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Respirantins are 18-membered antimycin-type depsipeptides produced by Streptomyces sp. and Kitasatospora sp. These compounds have shown extraordinary anticancer activities against a panel of cancer cell lines with nanomolar levels of IC50 values. However, further investigation has been impeded by the low titers of the natural producers and the challenging chemical synthesis due to their structural complexity. The biosynthetic gene cluster (BGC) of respirantin was previously proposed based on a bioinformatic comparison of the four members of antimycin-type depsipeptides. In this study, we report the first successful reconstitution of respirantin in Streptomyces albus using a synthetic BGC. This heterologous system serves as an accessible platform for the production and diversification of respirantins. Through polyketide synthase pathway engineering, biocatalysis, and chemical derivatization, we generated nine respirantin compounds, including six new derivatives. Cytotoxicity screening against human MCF-7 and Hela cancer cell lines revealed a unique biphasic dose-response profile of respirantin. Furthermore, a structure-activity relationship study has elucidated the essential functional groups that contribute to its remarkable cytotoxicity. This work paves the way for respirantin-based anticancer drug discovery and development.

Nocardiopsis sp. strain LC-9 was isolated from freshwater sediments and explored for its varied bioactive traits. Initially, ethyl acetate extract of strain LC-9 at varied concentrations showed pronounced antibacterial activities. After column chromatography, fraction F2 and F3 of the extract were identified as prominent fractions in terms of antimicrobial activities with low minimum inhibitory concentration values. Antioxidant activities of fraction F2 and F3 revealed remarkable scavenging of free radicals with low IC50 values (DPPH - 417.86 ± 0.24 µg/mL, ABTS - 431.6 ± 0.90 µg/mL, and FRAP - 404.36 ± 0.18 µg/mL). Fractions F2 and F3 were further characterized by UV spectrum, Fourier transform infrared spectroscopy, nuclear magnetic resonance, and liquid chromatography-mass spectrometry, and were identified as Antimycin A and 4-hydroxybenzoic acid. The compounds were further tested for anticancer activity against MCF-7 cells. The MTT assay showed reduced viability of MCF-7 cells with an increase in concentration of compounds. The IC50 values for Antimycin A and 4-hydroxybenzoic acid were 9.6 ± 0.7 µg/mL and 20.8 ± 0.4 µg/mL, respectively. Staining techniques confirmed the apoptosis mechanism. Finally, molecular docking (against targeted proteins of bacteria, fungus, and cancer cells) and molecular dynamics confirmed the pharmaceutical efficacy of the purified compounds. [ABSTRACT FROM AUTHOR]

Nocardiopsis sp. strain LC-9 was isolated from freshwater sediments and explored for its varied bioactive traits. Initially, ethyl acetate extract of strain LC-9 at varied concentrations showed pronounced antibacterial activities. After column chromatography, fraction F2 and F3 of the extract were identified as prominent fractions in terms of antimicrobial activities with low minimum inhibitory concentration values. Antioxidant activities of fraction F2 and F3 revealed remarkable scavenging of free radicals with low IC50 values (DPPH - 417.86 ± 0.24 μg/mL, ABTS - 431.6 ± 0.90 μg/mL, and FRAP - 404.36 ± 0.18 μg/mL). Fractions F2 and F3 were further characterized by UV spectrum, Fourier transform infrared spectroscopy, nuclear magnetic resonance, and liquid chromatography-mass spectrometry, and were identified as Antimycin A and 4-hydroxybenzoic acid. The compounds were further tested for anticancer activity against MCF-7 cells. The MTT assay showed reduced viability of MCF-7 cells with an increase in concentration of compounds. The IC50 values for Antimycin A and 4-hydroxybenzoic acid were 9.6 ± 0.7 μg/mL and 20.8 ± 0.4 μg/mL, respectively. Staining techniques confirmed the apoptosis mechanism. Finally, molecular docking (against targeted proteins of bacteria, fungus, and cancer cells) and molecular dynamics confirmed the pharmaceutical efficacy of the purified compounds.

Dysfunction of the mitochondrial electron transport chain (mETC) is a major cause of human mitochondrial diseases. To identify determinants of mETC function, we screened a genome-wide human CRISPRi library under oxidative metabolic conditions with selective inhibition of mitochondrial Complex III and identified ovarian carcinoma immunoreactive antigen (OCIA) domain-containing protein 1 (OCIAD1) as a Complex III assembly factor. We find that OCIAD1 is an inner mitochondrial membrane protein that forms a complex with supramolecular prohibitin assemblies. Our data indicate that OCIAD1 is required for maintenance of normal steady-state levels of Complex III and the proteolytic processing of the catalytic subunit cytochrome c1 (CYC1). In OCIAD1 depleted mitochondria, unprocessed CYC1 is hemylated and incorporated into Complex III. We propose that OCIAD1 acts as an adaptor within prohibitin assemblies to stabilize and/or chaperone CYC1 and to facilitate its proteolytic processing by the IMMP2L protease.

Glioblastoma Multiforme (GBM) tumors contain a small population of glioma stem-like cells (GSCs) among the various differentiated GBM cells (d-GCs). GSCs drive tumor recurrence, and resistance to Temozolomide (TMZ), the standard of care (SoC) for GBM chemotherapy. In order to investigate a potential link between GSC specific mitochondria function and SoC resistance, two patient-derived GSC lines were evaluated for differences in their mitochondrial metabolism. In both the lines, GSCs had significantly lower mitochondrial -content, and -function compared to d-GCs. In vitro, the standard mitochondrial-specific inhibitors oligomycin A, antimycin A, and rotenone selectively inhibited GSC proliferation to a greater extent than d-GCs and human primary astrocytes. These findings indicate that mitochondrial inhibition can be a potential GSC-targeted therapeutic strategy in GBM with minimal off-target toxicity. Mechanistically the standard mitochondrial inhibitors elicit their GSC-selective cytotoxic effects through the induction of apoptosis or autophagy pathways. We tested for GSC proliferation in the presence of 3 safe FDA-approved drugs--trifluoperazine, mitoxantrone, and pyrvinium pamoate, all of which are also known mitochondrial-targeting agents. The SoC GBM therapeutic TMZ did not trigger cytotoxicity in glioma stem cells, even at 100 μM concentration. By contrast, trifluoperazine, mitoxantrone, and pyrvinium pamoate exerted antiproliferative effects in GSCs about 30-50 fold more effectively than temozolomide. Thus, we hereby demonstrate that FDA-approved mitochondrial inhibitors induce GSC-selective cytotoxicity, and targeting mitochondrial function could present a potential therapeutic option for GBM treatment.

Small-molecule natural products have been an essential source of pharmaceuticals to treat human diseases, but very little is known about their behavior inside dynamic, live human cells. Here, we demonstrate the first structure-activity-distribution relationship (SADR) study of complex natural products, the anti-cancer antimycin-type depsipeptides, using the emerging bioorthogonal Stimulated Raman Scattering (SRS) Microscopy. Our results show that the intracellular enrichment and distribution of these compounds are driven by their potency and specific protein targets, as well as the lipophilic nature of compounds.

While irradiation with red LED light has been reported to modulate sperm function in different mammalian species, the mechanisms underlying their response are poorly understood. This work sought to provide new insights into whether this effect relies on a direct action upon mitochondrial electron chain and/or on PKC-linked mechanisms such as those related to opsins. For this purpose, pig semen was light-stimulated for 1, 5 or 10 min in the presence/absence of antimycin A, an inhibitor of the mitochondrial electron chain, or PKC 20–28® (PKCi), a PKC inhibitor. Antimycin A completely blocked the effects of light at all the performed irradiation patterns. This effect was linked to a complete immobility of sperm, which was accompanied with a significant (p < 0.05) drop in several markers of mitochondrial activity, such as JC-1 staining and O2 consumption rate. Antimycin A, however, did not affect intracellular ATP levels, intramitochondrial calcium, total ROS, superoxides or cytochrome C oxidase (CCO) activity. In the case of PKCi, it did also counteract the effects of light on motility, O2 consumption rate and CCO activity, but not to the same extent than that observed for antimycin A. Finally, the effects observed when sperm were co-incubated with antimycin A and PKCi were similar to those observed with antimycin A alone. In conclusion, red LED light acts on sperm function via a direct effect on mitochondrial electron chain. Additionally, light-activated PKC pathways have a supplementary effect to that observed in the electron chain, thereby modulating sperm parameters such as motility and CCO activity.

Mitochondrial electron transport chain (ETC) inhibition is a phenomenon interesting in itself and serves as a tool for studying various cellular processes. Despite the fact that searching the term "rotenone" in PubMed returns more than 6900 results, there are many discrepancies regarding the directions of changes reported to be caused by this RTC inhibitor in the delicate redox balance of the cell. Here, we performed a multifaceted study of the popular ETC inhibitors rotenone and antimycin A, involving assessment of mitochondrial membrane potential and the production of hydrogen peroxide and superoxide anions at cellular and mitochondrial levels over a wide range of inhibitor concentrations (1 nmol/dm3–100 µmol/dm3). All measurements were performed with whole cells, with accompanying control of ATP levels. Antimycin A was more potent in hindering HepG2 cells' abilities to produce ATP, decreasing ATP levels even at a 1 nmol/dm3 concentration, while in the case of rotenone, a 10,000-times greater concentration was needed to produce a statistically significant decrease. The amount of hydrogen peroxide produced in the course of antimycin A biological activity increased rapidly at low concentrations and decreased below control level at a high concentration of 100 µmol/dm3. While both inhibitors influenced cellular superoxide anion production in a comparable manner, rotenone caused a greater increase in mitochondrial superoxide anions compared to a modest impact for antimycin A. IC50 values for rotenone and antimycin A with respect to HepG2 cell survival were of the same order of magnitude, but the survival curve of cells treated with rotenone was clearly biphasic, suggesting a concentration-dependent mode of biological action. We propose a clear experimental setup allowing for complete and credible analysis of the redox state of cells under stress conditions which allows for better understanding of the effects of ETC inhibition. [ABSTRACT FROM AUTHOR]

Oxidative stress has been causally linked to various diseases. Electron transport chain (ETC) inhibitors such as rotenone and antimycin A are frequently used in model systems to study oxidative stress. Oxidative stress that is provoked by ETC inhibitors can be visualized using the fluorogenic probe 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH2-DA). Non-fluorescent DCFH2-DA crosses the plasma membrane, is deacetylated to 2′,7′-dichlorodihydrofluorescein (DCFH2) by esterases, and is oxidized to its fluorescent form 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence can, therefore, be used as a semi-quantitative measure of general oxidative stress. However, the use of DCFH2-DA is complicated by various protocol-related factors that mediate DCFH2-to-DCF conversion independently of the degree of oxidative stress. This study therefore analyzed the influence of ancillary factors on DCF formation in the context of ETC inhibitors. It was found that ETC inhibitors trigger DCF formation in cell-free experiments when they are co-dissolved with DCFH2-DA. Moreover, the extent of DCF formation depended on the type of culture medium that was used, the pH of the assay system, the presence of fetal calf serum, and the final DCFH2-DA solvent concentration. Conclusively, experiments with DCFH2-DA should not discount the influence of protocol-related factors such as medium and mitochondrial inhibitors (and possibly other compounds) on the DCFH2-DA-DCF reaction and proper controls should always be built into the assay protocol. [ABSTRACT FROM AUTHOR]

BACKGROUND: Fenpicoxamid and florylpicoxamid are picolinamide fungicides targeting the Qi site of the cytochrome bc1 complex, via their primary metabolites UK‐2A and CAS‐649, respectively. We explore binding interactions and resistance mechanisms for picolinamides, antimycin A and ilicicolin H in yeast by testing effects of cytochrome b amino acid changes on fungicide sensitivity and interpreting results using molecular docking. RESULTS: Effects of amino acid changes on sensitivity to UK‐2A and CAS‐649 were similar, with highest resistance associated with exchanges involving G37 and substitutions N31K and L198F. These changes, as well as K228M, also affected antimycin A, while ilicicolin H was affected by changes at G37 and L198, as well as Q22E. N31 substitution patterns suggest that a lysine at position 31 introduces an electrostatic interaction with neighbouring D229, causing disruption of a key salt‐bridge interaction with picolinamides. Changes involving G37 and L198 imply resistance primarily through steric interference. G37 changes also showed differences between CAS‐649 and UK‐2A or antimycin A with respect to branched versus unbranched amino acids. N31K and substitution of G37 by large amino acids reduced growth rate substantially while L198 substitutions showed little effect on growth. CONCLUSION: Binding of UK‐2A and CAS‐649 at the Qi site involves similar interactions such that general cross‐resistance between fenpicoxamid and florylpicoxamid is anticipated in target pathogens. Some resistance mutations reduced growth rate and could carry a fitness penalty in pathogens. However, certain changes involving G37 and L198 carry little or no growth penalty and may pose the greatest risk for resistance development in the field. © 2022 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The present study was conducted to investigate the evaluation of antimycin A (AA) as a modulator of the alternative respiratory pathway (AP) on the intensity of growth processes and some physiological parameters of etiolated wheat seedlings, such as maximum length of root, length of first leaf and coleoptile, fresh and dry weights at the whole plant level, leaf area as well as relative water content, saturation water deficit, degree of leaf succulence and sclerophylly. Generally, exposure to heat stress (HS) alone caused a noticeable reduction in root length (59%), leaf length (30%), plant fresh mass (10%), dry mass (14%), and leaf area (25%) which was consistent with the progressive alteration in water relations. Furthermore, the degree of leaf succulence and degree of leaf sclerophylly were severely affected by HS in wheat seedlings. However, the results demonstrated that the application of AA did not induce such a significant reduction in the leaf length (23%), coleoptile length (5%), plant fresh weight (7%), plant dry weight (3%), and leaf area (21%) as well as water relations in etiolated wheat seedlings compared to heat-stressed plants. Taken together, the activation of AP promoted a mitigating of the damaging effect of HS in wheat seedlings supporting growth processes.

Recent evidence has implicated succinate-driven reverse electron transport (RET) through complex I as a major source of damaging reactive oxygen species (ROS) underlying reperfusion injury after prolonged cardiac ischemia. However, this explanation may be incomplete, because RET on reperfusion is self-limiting and therefore transient. RET can only generate ROS when mitochondria are well polarized, and it ceases when permeability transition pores (PTP) open during reperfusion. Because prolonged ischemia/reperfusion also damages electron transport complexes, we investigated whether such damage could lead to ROS production after PTP opening has occurred. Using isolated cardiac mitochondria, we demonstrate a novel mechanism by which antimycin-inhibited complex III generates significant amounts of ROS in the presence of Mg2+ and NAD+ and the absence of exogenous substrates upon inner membrane pore formation by alamethicin or Ca2+-induced PTP opening. We show that H2O2 production under these conditions is related to Mg2+-dependent NADH generation by malic enzyme. H2O2 production is blocked by stigmatellin, indicating its origin from complex III, and by piericidin, demonstrating the importance of NADH-related ubiquinone reduction for ROS production under these conditions. For maximal ROS production, the rate of NADH generation has to be equal or below that of NADH oxidation, as further increases in [NADH] elevate ubiquinol-related complex III reduction beyond the optimal range for ROS generation. These results suggest that if complex III is damaged during ischemia, PTP opening may result in succinate/malate-fueled ROS production from complex III due to activation of malic enzyme by increases in matrix [Mg2+], [NAD+], and [ADP].

Key message: Already a short-term Cd treatment induces changes in gene expression in barley root tips via IAA and ROS signaling during mild and severe Cd stress, respectively. Even a short, 30 min, Cd treatment of roots induced a considerable alteration in gene expression in the barley root tips within an hour after the treatments. The very early activation of MYB1 transcription factor expression is partially regulated by auxin signaling in mildly stressed seedlings. An increase in allene oxide cyclase and NADPH oxidase expression was a distinguishing feature of root tips response to mild Cd stress and their expression is activated via IAA signaling. Meanwhile, early changes in the level of dehydrin transcripts were detected in moderately and severely stressed root tips, and their induction is related to altered ROS homeostasis in cells. The early activation of glutathione peroxidase expression by mild Cd stress indicates the involvement of IAA in the signaling process. In contrast, early ascorbate peroxidase expression was induced only with Cd treatment causing severe stress and ROS play central roles in its induction. The expression of cysteine protease was activated similarly in both mildly and severely Cd-stressed roots; consequently, both increased IAA and ROS levels take part in the regulation of cysteine protease expression. The Cd-evoked accumulation of BAX Inhibitor-1 mRNA was characteristic for moderately and severely stressed roots. Whereas decreased IAA level did not affect its expression, rotenone-mediated ROS depletion markedly reduced the Cd-induced expression of BAX Inhibitor-1. An early increase of alternative oxidase levels in the root tip cells indicated that the reduction of mitochondrial superoxide generation is an important component of barley root response to severe Cd stress. [ABSTRACT FROM AUTHOR]

Antimycins are a family of natural products generated from a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line. Although they possess an array of useful biological activities, their structural complexity makes chemical synthesis challenging, and their biosynthesis has thus far been dependent on slow-growing source organisms. Here, we reconstituted the biosynthesis of antimycins in Escherichia coli, a versatile host that is robust and easy to manipulate genetically. Along with Streptomyces genetic studies, the heterologous expression of different combinations of ant genes enabled us to systematically confirm the functions of the modification enzymes, AntHIJKL and AntO, in the biosynthesis of the 3-formamidosalicylate pharmacophore of antimycins. Our E. coli-based antimycin production system can not only be used to engineer the increased production of these bioactive compounds, but it also paves the way for the facile generation of novel and diverse antimycin analogues through combinatorial biosynthesis.

Plant oxylipins are signaling molecules produced from fatty acids by oxidative pathways, mainly initiated by 9- and 13-lipoxygenases (9-LOX and 13-LOX), alpha-dioxygenases or non-enzymatic oxidation. Oxylipins from the 9-LOX pathway induce oxidative stress and control root development and plant defense. These activities have been associated with mitochondrial processes, but precise cellular targets and pathways remain unknown. In order to study oxylipin signaling, we previously generated a collection of Arabidopsis thaliana mutants that were insensitive to the 9-LOX products 9(S)-hydroxy-10,12, 15-octadecatrienoic acid (9-HOT) and its ketone derivative 9-KOT (noxy mutants). Here, we describe noxy1, noxy3, noxy5, noxy23, and noxy54 mutants, all affected in nucleus-encoded mitochondrial proteins, and use them to study the role of mitochondria in oxylipin signaling. Functional and phenotypic analyses showed that noxy plants displayed mitochondrial aggregation, reduced respiration rates and resistance to the complex III inhibitor Antimycin A (AA), thus indicating a close similarity of the oxylipin signaling and mitochondrial stress. Application of 9-HOT and 9-KOT protected plants against subsequent mitochondrial stress, whereas they boosted root growth reduction when applied in combination with complex III inhibitors but did not with inhibitors of other respiratory complexes. A similar effect was caused by linear-chain oxylipins from 13-LOX or non-enzymatic pathways having α,β-unsaturated hydroxyl or keto groups in their structure. Studies to investigate 9-HOT and 9-KOT activity indicated that they do not reduce respiration rates, but their action is primarily associated with enhanced ROS responses. This was supported by the results showing that 9-HOT or 9-KOT combined with AA amplified the expression of oxylipin- and ROS-responding genes but not of the AA marker AOX1a, thus implying the activation of a specific mitochondria retrograde signaling pathway. Our results implicate mitochondrial complex III as a hub in the signaling activity of multiple oxylipin pathways and point at downstream ROS responses as components of oxylipin function.

Plant oxylipins are signaling molecules produced from fatty acids by oxidative pathways, mainly initiated by 9- and 13-lipoxygenases (9-LOX and 13-LOX), alpha-dioxygenases or non-enzymatic oxidation. Oxylipins from the 9-LOX pathway induce oxidative stress and control root development and plant defense. These activities have been associated with mitochondrial processes, but precise cellular targets and pathways remain unknown. In order to study oxylipin signaling, we previously generated a collection of Arabidopsis thaliana mutants that were insensitive to the 9-LOX products 9(S)-hydroxy-10,12, 15-octadecatrienoic acid (9-HOT) and its ketone derivative 9-KOT (noxy mutants). Here, we describe noxy1, noxy3, noxy5, noxy23 , and noxy54 mutants, all affected in nucleus-encoded mitochondrial proteins, and use them to study the role of mitochondria in oxylipin signaling. Functional and phenotypic analyses showed that noxy plants displayed mitochondrial aggregation, reduced respiration rates and resistance to the complex III inhibitor Antimycin A (AA), thus indicating a close similarity of the oxylipin signaling and mitochondrial stress. Application of 9-HOT and 9-KOT protected plants against subsequent mitochondrial stress, whereas they boosted root growth reduction when applied in combination with complex III inhibitors but did not with inhibitors of other respiratory complexes. A similar effect was caused by linear-chain oxylipins from 13-LOX or non-enzymatic pathways having α,β-unsaturated hydroxyl or keto groups in their structure. Studies to investigate 9-HOT and 9-KOT activity indicated that they do not reduce respiration rates, but their action is primarily associated with enhanced ROS responses. This was supported by the results showing that 9-HOT or 9-KOT combined with AA amplified the expression of oxylipin- and ROS-responding genes but not of the AA marker AOX1a , thus implying the activation of a specific mitochondria retrograde signaling pathway. Our results implicate mitochondrial complex III as a hub in the signaling activity of multiple oxylipin pathways and point at downstream ROS responses as components of oxylipin function. [ABSTRACT FROM AUTHOR]

SUMMARY: To examine the effect of mitochondrial function on photosynthesis, wild‐type and transgenic Nicotiana tabacum with varying amounts of alternative oxidase (AOX) were treated with different respiratory inhibitors. Initially, each inhibitor increased the reduction state of the chloroplast electron transport chain, most severely in AOX knockdowns and least severely in AOX overexpressors. This indicated that the mitochondrion was a necessary sink for photo‐generated reductant, contributing to the 'P700 oxidation capacity' of photosystem I. Initially, the Complex III inhibitor myxothiazol and the mitochondrial ATP synthase inhibitor oligomycin caused an increase in photosystem II regulated non‐photochemical quenching not evident with the Complex III inhibitor antimycin A (AA). This indicated that the increased quenching depended upon AA‐sensitive cyclic electron transport (CET). Following 12 h with oligomycin, the reduction state of the chloroplast electron transport chain recovered in all plant lines. Recovery was associated with large increases in the protein amount of chloroplast ATP synthase and mitochondrial uncoupling protein. This increased the capacity for photophosphorylation in the absence of oxidative phosphorylation and enabled the mitochondrion to act again as a sink for photo‐generated reductant. Comparing the AA and myxothiazol treatments at 12 h showed that CET optimized photosystem I quantum yield, depending upon the P700 oxidation capacity. When this capacity was too high, CET drew electrons away from other sinks, moderating the P700+ amount. When P700 oxidation capacity was too low, CET acted as an electron overflow, moderating the amount of reduced P700. This study reveals flexible chloroplast–mitochondrion interactions able to overcome lesions in energy metabolism. Significance Statement: Optimal leaf energy metabolism requires interactions between chloroplast photosynthesis and mitochondrial respiration, but the extent and importance of these interactions is not fully understood. This study reveals flexible chloroplast–mitochondrion interactions able to overcome major lesions in energy metabolism, but also shows that the electron sink function of mitochondria is essential for photosynthesis, particularly if chloroplast antimycin A‐sensitive cyclic electron transport is constrained. [ABSTRACT FROM AUTHOR]

Nano- and micro-scale engineered surface structures are often used to control cell morphology and mimic the extracellular matrix in tissue engineering. However, there is little understanding of how toxins produced by common bacteria might affect cell adhesion to these structures. In this study, human dermal fibroblast (GM5565) cells were incubated on patterned tungsten/silicon oxide nanocomposite in media in the presence or absence of Antimycin A. This composite consists of parallel tungsten and silicon oxide lines with identical widths in the range of 0.18 and 50 μm. The morphology of the cells and of their mitochondria was characterized by using high-resolution scanning electron microscopy and fluorescence confocal microscopy. Results show that cells preferentially align along the line axes in a pattern-dependent manner, with a maximum population of cells oriented within 10° of the line axes on the structures containing 10 μm wide lines. Cells treated with Antimycin A, however, show a smaller proportion of cells oriented in this direction as compared to cells cultured in Antimycin A-free media (34.4% vs 53.0%). The majority of mitochondria in cells growing in Antimycin A-free media are tubular in shape and are preferentially positioned on the tungsten lines, whereas these organelles exhibit a circular geometry and are less attracted to the metal lines in the presence of Antimycin A.

Oxidative stress has been causally linked to various diseases. Electron transport chain (ETC) inhibitors such as rotenone and antimycin A are frequently used in model systems to study oxidative stress. Oxidative stress that is provoked by ETC inhibitors can be visualized using the fluorogenic probe 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH2-DA). Non-fluorescent DCFH2-DA crosses the plasma membrane, is deacetylated to 2′,7′-dichlorodihydrofluorescein (DCFH2) by esterases, and is oxidized to its fluorescent form 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence can, therefore, be used as a semi-quantitative measure of general oxidative stress. However, the use of DCFH2-DA is complicated by various protocol-related factors that mediate DCFH2-to-DCF conversion independently of the degree of oxidative stress. This study therefore analyzed the influence of ancillary factors on DCF formation in the context of ETC inhibitors. It was found that ETC inhibitors trigger DCF formation in cell-free experiments when they are co-dissolved with DCFH2-DA. Moreover, the extent of DCF formation depended on the type of culture medium that was used, the pH of the assay system, the presence of fetal calf serum, and the final DCFH2-DA solvent concentration. Conclusively, experiments with DCFH2-DA should not discount the influence of protocol-related factors such as medium and mitochondrial inhibitors (and possibly other compounds) on the DCFH2-DA-DCF reaction and proper controls should always be built into the assay protocol.

Scheffersomyces stipitis is a Crabtree-negative pentose fermenting yeast, which shows a complex respiratory system involving a cytochrome and an alternative salicylhydroxamic acid (SHAM)-sensitive respiration mechanism that is poorly understood. This work aimed to investigate the role of the antimycin A (AA) sensitive respiration and SHAM-sensitive respiration in the metabolism of xylose and glucose by S. stipitis , upon different agitation conditions. Inhibition of the SHAM-sensitive respiration caused a significant (P  < 0.05) decrease in glycolytic flux and oxygen consumption when using glucose and xylose under agitation conditions, but without agitation, only a mild reduction was observed. The combination of SHAM and AA abolished respiration, depleting the glycolytic flux using both carbon sources tested, leading to increased ethanol production of 21.05 g/L at 250 rpm for 0.5 M glucose, and 8.3 g/L ethanol using xylose. In contrast, inhibition of only the AA-sensitive respiration, caused increased ethanol production to 30 g/L using 0.5 M glucose at 250 rpm, and 11.3 g/L from 0.5 M xylose without agitation. Results showed that ethanol production can be induced by respiration inhibition, but the active role of SHAM-sensitive respiration should be considered to investigate better conditions to increase and optimize yields. [ABSTRACT FROM AUTHOR]

The present study was conducted to investigate the evaluation of antimycin A (AA) as a modulator of the alternative respiratory pathway (AP) on the intensity of growth processes and some physiological parameters of etiolated wheat seedlings, such as maximum length of root, length of first leaf and coleoptile, fresh and dry weights at the whole plant level, leaf area as well as relative water content, saturation water deficit, degree of leaf succulence and sclerophylly. Generally, exposure to heat stress (HS) alone caused a noticeable reduction in root length (59%), leaf length (30%), plant fresh mass (10%), dry mass (14%), and leaf area (25%) which was consistent with the progressive alteration in water relations. Furthermore, the degree of leaf succulence and degree of leaf sclerophylly were severely affected by HS in wheat seedlings. However, the results demonstrated that the application of AA did not induce such a significant reduction in the leaf length (23%), coleoptile length (5%), plant fresh weight (7%), plant dry weight (3%), and leaf area (21%) as well as water relations in etiolated wheat seedlings compared to heat-stressed plants. Taken together, the activation of AP promoted a mitigating of the damaging effect of HS in wheat seedlings supporting growth processes. [ABSTRACT FROM AUTHOR]

Metabolic reprogramming of tumour cells sustains cancer progression. Similar to other cancer cells, glioblastoma cells exhibit an increased glycolytic flow, which encourages the use of antiglycolytics as an effective complementary therapy. We used the antiglycolytic 3-bromopyruvate (3BP) as a metabolic modifier to treat U118 glioblastoma cells and investigated the toxic effects and the conditions to increase drug effectiveness at the lowest concentration. Cellular vitality was not affected by 3BP concentrations lower than 40 μM, although p-Akt dephosphorylation, p53 degradation, and ATP reduction occurred already at 30 μM 3BP. ROS generated in mitochondria were enhanced at 30 μM 3BP, possibly by unbalancing their generation and their disposal because of glutathione peroxidase inhibition. ROS triggered JNK and ERK phosphorylation, and cyt c release outside mitochondria, not accompanied by caspases-9 and -3 activation, probably due to 3BP-dependent alkylation of cysteine residues at caspase-9 catalytic site. To explore the possibility of sensitizing cells to 3BP treatment, we exploited 3BP effects on mitochondria by using 30 μM 3BP in association with antimycin A or menadione concentrations that in themselves exhibit poor toxicity. 3BP effect on cyt c release and cell vitality loss was potentiated due the greater oxidative stress induced by antimycin or menadione association with 3BP, supporting a preeminent role of mitochondrial ROS in 3BP toxicity. Indeed, the scavenger of mitochondrial superoxide MitoTEMPO counteracted 3BP-induced cyt c release and weakened the potentiating effect of 3BP/antimycin association. In conclusion, the biochemical mechanisms leading U118 glioblastoma cells to viability loss following 3BP treatment rely on mitochondrial ROS-dependent pathways. Their potentiation at low 3BP concentrations is consistent with the goal to minimize the toxic effect of the drug towards non-cancer cells.

The present study was aimed to study the plasticity of the antioxidative defense system in the etiolated leaves of Triticum aestivum L. and grown under normal daylight conditions following short-term and long-term high temperature (HT), as well as to investigate the role of antimycin A (AA) as an activator of the alternative respiratory pathway (AP) under stressful conditions. Wheat seedlings exposed to short-term HT (42°C) showed no significant changes in Mn-superoxide dismutase (SOD) activity but following prolonged HT a pronounced induction of CuZn-SOD activity was detected. Catalase and ascorbate peroxidase activities increased in response to short-term and long-term HT treatments by 24 and 42%, respectively. Treatment with AA showed a significant decrease in the activities of antioxidative enzymes in etiolated leaves and grown under normal daylight. Modulation of the AP by AA in leaves of wheat seedlings might be responsible for conferring tolerance to HT in wheat seedlings preventing oxidative stress [ABSTRACT FROM AUTHOR]

ABSTRACT Candida albicans adapts to various conditions in different body niches by regulating gene expression, protein synthesis, and metabolic pathways. These adaptive reactions not only allow survival but also influence the interaction with host cells, which is governed by the composition and structure of the fungal cell wall. Numerous studies had shown linkages between mitochondrial functionality, cell wall integrity and structure, and pathogenicity. Thus, we decided to inhibit single complexes of the respiratory chain of C. albicans and to analyze the resultant interaction with macrophages via their phagocytic activity. Remarkably, inhibition of the fungal bc1 complex by antimycin A increased phagocytosis, which correlated with an increased accessibility of β-glucans. To contribute to mechanistic insights, we performed metabolic studies, which highlighted significant changes in the abundance of constituents of the plasma membrane. Collectively, our results reinforce the strong linkage between fungal energy metabolism and other components of fungal physiology, which also determine the vulnerability to immune defense reactions. IMPORTANCE The yeast Candida albicans is one of the major fungal human pathogens, for which new therapeutic approaches are required. We aimed at enhancements of the phagocytosis efficacy of macrophages by targeting the cell wall structure of C. albicans, as the coverage of the β-glucan layer by mannans is one of the immune escape mechanisms of the fungus. We unambiguously show that inhibition of the fungal bc1 complex correlates with increased accessibilities of β-glucans and improved phagocytosis efficiency. Metabolic studies proved not only the known direct effects on reactive oxygen species (ROS) production and fermentative pathways but also the clear downregulation of the ergosterol pathway and upregulation of unsaturated fatty acids. The changed composition of the plasma membrane could also influence the interaction with the overlying cell wall. Thus, our work highlights the far-reaching relevance of energy metabolism, indirectly also for host-pathogen interactions, without affecting viability.

Summary: Plants need to attune their stress responses to the ongoing developmental programmes to maximize their efficacy. For instance, successful submergence adaptation is often associated with a delicate balance between saving resources and their expenditure to activate measures that allow stress avoidance or attenuation. We observed a significant decrease in submergence tolerance associated with ageing in Arabidopsis thaliana, with a critical step between 2 and 3 weeks of post‐germination development. This sensitization to flooding was concomitant with the transition from juvenility to adulthood. Transcriptomic analyses indicated that a group of genes related to abscisic acid and oxidative stress response was more highly expressed in juvenile plants than in adult ones. These genes are induced by the endomembrane tethered transcription factor ANAC017 that was in turn activated by submergence‐associated oxidative stress. A combination of molecular, biochemical and genetic analyses showed that these genes are located in genomic regions that move towards a heterochromatic state with adulthood, as marked by lysine 4 trimethylation of histone H3. We concluded that, while the mechanisms of flooding stress perception and signal transduction were unaltered between juvenile and adult phases, the sensitivity that these mechanisms set into action is integrated, via epigenetic regulation, into the developmental programme of the plant. Significance Statement: This study reports the superior submergence tolerance of juvenile Arabidopsis plants in comparison to adult ones, and investigates the molecular determinants of such features. Here, we highlight the role of mitochondrial retrograde signalling during submergence, pointing at the post‐translationally regulated transcription factor ANAC017 and the epigenetic regulation of downstream target genes. [ABSTRACT FROM AUTHOR]

The flexible plant mitochondrial electron transport chain with cytochrome c oxidase (COX) and alternative oxidase (AOX) pathways is known to be modulated by abiotic stress conditions. The effect of salinity stress on the mitochondrial electron transport chain and the importance of COX and AOX pathways for optimization of photosynthesis under salinity stress conditions is not clearly understood. In the current study, importance of COX and AOX pathways for photosynthetic performance of pea plants (Pisum sativum L. Pea Arkel cv) was analysed by using the mitochondrial electron transport chain inhibitors Antimycin A (AA) and salicylhydroxamic acid (SHAM) which restrict the electron flow through COX and AOX pathways respectively. Salinity stress resulted in decreased CO 2 assimilation rates, leaf stomatal conductance, transpiration and leaf intercellular CO 2 concentration in a stress dependent manner. Superimposition of leaves of salt stressed plants with AA and SHAM caused cellular H 2 O 2 and O 2 − accumulation along with cell death. Additionally, aggravation in decrease of CO 2 assimilation rates, leaf stomatal conductance, transpiration and leaf intercellular CO 2 concentration upon superimposition with AA and SHAM during salinity stress suggests the importance of mitochondrial oxidative electron transport for photosynthesis. Increased expression of AOX1a and AOX2 transcripts along with AOX protein levels indicated up regulation of AOX pathway in leaves during salinity stress. Chlorophyll fluorescence measurements revealed enhanced damage to Photosystem (PS) II in the presence of AA and SHAM during salinity stress. Results suggested the beneficial role of COX and AOX pathways for optimal photosynthetic performance in pea leaves during salinity stress conditions. • COX and AOX pathway inhibition led to aggravation in the decrease of CO 2 assimilation rates in a stress dependent manner. • COX and AOX pathways play an important role in cellular ROS homeostasis during salinity stress. • Decrease in chlorophyll fluorescence parameters upon COX and AOX pathway inhibition during salinity stress is observed. • The COX and AOX pathways play an important role in protecting photosynthesis during salinity stress. [ABSTRACT FROM AUTHOR]

The role of furostanol glycosides (FGs) in the intensification of main metabolic processes in cultured yam (Dioscorea deltoidea Wall.) cells was shown. The effects of FGs were associated with changes in the complicated network of intracellular enzymatic reactions. This was evidenced by an increase in the activities of the enzymes from the glucose glycolytic oxidation pathway (hexokinase and fructose-1,6-difosphate aldolase) and the malate dehydrogenase complex (oxidoreductive—NADH/NAD-malate dehydrogenase and malic enzyme—decarboxylating NADP-malate dehydrogenase) as well as the stimulation of mitochondrial respiration accompanied by changes in activity of the alternative respiratory pathway. At the lag-phase of the growth cycle, the cell treatment with FGs was capable to guide glucose conversion through the glycolytic pathway. This was witnessed by a decrease in the activity of glucose-6-phosphate dehydrogenase and increase in that of hexokinase. Inhibition of NADPH oxidase afforded by diphenyliodonium chloride resulted in a decrease in activities of the key enzymes of glycolysis and the pentose phosphate cycle of glucose oxidation. Simultaneously, the aldolase activity increased and raised the content of its product glyceraldehyde 3-phosphate. The effect may explain the earlier-observed activation of the aldehyde-utilizing enzymes diminishing the initial POL level. The modulating action of FGs on activities of the enzymes of sugar glycolytic oxidation was shown. This was expressed as the activation of the tested enzymes at the lag- and exponential phases of the cellular growth and as the absence of the FG effect at the stationary phase. The short-term exposure of yam cells to FGs intensified the total mitochondrial respiration. An inhibitory analysis revealed the dominance of the cytochrome respiration pathway at the lag-phase, while its contribution became considerably lower at the later growth phases. FGs did not affect the rate of this respiration path but increased the potential activity of alternative oxidase (AO) during the lag- and exponential phases of the growth cycle. The FG-induced activation of AO, which may be intended to maintain a balance between carbohydrate metabolism and the rate of electron transport, is discussed. [ABSTRACT FROM AUTHOR]

Background: The ischemic environment of the receiving area compromises the outcome of autologous fat grafts. The aim of this study was to isolate and expand the stromal vascular fraction from patient lipoaspirates and investigate the gain in cell viability exerted by some protective agents against the blockage of mitochondrial respiration. Methods: The aspirates were (1) washed, using the "Lull pgm system," (2) centrifuged and (3) decanted. The corresponding stromal vascular fractions were isolated, and after cell adherence selection, the stromal/stem cell subpopulations were exposed to Antimycin A for 1 h. Then, the protection induced by cell pretreatment with deferoxamine, diazoxide and IGF-1 was evaluated. Results: The residual cell viability of the "Lull pgm system"-washed samples was greater than that of the centrifuged samples (p < 0.05), and this advantage was maintained during the following 12 days of culture. The administration of 400 μM deferoxamine before Antimycin A treatment increased the number of viable cells from 56.5 to 80.8% (p < 0.05). On the contrary, the pretreatment with 250 μM diazoxide or 0.1 μg/ml IGF-1 did not exert any significant pro-survival action. Echinomycin abolished the positive effect of deferoxamine, suggesting that its protection involved HIF-1α. Conclusions: Adipose-derived stromal–stem cells survive the inhibition of mitochondrial respiration better if the lipoaspirate is washed using the "Lull pgm system" rather than centrifuged. Moreover, a significant contribution to cell survival can be obtained by preconditioning stromal–stem cells with deferoxamine. In a clinical perspective, this drug could be safely administered before surgery to patients undergoing autologous fat transfer. No Level Assigned: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266. [ABSTRACT FROM AUTHOR]

Nano- and micro-scale engineered surface structures are often used to control cell morphology and mimic the extracellular matrix in tissue engineering. However, there is little understanding of how toxins produced by common bacteria might affect cell adhesion to these structures. In this study, human dermal fibroblast (GM5565) cells were incubated on patterned tungsten/silicon oxide nanocomposite in media in the presence or absence of Antimycin A. This composite consists of parallel tungsten and silicon oxide lines with identical widths in the range of 0.18 and 50 μm. The morphology of the cells and of their mitochondria was characterized by using high-resolution scanning electron microscopy and fluorescence confocal microscopy. Results show that cells preferentially align along the line axes in a pattern-dependent manner, with a maximum population of cells oriented within 10° of the line axes on the structures containing 10 μm wide lines. Cells treated with Antimycin A, however, show a smaller proportion of cells oriented in this direction as compared to cells cultured in Antimycin A-free media (34.4% vs 53.0%). The majority of mitochondria in cells growing in Antimycin A-free media are tubular in shape and are preferentially positioned on the tungsten lines, whereas these organelles exhibit a circular geometry and are less attracted to the metal lines in the presence of Antimycin A. [ABSTRACT FROM AUTHOR]

The combination of laser tweezers, fluorescent imaging, and real-time automated tracking and trapping (RATTS) can measure sperm swimming speed and swimming force simultaneously with mitochondrial membrane potential (MMP). This approach is used to study the roles of two sources of ATP in sperm motility: oxidative phosphorylation, which occurs in the mitochondria located in the sperm midpiece and glycolysis, which occurs along the length of the sperm tail (flagellum). The relationships between (a) swimming speed and MMP and (b) swimming force and MMP are studied in dog and human sperm. The effects of glucose, oxidative phosphorylation inhibitors and glycolytic inhibitors on human sperm motility are examined. The results indicate that oxidative phosphorylation does contribute some ATP for human sperm motility, but not enough to sustain high motility. The glycolytic pathway is shown to be a primary source of energy for human sperm motility.

The current study investigated the physiological effects of flavonoids found in daily consumed rooibos tea, aspalathin, isoorientin, and orientin on improving processes involved in mitochondrial function in C2C12 myotubes. To achieve this, C2C12 myotubes were exposed to a mitochondrial channel blocker, antimycin A (6.25 µM), for 12 h to induce mitochondrial dysfunction. Thereafter, cells were treated with aspalathin, isoorientin, and orientin (10 µM) for 4 h, while metformin (1 µM) and insulin (1 µM) were used as comparators. Relevant bioassays and real-time PCR were conducted to assess the impact of treatment compounds on some markers of mitochondrial function. Our results showed that antimycin A induced alterations in the mitochondrial respiration process and mRNA levels of genes involved in energy production. In fact, aspalathin, isoorientin, and orientin reversed such effects leading to the reduced production of intracellular reactive oxygen species. These flavonoids further enhanced the expression of genes involved in mitochondrial function, such as Ucp 2, Complex 1/3, Sirt 1, Nrf 1, and Tfam. Overall, the current study showed that dietary flavonoids, aspalathin, isoorientin, and orientin, have the potential to be as effective as established pharmacological drugs such as metformin and insulin in protecting against mitochondrial dysfunction in a preclinical setting; however, such information should be confirmed in well-established in vivo disease models.

Vertebrate photoreceptors consist of strictly delimited subcellular domains: the outer segment, ellipsoid, cell body and synaptic terminal, each hosting crucial cellular functions, including phototransduction, oxidative metabolism, gene expression and transmitter release. We used optical imaging to explore the spatiotemporal dynamics of Ca2+ signaling in non-outer segment regions of rods and cones. Sustained depolarization, designed to emulate photoreceptor activation in the darkness, evoked a standing Ca2+ gradient in tiger salamander photoreceptors with spatially-averaged intracellular Ca2+ concentration within synaptic terminals of similar to 2 mu M and lower (similar to 750 nM) intracellular calcium concentration in the ellipsoid. Measurements from axotomized cell bodies and isolated ellipsoids showed that Ca2+ enters the two compartments via both local L-type Ca2+ channels and diffusion. The results from optical imaging studies were supported by immunostaining analysis. L-type voltage-operated Ca2+ channels and plasma membrane Ca2+ ATPases were highly expressed in synaptic terminals with progressively lower expression levels in the cell body and ellipsoid. These results show photoreceptor Ca2+ homeostasis is controlled in a region-specific manner by direct Ca2+ entry and diffusion as well as Ca2+ extrusion. Moreover, quantitative measurement of intracellular calcium concentration levels in different photoreceptor compartments indicates that the dynamic range of Ca2+ signaling in photoreceptors is approximately 40-fold, from similar to 50 nM in the light to similar to 2 mu M in darkness. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

The objective of the present investigation was to identify the effect of antimycin A (AA) as an activator of the alternative pathway (AP) of respiration, on oxidative stress intensity, the level of lipid peroxidation (LPO) and activities of H2O2 scavenging enzymes in functionally different organs of Triticum aestivum L. subjected to short- and long-term exposure to high temperature (HT). The level of LPO was assessed in terms of malondialdehyde (MDA), an indicator of oxidative injury. The results demonstrated increases in the total content of reactive oxygen species (ROS) and MDA production in developing and senescent organs of wheat seedlings, and significant augmentation of the activities of the antioxidant enzymes, catalase and ascorbate peroxidase, in different organs in response to exposure to HT. The activation of the AP by AA restrained ROS production in the mitochondrial electron transport chain (mETC) under exposure to HT.

Oxidative stress plays a crucial role in the development and progression of various kidney diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is the primary transcription factor that protects cells from oxidative stress by regulating cytoprotective genes including those involved in the antioxidant glutathione (GSH) pathway. GSH maintains cellular redox status and affects redox signaling, cell proliferation, and cell death. Antimycin A, an inhibitor of complex III of the electron transport chain, causes oxidative stress and reduces GSH levels. In this study, we induced mitochondrial damage in rat renal proximal tubular cells using antimycin A and investigated cellular viability and levels of NRF2 and GSH. Treatment with antimycin A altered the expression of antioxidant genes, including reduction in the transcription of glutathione-cysteine ligase subunits (Gclc and Gclm) and glutathione reductase (Gsr1), followed by a reduction in total GSH content with a concomitant decrease in NRF2 protein expression. AR-20007, previously described as an NRF2 activator, stabilizes and increases NRF2 protein expression in cells. By stimulating NRF2, AR-20007 increased the expression of antioxidant and detoxifying enzymes, thereby enhancing protection against oxidative stress induced by antimycin A. These data suggest that NRF2 activation effectively inhibits antimycin A-induced oxidative stress and that NRF2 may be a promising therapeutic target for preventing cell death during acute kidney injury.

The strain of Cellulomonas fimi NBRC 15513 can generate electricity with cellulose as fuel without mediator using a single chamber type microbial fuel cell (MFC) which had 100 mL of chamber and 50 cm2 of the air cathode. The MFCs were operated over five days and showed the maximum current density of 10.0 ± 1.8 mA/m2, the maximum power density of 0.74 ± 0.07 mW/m2 and the ohmic resistance of 6.9 kΩ. According to the results of cyclic voltammetry, the appearance of the oxidation or reduction peak was not observed from the cell removed solution. The fact is that C. fimi does not secrete mediator-like compounds, while the oxidation peak was observed at +0.68 V from the phosphate buffer containing the washed cell. The peak appearance was caused by the electron transfer activity of which corresponds to cytochrome c , and disappeared after adding antimycin A which inhibits the electron transfer activity. The cell was alive throughout the experiment as the result of a colony forming unit on Luria–Bertani agar plates. This was thought that cytochrome c was on the membrane surface of the living cell and played a role in the direct electron transfer between the cells and anode. [ABSTRACT FROM AUTHOR]

We studied the contribution of NADH oxidase to mitochondrial respiration in a suspension under extreme conditions: in a hypotonic medium and with respiratory poisons rotenone, antimycin, and sodium azide. The experiment was carried out with rat liver mitochondria. It was shown that not only cytochrome oxidase but also NADH dehydrogenase of the respiratory chain notably consume oxygen to oxidize exogenous NADH. For this reason NADH-associated mitochondrial respiration is not completely inhibited by respiratory poisons. The insensitivity of respiration to poisoning is a result of a shunt of electron transfer from NADH to oxygen directly via NADH dehydrogenase. This residual respiration via NADH oxidase, which is insensitive to respiratory chain inhibitors, may account for one-half of the total oxygen consumption under extreme conditions. [ABSTRACT FROM AUTHOR]

Photosynthetic induction, a gradual increase in photosynthetic rate on a transition from darkness or low light to high light, has ecological significance, impact on biomass accumulation in fluctuating light and relevance to photoprotection in strong light. However, the experimental quantification of the component electron fluxes in and around both photosystems during induction has been rare. Combining optimized chlorophyll fluorescence, the redox kinetics of P700 [primary electron donor in Photosystem I (PSI)] and membrane inlet mass spectrometry in the absence/presence of inhibitors/mediator, we partially estimated the components of electron fluxes in spinach leaf disks on transition from darkness to 1,000 µmol photons·m−2·s−1 for up to 10 min, obtaining the following findings: (i) the partitioning of energy between both photosystems did not change noticeably; (ii) in Photosystem II (PSII), the combined cyclic electron flow (CEF2) and charge recombination (CR2) to the ground state decreased gradually toward 0 in steady state; (iii) oxygen reduction by electrons from PSII, partly bypassing PSI, was small but measurable; (iv) cyclic electron flow around PSI (CEF1) peaked before becoming somewhat steady; (v) peak magnitudes of some of the electron fluxes, all probably photoprotective, were in the descending order: CEF1 > CEF2 + CR2 > chloroplast O2 uptake; and (vi) the chloroplast NADH dehydrogenase-like complex appeared to aid the antimycin A-sensitive CEF1. The results are important for fine-tuning in silico simulation of in vivo photosynthetic electron transport processes; such simulation is, in turn, necessary to probe partial processes in a complex network of interactions in response to environmental changes. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction impairs muscle health and causes subsequent muscle wasting. This study explores the role of mitochondrial dysfunction as an intramuscular signal for the extracellular matrix (ECM)-based proteolysis and, consequentially, muscle cell dystrophy. We found that inhibition of the mitochondrial electron transport chain causes paralysis as well as muscle structural damage in the nematode Caenorhabditis elegans. This was associated with a significant decline in collagen content. Both paralysis and muscle damage could be rescued with collagen IV overexpression, matrix metalloproteinase (MMP), and Furin inhibitors in Antimycin A-treated animal as well as in the C. elegans Duchenne muscular dystrophy model. Additionally, muscle cytosolic calcium increased in the Antimycin A-treated worms, and its down-regulation rescued the muscle damage, suggesting that calcium overload acts as one of the early triggers and activates Furin and MMPs for collagen degradation. In conclusion, we have established ECM degradation as an important pathway of muscle damage. [ABSTRACT FROM AUTHOR]

• Antimycin A-induced mitochondrial dysfunction activates a subset of vagal nociceptors. • Antimycin A-evoked Ca2+ fluxes correlate with capsaicin (TRPV1) responses. • TRPV1 and TRPA1 contribute to distinct Ca2+ responses. • ROS are required for antimycin A-evoked activation of TRPA1 but not TRPV1. Inflammation causes activation of nociceptive sensory nerves, resulting in debilitating sensations and reflexes. Inflammation also induces mitochondrial dysfunction through multiple mechanisms. Sensory nerve terminals are densely packed with mitochondria, suggesting that mitochondrial signaling may play a role in inflammation-induced nociception. We have previously shown that agents that induce mitochondrial dysfunction, such as antimycin A, activate a subset of nociceptive vagal sensory nerves that express transient receptor potential (TRP) channels ankyrin 1 (A1) and vanilloid 1 (V1). However, the mechanisms underlying these responses are incompletely understood. Here, we studied the contribution of TRPA1, TRPV1 and reactive oxygen species (ROS) to antimycin A-induced vagal sensory nerve activation in dissociated neurons and at the sensory terminals of bronchopulmonary C-fibers. Nociceptive neurons were defined chemically and genetically. Antimycin A-evoked activation of vagal nociceptors in a Fura2 Ca2+ assay correlated with TRPV1 responses compared to TRPA1 responses. Nociceptor activation was dependent on both TRP channels, with TRPV1 predominating in a majority of responding nociceptors and TRPA1 predominating only in nociceptors with the greatest responses. Surprisingly, both TRPA1 and TRPV1 were activated by H 2 O 2 when expressed in HEK293. Nevertheless, targeting ROS had no effect of antimycin A-evoked TRPV1 activation in either HEK293 or vagal neurons. In contrast, targeting ROS inhibited antimycin A-evoked TRPA1 activation in HEK293, vagal neurons and bronchopulmonary C-fibers, and a ROS-insensitive TRPA1 mutant was completely insensitive to antimycin A. We therefore conclude that mitochondrial dysfunction activates vagal nociceptors by ROS-dependent (TRPA1) and ROS-independent (TRPV1) mechanisms. [ABSTRACT FROM AUTHOR]

The effect of antimycin A (AA) as a possible activator of the alternative pathway (AP) was assessed on photosynthetic pigment composition and chlorophyll (Chl) fluorescence parameters in response to heat stress (HS) (42°C) in etiolated wheat (Triticum aestivum L., var. Harmony) seedlings. Results indicate that pigment concentrations significantly decreased (20-70%) following long-term HS, while the short-term exposure resulted in a 16-56% decline in etiolated wheat seedlings. It was observed that short-term and long-term HS altered photosystem (PSII) functionality in leaves, which was evidenced by a low maximum photochemical efficiency of PS II (Fv/Fm), a maximum fluorescence yield in the dark-adapted state (Fm), and by a high minimal fluorescence yield in the dark-adapted state (F0). However, at the same time AA reduced the negative effect of HS, suggesting that the activation of the AP induced by AA displayed tolerance to stress contributing the stabilization of the photosynthetic apparatus in etiolated wheat seedlings under HS. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are by‐products of physiological mitochondrial metabolism that are involved in several cellular signaling pathways as well as tissue injury and pathophysiological processes, including brain ischemia/reperfusion injury. The mitochondrial respiratory chain is considered a major source of ROS; however, there is little agreement on how ROS release depends on oxygen concentration. The rate of H2O2 release by intact brain mitochondria was measured with an Amplex UltraRed assay using a high‐resolution respirometer (Oroboros) equipped with a fluorescent optical module and a system of controlled gas flow for varying the oxygen concentration. Three types of substrates were used: malate and pyruvate, succinate and glutamate, succinate alone or glycerol 3‐phosphate. For the first time we determined that, with any substrate used in the absence of inhibitors, H2O2 release by respiring brain mitochondria is linearly dependent on the oxygen concentration. We found that the highest rate of H2O2 release occurs in conditions of reverse electron transfer when mitochondria oxidize succinate or glycerol 3‐phosphate. H2O2 production by complex III is significant only in the presence of antimycin A and, in this case, the oxygen dependence manifested mixed (linear and hyperbolic) kinetics. We also demonstrated that complex II in brain mitochondria could contribute to ROS generation even in the absence of its substrate succinate when the quinone pool is reduced by glycerol 3‐phosphate. Our results underscore the critical importance of reverse electron transfer in the brain, where a significant amount of succinate can be accumulated during ischemia providing a backflow of electrons to complex I at the early stages of reperfusion. Our study also demonstrates that ROS generation in brain mitochondria is lower under hypoxic conditions than in normoxia. Open science badges: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Oxidative stress plays an important role in ischemia/reperfusion injury when tissue oxygen level dramatically changes. We determined that H2O2 release by respiring brain mitochondria depends linearly on oxygen concentration when substrates of complex I, II, or glycerol 3‐phosphate dehydrogenase are oxidized in phosphorylating (State 3) or non‐phosphorylating (State 4) conditions. The highest rate of H2O2 release occurs at complex I in reverse electron transfer when mitochondria oxidize succinate or glycerol 3‐phosphate. H2O2 production by complex III is significant only in the presence of antimycin A, and in this case, the oxygen dependence manifests mixed (hyperbolic+linear) kinetics. Our study demonstrates that H2O2 release by brain mitochondria is lower in hypoxia than in normoxia. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ [ABSTRACT FROM AUTHOR]

The light reactions of photosynthesis are known to comprise both linear and cyclic electron flow in order to convert light energy into chemical energy in the form of NADPH and ATP. Antimycin A (AA) has been proposed as an inhibitor of ferredoxin-dependent cyclic electron flow around photosystem I (CEF-PSI) in photosynthesis research. However, its precise inhibitory mechanism and target site had not been elucidated yet. Here we show that AA inhibits the cyclic (alternative) electron flow via cytochrome b559 (Cyt b559) within photosystem II (CEF-PSII). When AA was applied to thylakoid membranes isolated from spinach leaves, the high potential form of Cyt b559, which was reduced in the dark, was transformed into the lower potential forms and readily oxidized by molecular oxygen. In the absence of AA, the reduced Cyt b559 was oxidized by P680+ upon light illumination and re-reduced in the dark, mainly by the electron from the QB site on the acceptor side of PSII. In contrast, AA suppressed the oxidation of Cyt b559 and induced its reduction under the illumination. This inhibition of Cyt b559 oxidation by AA enhanced photoinhibition of PSII. Based on the above results, we propose caution regarding the use of AA for evaluating CEF-PSI per se and concurrently propose that AA provides for new insights into, and interpretations of, the physiological importance of Cyt b559, rather than that of CEF-PSI in photosynthetic organisms. [ABSTRACT FROM AUTHOR]

The impact of antimycin A (AA) as a possible modulator of the alternative pathway (AP) was investigated on some biochemical parameters such as the rate of superoxide (O2-) production and antioxidant status in response to high temperature (HT) (42°C) in the leaves of wheat seedlings at the early and late stages of development. It was observed that the rate of O2- production and activities of the antioxidative defence system significantly increased following HT. However, a significant decrease of the rate of O2- production and activities of antioxidative enzymes was detected in the leaves of wheat seedlings under the combined effect of AA and HT. This finding suggests the activation of the AP by AA in leaves of wheat seedlings that prevented oxidative stress caused by HT. [ABSTRACT FROM AUTHOR]

Using some solutions activated by irradiation with non-thermal atmospheric pressure plasma (NTAPP), we had discovered that a new and distinctive mode of cell death, named spoptosis, exists in cells, the induction of which involves the action of reactive oxygen species (ROS). However, it was unknown what types of ROS and how they trigger the cell death. When cells were treated with a higher dose of Ascorbic acid (AA) generating O 2 − and H 2 O 2 or Antimycin A (AM) generating O 2 −, cell death occurred along with cellular shrinkage, Pdcd4 disappearance, and vesicle formation. Only in cells treated with AA, genomic DNA was digested irregularly and membrane permeability increased aberrantly. On the other hand, cells treated with a higher dose of H 2 O 2 displayed cell death and cellular shrinkage but not the other events, and those treated with a lower dose of H 2 O 2 displayed cell death but not the other events. Strikingly, when cells underwent double treatment with AM and H 2 O 2 , the events, which had not been observed by their single treatment, became compensated. All the events were suppressed with an antioxidant, confirming that they were mediated by ROS. Thus, the mode of cell death induced by AA or combination of AM and H 2 O 2 was consistent with that of cell death by NTAPP-activated solutions. These results suggested that O 2 − and H 2 O 2 collaboratively trigger spoptotic cell death with the associated events, and that AA and combination of AM and H 2 O 2 are functionally alternative in place of NTAPP-activated solutions. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) play a vital role in cell signaling and redox regulation, but when present in excess, lead to numerous pathologies. Detailed quantitative characterization of mitochondrial superoxide anion (O2•−) production in fetal pulmonary artery endothelia cells (PAECs) has never been reported. The aim of this study is to assess mitochondrial O2•− production in cultured PAECs over time using a novel quantitative optical approach. The rate, the sources, and the dynamics of O2•− production were assessed using targeted metabolic modulators of the mitochondrial electron transport chain (ETC) complexes, specifically an uncoupler and inhibitors of the various ETC complexes, and inhibitors of extra-mitochondrial sources of O2•−. After stabilization, the cells were loaded with nanomolar mitochondrial-targeted hydroethidine (Mito-HE, MitoSOX) online during the experiment without washout of the residual dye. Time-lapse fluorescence microscopy was used to monitor the dynamic changes in O2•− fluorescence intensity over time in PAECs. The transient behaviors of the fluorescence time course showed exponential increases in the rate of O2•− production in the presence of the ETC uncoupler or inhibitors. The most dramatic and the fastest increase in O2•− production was observed when the cells were treated with the uncoupling agent, PCP. We also showed that only the complex IV inhibitor, KCN, attenuated the marked surge in O2•− production induced by PCP. The results showed that mitochondrial respiratory complexes I, III and IV are sources of O2•− production in PAECs, and a new observation that ROS production during uncoupling of mitochondrial respiration is mediated in part via complex IV. This novel method can be applied in other studies that examine ROS production under stress condition and during ROS-mediated injuries in vitro.