Presence Of Menadione Research Articles

Abstract Tu123: Redox Biology of the Mitochondrial Protein mitoNEET and Ascorbate

Background: It has been proposed that mitoNEET is an Fe-S transfer/repair enzyme, playing a role under conditions of oxidative stress. mitoNEET protects mitochondria against oxidative stress in cardiomyocytes. Loss of mitoNEET is linked with age-related heart failure. Oxidized mitoNEET, [2Fe-2S] 2+ , exports Fe-S cluster from mitochondria to cytosol. Redox control of mitochondrion is essential for the proper functioning of the organelle/mitochondria. However, the molecular mechanisms involved in the redox biology of mitoNEET is not fully studied. Hypothesis: Ascorbate regulates redox reactions and Fe-S cluster transport function of mitoNEET in the mitochondria. Aims: The objective of this work is to evaluate the redox reactions between mitoNEET and ascorbate. Methods: Room temperature and low temperature (77 K) electron paramagnetic resonance (EPR) spectroscopy techniques were used to measure the oxidation of ascorbate and reduced mitoNEET, respectively. Results: EPR spectroscopic study shows that mitoNEET oxidizes ascorbate to ascorbate radical. The formation of ascorbate radical increases with increasing concentration of ascorbate. The formation of ascorbate radical is decreased in the presence of menadione. Low temperature EPR study shows that one electron reduction of mitoNEET by ascorbate. Conclusion: This study suggest that ascorbate is a substrate/cofactor for mitoNEET. Ascorbate regulates the Fe-S cluster transport function of mitoNEET. Ascorbate plays an important role in the redox biology of mitoNEET under physiological and pathophysiological conditions.

Docking Complexes of Active Phytochemicals with VK-deficient Genes

Background:: Vitamin K (VK) deficiency occurs when the body does not have enough vitamin K to produce proteins that are essential for blood clotting and bone health. Vitamin K is a cofactor that plays a major role in various comorbidities. Over the years, efforts have been made to identify the interaction between natural compounds, such as K vitamers, that could play a sig-nificant role in regulation of the blood coagulation. We intended to obtain insights into the poten-tial therapeutic implications of phytochemicals for treating VK deficiency-related diseases by in-vestigating the interactions between phytochemicals and VK-deficient genes. Methods:: On active phytochemical docking complexes with VK-deficient genes, there is no spe-cific information available as of yet. In this computationally aided docking study, we were inter-ested in finding the pathogenic blood coagulation-related genes that are linked to VK deficiency. Based on literature reviews and databases, bioactive phytochemicals and other ligands were con-sidered. To provide precise predictions of ligand-protein interactions, docking parameters and scoring algorithms were thoroughly optimized. We have performed molecular docking studies and observed the way the complexes interact. Results:: Specific binding interactions between active phytochemicals and VK pathogenic muta-tions have been identified by the docking study. Hydrogen bonds, van der Waals interactions, and hydrophobic contacts, which are indications of high binding affinities, have been observed in the ligand-protein complexes. Few phytochemicals have demonstrated the ability to interact with the targets of VK-deficient genes, indicating their capacity to modify pathways relevant to VK defi-ciency. The results of the docking study have explained the three pathogenic genes, viz. VWF, F8, and CFTR, wherein VWF and F8 play important roles in blood coagulation and people with cyst-ic fibrosis, to have a deficiency in vitamin K. Thirty-five compounds from different plant and natural sources were screened through molecular docking, out of which two compounds have been considered as controls, including curcumin and warfarin (R-warfarin and S-warfarin), which are the most common anticoagulants readily available in the market. They act by inhibiting vita-min K epoxide reductase (VKOR), which is needed for the gamma-carboxylation of vitamin K-dependent factors. Conclusion:: A focus on other compounds, like theaflavin, ellagic acid, myricetin, and catechin was also made in this study as they show more binding affinity with the three pathogenic proteins. Based on the results, the complexes have been found to possess great potential and thus may be considered for further interaction studies. The potential for active phytochemicals to generate docking complexes with VK-deficient genes is highlighted in this computational analysis. Health disorders related to VK insufficiency may be significantly impacted by these interactions. To val-idate the expected interactions and determine the therapeutic potential of the identified phyto-chemicals, more experimental research, including in vitro and in vivo experiments, is needed.

Antibacterial and Antibiofilm Potency of Menadione Against Multidrug-Resistant S. aureus.

Menadione is an analogue of 1,4-naphthoquinone (1,4-NQ) that possesses enormous pharmaceutical potential. The minimum inhibitory concentration (MIC) of menadione was determined against eighteen pathogens of the ESKAPE category, including thirteen multidrug-resistant (MDR) and five standard strains. From a total of eighteen pathogens, five strains of S. aureus (four: MDR and one: Standard strain) were considered further for detailed studies. This study included the determination of minimum bactericidal concentration (MBC), time-kill assay, scanning electron microscopic technique (SEM), and detection of reactive oxygen species (ROS). Additionally, the effect of menadione on biofilms of three strains of S. aureus was performed through crystal violet assay, SEM, and confocal laser scanning microscopy (CLSM). Menadione exerted substantial antibacterial activity against S. aureus (S8, S9, NCIM 5021) at a lower MIC (64µg/mL). Whereas, the MIC of 256µg/mL was displayed against J2 and J4 (MDR and biofilm-forming strains). The time-killing effect of menadione against S. aureus strains was observed after 9h at MBCs of 64µg/mL (NCIM 5021), 128µg/mL (S8, S9), and 512µg/mL (J2, J4). Enhanced levels of ROS in all five S. aureus were observed in presence of menadione (MICs and MBCs). The relation of enhanced ROS due to menadione activity invigorated us to explore its effect on S. aureus biofilms. We report menadione-mediated inhibition (> 90%) of biofilm formation (at respective MICs) and effect on preformed biofilms (> 85%) at 1024µg/mL. Menadione possessing antibacterial and antibiofilm potentials are imperative in the era of multidrug resistance developed by bacterial pathogens.

FadACB and smeU1VWU2X Contribute to Oxidative Stress-Mediated Fluoroquinolone Resistance in Stenotrophomonas maltophilia.

Pathogenic bacteria experience diverse stresses induced by host cells during infection and have developed intricate systems to trigger appropriate responses. Bacterial stress responses have been reported to defend against these stresses and cross-protect bacteria from antibiotic attack. In this study, we aimed to assess whether oxidative stress affects bacterial susceptibility to fluoroquinolone (FQ) and the underlying mechanism. Stenotrophomonas maltophilia, a species with high genetic diversity, is distributed ubiquitously and is an emerging multidrug-resistant opportunistic pathogen. FQs are among the limited antibiotic treatment options for S. maltophilia infection. The minimum inhibitory concentrations (MICs) of 103 S. maltophilia clinical isolates against ciprofloxacin (CIP) and levofloxacin (LVX) were determined using the agar dilution method in Mueller-Hinton plates with or without menadione (MD), a superoxide generator. The resistance rates for ciprofloxacin and levofloxacin were 40% and 18% in the MD-null group and increased to 91% and 23%, respectively, in the MD-treated group. Of the 103 isolates tested, 54% and 27% had elevated MICs against ciprofloxacin and levofloxacin, respectively, in the presence of MD. The involvement of oxidative stress responses in the MD-mediated FQ resistance was further assessed by mutants construction and viability assay. Among the 16 oxidative stress alleviation systems evaluated, fadACB and smeU1VWU2X contributed to MD-mediated FQ resistance. The antibiotic susceptibility test is an accredited clinical method to evaluate bacterial susceptibility to antibiotics in clinical practice. However, oxidative stress-mediated antibiotic resistance was not detected using this test, which may lead to treatment failure.

Association of NQO2 With UDP-Glucuronosyltransferases Reduces Menadione Toxicity in Neuroblastoma Cells.

The balance between detoxification and toxicity is linked to enzymes of the drug metabolism Phase I (cytochrome P450 or oxidoreductases) and phase II conjugating enzymes (such as the UGTs). After the reduction of quinones, the product of the reaction, the quinols—if not conjugated—re-oxidizes spontaneously to form the substrate quinone with the concomitant production of the toxic reactive oxygen species (ROS). Herein, we documented the modulation of the toxicity of the quinone menadione on a genetically modified neuroblastoma model cell line that expresses both the quinone oxidoreductase 2 (NQO2, E.C. 1.10.5.1) alone or together with the conjugation enzyme UDP-glucuronosyltransferase (UGT1A6, E.C. 2.4.1.17), one of the two UGT isoenzymes capable to conjugate menadione. As previously shown, NQO2 enzymatic activity is concomitant to massive ROS production, as previously shown. The quantification of ROS produced by the menadione metabolism was probed by electron-paramagnetic resonance (EPR) on cell homogenates, while the production of superoxide was measured by liquid chromatography coupled to mass spectrometry (LC-MS) on intact cells. In addition, the dysregulation of the redox homeostasis upon the cell exposure to menadione was studied by fluorescence measurements. Both EPR and LCMS studies confirmed a significant increase in the ROS production in the NQO2 overexpressing cells due to the fast reduction of quinone into quinol that can re-oxidize to form superoxide radicals. However, the effect of NQO2 inhibition was drastically different between cells overexpressing only NQO2 vs. both NQO2 and UGT. Whereas NQO2 inhibition decreases the amount of superoxide in the first case by decreasing the amount of quinol formed, it increased the toxicity of menadione in the cells co-expressing both enzymes. Moreover, for the cells co-expressing QR2 and UGT the homeostasis dysregulation was lower in presence of menadione than for the its counterpart expressing only QR2. Those results confirmed that the cooperation of the two enzymes plays a fundamental role during the cells’ detoxification process. The fluorescence measurements of the variation of redox homeostasis of each cell line and the detection of a glucuronide form of menadiol in the cells co-expressing NQO2 and UGT1A6 enzymes further confirmed our findings.

Reactive oxygen species production and the influence of exogenous copper in yeast mitochondria

Mitochondria are essential organelles within eukaryotic cells due to their role in metabolism, ATP production via the oxidative phosphorylation (OXPHOS) pathway, and other regulatory cellular processes. Our aim is to understand how copper in the mitochondria of yeast affects the production of reactive oxygen species due to its influence on the electron transport chain (ETC) and the localization of reactive oxygen species production during yeast lifespan. Reactive oxygen species (ROS) provide signaling in the cells of organisms which is beneficial for cells to maintain homeostasis. However, when there is a surplus of ROS this can lead to harmful and/or lethal results for the cell. To detect ROS, yeast cultures were grown for one to three days and treated with the fluorescent ROS indicator dihydroethidium (DHE), as well as the fluorescent indicators SYTO and DAPI which stain mitochondrial DNA and adenine-thymine rich regions in the DNA respectively. The ROS generated from the yeast was then analyzed via a fluorescence plate reader, fluorescent activated cell sorting (FACS), and fluorescent microscopy to compare methods of determining ROS in the samples. To investigate mitochondrial ROS production, cells were grown in the presence of antimycin A (a Complex III inhibitor), oligomycin (a Complex V inhibitor), or menadione (a futile redox cycling molecule). Detectable increases in ROS were observed through the inhibition of Complex III and in the presence of menadione. We are now using these methods in yeast with a superoxide dismutase 1 (SOD1) gene deletion, to understand the influence of exogenous copper on the generation of ROS. Copper is worth investigating in conjunction with ROS because the mitochondria in mammals and yeast cells contain a labile copper pool located in the matrix, which is used in the metalation of the copper containing enzymes Complex IV, Cytochrome C Oxidase(CcO), and superoxide dismutase (Sod1). It has been established that supplementing cultures with copper increases CcO and Sod1 activity, supporting previous results that exogenous copper contributes to a labile copper pool within the mitochondria to increase enzyme activity. Our results show that small amounts exogenous copper (0.25 mM copper sulfate in restricted nutrient media) extend yeast chronological lifespan, but a dramatic reduction in lifespan is observed when copper levels are increased to 2.0 mM copper sulfate or higher. These data indicates that low levels of exogenous copper in the media is beneficial for yeast in restricted media conditions. Our current work assesses how exogenous copper changes ETC protein complex expression, specifically looking at CcO subunits, and the production of ROS under these conditions.

Exposure to a sublethal menadione concentration modifies the mycelial secretome and conidial enzyme activities of Metarhizium anisopliae sensu lato and increases its virulence against Rhipicephalus microplus

Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC50 by evaluating conidia germination (38μM). To ensure high cell integrity, a sublethal dose of MND (half of LC50) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC50 and LT50, particularly at 108 conidia mL−1. The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC50), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species.

Interface and doping in carbon dots influence charge transfer and transport

Nanoscale carbon-based materials have the potential for large-scale optoelectronic and biomedical applications due to their tunable photo-physics. Here, we explore the impact of doping and the role of intervening media on both electron transfer and transport from carbon dots (CD) to an acceptor menadione (MD). In a binary mixture compared to undoped-CD (UCD), the amine-based nitrogen-doped-CD (ACD) reflects significant changes in its photo-physics like excitation-independent fluorescence, enhanced lifetime, along with a distinct drop in electron transfer rate (kET) to MD. With micro-emulsion, kET from UCD to MD is enhanced while a substantial reduction is observed for ACDs, emphasizing the critical role of doping, surface states, encapsulation, and interfaces of CTAB micelles. The impact of doping and Coulombic interaction at interfaces on the charge transport phenomena with redox-active MD is further investigated by current-sensing atomic force microscopy. For similar bias-voltage, ACD reflects a higher conductance than UCD, and the conductance is enhanced in the presence of CTAB micelles. Moreover, at a low voltage regime, the current-voltage curve shows a linear growth, while at higher voltage, Fowler-Nordheim (F–N) tunneling is observed. The presence of MD, however, leads to an abrupt rise in conductivity resulting in a transition from F–N to direct tunneling.

Effect of Vitamin K3 Inhibiting the Function of NorA Efflux Pump and Its Gene Expression on Staphylococcus aureus.

Resistance to antibiotics has made diseases that previously healed easily become more difficult to treat. Staphylococcus aureus is an important cause of hospital-acquired infections and multi-drug resistant. NorA efflux pump, present in bacteria S. aureus, is synthesized by the expression of the norA gene. Menadione, also known as vitamin K3, is one of the synthetic forms of vitamin K. Therefore, the aim of this study is to verify the menadione effect on efflux inhibition through NorA pump gene expression inhibition and assess the effects of menadione in bacterial membrane. The effect of menadione as an efflux pump inhibitor (EPI) was evaluated by the microdilution method, fluorimetry, electron microscopy, and by RT-qPCR to evaluate gene expression. In the molecular docking, association with menadione induces increased fluorescence intensity. Menadione was observed (100% of the clusters) interacting with residues ILE12, ILE15, PHE16, ILE19, PHE47, GLN51, ALA105, and MET109 from NorA. The results showed the norA gene had its expression significantly diminished in the presence of menadione. The simulation showed that several menadione molecules were able to go through the bilayer and allow the entry of water molecules into the hydrophobic regions of the bilayer. When present within membranes, menadione may have caused membrane structural changes resulting in a decline of the signaling pathways involved in norA expression. Menadione demonstrated to be an efflux pump inhibitor with dual mechanism: affecting the efflux pump by direct interaction with protein NorA and indirectly inhibiting the norA gene expression, possibly by affecting regulators present in the membrane altered by menadione.

Modulation of Oxidative Stress by 17 β-Estradiol and Genistein in Human Hepatic Cell Lines In Vitro

Background/Aims: estrogens and phytoestrogens exert hepatoprotection through mechanisms not clearly examined yet. Here, we investigated the protective effects exerted by 17β-estradiol and genistein against oxidative stress in hepatocytes and hepatic stellate cells (HSCs) and the involvement of specific receptors and the intracellular signalling. Methods: Huh7.5 and LX-2, alone or in co-culture with Huh7.5, were treated with 17β-estradiol and genistein alone or in the presence of menadione and of estrogen receptors (ERs) and G protein-coupled-estrogenic-receptors (GPER) blockers. Cell viability, mitochondrial membrane potential and oxidant/antioxidant system were measured by specific kits. Western Blot was used for the analysis of Akt and p38-mitogen-activated-protein kinases (MAPK) activation and α-smooth-muscle actin expression. Results: In Huh7.5, 17β-estradiol and genistein prevented the effects of peroxidation by modulating Akt and p38MAPK activation. Similar antioxidant and protective findings were obtained in LX-2 of co-culture experiments, only. ERs and GPER blockers were able to prevent the effects of 17β-estradiol and genistein. Conclusion: In Huh7.5 and LX-2, 17β-estradiol and genistein counteract the effects of peroxidation through the involvement of ERs and GPER and by an intracellular signalling related to Akt and p38MAPK. As concerning LX-2, paracrine factors released by Huh7.5 play a key role in protection against oxidative stress.

Drug-enhanced carbon monoxide production from heme by cytochrome P450 reductase.

Carbon monoxide (CO) formed endogenously is considered to be cytoprotective, and the vast majority of CO formation is attributed to the degradation of heme by heme oxygenases-1 and -2 (HO-1, HO-2). Previously, we observed that brain microsomes containing HO-2 produced many-fold more CO in the presence of menadione and its congeners; herein we explored these observations further. We determined the effects of various drugs on CO production of rat brain microsomes and recombinant human cytochrome P450 reductase (CPR); CO was measured by gas chromatography with reductive detection. Brain microsomes of Sprague-Dawley rats or recombinant human cytochrome P450 reductase (CPR) were incubated with NADPH and various drugs in closed vials in phosphate buffer at pH 7.4 and 37°C. After 15 minutes, the reaction was stopped by cooling in dry ice, and the headspace gas was analyzed for CO production using gas chromatography with reductive (mercuric oxide) detection. We observed drug-enhanced CO production in the presence of both microsomes and recombinant CPR alone; the presence of HO was not required. A range of structurally diverse drugs were capable of amplifying this CO formation; these molecules had structures consistent with redox cycling capability. The addition of catalase to a reaction mixture, that contained activating drugs, inhibited the production of CO. Drug-enhanced CO formation can be catalyzed by CPR. The mechanism of CPR activation was not through classical drug-receptor mediation. Redox cycling may be involved in the drug-induced amplification of CO production by CPR through the production of reactive oxygen species.

Superoxide radical-generating compounds activate a predicted promoter site for paraquat-inducible genes of the Chromobacterium violaceum bacterium in a dose-dependent manner.

The purpose of the present study was to functionally evaluate the influence of superoxide radical-generating compounds on the heterologous induction of a predicted promoter region of open reading frames for paraquat-inducible genes (pqi genes) revealed during genome annotation analyses of the Chromobacterium violaceum bacterium. A 388-bp fragment corresponding to a pqi gene promoter of C. violaceum was amplified using specific primers and cloned into a conjugative vector containing the Escherichia coli lacZ gene without a promoter. Assessments of the expression of the β-galactosidase enzyme were performed in the presence of menadione (MEN) and phenazine methosulfate (PMS) compounds at different final concentrations to evaluate the heterologous activation of the predicted promoter region of interest in C. violaceum induced by these substrates. Under these experimental conditions, the MEN reagent promoted highly significant increases in the expression of the β-galactosidase enzyme modulated by activating the promoter region of the pqi genes at all concentrations tested. On the other hand, significantly higher levels in the expression of the β-galactosidase enzyme were detected exclusively in the presence of the PMS reagent at a final concentration of 50 μg/mL. The findings described in the present study demonstrate that superoxide radical-generating compounds can activate a predicted promoter DNA motif for pqi genes of the C. violaceum bacterium in a dose-dependent manner.

227 SUPPLEMENTATION WITH CYSTEINE, CYSTEAMINE, AND CATALASE DURING IN VITRO MATURATION PROTECTS BOVINE PRE-IMPLANTATION EMBRYOS FROM ANTI-DEVELOPMENTAL ACTIONS OF MENADIONE

Menadione (MD), a naphthoquinone used as a vitamin K source in animal feed, can generate reactive oxygen species (ROS) and cause apoptosis. Hence, we examined whether MD reduces development of pre-implantation bovine embryos by inducing ROS production and apoptosis and tested the hypothesis that the anti-developmental actions of MD would be reduced by supplementation of the in vitro maturation (IVM) medium with antioxidants (a mixture of 0.6 mM cysteine + 100 μM cysteamine + 100 UI mL–1 catalase). Cumulus-oocyte complexes (COC; n = 3334; 50 per well in 11 replications) were matured in IVM medium (TCM-199 with bicarbonate, hormones, and 10% FCS) for 22 h. After fertilization, presumptive zygotes were cultured in synthetic oviduct fluid medium (SOF) with 2.5% FCS and 0.5% BSA up to 7 days (Day 0 = IVF). On Day 6, MD was included in the SOF medium (0 μM: Control; or 5.0 μM: MD5) during 24 h. All cultures were conducted at 38.5°C in 5% CO2 in air. In a second experiment, COC were matured in TCM-199 supplemented with or without the mixture of antioxidants (Antiox; only during IVM) and with or without MD at Day 6 in a 2 × 2 factorial design (Control, Control/Antiox, MD5, MD5/Antiox). The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively. A sample of the blastocysts (n = 1112) was stained for TUNEL (In Situ Cell Death Detection Kit, Roche, Indianapolis, IN, USA) to detect the apoptotic cells or with 5 μM H2DCFDA (Molecular Probes, Canada) to evaluate the ROS levels. Stained embryos were evaluated under an epifluorescence microscope and the images of embryos stained with H2DCFDA were analysed by Q-Capture Pro image software for determining the fluorescent intensity. The means (±s.e.m.) were compared by ANOVA followed by Tukey's test (P < 0.05). In the first experiment, and the cleavage rates were similar (P > 0.05) for Control (80.5% ± 0.9) and MD5 groups (78.9% ± 1.2), but the blastocyst rates were lower (P < 0.05) in the MD5 group (25.9% ± 2.1) when compared with Control (36.4% ± 1.5). The rate of apoptotic cells in blastocysts was higher (P < 0.05) in the MD5 group (19.2% ± 0.7) than in Control (10.0% ± 0.5), as well as the fluorescence intensity for ROS quantification (0.7 ± 0.1 v. 1.1 ± 0.1, respectively, for Control and MD5 groups). In an effort to rescue the impaired embryonic development, a mixture of antioxidants was added to the IVM medium and the results were transitional in response in which MD5/Antiox (28.3% ± 3.5) was intermediate (P > 0.05) between the Controls [Control (38.2% ± 2.5) and Control/Antiox (34.7% ± 1.9) groups] and the MD5 (21.4% ± 2.9) group. Principal effects evaluated were the presence of MD (without: 35.9% ± 2.3 v. with: 24.8% ± 2.3; P < 0.05) or Antiox (without: 28.4% ± 2.3 v. with: 32.3% ± 2.3; P > 0.05). In conclusion, addition of MD to embryonic culture reduces the blastocyst development and increases intracellular levels of ROS and the occurrence of apoptosis. Anti-developmental actions of MD on embryonic development can be partially blocked by treating oocytes with a mixture of antioxidants during IVM.Financial support was provided by FAPESP (2012/10083-8 and 2013/07382-6).

Multiple signals modulate the activity of the complex sensor kinase TodS.

The reason for the existence of complex sensor kinases is little understood but thought to lie in the capacity to respond to multiple signals. The complex, seven-domain sensor kinase TodS controls in concert with the TodT response regulator the expression of the toluene dioxygenase pathway in Pseudomonas putida F1 and DOT-T1E. We have previously shown that some aromatic hydrocarbons stimulate TodS activity whereas others behave as antagonists. We show here that TodS responds in addition to the oxidative agent menadione. Menadione but no other oxidative agent tested inhibited TodS activity in vitro and reduced PtodX expression in vivo. The menadione signal is incorporated by a cysteine-dependent mechanism. The mutation of the sole conserved cysteine of TodS (C320) rendered the protein insensitive to menadione. We evaluated the mutual opposing effects of toluene and menadione on TodS autophosphorylation. In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione. It was shown by others that menadione increases expression of glucose metabolism genes. The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways. This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules.

HSF1-mediated oxidative stress response to menadione in <i>Saccharomyces cerevisiae</i> KNU5377Y3 by using proteomic approach

The hat shock transcription factor HSF1 inthe yeast Saccharomyces cerevisiae regulates a wide range of genes and functions in diverse cellular reactions. To investigate the physiological response of HSF1 inthe presence of menadione (MD) in S. cerevisiae KNU 5377Y3, wild-type (k3wt) and isogenic hsf1 mutant (k3h1) cells were introduced. HSF1 was induced when k3wt cells were exposed to the superoxide-generating agent MD and k3h1 cells were hypersensitive to MD. Under MD stress, k3h1 cells down-regulated the expression of metabolic enzymes (Hxk, Fba1, Pgk1, Eno2, and Adh1), antioxidant enzymes (Trx2 and porin), and molecular chaperones and their cofactors (Hsp104, Ssb1, Hsp60, Hsp42, Hsp26, Hsp12, Cpr1, and Sti1). In addition, k3h1 cells increased cellular hydroperoxide levels and protein carbonylation under MD stress as compared to k3wt cells. However, there was a moderate difference in the wild-type (b3wt) and mutant (b3h1) cells derived from S. cerevisiae S288Cunder the same conditions. Thus, these results show that HSF1 is an important component of the stress response system, acting as an activator of cell rescue genes in S. cerevisiae KNU5377Y3, and its expression protects the cells from MD-induced oxidative damage by maintaining redox homeostasis and proteostasis in the presence of MD.

Cyclophilin A Cpr1 Protein Modulates the Response of Antioxidant Molecules to Menadione-induced Oxidative Stress in Saccharomyces cerevisiae KNU5377Y

ObjectivesThe cellular function of cyclophilin A (CypA) differs between organisms, even though CypA is conserved in both prokaryotes and eukaryotes. The purpose of this study was to elucidate the role of activated CypA isoform CPR1 in the antioxidative mechanisms of Saccharomyces cerevisiae under menadione (MD)-induced oxidative stress. MethodsFour S. cerevisiae strains, KNU5377Y (kwt) and BY4741 (bwt), and their isogenic cpr1Δ mutant strains (kc1 and bc1), were treated with MD, at a concentration ranging between 0.25 and 0.4 mM. Cpr1-mediated antioxidative effects were analyzed by measuring the levels of cellular glutathione (GSH) and ascorbate (AsA)-like molecules in yeast. ResultsGSH and AsA-like reductant molecule concentrations were more reduced in the presence of MD in the kc1 strain than in the kwt strain; whereas, there was no significant difference between the bwt and bc1 strains under the same conditions. In kc1 strain samples, we observed a reduction in the expression of proteins related both to GSH synthesis and the recycling system, and simultaneously, downregulation of GSH synthetase and GSH reductase activities were also evident. Oxidative stress in the kc1 strain was alleviated by the application of the GSH and AsA analog. ConclusionThese results indicate that activated Cpr1 modulates the response of antioxidant molecules involved in cellular redox homeostasis of KNU5377Y during oxidative stress induced by MD.

Human aldo–keto reductase AKR7A2 protects against the cytotoxicity and mutagenicity of reactive aldehydes and lowers intracellular reactive oxygen species in hamster V79-4 cells

Aldo–keto reductase (AKR) enzymes are critical for the detoxication of endogenous and exogenous aldehydes. Previous studies have shown that the AKR7A2 enzyme is catalytically active toward aldehydes arising from lipid peroxidation, suggesting a potential role against the consequences of oxidative stress, and representing an important detoxication route in mammalian cells. The aim of this study was to determine the ability of AKR7A2 to protect cells against aldehyde cytotoxicity and genotoxicity and elucidate its potential role in providing resistance to oxidative stress. A transgenic mammalian cell model was developed in which AKR7A2 was overexpressed in V79-4 cells and used to evaluate the ability of AKR7A2 to provide resistance against toxic aldehydes. Results show that AKR7A2 provides increased resistance to the cytotoxicity of 4-hydroxynonenal (HNE) and modest resistance to the cytotoxicity of trans, trans-muconaldehyde (MUC) and methyglyoxal, but provided no protection against crotonaldehyde and acrolein. Cells expressing AKR7A2 were also found to be less susceptible to DNA damage, showing a decrease in mutation rate cause by 4-HNE compared to control cells. Furthermore, the role of the AKR7A2 enzyme on the cellular capability to cope with oxidative stress was assessed. V79 cells expressing AKR7A2 were more resistant to the redox-cycler menadione and were able to lower menadione-induced ROS levels in both a time and dose dependent manner. In addition, AKR7A2 was able to maintain intracellular GSH levels in the presence of menadione. Together these findings indicate that AKR7A2 is involved in cellular detoxication pathways and may play a defensive role against oxidative stress in vivo.

Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes

Production of reactive oxygen species (hydroxyl radicals, superoxide radicals and hydrogen peroxide) was studied using EPR spin-trapping techniques and specific dyes in isolated plasma membranes from the growing and the non-growing zones of hypocotyls and roots of etiolated soybean seedlings as well as coleoptiles and roots of etiolated maize seedlings. NAD(P)H mediated the production of superoxide in all plasma membrane samples. Hydroxyl radicals were only produced by the membranes of the hypocotyl growing zone when a Fenton catalyst (FeEDTA) was present. By contrast, in membranes from other parts of the seedlings a low rate of spontaneous hydroxyl radical formation was observed due to the presence of small amounts of tightly bound peroxidase. It is concluded that apoplastic hydroxyl radical generation depends fully, or for the most part, on peroxidase localized in the cell wall. In soybean plasma membranes from the growing zone of the hypocotyl pharmacological tests showed that the superoxide production could potentially be attributed to the action of at least two enzymes, an NADPH oxidase and, in the presence of menadione, a quinone reductase.

A novel screening method based on menadione mediated rapid reduction of tetrazolium salt for testing of anti-mycobacterial agents

A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determine the viability of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis bacilli in microplate format. In general, XTT reduction is an extremely slow process which takes almost 24 h to produce a detectable signal. Menadione could drastically induce this reduction to an almost equal extent within a few minutes in a dose dependent manner. The reduction of XTT is directly proportional to the cell concentration in the presence of menadione. The standardized protocol used 200 μM of XTT and 60 μM of menadione in 250 μl of cell suspension grown either in aerobic or anaerobic conditions. The cell suspension of M. bovis BCG and M. tuberculosis were incubated for 40 min before reading the optical density at 470 nm whereas M. smegmatis was incubated for 20 min. Calculated Signal/Noise (S/N) ratios obtained by applying this protocol were 5.4, 6.4 and 9.4 using M. bovis BCG, M. tuberculosis and M. smegmatis respectively. The calculated Z′ factors were > 0.8 for all mycobacterium bacilli indicating the robustness of the XTT Reduction Menadione Assay (XRMA) for rapid screening of inhibitors. The assay protocol was validated by applying 10 standard anti-tubercular agents on M. tuberculosis, M. bovis BCG and M. smegmatis. The Minimum Inhibitory Concentration (MIC) values were found to be similar to reported values from Colony Forming Unit (CFU) and REMA (resazurin microplate assay) assays. Altogether, XRMA is providing a novel anti-tubercular screening protocol which could be useful in high throughput screening programs against different physiological stages of the bacilli.

Decarbonylated cyclophilin A Cpr1 protein protects KNU5377Y when exposed to stress induced by menadione

Cyclophilins are conserved cis-trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. The accumulation of Cpr1 protein to menadione in Saccharomyces cerevisiae KNU5377Y suggests a possibility that this protein may participate in the mechanism of stress tolerance. Stress response of S. cerevisiae KNU5377Y cpr1Δ mutant strain was investigated in the presence of menadione (MD). The growth ability of the strain was confirmed in an oxidant-supplemented medium, and a relationship was established between diminishing levels of cell rescue enzymes and MD sensitivity. The results demonstrate the significant effect of CPR1 disruption in the cellular growth rate, cell viability and morphology, and redox state in the presence of MD and suggest the possible role of Cpr1p in acquiring sensitivity to MD and its physiological role in cellular stress tolerance. The in vivo importance of Cpr1p for antioxidant-mediated reactive oxygen species (ROS) neutralization and chaperone-mediated protein folding was confirmed by analyzing the expression changes of a variety of cell rescue proteins in a CPR1-disrupted strain. The cpr1Δ to the exogenous MD showed reduced expression level of antioxidant enzymes, molecular chaperones, and metabolic enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH)- or adenosine triphosphate (ATP)-generating systems. More importantly, it was shown that cpr1Δ mutant caused imbalance in the cellular redox homeostasis and increased ROS levels in the cytosol as well as mitochondria and elevated iron concentrations. As a result of excess ROS production, the cpr1Δ mutant provoked an increase in oxidative damage and a reduction in antioxidant activity and free radical scavenger ability. However, there was no difference in the stress responses between the wild-type and the cpr1Δ mutant strains derived from S. cerevisiae BY4741 as a control strain under the same stress. Unlike BY4741, KNU5377Y Cpr1 protein was decarbonylated during MD stress. Decarbonylation of Cpr1 protein in KNU5377Y strain seems to be caused by a rapid and efficient gene expression program via stress response factors Hsf1, Yap1, and Msn2. Hence, the decarbonylated Cpr1 protein may be critical in cellular redox homeostasis and may be a potential chaperone to menadione.