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1.
Exp Cell Res ; 430(1): 113713, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37422059

RESUMO

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 O2- and H2O2 or Antimycin A (AM) generating O2-, 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 H2O2 displayed cell death and cellular shrinkage but not the other events, and those treated with a lower dose of H2O2 displayed cell death but not the other events. Strikingly, when cells underwent double treatment with AM and H2O2, 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 H2O2 was consistent with that of cell death by NTAPP-activated solutions. These results suggested that O2- and H2O2 collaboratively trigger spoptotic cell death with the associated events, and that AA and combination of AM and H2O2 are functionally alternative in place of NTAPP-activated solutions.


Assuntos
Peróxido de Hidrogênio , Morte Celular Regulada , Espécies Reativas de Oxigênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Morte Celular
2.
Pharmacol Res ; 189: 106697, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36796462

RESUMO

Necroptosis has been implicated in various inflammatory diseases including tumor-necrosis factor-α (TNF-α)-induced systemic inflammatory response syndrome (SIRS). Dimethyl fumarate (DMF), a first-line drug for treating relapsing-remitting multiple sclerosis (RRMS), has been shown to be effective against various inflammatory diseases. However, it is still unclear whether DMF can inhibit necroptosis and confer protection against SIRS. In this study, we found that DMF significantly inhibited necroptotic cell death in macrophages induced by different necroptotic stimulations. Both the autophosphorylation of receptor-interacting serine/threonine kinase 1 (RIPK1) and RIPK3 and the downstream phosphorylation and oligomerization of MLKL were robustly suppressed by DMF. Accompanying the suppression of necroptotic signaling, DMF blocked the mitochondrial reverse electron transport (RET) induced by necroptotic stimulation, which was associated with its electrophilic property. Several well-known anti-RET reagents also markedly inhibited the activation of the RIPK1-RIPK3-MLKL axis accompanied by decreased necrotic cell death, indicating a critical role of RET in necroptotic signaling. DMF and other anti-RET reagents suppressed the ubiquitination of RIPK1 and RIPK3, and they attenuated the formation of necrosome. Moreover, oral administration of DMF significantly alleviated the severity of TNF-α-induced SIRS in mice. Consistent with this, DMF mitigated TNF-α-induced cecal, uterine, and lung damage accompanied by diminished RIPK3-MLKL signaling. Collectively, DMF represents a new necroptosis inhibitor that suppresses the RIPK1-RIPK3-MLKL axis through blocking mitochondrial RET. Our study highlights DMF's potential therapeutic applications for treating SIRS-associated diseases.


Assuntos
Proteínas Quinases , Fator de Necrose Tumoral alfa , Camundongos , Animais , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases/metabolismo , Fumarato de Dimetilo , Necroptose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Síndrome de Resposta Inflamatória Sistêmica , Fosforilação Oxidativa , Apoptose
3.
Plant J ; 106(6): 1625-1646, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33811402

RESUMO

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.


Assuntos
Cloroplastos/fisiologia , Mitocôndrias/fisiologia , Nicotiana/genética , Nicotiana/metabolismo , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Oxirredução , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo , Água/administração & dosagem
4.
Plant Mol Biol ; 108(1-2): 145-155, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34928487

RESUMO

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.


Assuntos
Cádmio/toxicidade , Hordeum/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Western Blotting , Expressão Gênica/efeitos dos fármacos , Hordeum/anatomia & histologia , Hordeum/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/metabolismo , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismo
5.
Int J Mol Sci ; 23(16)2022 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-36012337

RESUMO

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.


Assuntos
Peróxido de Hidrogênio , Superóxidos , Trifosfato de Adenosina/metabolismo , Antimicina A/farmacologia , Transporte de Elétrons , Peróxido de Hidrogênio/metabolismo , Rotenona/farmacologia , Superóxidos/metabolismo
6.
Plant J ; 104(4): 979-994, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32860440

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Epigênese Genética , Oxigênio/metabolismo , Fatores de Transcrição/metabolismo , Ácido Abscísico/metabolismo , Adaptação Fisiológica , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Perfilação da Expressão Gênica , Germinação , Estresse Oxidativo , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas , Estresse Fisiológico , Fatores de Transcrição/genética
7.
FEMS Yeast Res ; 21(2)2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33493281

RESUMO

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.


Assuntos
Etanol/análise , Fermentação , Glucose/metabolismo , Consumo de Oxigênio , Saccharomycetales/metabolismo , Xilose/metabolismo , Antifúngicos/farmacologia , Antimicina A/farmacologia , Etanol/metabolismo , Saccharomycetales/efeitos dos fármacos
8.
Pharmacol Res ; 164: 105326, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33338625

RESUMO

The two-pore potassium channel TASK-3 has been shown to localize to both the plasma membrane and the mitochondrial inner membrane. TASK-3 is highly expressed in melanoma and breast cancer cells and has been proposed to promote tumor formation. Here we investigated whether pharmacological modulation of TASK-3, and specifically of mitochondrial TASK-3 (mitoTASK-3), had any effect on cancer cell survival and mitochondrial physiology. A novel, mitochondriotropic version of the specific TASK-3 inhibitor IN-THPP has been synthesized by addition of a positively charged triphenylphosphonium moiety. While IN-THPP was unable to induce apoptosis, mitoIN-THPP decreased survival of breast cancer cells and efficiently killed melanoma lines, which we show to express mitoTASK-3. Cell death was accompanied by mitochondrial membrane depolarization and fragmentation of the mitochondrial network, suggesting a role of the channel in the maintenance of the correct function of this organelle. In accordance, cells treated with mitoIN-THPP became rapidly depleted of mitochondrial ATP which resulted in activation of the AMP-dependent kinase AMPK. Importantly, cell survival was not affected in mouse embryonic fibroblasts and the effect of mitoIN-THPP was less pronounced in human melanoma cells stably knocked down for TASK-3 expression, indicating a certain degree of selectivity of the drug both for pathological cells and for the channel. In addition, mitoIN-THPP inhibited cancer cell migration to a higher extent than IN-THPP in two melanoma cell lines. In summary, our results point to the importance of mitoTASK-3 for melanoma cell survival and migration.


Assuntos
Mitocôndrias/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Pirimidinas/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/fisiologia , Bloqueadores dos Canais de Potássio/síntese química , Pirimidinas/síntese química , Espécies Reativas de Oxigênio/metabolismo
9.
Pharmacol Res ; 163: 105248, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33065283

RESUMO

The ubiquitin-proteasome system constitutes a major pathway for protein degradation in the cell. Therefore the crosstalk of this pathway with mitochondria is a major topic with direct relevance to many mitochondrial diseases. Proteasome dysfunction triggers not only protein toxicity, but also mitochondrial dysfunction. The involvement of proteasomes in the regulation of protein transport into mitochondria contributes to an increase in mitochondrial function defects. On the other hand, mitochondrial impairment stimulates reactive oxygen species production, which increases protein damage, and protein misfolding and aggregation leading to proteasome overload. Concurrently, mitochondrial dysfunction compromises cellular ATP production leading to reduced protein ubiquitination and proteasome activity. In this review we discuss the complex relationship and interdependence of the ubiquitin-proteasome system and mitochondria. Furthermore, we describe pharmacological inhibition of proteasome activity as a novel strategy to treat a group of mitochondrial diseases.


Assuntos
Mitocôndrias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Humanos , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/metabolismo , Peptídeos/metabolismo
10.
Molecules ; 26(20)2021 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-34684871

RESUMO

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.


Assuntos
Antimicina A/toxicidade , Aspalathus/química , Chalconas/farmacologia , Flavonoides/farmacologia , Glucosídeos/farmacologia , Luteolina/farmacologia , Mitocôndrias/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Animais , Antibacterianos/toxicidade , Linhagem Celular , Células Cultivadas , Camundongos , Mitocôndrias/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Chá/química
11.
FASEB J ; 33(8): 9540-9550, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31162948

RESUMO

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.-Sudevan, S., Takiura, M., Kubota, Y., Higashitani, N., Cooke, M., Ellwood, R. A., Etheridge, T., Szewczyk, N. J., Higashitani, A. Mitochondrial dysfunction causes Ca2+ overload and ECM degradation-mediated muscle damage in C. elegans.


Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Animais , Antimicina A/farmacologia , Western Blotting , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Furina/metabolismo , Metaloproteinases da Matriz/metabolismo , Mitocôndrias/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal , Distrofia Muscular de Duchenne
12.
Aesthetic Plast Surg ; 44(1): 168-176, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31741067

RESUMO

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.


Assuntos
Tecido Adiposo , Desferroxamina , Animais , Desferroxamina/farmacologia , Humanos , Respiração , Células-Tronco , Células Estromais
13.
J Neurochem ; 148(6): 731-745, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30582748

RESUMO

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 H2 O2 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, H2 O2 release by respiring brain mitochondria is linearly dependent on the oxygen concentration. We found that the highest rate of H2 O2 release occurs in conditions of reverse electron transfer when mitochondria oxidize succinate or glycerol 3-phosphate. H2 O2 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/.


Assuntos
Encéfalo/metabolismo , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Antimicina A/farmacologia , Encéfalo/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Flavoproteínas Transferidoras de Elétrons/efeitos dos fármacos , Flavoproteínas Transferidoras de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio/fisiologia
14.
Plant Cell Physiol ; 60(10): 2206-2219, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31271439

RESUMO

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 �mol 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.


Assuntos
Transporte de Elétrons , Oxigênio/metabolismo , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Spinacia oleracea/fisiologia , Antimicina A/farmacologia , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Escuridão , Fluorescência , Cinética , Luz , Oxirredução , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Spinacia oleracea/efeitos da radiação
15.
Photosynth Res ; 139(1-3): 487-498, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29790043

RESUMO

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.


Assuntos
Antimicina A/farmacologia , Grupo dos Citocromos b/metabolismo , Complexo de Proteína do Fotossistema II/efeitos dos fármacos , Complexo de Proteína do Fotossistema II/metabolismo , Complexo de Proteína do Fotossistema I/efeitos dos fármacos , Complexo de Proteína do Fotossistema I/metabolismo
16.
J Cell Sci ; 129(23): 4411-4423, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27793977

RESUMO

Mitochondria play a central role in cellular energy production, and their dysfunction can trigger a compensatory increase in glycolytic flux to sustain cellular ATP levels. Here, we studied the mechanism of this homeostatic phenomenon in C2C12 myoblasts. Acute (30 min) mitoenergetic dysfunction induced by the mitochondrial inhibitors piericidin A and antimycin A stimulated Glut1-mediated glucose uptake without altering Glut1 (also known as SLC2A1) mRNA or plasma membrane levels. The serine/threonine liver kinase B1 (LKB1; also known as STK11) and AMP-activated protein kinase (AMPK) played a central role in this stimulation. In contrast, ataxia-telangiectasia mutated (ATM; a potential AMPK kinase) and hydroethidium (HEt)-oxidizing reactive oxygen species (ROS; increased in piericidin-A- and antimycin-A-treated cells) appeared not to be involved in the stimulation of glucose uptake. Treatment with mitochondrial inhibitors increased NAD+ and NADH levels (associated with a lower NAD+:NADH ratio) but did not affect the level of Glut1 acetylation. Stimulation of glucose uptake was greatly reduced by chemical inhibition of Sirt2 or mTOR-RAPTOR. We propose that mitochondrial dysfunction triggers LKB1-mediated AMPK activation, which stimulates Sirt2 phosphorylation, leading to activation of mTOR-RAPTOR and Glut1-mediated glucose uptake.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Metabolismo Energético/efeitos dos fármacos , Glucose/farmacologia , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sirtuína 2/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Antioxidantes/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Transportador de Glucose Tipo 1/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Modelos Biológicos , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteína Regulatória Associada a mTOR
17.
Plant Cell Environ ; 40(7): 1074-1085, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27987212

RESUMO

The plant mitochondrial electron transport chain (ETC) is bifurcated such that electrons from ubiquinol are passed to oxygen via the usual cytochrome path or through alternative oxidase (AOX). We previously showed that knockdown of AOX in transgenic tobacco increased leaf concentrations of nitric oxide (NO), implying that an activity capable of generating NO had been effected. Here, we identify the potential source of this NO. Treatment of leaves with antimycin A (AA, Qi -site inhibitor of Complex III) increased NO amount more than treatment with myxothiazol (Myxo, Qo -site inhibitor) despite both being equally effective at inhibiting respiration. Comparison of nitrate-grown wild-type with AOX knockdown and overexpression plants showed a negative correlation between AOX amount and NO amount following AA. Further, Myxo fully negated the ability of AA to increase NO amount. With ammonium-grown plants, neither AA nor Myxo strongly increased NO amount in any plant line. When these leaves were supplied with nitrite alongside the AA or Myxo, then the inhibitor effects across lines mirrored that of nitrate-grown plants. Hence the ETC, likely the Q-cycle of Complex III generates NO from nitrite, and AOX reduces this activity by acting as a non-energy-conserving electron sink upstream of Complex III.


Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Nicotiana/metabolismo , Óxido Nítrico/metabolismo , Antimicina A/farmacologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Regulação da Expressão Gênica de Plantas , Metacrilatos/farmacologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Tiazóis/farmacologia , Nicotiana/efeitos dos fármacos , Nicotiana/genética
18.
Molecules ; 22(6)2017 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-28604620

RESUMO

A reaction of bis[(2-chlorocarbonyl)phenyl] diselenide with various mono and bisnucleophiles such as aminophenols, phenols, and amines have been studied as a convenient general route to a series of new antimicrobial and antiviral diphenyl diselenides. The compounds, particularly bis[2-(hydroxyphenylcarbamoyl)]phenyl diselenides and reference benzisoselenazol-3(2H)-ones, exhibited high antimicrobial activity against Gram-positive bacterial species (Enterococcus spp., Staphylococcus spp.), and some compounds were also active against Gram-negative E. coli and fungi (Candida spp., A.niger). The majority of compounds demonstrated high activity against human herpes virus type 1 (HHV-1) and moderate activity against encephalomyocarditis virus (EMCV), while they were generally inactive against vesicular stomatitis virus (VSV).


Assuntos
Anti-Infecciosos/farmacologia , Antifúngicos/farmacologia , Antivirais/farmacologia , Derivados de Benzeno/farmacologia , Compostos Organosselênicos/farmacologia , Aminas/síntese química , Aminas/química , Aminofenóis/síntese química , Aminofenóis/química , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Antifúngicos/síntese química , Antifúngicos/química , Antioxidantes/síntese química , Antioxidantes/química , Antivirais/síntese química , Antivirais/química , Derivados de Benzeno/síntese química , Derivados de Benzeno/química , Candida/efeitos dos fármacos , Candida/patogenicidade , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/patogenicidade , Humanos , Testes de Sensibilidade Microbiana , Compostos Organosselênicos/síntese química , Compostos Organosselênicos/química , Fenóis/síntese química , Fenóis/química
19.
Photosynth Res ; 129(3): 231-8, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26781235

RESUMO

Cyclic electron flow has puzzled and divided the field of photosynthesis researchers for decades. This mainly concerns the proportion of its overall contribution to photosynthesis, as well as its components and molecular mechanism. Yet, it is irrefutable that the absence of cyclic electron flow has severe effects on plant growth. One of the two pathways mediating cyclic electron flow can be inhibited by antimycin A, a chemical that has also widely been used to characterize the mitochondrial respiratory chain. For the characterization of cyclic electron flow, antimycin A has been used since 1963, when ferredoxin was found to be the electron donor of the pathway. In 2013, antimycin A was used to identify the PGRL1/PGR5 complex as the ferredoxin:plastoquinone reductase completing the last puzzle piece of this pathway. The controversy has not ended, and here, we review the history of research on this process using the perspective of antimycin A as a crucial chemical for its characterization.


Assuntos
Antimicina A/farmacologia , Ferredoxinas/metabolismo , Fotossíntese/efeitos dos fármacos , Plantas/efeitos dos fármacos , Quinona Redutases/metabolismo , Antimicina A/química , Transporte de Elétrons/efeitos dos fármacos , Elétrons , Complexo de Proteína do Fotossistema I/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo
20.
Pharmacol Res ; 103: 300-17, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26687095

RESUMO

We have investigated the growth-suppressive action of epigallocatechin-3-gallate (EGCG) on human leukemia Jurkat T cells. Results show a strong correlation between the dose-dependent reduction of clonogenic survival following acute EGCG treatments and the EGCG-induced decline of the mitochondrial level of Ca(2+). The cell killing ability of EGCG was synergistically enhanced by menadione. In addition, the cytotoxic effect of EGCG applied alone or in combination with menadione was accompanied by apoptosis induction. We also observed that in acute treatments EGCG displays strong antioxidant properties in the intracellular milieu, but concurrently triggers some oxidative stress generating mechanisms that can fully develop on a longer timescale. In parallel, EGCG dose-dependently induced mitochondrial depolarization during exposure, but this condition was subsequently reversed to a persistent hyperpolarized mitochondrial state that was dependent on the activity of respiratory Complex I. Fluorimetric measurements suggest that EGCG is a mitochondrial Complex III inhibitor and indicate that EGCG evokes a specific cellular fluorescence with emission at 400nm and two main excitation bands (at 330nm and 350nm) that may originate from a mitochondrial supercomplex containing dimeric Complex III and dimeric ATP-synthase, and therefore could provide a valuable means to characterize the functional properties of the respiratory chain.


Assuntos
Catequina/análogos & derivados , Células Jurkat/efeitos dos fármacos , Vitamina K 3/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Catequina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células Jurkat/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
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