NADH Oxidase Activity Research Articles

Assessment of myco-fabricated Al2O3 NPs toxicity on cancer cells and pathogenic microbes by suppression of bacterial metabolic key enzymes.

There has been a recent change in global attention towards addressing antimicrobial resistance (AMR) as a result of the concerning increase in mortality rates. Nanomaterials have become highly favorable options for a wide range of industrial and biological uses. The objective of this study was to produce aluminum oxide nanoparticles (Al2O3 NPs) using a crude extract from the fungus Aspergillus sp. WAH23, and then analyze the nanoparticles using UV-analysis, electron microscopy (TEM and SEM), and (FT-IR) and X-ray diffraction (XRD). Results revealed that formed nanoparticles are spherical with an average size of 8.5 nm. XRD analysis confirmed the crystalline nature of the synthesized Al2O3 NPs. The Al2O3 nanoparticles exhibited antibacterial properties against a wide range of pathogenic microbes. The antibacterial efficacy of these nanoparticles on the examined bacterial strains was exhibited through their ability to hinder several metabolic processes, including phosphofructokinase (PFK), enolase, and glutamine synthetase. Additionally, the nanoparticles increased the activity of NADH-oxidase, the content of MDA, and the formation of H2O2. The study also examined the anticancer properties of Al2O3 nanoparticles on various types of cancer cells.

Photothermally Reinforced Nanozyme Remodeling Tumor Microenvironment of Redox and Metabolic Homeostasis to Enhance Ferroptosis in Tumor Therapy.

The acidity and high GSH level in the tumor microenvironment (TME) greatly limit the antitumor activity of nanozymes. Thus, enhancing nanozymes' activity is fundamentally challenging in tumor therapy. Although the combination of photothermal therapy (PTT) and nanozymes can enhance the catalytic activity, cancer cells will overexpress heat shock proteins (HSPs) at high temperature, aggravating the heat resistance of tumor cells, which in turn compromises the outcome of chemodynamic therapy. Herein, we propose an iron-doped metal-organic framework nanozyme (IB@Fe-ZIF8@PDFA) that can be activated under the weak acidity and high level of GSH, demonstrating the activities of GSH oxidation (GSH-OXD), peroxidase (POD), and NADH oxidase (NADH-OXD). Under laser irradiation, it displays photothermal-enhanced multienzyme activities to simultaneously eliminate tumors and inhibit tumor metastasis. While consuming endogenous GSH, IB@Fe-ZIF8@PDFA promotes the decomposition of H2O2 into ·OH, enhancing ferroptosis in tumor cells. Surprisingly, IB@Fe-ZIF8@PDFA nanozyme can oxide NADH and subsequently limit the ATP supply, reducing the expression of HSPs and significantly weakening the heat resistance of tumor cells during PTT. Meanwhile, H2O2 is generated during this procedure, which can endogenously replenish the consumed H2O2. Thus, this IB@Fe-ZIF8@PDFA nanozyme constitutes a self-cascading platform to consume GSH and NADH, endogenously replenish the H2O2 and continuously generate ·OH to facilitate ferroptosis by disrupting the redox and metabolic homeostasis in tumor cells, achieving tumor elimination and tumor metastasis inhibition.

The Role of Propionate-Induced Rearrangement of Membrane Proteins in the Formation of the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Adhesive-invasive E. coli has been suggested to be associated with the development of Crohn's disease (CD). It is assumed that they can provoke the onset of the inflammatory process as a result of the invasion of intestinal epithelial cells and then, due to survival inside macrophages and dendritic cells, stimulate chronic inflammation. In previous reports, we have shown that passage of the CD isolate ZvL2 on minimal medium M9 supplemented with sodium propionate (PA) as a carbon source stimulates and inhibits the adherent-invasive properties and the ability to survive in macrophages. This effect was reversible and not observed for the laboratory strain K12 MG1655. We were able to compare the isogenic strain AIEC in two phenotypes-virulent (ZvL2-PA) and non-virulent (ZvL2-GLU). Unlike ZvL2-GLU, ZvL2-PA activates the production of ROS and cytokines when interacting with neutrophils. The laboratory strain does not cause a similar effect. To activate neutrophils, bacterial opsonization is necessary. Differences in neutrophil NADH oxidase activation and ζ-potential for ZvL2-GLU and ZvL2-PA are associated with changes in membrane protein abundance, as demonstrated by differential 2D electrophoresis and LC-MS. The increase in ROS and cytokine production during the interaction of ZvL2-PA with neutrophils is associated with a rearrangement of the abundance of membrane proteins, which leads to the activation of Rcs and PhoP/Q signaling pathways and changes in the composition and/or modification of LPS. Certain isoforms of OmpA may play a role in the formation of the virulent phenotype of ZvL2-PA and participate in the activation of NADPH oxidase in neutrophils.

Engineering Oxygen-Independent NADH Oxidase Integrated with Electrocatalytic FAD Cofactor Regeneration.

An electrochemically mediated enzyme process for nicotinamide adenine dinucleotide (NADH) oxidation and biosensing has been developed in which the oxygen-dependent activities of wild-type NADH oxidase are replaced by electrochemical regeneration of the flavin adenine dinucleotide (FAD) cofactor in the active site. Consequently, the present bioelectrocatalysis does not rely on a continuous oxygen supply through bubbling air or pure oxygen in biosynthetic applications, which reduces enzyme stability. The coupled electrochemical and enzymatic catalysis is achieved through a combination of enzyme immobilization on the electrode and electrochemical oxidation of FADH2 in the active site mediated by the electron transfer mediator ferrocene carboxylic acid (FcCA). Furthermore, to minimize the effect of dissolved oxygen when the electrocatalytic process is exposed to air, we successfully designed mutations at the Leu40 and Cys42 sites of Leuconostoc mesenteroides (LmNOx) to block the oxygen passage into the active site and to eliminate the native FAD cofactor regeneration half-reaction. The engineered enzymes, whose activities are significantly reduced or inactive in solution, are electrocatalytically active toward conversion of NADH to NAD+, demonstrating successful FAD cofactor regeneration in the active site via electrochemistry. Finally, we developed two highly responsive electrochemical biosensors for NADH detection which has a superior substrate specific to standard detectors using metal electrodes, and comparable detection range and detection limit (1-3 μM).

Water-accommodated fractions of crude oil and its mixture with chemical dispersant impairs oxidase stress and energy metabolism disorders in Oryzias melastigma embryos

In this study, marine medaka (Oryzias melastigma) embryos were exposed to different concentrations of water-accommodated fractions (WAFs) and chemically enhanced water-accommodated fractions (CEWAFs) of Oman crude oil for 14 d by semi-static exposure methods. The effects on growth and development and energy metabolism process were evaluated. Results showed that embryo survival and hatchability were decreased in a dose-dependent manner with an increase in the concentration of petroleum hydrocarbon compounds, whereas the malformation exhibited a dose-dependent increase. Compared to the control, the adenosine triphosphate (ATP) content and Na+-K+–ATPase (NKA) activities of embryos exposed to both WAFs and CEWAFs were reduced, while intracellular reactive oxygen species (ROS) levels and NADH oxidase (NOX) activities were increased. Our study demonstrated that exposure to crude oil dispersed by chemical dispersant affected the growth and development of marine medaka embryos, caused oxidative stress while produced a series of malformations in the body and dysregulation in energy metabolism. In comparison, the toxic effects of chemically dispersed crude oil might be more severe than the oil itself in the equivalent diluted concentration treatment solution. These would provide more valuable and reliable reference data for the use of chemical dispersants in oil spills.

Catalytic Differences between Flavohemoglobins of Giardia intestinalis and E. coli.

The sole known heme enzyme of the parasitic protist Giardia intestinalis is a flavohemoglobin (gFlHb) that acts as a nitric oxide dioxygenase (NOD) and protects the organism from the free radical nitric oxide. To learn more about the properties of this enzyme, we measured its nitric oxide dioxygenase, NADH oxidase, and cytochrome c reductase activities and compared these to the activities of the E. coli flavohemoglobin (Hmp). The turnover number for the NOD activity of gFlHb (23 s-1) is about two-thirds of that of Hmp (34 s-1) at pH 6.5 and 37 °C. The two enzymes differ in their sensitivity towards molecules that act as heme ligands. For both gFlHb and Hmp, inhibition with miconazole, a large imidazole ligand, is adequately described by simple competitive inhibition, with KI = 10 μM and 0.27 μM for gFlHb and Hmp, respectively. Inhibition plots with the small ligand imidazole were biphasic, which is consistent with previous experiments with carbon monoxide as a probe that show that the active site of flavohemoglobins exists in two conformations. Interestingly, the largest difference is observed with nitrite, which, like imidazole, also shows a biphasic inhibition plot; however, nitrite inhibits gFlHb at sub-millimolar concentrations while Hmp is not significantly affected. NADH oxidase activity measured under aerobic conditions in the absence of nitric oxide for Hmp was more than twice the activity of gFlHb. The addition of 1 mM hydrogen peroxide in these assays stimulated the NADH oxidase activity of gFlHb but not Hmp. Both enzymes had nearly identical cytochrome c reductase activities but the extent of the contribution of indirect reduction by flavohemoglobin-generated superoxide was much lower with gFlHb (4% SOD-inhibited) than with Hmp (17% SOD-inhibited). Although the active sites of the two enzymes share the same highly conserved residues that are important for catalysis, differences in the distal ligand binding site may account for these differences in activity and sensitivity towards NOD inhibitors. The differences observed in the NADH oxidase and cytochrome c reductase assays suggest that gFlHb may have evolved to protect the protist, which lacks both superoxide dismutase and catalase, from the damaging effects of superoxide by minimizing its production and from peroxide by actively reducing it.

Antioxidant, anti-amylase, anti-lipase, and efficiency of Satureja fatty acid on the anti-inflammatory parameters in lipopolysaccharide-stimulated macrophage through Nrf2/NF-kB/NADH oxidase pathway

Satureja is an aromatic plant that is used for flavoring, perfume, and food manufacturing due to its pleasant essential oil. Modern medicine research revealed several biological activities of Satureja essential oil, including antifungal, antibacterial, antiviral, antioxidant, anticancer, and anti-inflammatory. However, the functional properties of Satureja fatty acid have not been explored. This study examined the fatty acid profile, lipid nutritional quality, antioxidant, anti-amylase, and anti-lipase capacities of Satureja. The efficiency of Satureja fatty acid on the anti-oxidative and anti-inflammatory parameters in LPS-induced macrophage through the Nrf2/NF-kB/NADH oxidase pathway was examined. The whole lipid extract was prepared with chloroform/methanol/water solution. Fatty acids methyl ester from whole lipid extract were prepared with methanol/sulfuric acid reagent. The fatty acid profile was analyzed using gas chromatography-mass spectrometry. Total antioxidant was determined by ABTS decolorization. Lipase and amylase activities were determined by monitoring the decomposition of p-nitrophenyl butyrate and starch. The macrophage cell line was grown in DMEM media in the presence of fatty acid. The hydrogen peroxide production in treated cells was monitored using the FOX reagent. NADH oxidase activity was measured by monitoring NADH breakdown. The expression of NOX, NF-kB, and NRF2, were tested in the treated cells by real-time PCR. The main components of the Satureja fatty acid were linolenic acid (24.67–37.32%), palmitic acid (10.65–20.29%), linoleic acid (8.31–13.39%), oleic acid (4.42–14.35%), stearic acid (2.76–8.77%) and palmitoleic acid (1.77–4.95%). Given the nutritional quality, omega-3 PUFA (23.58–37.32%), SFA (21.53–26.70%), omega-6 PUFA (10.86–16.14%), omega-9 MUFA (4.42–14.35%), and omega-7 MUFA (1.77–4.95%) comprise the majority of fatty acids. Satureja fatty acid has a promising unsaturation index (120.77–164.27), PUFA/MUFA (2.07–6.41), hypocholesterolemic index (2.44–3.47), health-promoting index (2.03–2.42), PUFA/SFA (1.37–1.94), nutritive value index (0.53–1.71), MUFA/SFA (0.30–0.80) omega-6/omega-3 (0.34–0.65), atherogenicity index (0.41–0.49), and thrombogenicity index (0.17–0.27). Satureja fatty acid displayed strong antioxidant capacity (with IC50 ranging from 354 to 428 µg/mL), anti-lipase capacity (with IC50 ranging from 354 to 428 µg/mL), and anti-amylase capacity (with IC50 ranging from 370 to 390 µg/mL). LPS induced the expression of NOX, NRF2, and NF-kB and the synthesis of hydrogen peroxide in macrophage cells. In LPS-stimulated macrophages, Satureja fatty acid reduced NOX expression, hydrogen peroxide, and NF-kB expression and increased NRF2 at 0.04 mg/mL. In conclusion, Satureja fatty acids have potent antioxidant, anti-amylase, anti-lipase, and anti-inflammatory activities. The mechanisms in lowering oxidative stress markers depended on down-regulating superoxide-producing enzymes at gene and protein levels. Satureja polyunsaturated omega-3 fatty acids could be recommended for healthy products combined with dietary therapy to treat obesity, diabetes, and oxidative stress.

Antioxidant and immunomodulatory potential of cell wall polysaccharides from Harungana madagascariensis Lam

Numerous plant polysaccharides have demonstrated immunomodulatory and antioxidant potentials, and can be investigated as novel molecules with biological properties that may enhance immune function. The aim of this study was to assess the antioxidant and immunomodulatory properties of cell wall polysaccharides fractions isolated from Harungana madagascariensis (HM). Pectin fraction (PF) and hemicellulose fraction (HCF) were extracted and tested for their antioxidant properties using reducing power, 1,1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2′-azino-bis-3-éthylbenzylthiazoline6-sulphonic acid (ABTS) radical scavenging assays, chelating activity and inhibition of lipid peroxidation. Their immunomodulatory activities were carried out on peritoneal macrophages targeting NADH oxidase and lysosomal enzyme activities as well as the production of nitric oxide (NO) and hydroperoxides (H2O2) during phagocytosis of Saccharomyces cereviceae. The polysaccharides fractions showed significant antioxidant and immunostimulatory activities via stimulation of NADH oxidase and lysosomal enzyme of peritoneal macrophages and production of NO and H2O2. Taken together, the results of this study indicate the potential of polysaccharides from H. madagascariensis as antioxidant and immunomodulator agents.

Chronic stress-induced anxiety-like behavior, hippocampal oxidative, and endoplasmic reticulum stress are reversed by young plasma transfusion in aged adult rats.

Aging and stress synergistically induce behavioral dysfunctions associated with oxidative and endoplasmic reticulum (ER) stress in brain regions. Considering the rejuvenating effects of young plasma on aging brain function, in the current study, we examined the effects of young plasma administration on anxiety-like behavior, NADH oxidase, NADPH oxidase, and ER stress markers in the hippocampus of old male rats. Young (3 months old) and aged (22 months old) rats were randomly assigned into five groups: young control (Y), aged control (A), aged rats subjected to chronic stress for four weeks (A+S), aged rats subjected to chronic stress and treated with old plasma (A+S+OP), and aged rats subjected to chronic stress and treated with young plasma (A+S+YP). Systemic injection of (1 ml) young and old plasma was performed for four weeks (3 times/week). Young plasma transfusion significantly improved anxiety-like behavior in aged rats and modulated oxidative stress in the hippocampus, evidenced by the increased NADH oxidase (NOX) activity and the reduced NADPH oxidase. In addition, the levels of C/EBP homologous protein (CHOP) and Glucose-Regulated Protein 78 (GRP-78), as ER stress markers, markedly reduced in the hippocampus following the administration of young plasma. These findings suggest that young plasma transfusion could reverse anxiety-like behavior in stress-exposed aged rats by modulating the hippocampal oxidative and ER stress markers.

Structural and Functional Analyses of the Flavoprotein Disulfide Reductase FN0820 of Fusobacterium nucleatum.

Escherichia coli RclA and Staphylococcus aureus MerA are part of the Group I flavoprotein disulfide reductase (FDR) family and have been implicated in the contribution to bacterial pathogenesis by defending against the host immune response. Fusobacterium nucleatum is a pathogenic, anaerobic Gram-negative bacterial species commonly found in the human oral cavity and gastrointestinal tract. In this study, we discovered that the F. nucleatum protein FN0820, belonging to the Group I FDR family, exhibited a higher activity of a Cu2+-dependent NADH oxidase than E. coli RclA. Moreover, FN0820 decreased the dissolved oxygen level in the solution with higher NADH oxidase activity. We found that L-tryptophan and its analog 5-hydroxytryptophan inhibit the FN0820 activities of NADH oxidase and the concomitant reduction of oxygen. Our results have implications for developing new treatment strategies against pathogens that defend the host immune response with Group I FDRs.

Prolonged stress-induced depression-like behaviors in aged rats are mediated by endoplasmic reticulum stress and apoptosis in the hippocampus

Structural and functional recovery from stress-induced depression is impaired in the context of aging brain. Since investigating the molecular substrates that facilitate behavioral recovery may have important implications for understanding brain plasticity and resilience of individuals, we studied depressive-like behaviors in young and aged rats 6 weeks after chronic stress exposure as a recovery period and examined the levels of TNF-α and IL-6 inflammatory cytokines, NADH oxidase activity, NADPH oxidase, endoplasmic reticulum (ER) stress markers, and apoptosis in the hippocampus. Young (3 months old) and aged (22 months old) male Wistar rats were divided into four groups; young control (Young), depression model of young rats that received chronic stress procedure followed by a 6-week recovery period (Young+S), aged control (Aged), and depression model of aged rats that received chronic stress procedure followed by a 6-week recovery period (Aged+S). After the recovery period, aged but not young rats showed depression-like behaviors, evaluated by the sucrose preference test (SPT) and forced swimming test (FST), coincided with the altered levels of TNF-α, IL-6, NADH oxidase activity, NADPH oxidase, GRP78, CHOP, and cleaved caspase-12 in the hippocampus of these animals. These data suggested that oxidative and ER stress-induced apoptosis in the aging hippocampus may affect the recovery-related outcomes after the stress paradigm.

Antimicrobial mechanisms and secondary metabolite profiles of Streptomyces hygroscopicus subsp. hygroscopicus 5-4 against banana fusarium wilt disease using metabolomics.

Fusarium wilt of bananas (FWB) is seriously affecting the sustainable development of the banana industry and is caused by the devastating soil-borne fungus Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). Biological control is a promising strategy for controlling Fusarium wilt in bananas. We previously identified Streptomyces hygroscopicus subsp. hygroscopicus 5-4 with strong antifungal activity against the FWB. The most possible antimicrobial mechanism of strain 5-4 was explored using the metabolomics approach, light microscopy imaging, and transmission electron microscopy (TEM). The membrane integrity and ultrastructure of Foc TR4 was damaged after extract treatment, which was supported by the degradation of mycelium, soluble protein content, extracellular reducing sugar content, NADH oxidase activity, malondialdehyde content, mitochondrial membrane potential, and mitochondrial respiratory chain complex enzyme activity. The extracts of strain 5-4 cultivated at different times were characterized by a liquid chromatography-mass spectrometer (LC-MS). 647 known metabolites were detected in the extracts of strains 5-4. Hygromycin B, gluten exorphin B4, torvoside G, (z)-8-tetradecenal, piperitoside, sarmentosin, pubescenol, and other compounds were the main differential metabolites on fermentation culture for 7 days. Compared with strain 5-4 extracts, hygromycin B inhibited the mycelial growth of Foc TR4, and the EC50 concentration was 7.4 μg/mL. These results showed that strain 5-4 could destroy the cell membrane of Foc TR4 to inhibit the mycelial growth, and hygromycin B may be the key antimicrobial active metabolite. Streptomyces hygroscopicus subsp. hygroscopicus 5-4 might be a promising candidate strain to control the FWB and provide a scientific basis for the practical application of hygromycin B as a biological control agent.

Seed pretreatment with melatonin confers cadmium tolerance to chickpea seedlings through cellular redox homeostasis and antioxidant gene expression improvement.

Several phytoremediation strategies have been undertaken to alleviate cadmium (Cd)-mediated injury to crop yield resulting from agricultural land pollution. In the present study, the potentially beneficial effect of melatonin (Me) was appraised. Therefore, chickpea (Cicer arietinum L.) seeds were imbibed for 12 H in distilled water or Me (10µM) solution. Then, the seeds germinated in the presence or the absence of 200µM CdCl2 for 6days. Seedlings obtained from Me-pretreated seeds exhibited enhanced growth traits, reflected by fresh biomass and length increase. This beneficial effect was associated with a decreased Cd accumulation in seedling tissues (by 46 and 89% in roots and shoots, respectively). Besides, Me efficiently protected the cell membrane integrity of Cd-subjected seedlings. This protective effect was manifested by the decreased lipoxygenase activity and the subsequently reduced accumulation of 4-hydroxy-2-nonenal. Melatonin counteracted the Cd-mediated stimulation of the pro-oxidant NADPH-oxidase (90 and 45% decrease compared to non-pretreated Cd-stressed roots and shoots, respectively) and NADH-oxidase activities (almost 40% decrease compared to non-pretreated roots and shoots), preventing, thus, hydrogen peroxide overaccumulation (50 and 35% lesser than non-pretreated roots and shoots, respectively). Furthermore, Me enhanced the cellular content of pyridine nicotinamide reduced forms [NAD(P)H] and their redox state. This effect was associated with the Me-mediated stimulation of the glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase activities, concomitantly with the inhibition of NAD(P)H-consuming activities. These effects were accompanied by the up-regulation of G6PDH gene expression (45% increase in roots) and the down-regulation of the respiratory burst oxidase homolog protein F (RBOHF) gene expression (53% decrease in roots and shoots). Likewise, Me induced an increased activity and gene transcription of the Asada-Halliwell cycle, namely ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase, concomitantly with a reduction of the glutathione peroxidase activity. This modulating effect led to the restoration of the redox homeostasis of the ascorbate and the glutathione pools. Overall, current results attest that seed pretreatment with Me is effective in Cd stress relief and can be a beneficial crop-protective approach.

Correction of the Mitochondrial NADH Oxidase Activity, Peroxidation and Phospholipid Metabolism by Haplogenin-7-Glucoside in Hypoxia and Ischemia

In this study, it was used haplogenin-7-glucoside flavonol (C16H12O8) isolated from the plant Haplophyllum perforatum. It was studied the effect of haplogenin-7-glycoside on NADH oxidase activity, peroxidation, and phospholipid metabolism in rat liver mitochondria under in vitro conditions. Incubation of mitochondria at 37 ℃ leads to disruption of the integrity of mitochondrial membranes, as a result, in cytochrome с desorption from the inner membrane into the intermembrane space, which leads to a decrease in the activity of rotenone-sensitive NADH oxidase and an increase in the activity of rotenone-insensitive NADH oxidase. Haplogenin-7-glucoside exhibits membrane stabilization by reducing these alterations. Under ischemia conditions, the effect of haplogenin-7-glucoside on the LPO process in mitochondria decreases LPO in mitochondria. Quantitative changes in phosphatidylserine in mitochondrial membranes under LPO conditions during incubation of mitochondria at 37 ℃ were observed to accelerate controlled (auto-oxidation) LPO over time (0 - 90 min). Peroxidation of phosphatidylserine in mitochondria when the addition of haplogenin-7-glucoside to the incubation medium at 30, 60 and 90 min, LPO was found to be reduced. The increasing amount of phosphatidylinositol has not happened depend on time. However, a slight increase was found under the influence of haplogenin-7-glucoside.
 HIGHLIGHTS
 
 Cytochrome c desorption from the inner membrane into the interstitial space leads to a decrease in the activity of rotenone-sensitive NADH-oxidase and an increase in the activity of non-rotenone-sensitive NADH-oxidase
 Haplogenin-7-glycoside showed membrane-stabilizing properties by significantly reducing these changes; the addition of haplogenin-7-glucoside to mitochondria leads to a decrease for MDA, a decrease in the LPO process
 At incubation of mitochondria at 37℃, the amount of phosphatidic acid and lysophosphatidic acid in mitochondria under LPO conditions was also found to be less than control under the influence of haplogenin-7-glucoside. Thus, haplogenin-7-glucoside reduces the rate of peroxidation of phospholipids in liver mitochondria and has a corrective effect
 
 GRAPHICAL ABSTRACT

Giardia duodenalis: Flavohemoglobin is involved in drug biotransformation and resistance to albendazole.

Giardia duodenalis causes giardiasis, a major diarrheal disease in humans worldwide whose treatment relies mainly on metronidazole (MTZ) and albendazole (ABZ). The emergence of ABZ resistance in this parasite has prompted studies to elucidate the molecular mechanisms underlying this phenomenon. G. duodenalis trophozoites convert ABZ into its sulfoxide (ABZSO) and sulfone (ABZSOO) forms, despite lacking canonical enzymes involved in these processes, such as cytochrome P450s (CYP450s) and flavin-containing monooxygenases (FMOs). This study aims to identify the enzyme responsible for ABZ metabolism and its role in ABZ resistance in G. duodenalis. We first determined that the iron-containing cofactor heme induces higher mRNA expression levels of flavohemoglobin (gFlHb) in Giardia trophozoites. Molecular docking analyses predict favorable interactions of gFlHb with ABZ, ABZSO and ABZSOO. Spectral analyses of recombinant gFlHb in the presence of ABZ, ABZSO and ABZSOO showed high affinities for each of these compounds with Kd values of 22.7, 19.1 and 23.8 nM respectively. ABZ and ABZSO enhanced gFlHb NADH oxidase activity (turnover number 14.5 min-1), whereas LC-MS/MS analyses of the reaction products showed that gFlHb slowly oxygenates ABZ into ABZSO at a much lower rate (turnover number 0.01 min-1). Further spectroscopic analyses showed that ABZ is indirectly oxidized to ABZSO by superoxide generated from the NADH oxidase activity of gFlHb. In a similar manner, the superoxide-generating enzyme xanthine oxidase was able to produce ABZSO in the presence of xanthine and ABZ. Interestingly, we find that gFlHb mRNA expression is lower in albendazole-resistant clones compared to those that are sensitive to this drug. Furthermore, all albendazole-resistant clones transfected to overexpress gFlHb displayed higher susceptibility to the drug than the parent clones. Collectively these findings indicate a role for gFlHb in ABZ conversion to its sulfoxide and that gFlHb down-regulation acts as a passive pharmacokinetic mechanism of resistance in this parasite.

Covalent conjugation of ubiquitin-like ISG15 to apoptosis-inducing factor exacerbates toxic stimuli-induced apoptotic cell death

Apoptosis-inducing factor (AIF) is a mitochondrion-localized flavoprotein with NADH oxidase activity. AIF normally acts as an oxidoreductase to catalyze the transfer of electrons between molecules, but it can also kill cells when exposed to certain stimuli. For example, intact AIF is cleaved upon exposure to DNA-damaging agents such as etoposide, and truncated AIF (tAIF) is released from the mitochondria to the cytoplasm and translocated to the nucleus where it induces apoptosis. Although the serial events during tAIF-mediated apoptosis and the transition of AIF function have been widely studied from various perspectives, their underlying regulatory mechanisms and the factors involved are not fully understood. Here, we demonstrated that tAIF is a target of the covalent conjugation of the ubiquitin-like moiety ISG15 (referred to as ISGylation), which is mediated by the ISG15 E3 ligase HERC5. In addition, ISGylation increases the stability of tAIF protein as well as its K6-linked polyubiquitination. Moreover, we found that ISGylation increases the nuclear translocation of tAIF upon cytotoxic etoposide treatment, subsequently causing apoptotic cell death in human lung A549 carcinoma cells. Collectively, these results suggest that HERC5-mediated ISG15 conjugation is a key factor in the positive regulation of tAIF-mediated apoptosis, highlighting a novel role of posttranslational ISG15 modification as a switch that allows cells to live or die under the stress that triggers tAIF release.

Altered mitochondria oscillations and circadian changes in NADH levels in freshwaters mussels exposed to cadmium

The effects of contaminants on biological rhythms are largely unknown. The purpose of this study was to examine the effects of cadmium (Cd) and time of day on levels of defence mechanism (metallothionein-MT), mitochondria NADH oxidase activity and oscillations in NADH levels in freshwater mussels Dreissena bugensis. Following exposure of mussels to 50 and 100 μg/L Cd for 7 days, mussels were sampled at various times of the day (9, 12, 15, 18, 21 and 24 h) and immediately stored at −85 °C. Tissues were analyzed for MT levels, NADH oxidase and oscillatory changes in NADH in mitochondria assessments. The data revealed that exposure to Cd readily increased MT and NADH oxidase activity at each time of day. In control mussels, NADH oxidase activity changed during the day reaching a minimum and maximum at 15 h and 24 h respectively as with MT levels but the maximum occurred at 12 h. Exposure to Cd displaced the maximum values in NADH oxidase towards the night (≥18 h) while no change in the pattern in MT changes during the day albeit remained at high levels. NADH oscillations in mitochondria were complex but main changes occurred at periods of 60–90 s in control mussels. The strength of association (β) between amplitudes and frequencies of these oscillations was negatively associated with MT levels suggesting that activation of defence mechanism limited the propagation of amplitude changes at higher times. The study revealed that Cd exposure could disrupt not only the intensity of responses but at the levels of rhythms commonly found in biological systems.

Antifungal Activity of β-Carboline Alkaloids Compound and Its Resistance Mechanism on Peronophythora Litchii

ABSTRACT Peronophythora litchii (P. litchii) is the pathogenic factor of litchi downy blight. Derivatives extracted from harmine officinalis and modified, N-(2-pyridyl)-1-phenyl-9 H-pyrido [3, 4-b] indole-3-formamide (PPPIF), could inhibited P. litchii growth and development. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) analyses indicated that PPPIF severely damaged the mitochondria, cell wall and endomembrane system of P. litchii cells, thus abnormal morphology, as well as deformed mycelia. PPPIF could suppress mycelial growth, sporulation, sporangia germination and germ tube elongation as well. PPPIF also caused a series of physiological and biochemical changes of P. litchii, including the serious deviation of extracellular pH, the obvious increase of the content of extracellular reducing sugar and malondialdehyde, the notable decrease of soluble protein and the activity of NADH oxidase. Furthermore, PPPIF seriously raised openness of membrane permeability transporter (MPTP) and markedly decreased the transmembrane potential (Δψm) and the activity of enzyme complex I–complex V in respiratory chain of mitochondria. The current research suggests that PPPIF may act upon the mitochondrion of P. litchii, then respiratory chain complex activity was blocked and energy metabolism disrupted or inhibited, resulting in the growth inhibition of P. litchii. Above results have strengthened our understanding of P. litchii resistance mechanisms and may help in the development of more potent inhibitors against plant diseases in the fields.

Inhibition of respiratory complex I by 6-ketocholestanol: Relevance to recoupling action in mitochondria

6-Ketocholestanol (kCh) is known as a mitochondrial recoupler, i.e. it abolishes uncoupling of mitochondria by such potent agents as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3,5-di(tert-butyl)-4-hydroxybenzylidenemalononitril (SF6847) [Starkov et al., 1997]. Here, we report data on the kCh-induced inhibition of both NADH-oxidase and NADH-ubiquinone oxidoreductase activities of the respiratory complex I in bovine heart submitochondrial particles (SMP). Based on the absence of such inhibition with hexaammineruthenium (III) (HAR) as the complex I electron acceptor, the kCh effect could be associated with the ubiquinone-binding centre of this respiratory enzyme. In isolated rat liver mitochondria (RLM), kCh inhibited oxygen consumption with the glutamate/malate, substrates of NAD-linked dehydrogenases, while no inhibition of RLM respiration was observed with succinate, in agreement with the absence of the kCh effect on the succinate oxidase activity in SMP. Three kCh analogs (cholesterol, 6α-hydroxycholesterol, and 5α,6α-epoxycholesterol) exhibited no effect on the NADH oxidase activities in both SMP and RLM. Importantly, the kCh analogs were ineffective in the recoupling of RLM treated with CCCP or SF6847. Therefore, interaction of kCh with the complex I may be involved in the kCh-mediated mitochondrial recoupling.

A natural fusion of flavodiiron, rubredoxin, and rubredoxin oxidoreductase domains is a self-sufficient water-forming oxidase of Trichomonas vaginalis

Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. This oxygen consumption results from H2O-forming NADH oxidase (NOX) activity of two distinct flavin-containing systems: H2O-forming NOXes and multicomponent flavodiiron proteins (FDPs). Neither system is membrane bound, and both recycle NADH into oxidized NAD+ while simultaneously removing O2 from the local environment. However, little is known about the specific contributions of these systems in T. vaginalis. In this study, we use bioinformatics and biochemical analyses to show that T. vaginalis lacks a NOX–like enzyme and instead harbors three paralogous genes (FDPF1–3), each encoding a natural fusion product between the N-terminal FDP, central rubredoxin (Rb), and C-terminal NADH:Rb oxidoreductase domains. Unlike a “stand-alone” FDP that lacks Rb and oxidoreductase domains, this natural fusion protein with fully populated flavin redox centers directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of oxygen to water within a single polypeptide with an extremely high turnover. Furthermore, using single-particle cryo-EM, we present structural insights into the spatial organization of the FDP core within this multidomain fusion protein. Together, these results contribute to our understanding of systems that allow protozoan parasites to maintain optimal redox balance and survive transient exposure to oxic conditions.