Decreased H2O2 Production Research Articles

NADPH oxidase 4-derived hydrogen peroxide counterbalances testosterone-induced endothelial dysfunction and migration.

High levels of testosterone (Testo) are associated with cardiovascular risk by increasing reactive oxygen species (ROS) formation. NADPH oxidases (NOX) are the major source of ROS in the vasculature of cardiovascular diseases. NOX4 is a unique isotype, which produces hydrogen peroxide (H2O2), and its participation in cardiovascular biology is controversial. So far, it is unclear whether NOX4 protects from Testo-induced endothelial injury. Thus, we hypothesized that supraphysiological levels of Testo induce endothelial NOX4 expression to attenuate endothelial injury. Human mesenteric vascular endothelial cells (HMECs) and human umbilical vein endothelial cells (HUVEC) were treated with Testo (10-7 M) with or without a NOX4 inhibitor [GLX351322 (10-4 M)] or NOX4 siRNA. In vivo, 10-wk-old C57Bl/6J male mice were treated with Testo (10 mg/kg) for 30 days to study endothelial function. Testo increased mRNA and protein levels of NOX4 in HMECs and HUVECs. Testo increased superoxide anion (O2-) and H2O2 production, which were abolished by NOX1 and NOX4 inhibition, respectively. Testo also attenuated bradykinin-induced NO production, which was further impaired by NOX4 inhibition. In vivo, Testo decreased H2O2 production in aortic segments and triggered endothelial dysfunction [decreased relaxation to acetylcholine (ACh)], which was further impaired by GLX351322 and by a superoxide dismutase and catalase mimetic (EUK134). Finally, Testo led to a dysregulated endothelial cell migration, which was exacerbated by GLX351322. These data indicate that supraphysiological levels of Testo increase the endothelial expression and activity of NOX4 to counterbalance the deleterious effects caused by Testo in endothelial function.NEW & NOTEWORTHY By inducing ROS formation, high levels of testosterone play a major role in the pathogenesis of cardiovascular disease. NOXs are the major sources of ROS in the vasculature of cardiovascular diseases. Herein, we describe a novel compensatory mechanism by showing that NOX4 is a protective oxidant enzyme and counterbalances the deleterious effects of testosterone in endothelial cells by modulating hydrogen peroxide formation.

Praseodymium and warming interactions in mussels: Comparison between observed and predicted results

Being a crucial element for technological development, praseodymium (Pr) has been increasingly used, leading to a rise in its concentration in aquatic systems. However, its potential threats to organisms remain poorly understood. Besides contamination, organisms are also threatened by climate change-related factors, including warming. It is important to evaluate how climate change-related factors may influence the effects of contaminants. To address this, histopathological and biochemical analyses were performed in adult mussels of Mytilus galloprovincialis, following a 28-day exposure to Pr (10 μg/L) and warming (4 °C increase) separately, and in combination. Additionally, biochemical and physiological alterations were analysed in the sperm of mussels after 30-min exposure to the same treatments. Furthermore, it was used the Independent Action model to predict the interaction between Pr and warming. The results showed, in the case of adults exposed to Pr, an increase in superoxide dismutase (SOD) and glutathione S-transferases (GSTs) activities. However, it was insufficient, leading to histopathological injuries, redox imbalance, and cellular damage. In the case of sperm, Pr induced an increase of mitochondrial activity and respiration rate, in response to the increase in systemic metabolic rate and oxygen demand. Warming increased the metabolism, and induced redox imbalance and cellular damage in adults. In sperm, a rise in temperature induced lipid peroxidation and a decrease in velocity. Warming induced some alterations in how adult mussels responded to Pr, activating catalase instead of SOD, and in addition to GSTs, also activated carboxylesterases. However, it was not enough to avoid redox imbalance and cellular damage. In the case of sperm, the combination induced a decrease in H2O2 production, and higher oxygen demand, which prevented the decrease in motility and velocity. This study highlights the limitations of using models and emphasizes the importance of studying the impacts of emerging contaminants, such as rare earth elements, and their combination with climate change-related factors. Under environmental conditions, chronic exposure to the combined effect of different stressors might generate impacts at higher biological levels. This may affect organisms' respiratory and filtration capacity, nutrient absorption, defence capacity against infections or diseases, and sperm viability, ultimately resulting in reduced growth and reproduction, with consequences at the population level.

D-Amino acids differentially trigger an inflammatory environment in vitro

Studies in vivo have demonstrated that the accumulation of d-amino acids (d-AAs) is associated with age-related diseases and increased immune activation. However, the underlying mechanism(s) of these observations are not well defined. The metabolism of d-AAs by d-amino oxidase (DAO) produces hydrogen peroxide (H2O2), a reactive oxygen species involved in several physiological processes including immune response, cell differentiation, and proliferation. Excessive levels of H2O2 contribute to oxidative stress and eventual cell death, a characteristic of age-related pathology. Here, we explored the molecular mechanisms of d-serine (d-Ser) and d-alanine (d-Ala) in human liver cancer cells, HepG2, with a focus on the production of H2O2 the downstream secretion of pro-inflammatory cytokine and chemokine, and subsequent cell death. In HepG2 cells, we demonstrated that d-Ser decreased H2O2 production and induced concentration-dependent depolarization of mitochondrial membrane potential (MMP). This was associated with the upregulation of activated NF-кB, pro-inflammatory cytokine, TNF-α, and chemokine, IL-8 secretion, and subsequent apoptosis. Conversely, d-Ala-treated cells induced H2O2 production, and were also accompanied by the upregulation of activated NF-кB, TNF-α, and IL-8, but did not cause significant apoptosis. The present study confirms the role of both d-Ser and d-Ala in inducing inflammatory responses, but each via unique activation pathways. This response was associated with apoptotic cell death only with d-Ser. Further research is required to gain a better understanding of the mechanisms underlying d-AA-induced inflammation and its downstream consequences, especially in the context of aging given the wide detection of these entities in systemic circulation.

Impact of Endogenous Pneumococcal Hydrogen Peroxide on the Activity and Release of Pneumolysin.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. The pore-forming cholesterol-dependent cytolysin (CDC) pneumolysin (PLY) and the physiological metabolite hydrogen peroxide (H2O2) can greatly increase the virulence of pneumococci. Although most studies have focused on the contribution of both virulence factors to the course of pneumococcal infection, it is unknown whether or how H2O2 can affect PLY activity. Of note, S. pneumoniae exploits endogenous H2O2 as an intracellular signalling molecule to modulate the activity of several proteins. Here, we demonstrate that H2O2 negatively affects the haemolytic activity of PLY in a concentration-dependent manner. Prevention of cysteine-dependent sulfenylation upon substitution of the unique and highly conserved cysteine residue to serine in PLY significantly reduces the toxin's susceptibility to H2O2 treatment and completely abolishes the ability of DTT to activate PLY. We also detect a clear gradual correlation between endogenous H2O2 generation and PLY release, with decreased H2O2 production causing a decline in the release of PLY. Comparative transcriptome sequencing analysis of the wild-type S. pneumoniae strain and three mutants impaired in H2O2 production indicates enhanced expression of several genes involved in peptidoglycan (PG) synthesis and in the production of choline-binding proteins (CPBs). One explanation for the impact of H2O2 on PLY release is the observed upregulation of the PG bridge formation alanyltransferases MurM and MurN, which evidentially negatively affect the PLY release. Our findings shed light on the significance of endogenous pneumococcal H2O2 in controlling PLY activity and release.

TaUAM3, a UDP‐Ara mutases protein, positively regulates wheat resistance to the stripe rust fungus

AbstractPlant cell walls are composed of polysaccharides such as cellulose, hemicelluloses, and pectins, whose location and function differ depending on plant type. UDP‐Ara mutases (UAMs) have been reported to play important roles in plant development and response to various plant stresses (abiotic and biotic). However, little work has been reported on UAM in wheat. In this study, we dissected the role of the UAM family member, UAM3, during the interaction between wheat and the stripe rust fungus, Puccinia striiformis f. sp. tritici (Pst), and in response to treatment with salicylic acid (SA). RNA interference (RNAi)‐based stable silencing of TaUAM3 resulted in decreased resistance to Pst fungus. In addition, CRISPR‐mediated genome editing (GE) of TaUAM3 enhanced the susceptibility of wheat to Pst or compromised disease resistance accompanied by increased fungal growth and decreased H2O2 production in plant tissues. Moreover, the transcript levels of pathogenesis‐related (PR) genes and reactive oxygen species (ROS)‐generating genes were down‐regulated in both the RNAi‐silenced and CRISPR‐edited plants, while the ROS‐scavenging gene, TaCAT3, was up‐regulated. Therefore, TaUAM3 positively regulates the resistance of wheat to Pst.

Mitochondrial oxidative phosphorylation response overrides glucocorticoid-induced stress in a reptile.

Stress hormones and their impacts on whole organism metabolic rates are usually considered as appropriate proxies for animal energy budget that is the foundation of numerous concepts and models aiming at predicting individual and population responses to environmental stress. However, the dynamics of energy re-allocation under stress make the link between metabolism and corticosterone complex and still unclear. Using ectopic application of corticosterone for 3, 11 and 21days, we estimated a time effect of stress in a lizard (Zootoca vivipara). We then investigated whole organism metabolism, muscle cellular O2 consumption and liver mitochondrial oxidative phosphorylation processes (O2 consumption and ATP production) and ROS production. The data showed that while skeletal muscle is not impacted, stress regulates the liver mitochondrial functionality in a time-dependent manner with opposing pictures between the different time expositions to corticosterone. While 3days exposition is characterized by lower ATP synthesis rate and high H2O2 release with no change in the rate of oxygen consumption, the 11days exposition reduced all three fluxes of about 50%. Oxidative phosphorylation capacities in liver mitochondria of lizard treated with corticosterone for 21days was similar to the hepatic mitochondrial capacities in lizards that received no corticosterone treatment but with 40% decrease in H2O2 production. This new mitochondrial functioning allows a better capacity to respond to the energetic demands imposed by the environment but do not influence whole organism metabolism. In conclusion, global mitochondrial functioning has to be considered to better understand the proximal causes of the energy budget under stressful periods.

Proton Irradiations at Ultra-High Dose Rate vs. Conventional Dose Rate: Strong Impact on Hydrogen Peroxide Yield.

During ultra-high dose rate (UHDR) external radiation therapy, healthy tissues appear to be spared while tumor control remains the same compared to conventional dose rate. However, the understanding of radiochemical and biological mechanisms involved are still to be discussed. This study shows how the hydrogen peroxide (H2O2) production, one of the reactive oxygen species (ROS), could be controlled by early heterogenous radiolysis processes in water during UHDR proton-beam irradiations. Pure water was irradiated in the plateau region (track-segment) with 68 MeV protons under conventional (0.2 Gy/s) and several UHDR conditions (40 Gy/s to 60 kGy/s) at the ARRONAX cyclotron. Production of H2O2 was then monitored using the Ghormley triiodide method. New values of GTS(H2O2) were added in conventional dose rate. A substantial decrease in H2O2 production was observed from 0.2 to 1.5 kGy/s with a more dramatic decrease below 100 Gy/ s. At higher dose rate, up to 60 kGy/s, the H2O2 production stayed stable with a mean decrease of 38% ± 4%. This finding, associated to the decrease in the production of hydroxyl radical (•OH) already observed in other studies in similar conditions can be explained by the well-known spur theory in radiation chemistry. Thus, a two-step FLASH-RT mechanism can be envisioned: an early step at the microsecond scale mainly controlled by heterogenous radiolysis, and a second, slower, dominated by O2 depletion and biochemical processes. To validate this hypothesis, more measurements of radiolytic species will soon be performed, including radicals and associated lifetimes.

Role of cytochrome P450 2C8 genetic polymorphism and epoxygenase uncoupling in periodontal remodelling affecting orthodontic treatment.

In genome-wide association studies, the CYP2C8 gene locus has been reported to be associated with bisphosphonate-related osteonecrosis of the jaw, a severe devastating side effect of antiresorptive bone treatment. The aim of this study was to elucidate the putative pathomechanism explaining the association between the genetic polymorphism with the alleles CYP2C8*2 and *3 causing low CYP2C8 activity, and disturbed periodontal remodelling in periodontal fibroblasts cultured from patients undergoing orthodontic treatment. CYP2C8 activity, enzyme expression and substrate metabolism were detected in human periodontal fibroblast cultures. Zoledronic acid caused enhanced reactive oxygen species (ROS) production in periodontal fibroblasts, which was enhanced by arachidonic acid as inflammatory signal. Enhanced bisphosphonate-induced uncoupling of the CYP2C8 enzyme was detected in the variant allele (CYP2C8*3) with the result of increased H2 O2 production and lowered substrate oxidation. Conversely, substrate (amodiaquine) addition led to decreased H2 O2 production in isolated CYP2C8 enzymes, but in CYP2C8*3 enzyme, increased H2 O2 was still detected, especially in presence of arachidonic acid. CYP2C8 variants leading to decreased enzyme activity in substrate oxidation may enhance ROS production by reaction uncoupling, and thus, contribute to difficulties in orthodontic treatment and the risk of side effects of antiresorptive drugs.

Mild Mitochondrial Uncoupling Decreases Experimental Atherosclerosis, A Proof of Concept.

Aim: Atherosclerosis is responsible for high morbidity and mortality rates around the world. Local arterial oxidative stress is involved in all phases of atherosclerosis development. Mitochondria is a relevant source of the oxidants, particularly under certain risky conditions, such as hypercholesterolemia. The aim of this study was to test whether lowering the production of mitochondrial oxidants by induction of a mild uncoupling can reduce atherosclerosis in hypercholesterolemic LDL receptor knockout mice. Methods: The mice were chronically treated with very low doses of DNP (2,4-dinitrophenol) and metabolic, inflammatory and redox state markers and atherosclerotic lesion sizes were determined. Results: The DNP treatment did not change the classical atherosclerotic risk markers, such as plasma lipids, glucose homeostasis, and fat mass, as well as systemic inflammatory markers. However, the DNP treatment diminished the production of mitochondrial oxidants, systemic and tissue oxidative damage markers, peritoneal macrophages and aortic rings oxidants generation. Most importantly, development of spontaneous and diet-induced atherosclerosis (lipid and macrophage content) were significantly decreased in the DNP-treated mice. In vitro, DNP treated peritoneal macrophages showed decreased H2O2 production, increased anti-inflammatory cytokines gene expression and secretion, increased phagocytic activity, and decreased LDL-cholesterol uptake. Conclusions: These findings are a proof of concept that activation of mild mitochondrial uncoupling is sufficient to delay the development of atherosclerosis under the conditions of hypercholesterolemia and oxidative stress. These results promote future approaches targeting mitochondria for the prevention or treatment of atherosclerosis.

Glutathione S-transferase, catalase, and mitochondrial superoxide dismutase gene polymorphisms modulate redox potential in systemic lupus erythematosus patients from Manaus, Amazonas, Brazil.

To investigate the frequency of glutathione S-transferase (GST), catalase, and SOD2 genetic polymorphisms and their correlation with SLE. A total of 290 females (patients = 151; controls= 139) were recruited. Multiplex PCR was performed for genotyping GSTM1 and GSTT1 genes, whereas real-time qPCR was used for determination of SNPs: CAT C262T, SOD2 C47T, GSTP1 A313G and GSTP1 IVS6 -C16T. Thiol levels are decreased in SLE patients (p<0.001), while MDA levels were significantly higher (p<0.001) and those carrying the polymorphisms had higher rates of oxidative stress. Patients with double null deletion GSTT1null/GSTM1null had a frequency almost five times higher than the controls (p<0.001, OR 4.81, CI 1.98-12.11). SLE patients had a lower wild-type frequency of SOD2CC allele compared to controls (12.4% vs 27.3%). Statistical significances were observed on the association between the GSTT1null and GSTM1null with SOD2mut (p<0.001, OR 0.15, CI 0.05-0.47), with GSTP1 A303G (p=0.012, OR 0.19, CI 0.05-0.69), and with GSTP1 IVS6 (p=0.008, OR 0.14, CI 0.03-0.63). The same was observed between SOD2 C47T with GSTP1 A303G (p=0.09, OR 0.27, CI 0.09-0.74) and GSTP1 IVS6 (p=0.036, OR 0.41, CI 0.18-0.92). The deletion GSTT1null/GSTM1null may contribute to the increased of the oxidative stress in SLE patients. Isolated GSTP1 and CAT polymorphisms do not seem to influence the increased oxidative stress, neither SLE clinical manifestations. SOD2 47CT/TT allele may have greater oxidative stress due to structural change in the protein and decreased H2O2 production. The combination of polymorphic genes may be involved in the pathogenesis of the disease. Key points • Major question of our paper: Many studies have shown that the antioxidant status levels are decreased in patients with SLE, especially in severe stages of disease. We believe that this paper will be of interest to the readership of your journal had the involvement of polymorphisms and mutations in several genes that contribute to the genetic etiology of SLE, suggesting that these may influence the mechanisms of disease. • Our results. Thiol level was significantly (p<.001) lower and MDA level significantly increased (p<.001) among SLE patients. Those carrying the polymorphisms hadhigher rates of oxidative stress. SLE Patients had a frequency almost five times higher of double null deletion GSTT1null/GSTM1null than the controls.SLE Patients had a lower wild type frequency of SOD2CC allele compared to controls (12.4% vs 27.3%). We believed the deletion GSTT1null/GSTM1nullmay contribute to the increased of the oxidative stress in SLE patients while carriers of the mutant SOD2 47CT/TT allele may have greater oxidative stressdue to structural change in the protein and decreased H2O2 production. The combination of polymorphic genes may be involved in the pathogenesis of thedisease. • Implications of our results: Evidence for the involvement of genetic factors in severe clinical to lupus is compelling. This manuscript shows genetic insights in pathogenic pathways that may lead to severe clinical implications to LES. Therefore, it is necessary to understand their impact on overall disease pathogenesis and prognosis in these patients. We understand from general consensus about environmental factors can modify disease, however, maybe just in individuals who have a permissive genetic background. Even that no single gene predisposes some individuals to LES, we believe the genetic factors described in this manuscript are important elements in susceptibility to severe clinical to LES.

Ascorbate-glutathione pathways mediated by cytokinin regulate H2O2 levels in light-controlled rose bud burst.

Rosebush (Rosa “Radrazz”) plants are an excellent model to study light control of bud outgrowth since bud outgrowth only arises in the presence of light and never occurs in darkness. Recently, we demonstrated high levels of hydrogen peroxide (H2O2) present in the quiescent axillary buds strongly repress the outgrowth process. In light, the outgrowing process occurred after H2O2 scavenging through the promotion of Ascorbic acid–Glutathione (AsA–GSH)-dependent pathways and the continuous decrease in H2O2 production. Here we showed Respiratory Burst Oxidase Homologs expression decreased in buds during the outgrowth process in light. In continuous darkness, the same decrease was observed although H2O2 remained at high levels in axillary buds, as a consequence of the strong inhibition of AsA–GSH cycle and GSH synthesis preventing the outgrowth process. Cytokinin (CK) application can evoke bud outgrowth in light as well as in continuous darkness. Furthermore, CKs are the initial targets of light in the photocontrol process. We showed CK application to cultured buds in darkness decreases bud H2O2 to a level that is similar to that observed in light. Furthermore, this treatment restores GSH levels and engages bud burst. We treated plants with buthionine sulfoximine, an inhibitor of GSH synthesis, to solve the sequence of events involving H2O2/GSH metabolisms in the photocontrol process. This treatment prevented bud burst, even in the presence of CK, suggesting the sequence of actions starts with the positive CK effect on GSH that in turn stimulates H2O2 scavenging, resulting in initiation of bud outgrowth.

Overexpression of WssgtL3.1 gene from Withania somnifera confers salt stress tolerance in Arabidopsis.

Overexpression of Withania somnifera SGT gene (WssgtL3.1) in transgenic Arabidopsis improves various agronomic and physiological traits and alters conjugated sterol levels to mitigate the effect of salt stress. Sterols are essential constituents of cell membranes that are involved in several biological functions, including response to various biotic and abiotic stresses by altering membrane permeability and signaling pathways. Sterol glycosyltransferases (SGTs) are enzymes that are involved in sterol modification by converting sterols into sterol-conjugates to play essential roles in adaptive responses. However, their roles under abiotic stresses are lesser-known. Among abiotic stresses, salinity imposes serious threat to crop yield worldwide, hence the present study intends to investigate the role of WssgtL3.1-overexpressed Arabidopsis plants under salt stress indicating the crosstalk between SGT gene and salinity to develop improved crop varieties with better stress tolerance ability. The findings revealed that overexpression of WssgtL3.1 gene in A. thaliana improved the resistance against salt stress in the overexpressing lines. Transgenic lines showed significantly higher germination rate, increased plant growth with less chlorophyll damage compared to wild-type (WT) control plants. Moreover, better tolerance also correlated with enhanced osmolytes (proline and soluble sugar), better membrane integrity, decreased H2O2 production and lesser MDA accumulation and Na+/K+ ratio with more negative osmotic potential in overexpressed lines. Additionally, in sterol profiling, significant enhancement in stigmasterol was also observed in transgenic lines than WT plants. Furthermore, in expression profiling, salt responsive genes LEA 4-5, sucrose synthase, and transporter of monosaccharide (ERD) significantly upregulated in overexpressing lines as compared to WT. Thus our data strongly support the defensive role of Withania somnifera SGT gene (WssgtL3.1) against salt stress and contribute to improved salinity tolerance in plants through sterol modulation.

Integrated paper-based 3D platform for long-term cell culture and in situ cell viability monitoring of Alzheimer's disease cell model

Reactive oxygen species including superoxide anion, hydrogen peroxide (H2O2) and hydroxyl radicals, as a conflicting class of biological metabolites in living organism, act crucial effect on Alzheimer's disease (AD). In this work, a facile integrated platform composed of a paper-based three-dimension (3D) cell culture system and an electrochemical sensor was developed for the construction of AD cell model in third dimensional structure and in situ cell viability monitoring by H2O2 released from PC12 cells cultured on paper scaffold were divided into three groups containing control group, amyloid beta peptide 25–35 (Aβ25-35) group and Aβ25-35+curcumin (Aβ25-35+cur) group, respectively. In addition, the paper-based 3D platform displayed excellent properties, such as sensitivity, selectivity, reproducibility and stability. The levels of H2O2 expressed in PC12 cells of the three groups were monitored through a paper-based 3D platform. The viability of cells cultured on the 96-well plate was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Results of this paper-based platform are consistent with those of MTT, both displaying improved cell viability and decreased H2O2 production in Aβ25-35+cur group compared to Aβ25-35 group, which indicates that curcumin has effective cytoprotection. The paper-based 3D platform provides a convenient, economic and universal platform for in situ cell activity monitoring by key small molecules released from living cells.

Diazoxide Modulates Cardiac Hypertrophy by Targeting H2O2 Generation and Mitochondrial Superoxide Dismutase Activity.

Cardiac hypertrophy involves marked wall thickening or chamber enlargement. If sustained, this condition will lead to dysfunctional mitochondria and oxidative stress. Mitochondria have ATP-sensitive K+ channels (mitoKATP) in the inner membrane that modulate the redox status of the cell. We investigated the in vivo effects of mitoKATP opening on oxidative stress in isoproterenol- induced cardiac hypertrophy. Cardiac hypertrophy was induced in Swiss mice treated intraperitoneally with isoproterenol (ISO - 30 mg/kg/day) for 8 days. From day 4, diazoxide (DZX - 5 mg/kg/day) was used in order to open mitoKATP (a clinically relevant therapy scheme) and 5-hydroxydecanoate (5HD - 5 mg/kg/day) or glibenclamide (GLI - 3 mg/kg/day) were used as mitoKATP blockers. Isoproterenol-treated mice had elevated heart weight/tibia length ratios (HW/TL). Additionally, hypertrophic hearts had elevated levels of carbonylated proteins and Thiobarbituric Acid Reactive Substances (TBARS), markers of protein and lipid oxidation. In contrast, mitoKATP opening with DZX avoided ISO effects on gross hypertrophic markers (HW/TL), carbonylated proteins and TBARS, in a manner reversed by 5HD and GLI. Moreover, DZX improved mitochondrial superoxide dismutase activity. This effect was also blocked by 5HD and GLI. Additionally, ex vivo treatment of isoproterenol- induced hypertrophic cardiac tissue with DZX decreased H2O2 production in a manner sensitive to 5HD, indicating that this drug also acutely avoids oxidative stress. Our results suggest that diazoxide blocks oxidative stress and reverses cardiac hypertrophy. This pharmacological intervention could be a potential therapeutic strategy to prevent oxidative stress associated with cardiac hypertrophy.

Involvement of the Dectin-1 Receptor upon the Effector Mechanisms of Human Phagocytic Cells against Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America, occurs after inhalation of mycelial components of Paracoccidioides spp. When the fungus reaches the lungs and interacts with the alveolar macrophages and other cells, phagocytic cells such as neutrophils and monocytes are immediately recruited to the injured site. The interaction between surface molecules of pathogens and homologous receptors, present on the surface membrane of phagocytes, modulates the innate immune cell activation. Studies have shown the importance of fungal recognition by the Dectin-1 receptor, which can induce a series of cellular protective responses against fungi. The objective of the present study was to evaluate Dectin-1 receptor expression and the effector mechanisms of human monocytes and neutrophils activated or not with different cytokines, such as IFN-γ, TNF-α, and GM-CSF, followed by the challenge with Paracoccidioides brasiliensis (P. brasiliensis or Pb265). Therefore, analysis of Dectin-1 receptor expression was done by flow cytometry whereas the effector mechanisms were evaluated by fungal recovery by colony-forming unit (CFU) counting and hydrogen peroxide (H2O2) production. Our results showed that, after treatment with IFN-γ, TNF-α, and GM-CSF and challenge with Pb265, cells, especially monocytes, demonstrated an increase in Dectin-1 expression. Both types of cells treated with the cytokines exhibited a decreased fungal recovery and, conversely, an increased production of H2O2. However, when cultures were treated with an anti-Dectin-1 monoclonal antibody, to block the P. brasiliensis binding, a decrease in H2O2 production and an increase in fungal recovery were detected. This effect was observed in all cultures treated with the specific monoclonal antibody. These results show the involvement of the Dectin-1 receptor in fungal recognition and its consequent participation in the induction of the killing mechanisms against P. brasiliensis.

Dietary antioxidants as a source of hydrogen peroxide.

Studies of 54 antioxidants revealed that 27 of them, mainly polyphenols, generated hydrogen peroxide (H2O2) when added to Dulbecco's modified Eagle's medium (DMEM), other media used for culture of mammalian and yeast cells and phosphate-buffered saline. The most active antioxidants were: propyl gallate (PG), (-)-epigallocatechin gallate (EGCG) and quercetin (Q). Chelex treatment and iron chelators decreased H2O2 generation suggesting that transition metal ions catalyze antioxidant autoxidation and H2O2 production. Green tea also generated H2O2; tea prepared on tap water generated significantly more H2O2 than tea prepared on deionized water. Ascorbic acid decreased H2O2 production although it generated H2O2 itself, in the absence of other additives. Lemon added to the tea significantly reduced generation of H2O2. Hydrogen peroxide generated in the medium contributed to the cytotoxicity of PG, EGCG and Q to human prostate carcinoma DU-145 cells, since catalase increased the survival of the cells subjected to these compounds in vitro.

Crosstalk between the serine/threonine kinase StkP and the response regulator ComE controls the stress response and intracellular survival of Streptococcus pneumoniae.

Streptococcus pneumoniae is an opportunistic human bacterial pathogen that usually colonizes the upper respiratory tract, but the invasion and survival mechanism in respiratory epithelial cells remains elusive. Previously, we described that acidic stress-induced lysis (ASIL) and intracellular survival are controlled by ComE through a yet unknown activation mechanism under acidic conditions, which is independent of the ComD histidine kinase that activates this response regulator for competence development at pH 7.8. Here, we demonstrate that the serine/threonine kinase StkP is essential for ASIL, and show that StkP phosphorylates ComE at Thr128. Molecular dynamic simulations predicted that Thr128-phosphorylation induces conformational changes on ComE’s DNA-binding domain. Using nonphosphorylatable (ComET128A) and phosphomimetic (ComET128E) proteins, we confirmed that Thr128-phosphorylation increased the DNA-binding affinity of ComE. The non-phosphorylated form of ComE interacted more strongly with StkP than the phosphomimetic form at acidic pH, suggesting that pH facilitated crosstalk. To identify the ComE-regulated genes under acidic conditions, a comparative transcriptomic analysis was performed between the comET128A and wt strains, and differential expression of 104 genes involved in different cellular processes was detected, suggesting that the StkP/ComE pathway induced global changes in response to acidic stress. In the comET128A mutant, the repression of spxB and sodA correlated with decreased H2O2 production, whereas the reduced expression of murN correlated with an increased resistance to cell wall antibiotic-induced lysis, compatible with cell wall alterations. In the comET128A mutant, ASIL was blocked and acid tolerance response was higher compared to the wt strain. These phenotypes, accompanied with low H2O2 production, are likely responsible for the increased survival in pneumocytes of the comET128A mutant. We propose that the StkP/ComE pathway controls the stress response, thus affecting the intracellular survival of S. pneumoniae in pneumocytes, one of the first barriers that this pathogen must cross to establish an infection.

Post-ischemic administration of 5-methoxyindole-2-carboxylic acid at the onset of reperfusion affords neuroprotection against stroke injury by preserving mitochondrial function and attenuating oxidative stress

We previously reported that 5-methoxyindole-2-carboxylic acid (MICA) could induce preconditioning effect in the ischemic brain of rat. In the present study, we addressed the question of whether MICA could also trigger a postconditioning effect in ischemic stroke. To this end, MICA (100 mg/kg body weight) was injected intraperitoneally at the onset of 24 h reperfusion following 1 h ischemia in rat brain. Results indicate that stroked animals treated with MICA showed less brain infarction volume than that of vehicle-treated animals. Further experiments revealed that brain mitochondrial complexes I and IV showed elevated enzymatic activities in MICA treated group and the elevation in complex I activity was likely contributed by seemingly enhanced expression of many complex I subunits, which was determined by mass spectral peptide sequencing. When compared with vehicle-treated rats, the preservation of complexes I and IV activities was shown to be accompanied by enhanced mitochondrial membrane potential, increased ATP production, and decreased caspase-3 activity. Additional studies also indicate the involvement of NQO1 upregulation by the Nrf2 signaling pathway in this MICA postconditioning paradigm. Consequently, attenuated oxidative stress in the MICA treated group reflected by decrease in H2O2 production and protein carbonylation and lipid peroxidation was detected. Taken together, the present study demonstrates that MICA can also induce a postconditioning effect in the ischemic brain of rat and the underlying mechanism likely involves preservation of mitochondrial function, upregulation of cellular antioxidative capacity, and attenuation of oxidative stress.

Melatonin alleviates aluminium toxicity through modulating antioxidative enzymes and enhancing organic acid anion exudation in soybean.

Aluminium (Al) toxicity is a major chemical constraint limiting plant growth and production on acidic soils. Melatonin (N-acetyl-5-methoxytryptamine) is a ubiquitous molecule that plays crucial roles in plant growth and stress tolerance. However, there is no knowledge regarding whether melatonin is involved in plant responses to Al stress. Here, we show that optimal concentrations of melatonin could effectively ameliorate Al-induced phytotoxicity in soybean (Glycine max L.). The concentration of melatonin in roots was significantly increased by the 50μM Al treatment. Such an increase in endogenous melatonin coincided with the upregulation of the gene encoding acetyltransferase NSI-like (nuclear shuttle protein-interacting) in soybean roots. Supplementation with low concentrations of melatonin (0.1 and 1μM) conferred Al resistance as evident in partial alleviation of root growth inhibition and decreased H2O2 production: in contrast, high concentrations of melatonin (100 and 200μM) had an opposite effect and even decreased root growth in Al-exposed seedlings. Mitigation of Al stress by the 1μM melatonin root treatment was associated with enhanced activities of the antioxidant enzymes and increased exudation of malate and citrate. In conclusion, melatonin might play a critical role in soybean resistance to Al toxicity.

7 - Effect of Iron on Mitochondrial Function and RNA Oxidation

Objectives Iron is a redox reactive metal and causes reactive oxygen species formation via the Fenton reaction; Iron(II) is oxidized by hydrogen peroxide (H2O2) to iron(III), thereby forming the reactive hydroxyl radical. Subsequently, the hydroxyl radical can cause oxidative damage to cellular macromolecules. The mitochondria contribute to H2O2 formation and also take part in iron metabolism. We wanted to investigate if iron has an effect on mitochondrial function. Furthermore, we wanted to examine if iron increased RNA oxidation. Methods Experiments were performed on permeabilized mice muscle fibers. Mitochondrial function was measured by the Oxygraph-2k (Oroboros Instruments) as respiration and H2O2 production. Iron (iron(II)sulfate, 0.1mM or 1 mM) was added during OXPHOS state (maximal respiration). Effect of iron chelator DFOM (0.1mM) was tested. In separate experiments permeabilized fibers were incubated for an hour with iron (0.01mM, 0.1mM and 1mM) and RNA oxidation was measured as 8-oxo-7,8-dihydroguanosine (8-oxoGuo) per 106 guanosine by UPLC-MS/MS. Results Addition of iron did not change the mitochondrial respiration (p=0.9). Addition of DFOM did not have an effect on respiration when comparing post-iron and post-DFOM addition (p=0.4). H2O2 production increased only when 1mM iron was added (p=0.05), however this was also observed in experiments without a biopsy. DFOM decreased H2O2 production in experiments with the biopsies, but had surprisingly no effect when no biopsy was present. We saw a significant 8-oxoGuo increase when adding iron, the increase being higher, the higher the iron concentration (p Conclusions Iron does not have an acute effect on mitochondrial OXPHOS state or H2O2 production in mice biopsies. The observed increase in H2O2 production is suspected to be caused by a chemical reaction between iron and the used buffer. However, addition of iron causes RNA oxidation. The acute effect of iron on RNA oxidation does not seem to be mediated by mitochondrial damage.