NPQ mechanisms in algae involving proteins of the light-harvesting complex type and antioxidant protection of the thylakoid membrane by de-epoxidized carotenoids are discussed.     

Protein-mediated hydroxyl radical generation--the primary event in NADH oxidation and oxygen reduction by the granule rich fraction of human resting leukocytes.

The granule rich-fraction isolated from human resting polymorphonuclear leukocytes is capable of CN-insensitive NADH oxidation and O₂-uptake, accompanied by production of superoxide anion, hydroxyl radicals and H₂O₂. We showed that H₂O₂ initiates and maintains NADH oxidation and O₂-uptake but is also necessary for the formation of superoxide anion and hydroxyl radicals. It acts as a primary substrate for CN-insensitive protein-mediated formation of hydroxyl radicals, which in turn produce superoxide anions, probably through univalent oxidation of NADH as an intermediary.     

Reversible o(2) inhibition of nitrogenase activity in attached soybean nodules

Various forms of stress result in decreased O₂ permeability or decreased capacity to consume O₂ in legume root nodules. These changes alter the nodule interior O₂ concentration (O_i). To determine the relationship between O_i and nitrogenase activity in attached soybean (Glycine max) nodules, we controlled O_i by varying external pO₂ while monitoring internal H₂ concentration (H_i) with microelectrodes. O_i was monitored by noninvasive leghemoglobin spectrophotometry (nodule oximetry). After each step-change in O_i, H_i approached a new steady state, with a time constant averaging 23 s. The rate of H₂ production by nitrogenase was calculated as the product of H_i, nodule surface area, and nodule H₂ permeability. H₂ permeability was estimated from O₂ permeability (measured by nodule oximetry) by assuming diffusion through air-filled pores; support for this assumption is presented. O_i was nearly optimal for nitrogenase activity (H₂ production) between 15 and 150 nm. A 1- to 2-min exposure to elevated external pO₂ (40-100 kPa) reduced H_i to zero, but nitrogenase activity recovered quickly under air, often in <20 min. This rapid recovery contrasts with previous reports of much slower recovery with longer exposures to elevated pO₂. The mechanism of nitrogenase inhibition may differ between brief and prolonged O₂ exposures.     

The involvement of hydrogen peroxide in the differentiation of secondary walls in cotton fibers

H₂O₂ is a widespread molecule in many biological systems. It is created enzymatically in living cells during various oxidation reactions and by leakage of electrons from the electron transport chains. Depending on the concentration H₂O₂ can induce cell protective responses, programmed cell death, or necrosis. Here we provide evidence that H₂O₂ may function as a developmental signal in the differentiation of secondary walls in cotton (Gossypium hirsutum) fibers. Three lines of evidence support this conclusion: (a) the period of H₂O₂ generation coincided with the onset of secondary wall deposition, (b) inhibition of H₂O₂ production or scavenging the available H₂O₂ from the system prevented the wall differentiation process, and (c) exogenous addition of H₂O₂ prematurely promoted secondary wall formation in young fibers. Furthermore, we provide support for the concept that H₂O₂ generation could be mediated by the expression of the small GTPase Rac, the accumulation of which was shown previously to be strongly induced during the onset of secondary wall differentiation. In support of Rac's role in the activation of NADPH oxidase and the generation of reactive oxygen species, we transformed soybean (Glycine max) and Arabidopsis cells with mutated Rac genes. Transformation with a dominantly activated cotton Rac13 gene resulted in constitutively higher levels of H₂O₂, whereas transformation with the antisense and especially with dominant-negative Rac constructs decreased the levels of H₂O₂.     

Endogenous H2O2 produced by Streptococcus pneumoniae controls FabF activity

FabF elongation condensing enzyme is a critical factor in determining the spectrum of products produced by the FASII pathway. Its active site contains a critical cysteine-thiol residue, which is a plausible target for oxidation by H₂O₂. Streptococcus pneumoniae produces exceptionally high levels of H₂O₂, mainly through the conversion of pyruvate to acetyl-P via pyruvate oxidase (SpxB). We present evidence showing that endogenous H₂O₂ inhibits FabF activity by specifically oxidizing its active site cysteine-thiol residue. Thiol trapping methods revealed that one of the three FabF cysteines in the wild-type strain was oxidized, whereas in an spxB mutant, defective in H₂O₂ production, none of the cysteines was oxidized, indicating that the difference in FabF redox state originated from endogenous H₂O₂. In vitro exposure of the spxB mutant to various H₂O₂ concentrations further confirmed that only one cysteine residue was susceptible to oxidation. By blocking FabF active site cysteine with cerulenin we show that the oxidized cysteine was the catalytic one. Inhibition of FabF activity by either H₂O₂ or cerulenin resulted in altered membrane fatty acid composition. We conclude that FabF activity is inhibited by H₂O₂ produced by S. pneumoniae.     

Aquaporin as a membrane transporter of hydrogen peroxide in plant response to stresses

Hydrogen peroxide (H₂O₂) is a reactive oxygen species that signals between cells, and H₂O₂ signaling is essential for diverse cellular processes, including stress response, defense against pathogens, and the regulation of programmed cell death in plants. Although plasma membrane intrinsic proteins (PIPs) have been known to transport H₂O₂ across cell membranes, the permeability of each family member of PIPs toward H₂O₂ has not yet been determined in most plant species. In a recent study, we showed that certain isoforms of Arabidopsis thaliana AtPIPs, including AtPIP2;2, AtPIP2;4, AtPIP2;5, and AtPIP2;7, are permeable for H₂O₂ in yeast cells. Since the expression of PIPs is differently modulated in Arabidopsis by abiotic stress or H₂O₂ treatment, it is important to investigate the integrated regulation of aquaporin expression and their physiological significance in H₂O₂ transport and plant response to diverse abiotic stresses.     

Real-time monitoring of subcellular H2O2 distribution in Chlamydomonas reinhardtii

Hydrogen peroxide (H₂O₂) is recognized as an important signaling molecule in plants. We sought to establish a genetically encoded, fluorescent H₂O₂ sensor that allows H₂O₂ monitoring in all major subcompartments of a Chlamydomonas cell. To this end, we used the Chlamydomonas Modular Cloning toolbox to target the hypersensitive H₂O₂ sensor reduction–oxidation sensitive green fluorescent protein2-Tsa2ΔC_R to the cytosol, nucleus, mitochondrial matrix, chloroplast stroma, thylakoid lumen, and endoplasmic reticulum (ER). The sensor was functional in all compartments, except for the ER where it was fully oxidized. Employing our novel sensors, we show that H₂O₂ produced by photosynthetic linear electron transport (PET) in the stroma leaks into the cytosol but only reaches other subcellular compartments if produced under nonphysiological conditions. Furthermore, in heat-stressed cells, we show that cytosolic H₂O₂ levels closely mirror temperature up- and downshifts and are independent from PET. Heat stress led to similar up- and downshifts of H₂O₂ levels in the nucleus and, more mildly, in mitochondria but not in the chloroplast. Our results thus suggest the establishment of steep intracellular H₂O₂ gradients under normal physiological conditions with limited diffusion into other compartments. We anticipate that these sensors will greatly facilitate future investigations of H₂O₂ biology in plant cells.

The establishment of a hypersensitive H₂O₂ sensor in six major compartments of the Chlamydomonas cell reveals steep intracellular H₂O₂ gradients under normal physiological conditions with limited diffusion into other compartments.