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Lee 2012 Invest Ophthalmol Vis Sci + (''Purpose'': Following the recent demonstr … ''Purpose'': Following the recent demonstration of increased mitochondrial DNA mutations in lymphocytes of POAG patients, the authors sought to characterize mitochondrial function in a separate cohort of POAG.''Methods'': Using similar methodology to that previous applied to Leber's hereditary optic neuropathy (LHON) patients, maximal adenosine triphosphate (ATP) synthesis and cellular respiration rates, as well as cell growth rates in glucose and galactose media, were assessed in transformed lymphocytes from POAG patients (n = 15) and a group of age- and sex-matched controls (n = 15).''Results'': POAG lymphoblasts had significantly lower rates of complex-I-driven ATP synthesis, with preserved complex-II-driven ATP synthesis. Complex-I driven maximal respiration was also significantly decreased in patient cells. Growth in galactose media, where cells are forced to rely on mitochondrial ATP production, revealed no significant differences between the control and POAG cohort.''Conclusions'': POAG lymphoblasts in the study cohort exhibited a defect in complex-I of the oxidative phosphorylation pathway, leading to decreased rates of respiration and ATP production. Studies in LHON and other diseases have established that lymphocyte oxidative phosphorylation measurement is a reliable indicator of systemic dysfunction of this pathway. While these defects did not impact lymphoblast growth when the cells were forced to rely on oxidative ATP supply, the authors suggest that in the presence of a multitude of cellular stressors as seen in the early stages of POAG, these defects may lead to a bioenergetic crisis in retinal ganglion cells and an increased susceptibility to cell death.an increased susceptibility to cell death.)
Li 2018 Gene + (''SURF1'' is an assembly factor of mitocho … ''SURF1'' is an assembly factor of mitochondrial complex IV, and its mutations are the primary cause of Leigh syndrome in infants. To date, over 100 ''SURF1'' mutations have been reported worldwide, but the spectrum of the ''SURF1'' mutations in China remains unclear. Here, using next-generation sequencing targeting mitochondrial protein-coding sequences, we sequenced 178 patients suspected to have mitochondrial diseases. Fifteen ''SURF1'' mutations were identified in 12 Leigh syndrome patients, of which three, c.465_466delAA, c.532A > T, and c.826_827ins AGCATCTGCAGTACATCG, were newly described. The percentage of ''SURF1'' frameshift mutations (6/28, 21.4%) we detected in Chinese population is higher than other studies (21/106, 19.8%) with different populations, however, the percentage of missense mutations is lower in this study than others (4/28, 14.3% VS. 25/106, 23.6%). Since complex IV can be detected in cells carrying missense mutations (3/8) but not in cells carrying null mutations (0/4) by using cell model-based complementation assay, our results indicate that ''SURF1'' mutations may be associated with worse clinical outcome in Chinese patients than other populations. However, studies with larger sample size are needed to verify this conclusion. Additionally, we found that the frameshift mutations resulting in protein truncation closer to the C-terminus are not associated with better disease prognosis. Lastly, we found that determining the levels of complex IV assembly using cell models or lymphocyte analysis rather than invasive muscle and skin fibroblast biopsy, may help predict disease progression in Leigh syndrome patients.sease progression in Leigh syndrome patients.)
Rosenfeld 2003 Yeast + (''Saccharomyces cerevisiae'' is a facultat … ''Saccharomyces cerevisiae'' is a facultative anaerobe devoid of mitochondrial alternative oxidase. In this yeast, the structure and biogenesis of the respiratory chain, on the one hand, and the functional interactions of oxidative phosphorylation with the cellular energetic metabolism, on the other, are well documented. However, to our knowledge, the molecular aspects and the physiological roles of the non-respiratory pathways that utilize molecular oxygen have not yet been reviewed. In this paper, we review the various non-respiratory pathways in a global context of utilization of molecular oxygen in S. cerevisiae. The roles of these pathways are examined as a function of environmental conditions, using either physiological, biochemical or molecular data. Special attention is paid to the characterization of the so-called 'cyanide-resistant respiration' that is induced by respiratory deficiency, catabolic repression and oxygen limitation during growth. Finally, several aspects of oxygen sensing are discussed.l aspects of oxygen sensing are discussed.)
Oliveira 2016 PLOS ONE + (''Schistosoma mansoni'', one of the causat … ''Schistosoma mansoni'', one of the causative agents of human schistosomiasis, has a unique antioxidant network that is key to parasite survival and a valuable chemotherapeutic target. The ability to detoxify and tolerate reactive oxygen species increases along ''S. mansoni'' development in the vertebrate host, suggesting that adult parasites are more exposed to redox challenges than young stages. Indeed, adult parasites are exposed to multiple redox insults generated from blood digestion, activated immune cells, and, potentially, from their own parasitic aerobic metabolism. However, it remains unknown how reactive oxygen species are produced by ''S. mansoni'' metabolism, as well as their biological effects on adult worms. Here, we assessed the contribution of nutrients and parasite gender to oxygen utilization pathways, and reactive oxygen species generation in whole unpaired adult ''S. mansoni'' worms. We also determined the susceptibilities of both parasite sexes to a pro-oxidant challenge. We observed that glutamine and serum importantly contribute to both respiratory and non-respiratory oxygen utilization in adult worms, but with different proportions among parasite sexes. Analyses of oxygen utilization pathways revealed that respiratory rates were high in male worms, which contrast with high non-respiratory rates in females, regardless nutritional sources. Interestingly, mitochondrial complex I-III activity was higher than complex IV specifically in females. We also observed sexual preferences in substrate utilization to sustain hydrogen peroxide production towards glucose in females, and glutamine in male worms. Despite strikingly high oxidant levels and hydrogen peroxide production rates, female worms were more resistant to a pro-oxidant challenge than male parasites. The data presented here indicate that sexual preferences in nutrient metabolism in adult ''S. mansoni'' worms regulate oxygen utilization and reactive oxygen species production, which may differently contribute to redox biology among parasite sexes.ute to redox biology among parasite sexes.)
Konickova 2014 Annals Hepatol + (''Spirulina platensis'' is a blue-green al … ''Spirulina platensis'' is a blue-green alga used as a dietary supplement because of its hypocholesterolemic properties. Among other bioactive substances, it is also rich in tetrapyrrolic compounds closely related to bilirubin molecule, a potent antioxidant and anti-proliferative agent. The aim of our study was to evaluate possible anticancer effects of ''S. platensis'' and ''S. platensis''-derived tetrapyrroles using an experimental model of pancreatic cancer. The anti-proliferative effects of ''S. platensis'' and its tetrapyrrolic components [phycocyanobilin (PCB) and chlorophyllin, a surrogate molecule for chlorophyll A] were tested on several human pancreatic cancer cell lines and xenotransplanted nude mice. The effects of experimental therapeutics on mitochondrial reactive oxygen species (ROS) production and glutathione redox status were also evaluated. Compared to untreated cells, experimental therapeutics significantly decreased proliferation of human pancreatic cancer cell lines ''in vitro'' in a dose-dependent manner (from 0.16 g•L-1 [''S. platensis''], 60 μM [PCB], and 125 μM [chlorophyllin], ''p''<0.05). The anti-proliferative effects of ''S. platensis'' were also shown ''in vivo'', where inhibition of pancreatic cancer growth was evidenced since the third day of treatment (''p''<0.05). All tested compounds decreased generation of mitochondrial ROS and glutathione redox status (''p''=0.0006; 0.016; and 0.006 for ''S. platensis'', PCB, and chlorophyllin, respectively). In conclusion, ''S. platensis'' and its tetrapyrrolic components substantially decreased the proliferation of experimental pancreatic cancer. These data support a chemopreventive role of this edible alga. Furthermore, it seems that dietary supplementation with this alga might enhance systemic pool of tetrapyrroles, known to be higher in subjects with Gilbert syndrome.roles, known to be higher in subjects with Gilbert syndrome.)
Uribe-Alvarez 2016 Abstract MitoFit Science Camp 2016 + (''Staphylcoccus epidermidis'' does not inv … ''Staphylcoccus epidermidis'' does not invade healthy tissues, however, it has been identified as a cause of nosocomial infections due to its ability to form biofilms on polymer surfaces [1]. ''S. epidermidis'' can be grown at different oxygen concentrations ([O2]), including mammalian skin where [O2] ranges from 3-5% and in anaerobic altered tissues [2,3]. Biofilm formation of ''S. epidermidis'' and its respiratory chain components grown in aerobic, microaerobic and anaerobic conditions were evaluated by in-gel activities, enzymatic activities, spectrophotometry and oxymetry. Varying [O2] modified both biofilm formation and the components in the respiratory chain: At high [O2], little tendency to form biofilms was observed. ''S. epidermidis'' expressed glycerol-3-phosphate, pyruvate, ethanol and succinate dehydrogenases; and cyt bo and aa3. Under micro-aerobiosis, biofilm formation increased slightly; pyruvate, ethanol, glycerol-3-phosphate and succinate dehydrogenase decreased; aa3 cyt was not detected; Under anaerobiosis high biofilm-formation and low ethanol and pyruvate dehydrogenase activities were found; anaerobic nitrate dehydrogenase activity was detected. Aerobic-grown cells with cyanide increased biofilm formation. Anaerobic-grown cells with methylamine decreased biofilm formation. Thus, either a decrease in [O2] or the inhibition of the aerobic chain led ''S. epidermidis'' to associate into biofilms. In contrast, high [O2] or inhibition of the anaerobic nitrate reductase prevented biofilm formation suggesting that the enzymes expressed at low to null [O2] are therapeutic targets against biofilm formation by ''S. epidermidis''. expressed at low to null [O2] are therapeutic targets against biofilm formation by ''S. epidermidis''.)
Snow 2015 PLoS One + (''Trichodesmium'' is a biogeochemically im … ''Trichodesmium'' is a biogeochemically important marine cyanobacterium, responsible for a significant proportion of the annual 'new' nitrogen introduced into the global ocean. These non-heterocystous filamentous diazotrophs employ a potentially unique strategy of near-concurrent nitrogen fixation and oxygenic photosynthesis, potentially burdening Trichodesmium with a particularly high iron requirement due to the iron-binding proteins involved in these processes. Iron availability may therefore have a significant influence on the biogeography of Trichodesmium. Previous investigations of molecular responses to iron stress in this keystone marine microbe have largely been targeted. Here a holistic approach was taken using a label-free quantitative proteomics technique (MSE) to reveal a sophisticated multi-faceted proteomic response of Trichodesmium erythraeum IMS101 to iron stress. Increased abundances of proteins known to be involved in acclimation to iron stress and proteins known or predicted to be involved in iron uptake were observed, alongside decreases in the abundances of iron-binding proteins involved in photosynthesis and nitrogen fixation. Preferential loss of proteins with a high iron content contributed to overall reductions of 55-60% in estimated proteomic iron requirements. Changes in the abundances of iron-binding proteins also suggested the potential importance of alternate photosynthetic pathways as Trichodesmium reallocates the limiting resource under iron stress. ''Trichodesmium'' therefore displays a significant and integrated proteomic response to iron availability that likely contributes to the ecological success of this species in the ocean.ical success of this species in the ocean.)
Subrtova 2013 Abstract MiP2013 + (''Trypanosoma brucei'' is a parasitic flag … ''Trypanosoma brucei'' is a parasitic flagellate that causes devastating diseases of humans and lifestock. The infective form dwells in the glucose rich environment of mammalian blood and generate energy solely via glycolysis. In consequence, the bloodstream stage single mitochondrion is highly reduced lacking key Krebs cycle enzymes and traditional cytochrome mediated respiratory chain. Interestingly, the essential mitochondrial membrane potential (Δ''ψ''mt) is maintained by hydrolytic activity of the unique FoF1-ATPase, which contains several trypanosoma specific subunits of unknown function [1].We determined that one of the largest novel subunit, Tb2930 (43 kDa), is membrane-bound and localizes into monomeric and multimeric assemblies of the FoF1-ATPase. RNAi silencing of Tb2930 led to a significant decrease of Δ''ψ''mt and consequently to ''T. brucei'' growth inhibition, indicating that the FoF1-ATPase is not functioning properly even though its structural intergrity seems to be almost unchanged. To further explore the function of this protein, we employed naturally occuring trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) including subunit a, an essential component of the Fo-moiety and proton pore. These Dk cells maintain Δ''ψ''mt by electrogenic exchange of ATP4-/ADP3- by the ATP/ADP carrier (AAC) and hydrolytic activity of the soluble F1-ATPase [2]. So far, it has been assumed that only the F1-moiety subunits are present and will be essential for these parasites. Interestingly, glycerol gradient sedimentation and native electrophoresis of Dk mitochondria revealed the presence of high molecular weight ATPase complexes that correspond to the bloodstream stage monomeric and multimeric FoF1-ATPase. Furthermore, the Tb2930 subunit is expressed in Dk cells and co-sediments with these high molecular weight membrane bound complexes. The RNAi study demonstrated that Tb2930 subunit is essential for Dk trypanosoma cells and crucial for maintaining Δ''ψ''mt. Importantly, upon ablation of Tb2930 we observed a shift of the FoF1-ATPase complexes to the lower S-values on glycerol gradient, where the free F1-ATPase sediments, indicating changes in the structural integrity of the Dk FoF1-ATPase. In conclusion, we propose that Tb2930 is responsible for connecting the Dk F1-ATPase to the mitochondrial membrane in the absence of subunit a of the Fo-moiety, thus increasing the efficiency of the functional association between F1-ATPase and AAC.y, thus increasing the efficiency of the functional association between F1-ATPase and AAC.)
Dolezelova 2017 Abstract IOC122 + (''Trypanosoma brucei'' undergoes a complex … ''Trypanosoma brucei'' undergoes a complex life cycle as it alternates between a mammalian host and the blood-feeding insect vector, a tsetse fly. Due to the different environments, the distinct life stages differ in their energy metabolism, i.e. insect stage (procyclic cells, PS) depends on mitochondrial oxidative phosphorylation (OXPHOS) for ATP production while the bloodstream stage (BS) gains energy by aerobic glycolysis. The dramatic switch from the OXPHOS to glycolysis happens during the complex development of the PS in the tsetse fly. This development differentiation is characterized by extensive remodeling of mitochondrion structure and changes in mitochondrial bioenergetics. Importantly, the molecular mechanism behind this process is completely unknown. We have established the ''in vitro'' differentiation system, in which the transition from PS to epimastigotes followed by differentiation to transmission-ready metacylic trypanosomes is triggered by RNA binding protein 6 (RBP6) expression. This ''in vitro'' induced differentiation of PF cells takes 8 days. The appearance of epimastigotes and metacyclic trypanosomes in the culture was mapped using light and fluorescent microscopy. The whole cell proteome of cell culture harvested every day after the RBP6 induction was identified by label-free quantitative mass spectrometry. This proteomic data serves as a resource for further detailed characterization of changes happening in the parasite mitochondrion as well as identification of possible candidates involved in the PS differentiation.idates involved in the PS differentiation.)
Paes 2014 Abstract IOC 2014-04 Schroecken + (''Trypanosoma cruzi'' has a single mitocho … ''Trypanosoma cruzi'' has a single mitochondrion, the main site of reactive oxygen species (ROS) production. Moreover, ''T. cruzi'' epimastigotes proliferate in the presence of heme, which induces ROS formation (Nogueira et al 2011; Lara et al 2007). Therefore, we evaluated heme effect upon mitochondrial ROS formation and mitochondrial membrane potential (ΔΨmt). For that, epimastigotes were incubated with DHE or TMRM with or without heme. After this, FCCP and antymicin A (Ama) were added. Mitochondrial ROS production and ΔΨmt were analyzed by flow cytometry. Our results showed that heme duplicated ROS production and induced a 4-fold increase of ΔΨmt. The FCCP addition reversed heme effects upon ROS generation and ΔΨm. Additionally, Ama induced a 2-fold increase of ROS production and 46% increment in ΔΨmt, while co-incubation with heme and AA presented a 3-fold increase upon ROS formation and increase ΔΨmt in 70%. In order to corroborate the involvement of heme in mitochondrial ROS, we incubated the parasites with heme, in the absence or in the presence of mitoTEMPO, a mitochondrial antioxidant. Our results showed that in the presence of this antioxidant greatly decreased heme induced ROS generation. Afterwards, we incubated epimastigotes with heme for 30 min and then, performed a substrate-uncoupler-inhibitor-tritation protocol with rotenone, succinate, ADP, cytocrome c, FCCP and Ama. We were able to detect a decrease in several states, mainly ROUTINE, OXPHOS and reserve capacity, compared to control cells. Finally, we evaluated epimastigotes proliferation with or without heme, H2O2, FCCP, Ama or mitoTEMPO. We observed that low concentrations of H2O2 increased proliferation, while higher concentrations showed deleterious effects upon the cells. FCCP and mitoTEMPO also reversed heme-induced proliferation, whereas, Ama promoted a tripanostatic effect. Taken together, our results strongly suggest that heme modulates ''T. cruzi'' mitochondrial physiology since it promotes mitochondrial ROS production, decreasing mitochondrial states, and enhances the ΔΨmt.tochondrial states, and enhances the ΔΨmt.)
Goncalves 2011 Abstract IOC65 + (''Trypanosoma cruzi'' is a hemoflagellate … ''Trypanosoma cruzi'' is a hemoflagellate protozoan that causes Chagas’ disease. ''T. cruzi'' life-cycle is complex involving different evolutive forms that experience striking differences in their environmental condition. Here we carried out a functional assessment of mitochondrial function in two distinct ''T. cruzi'' forms: the insect stage, epimastigote and the freshly isolated bloodstream trypomastigote. We observed that in comparison to epimastigotes, bloodstream trypomastigotes facilitate electrons entry into the electron transport chain increasing Complex II-III activity. Curiously, cytochrome c oxidase (CIV) activity and the expression of CIV subunit IV were reduced in bloodstream forms, creating an “electron bottleneck” that favored increased electron leak and H2O2 formation. We propose that the oxidative preconditioning provided by this mechanism would confer a protection to the bloodstream trypomastigotes against host immune response. Thus, mitochondrial remodeling during the ''T. cruzi'' life-cycle can represent a key metabolic adaptation for parasite survival in different environments.rasite survival in different environments.)
Santos Bertolini 2018 Thesis + (''Trypanosoma cruzi'' is the etiologic age … ''Trypanosoma cruzi'' is the etiologic agent of Chagas disease, a disorder affecting thousands of people, for which an effective treatment is not available for the chronic phase. Calcium signaling is important for host cell invasion, differentiation, osmoregulation, cell death and flagellar function in trypanosomatids. The influx of calcium into the mitochondria, which is important for intracellular calcium homeostasis, occurs through a mitochondrial calcium uniporter complex (MCUC) and this complex consists of several components, including two regulatory proteins named mitochondrial calcium uptake 1 and 2 (MICU1 and MICU2). In mammalian cells, these proteins are located in the mitochondrial intermembrane space and play a role in sensing cytosolic calcium levels and regulating the MCU opening. Although several MCUC components have been identified in trypanosomes, the mechanism by which it is regulated is still unknown. In this work, we aimed at studying the role of MICU1 and MICU2 in the mitochondrial calcium uptake of ''T. cruzi''. The predicted TcMICU1 and TcMICU2 proteins displayed a mitochondrial targeting signal and EF-hands domains that could be sensitive to changes in cytosolic calcium. We obtained TcMICU1 (MICU1-KO) and TcMICU2 (MICU2-KO) knockout cell lines using the CRISPR/Cas9 system by co-transfecting ''T. cruzi'' epimastigotes with the Cas9/pTREX-n vector (containing a specific sgRNA) and a DNA donor cassette with a blasticidin resistance marker to induce the DNA double-strand break repair by homologous recombination. Additionally, we generated a cell line of ''T. cruzi'' epimastigotes overexpressing TcMICU2 tagged with 2xHA (MICU2-OE) using pTREX-n vector. Such molecular constructs were used to analyze the mutant phenotypes and indicate the functions of these proteins. Our results show that MICU1-KO and MICU2-KO have a significant decrease in the capacity to take up calcium, showing a different regulation when we compared to what has already been described previously in mammals. In the absence of these proteins there is a decrease in the growth rate and respiration rates of epimastigotes, showing how important these two proteins are to this stage of ''T. cruzi''. In addition, MICU1-KO epimastigotes are able to differentiate to metacyclic trypomastigotes in a greater proportion than the control cells while the metacyclogenesis capacity was reduced in MICU2-KO cells. Using the MICU2-OE cell line we demonstrated by immunofluorescence microscopy the mitochondrial localization of MICU2 and that its overexpression does not alter the capacity to take up calcium, besides that it does not affect the mitochondrial membrane potential and parasite growth. We can conclude that the TcMICU1 and TcMICU2 proteins are essential for the regulation of mitochondrial calcium uptake by MCU in ''T. cruzi''. Likewise, the results suggest that both proteins play an important role in the growth and differentiation of epimastigotes.owth and differentiation of epimastigotes.)
Barison 2016 J Bioenerg Biomembr + (''Trypanosoma cruzi'', the aetiological ag … ''Trypanosoma cruzi'', the aetiological agent of Chagas's disease, metabolizes glucose, and after its exhaustion, degrades amino acids as energy source. Here, we investigate histidine uptake and its participation in energy metabolism. No putative genes for the histidine biosynthetic pathway have been identified in genome databases of ''T. cruzi'', suggesting that its uptake from extracellular medium is a requirement for the viability of the parasite. From this assumption, we characterized the uptake of histidine in ''T. cruzi'', showing that this amino acid is incorporated through a single and saturable active system. We also show that histidine can be completely oxidised to CO2. This finding, together with the fact that genes encoding the putative enzymes for the histidine - glutamate degradation pathway were annotated, led us to infer its participation in the energy metabolism of the parasite. Here, we show that His is capable of restoring cell viability after long-term starvation. We confirm that as an energy source, His provides electrons to the electron transport chain, maintaining mitochondrial inner membrane potential and O2 consumption in a very efficient manner. Additionally, ATP biosynthesis from oxidative phosphorylation was found when His was the only oxidisable metabolite present, showing that this amino acid is involved in bioenergetics and parasite persistence within its invertebrate host.oenergetics and parasite persistence within its invertebrate host.)
Saraiva 2022 Pathogens + (''Trypanosoma cruzi'', the causative agent … ''Trypanosoma cruzi'', the causative agent of Chagas disease, faces changes in redox status and nutritional availability during its life cycle. However, the influence of oxygen fluctuation upon the biology of ''T. cruzi'' is unclear. The present work investigated the response of ''T. cruzi'' epimastigotes to hypoxia. The parasites showed an adaptation to the hypoxic condition, presenting an increase in proliferation and a reduction in metacyclogenesis. Additionally, parasites cultured in hypoxia produced more reactive oxygen species (ROS) compared to parasites cultured in normoxia. The analyses of the mitochondrial physiology demonstrated that hypoxic condition induced a decrease in both oxidative phosphorylation and mitochondrial membrane potential (ΔΨm) in epimastigotes. In spite of that, ATP levels of parasites cultivated in hypoxia increased. The hypoxic condition also increased the expression of the hexokinase and NADH fumarate reductase genes and reduced NAD(P)H, suggesting that this increase in ATP levels of hypoxia-challenged parasites was a consequence of increased glycolysis and fermentation pathways. Taken together, our results suggest that decreased oxygen levels trigger a shift in the bioenergetic metabolism of ''T. cruzi'' epimastigotes, favoring ROS production and fermentation to sustain ATP production, allowing the parasite to survive and proliferate in the insect vector.vive and proliferate in the insect vector.)
Souza 2021 PLoS Pathog + (''Trypanosoma cruzi'', the parasite causin … ''Trypanosoma cruzi'', the parasite causing Chagas disease, is a digenetic flagellated protistthat infects mammals (including humans) and reduviid insect vectors. Therefore, ''T. cruzi''must colonize different niches in order to complete its life cycle in both hosts. This fact determines the need of adaptations to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. In this regard,it is well known that ''T. cruzi'' has a flexible metabolism able to rapidly switch from carbohydrates (mainly glucose) to amino acids (mostly proline) consumption. Also established hasbeen the capability of ''T. cruzi'' to use glucose and amino acids to support the differentiationprocess occurring in the insect, from replicative non-infective epimastigotes to non-replicative infective metacyclic trypomastigotes. However, little is known about the possibilities ofusing externally available and internally stored fatty acids as resources to survive in nutrient-poor environments, and to sustain metacyclogenesis. In this study, we revisit the metabolic fate of fatty acid breakdown in ''T. cruzi''. Herein, we show that during parasiteproliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can betaken up from the medium, leading to CO2 production. Additionally, we show that electronsare fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylicacid (TCA) cycle, which can be used to feed anabolic pathways such as the ''de novo'' biosynthesis of fatty acids. Finally, we show as well that the inhibition of fatty acids mobilization intothe mitochondrion diminishes the survival to severe starvation, and impairsmetacyclogenesis.s the survival to severe starvation, and impairs metacyclogenesis.)
Uribe-Alvarez 2018 Microbiologyopen + (''Wolbachia sp.'' has colonized over 70% o … ''Wolbachia sp.'' has colonized over 70% of insect species, successfully manipulatinghost fertility, protein expression, lifespan, and metabolism. Understanding and engineeringthe biochemistry and physiology of ''Wolbachia'' holds great promise for insectvector-borne disease eradication. ''Wolbachia'' is cultured in cell lines, which have longduplication times and are difficult to manipulate and study. The yeast strain''Saccharomyces cerevisiae'' W303 was used successfully as an artificial host for''Wolbachia'' wAlbB. As compared to controls, infected yeast lost viability early, probablyas a result of an abnormally high mitochondrial oxidative phosphorylation activityobserved at late stages of growth. No respiratory chain proteins from ''Wolbachia''were detected, while several ''Wolbachia'' F1F0-ATPasesubunits were revealed. After 5 days outside the cell, Wolbachia remained fully infective against insect cells.the cell, Wolbachia remained fully infective against insect cells.)
38th Mahabaleshwar Seminar + ('Mitochondria, Metabolism and Energetics': [[Media:MiPNet18.14 IOC85 Mahabaleshwar.pdf|'''38th Mahabaleshwar Seminar''']], [http://www.tifr.res.in/~dbsconf/mito2014/Home.html mito2014], including '''[[MiPNet18.14 | 85th OROBOROS O2k-Workshop]]'''.)
Paeaebo 2014 Basic Books + ('The Neanderthals live on in many of us to … 'The Neanderthals live on in many of us today' (p 199).Neanderthal Man tells the story of geneticist Svante Pääbo's mission to answer this question, and recounts his ultimately successful efforts to genetically define what makes us different from our Neanderthal cousins. Beginning with the study of DNA in Egyptian mummies in the early 1980s and culminating in the sequencing of the Neanderthal genome in 2010, Neanderthal Man describes the events, intrigues, failures, and triumphs of these scientifically rich years through the lens of the pioneer and inventor of the field of ancient DNA.We learn that Neanderthal genes offer a unique window into the lives of our hominin relatives and may hold the key to unlocking the mystery of why humans survived while Neanderthals went extinct. Drawing on genetic and fossil clues, Pääbo explores what is known about the origin of modern humans and their relationship to the Neanderthals and describes the fierce debate surrounding the nature of the two species' interactions. His findings have not only redrawn our family tree, but recast the fundamentals of human history—the biological beginnings of fully modern ''Homo sapiens'', the direct ancestors of all people alive today.A riveting story about a visionary researcher and the nature of scientific inquiry, Neanderthal Man offers rich insight into the fundamental question of who we are.to the fundamental question of who we are.)
Sipos 2005 Cell Mol Neurobiol + ((1) Endothelial cells are permanently chal … (1) Endothelial cells are permanently challenged by altering pH in the blood, and oxidative damage could also influence the intracellular pH (pH(i)) of the endothelium. Cerebral microvascular endothelial cells form the blood-brain barrier (BBB) and pH(i) regulation of brain capillary endothelial cells is important for the maintenance of BBB integrity. The aim of this study was to address the pH regulatory mechanisms and the effect of an acute exposure to hydrogen peroxide (H2O2) on the pH regulation in primary rat brain capillary endothelial (RBCE) cells The RBCE monolayers were loaded with the fluorescent pH indicator BCECF and pH(i) was monitored by detecting the fluorescent changes. (2) The steady-state pH(i) of RBCE cells in HEPES-buffer (6.83 +/- 0.1) did not differ significantly from that found in bicarbonate-buffered medium (6.90 +/- 0.08). Cells were exposed to NH4CI to induce intracellular acidification and then the recovery to resting pH was studied. Half-recovery time after NH4Cl prepulse-induced acid load was significantly less in the bicarbonate-buffered medium than in the HEPES-medium, suggesting that in addition to the Na+ / H+ exchanger, HCO3- / Cl- exchange mechanism is also involved in the restoration of pH(i) after an intracellular acid load in primary RBCE cells. We used RT-PCR-reactions to detect the isoforms of Na+ / H+ exchanger gene family (NHE). NHE-1 -2, -3 and -4 were equally present, and there was no significant difference in the relative abundance of the four transcripts in these cells. (3) No pH(i) recovery was detected when the washout after an intracellular acid load occurred in nominally Na+ -free HEPES-buffered medium or in the presence of 10 microM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a specific inhibitor of Na+ / H+ exchanger. The new steady-state pH(i) were 6.37 +/- 0.02 and 6.60 +/- 0.02, respectively. (4) No detectable change was observed in the steady-state pH(i) in the presence of 100 microM H2O2; however, recovery from NH4Cl prepulse-induced intracellular acid load was inhibited when H2O2 was present in 50 or 100 microM concentration in the HEPES-buffered medium during NH4Cl washout. These data suggest that H2O2 is without effect on the activity of Na+ / H+ exchanger at rest, but could inhibit the function of the exchanger after an intracellular acid load.xchanger after an intracellular acid load.)
MiPNet25.14 TPP Analysis Template + ((2020) Excel template for TPP data analys … (2020) Excel template for TPP data analysis. Mitochondr Physiol Network 25.14(01):1-8. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>:» Product: [[DatLab]], [[Oroboros O2k]], [[Oroboros O2k-Catalogue |O2k-Catalogue]]oboros O2k-Catalogue |O2k-Catalogue]])
Kagawa 1971 J Biol Chem + ( * Amorphous membrane fragments depleted i … * Amorphous membrane fragments depleted in P-lipids and cytochrome oxidase were isolated from bovine heart mitochondria and were reconstituted with P-lipids and coupling factors to yield vesicular structures. These vesicles catalyzed a 32Pi—ATP exchange and showed an induced enhancement of anilinonaphthalene sulfonate fluorescence on addition of ATP* 32Pi—ATP exchange and fluorescence enhancement were abolished by uncouplers of oxidative phosphorylation and by energy transfer inhibitors. The ATPase activity was inhibited by energy transfer inhibitors, but stimulated by uncouplers or by the combined action of nigericin and valinomycin in the presence of K+. Both ATPase activity and 32Pi—ATP exchange were inhibited by a specific antibody against coupling factor 1.* It was shown that the reconstitution of vesicular structures with functional activity required several hours. Rapid reconstitution resulted in inactive vesicles. Evidence for the formation of new vesicles from solubilized P-lipids was obtained by demonstrating inclusion of macromolecules such as 14C-labeled inulin or ferritin which could not be removed by washing.itin which could not be removed by washing. )
Cannon 2015 Fatty Acid Oxidation O2k-Network Discussion Forum + ( *Saponin permeabilized skeletal muscle fi … *Saponin permeabilized skeletal muscle fiber bundles*'''Protocol''':#0.5mM Malate#50 µM palmitoyl-CoA + 2mM carnitine#5mM ADP#From here on out, various combinations for titration protocol*Coupling states:#LEAK_M+Palmitoylcarnitine#P_M+Palmitoylcarnitine#P_M+Palmitoylcarnitine+S#E_O+CCCP titrations#E_S+Rot#ROX_AntimycinA_O+CCCP titrations #E_S+Rot #ROX_AntimycinA )
Nernst 1921 Nobel Lecture + (.. From the start of my scientific career … .. From the start of my scientific career the galvanic cell, the first form of which, the Volta pile, popularized physics in a single stroke and at the same time presented us with so many problems, appeared to me to merit especially further study. ..It was particularly disillusioning to find a man like Helmholtz returning repeatedly throughout his scientific career to his first love, the galvanic cell, which he had courted in his great youthful work "Erhaltung der Kraft" (The conservation of energy), without however succeeding in finding a satisfactory solution.As often in natural science the picture changed quite suddenly. New fruitful concepts appeared, through the interplay and extension of which most of the darkness has been to a large extent dispelled in a single stroke. Such means were Van ’t Hoff’s theory of osmotic pressure, Arrhenius’ theory of electrolytic dissociation, and finally many new approaches to the treatment of chemical equilibria, which, brilliantly presented, are to be found scattered throughout the first edition of Ostwald’s "Lehrbuch der Allgemeinen Chemie" (Textbook of general chemistry). So there arose in 1889 the osmotic theory of galvanic current generation, which has not been seriously challenged since it was put forward more than thirty years ago and has undergone no appreciable elaboration since its acceptance, surely a clear sign that it has so far satisfied scientific needs. ..The osmotic theory of current generation stipulates moreover that when a metal ion concentration is higher than consistent with the solution tension of the particular metal, on immersion of the metal, ions of the relevant metal electrode must go into solution, while conversely they must settle on the electrode when the reverse is the case.he electrode when the reverse is the case.)
Estabrook 1967 Methods Enzymol + (.. The convenience and simplicity of the p … .. The convenience and simplicity of the polarographic 'oxygen electrode' technique for measuring rapid changes in the rate of oxygen utilization by cellular and subcellular systems is now leading to its more general application in many laboratories. The types and design of oxygen electrodes vary, depending on the invetigator's ingenuity and specific requirements of the system under investigation.rements of the system under investigation.)
Gnaiger 1997 Transplant Proc + (0RGAN PRESERVATION under hypothermic ische … 0RGAN PRESERVATION under hypothermic ischemia is enhanced by storage solutions that protect the vascular endothelium from ischemia-reperfusion injury. Ischemia-reperfusion injury leads to primary graft failure and chronic rejection, and is commonly assessed by measuring endothelial activation and damage of the endothelial plasma membrane. However, corresponding primary intracellular events are little understood compared with the secondary cytokine/adhesion molecule cascade and inflammatory responses.1, 2 Because protection of intracellular and cell membrane function is fundamental for further improvement of organ preservation, we developed highresolution respirometry as a sensitive diagnostic test for mitochondrial and plasma membrane competence.3 Whereas the plasma membrane remained impermeable after clinically relevant cold storage times of 8 hours and 20 to 60 minutes of reoxygenation, mitochondrial function was impaired at several steps of the respiratory chain.l function was impaired at several steps of the respiratory chain.)

(0RGAN PRESERVATION under hypothermic ischemia is enhanced by storage sol)

Majiene 2019 Nutrients + (1,4-naphthoquinones, especially juglone, a … 1,4-naphthoquinones, especially juglone, are known for their anticancer activity. However, plumbagin, lawsone, and menadione have been less investigated for these properties. Therefore, we aimed to determine the effects of plumbagin, lawsone, and menadione on C6 glioblastoma cell viability, ROS production, and mitochondrial function.Cell viability was assessed spectrophotometrically using metabolic activity method, and by fluorescent Hoechst/propidium iodide nuclear staining. ROS generation was measured fluorometrically using DCFH-DA. Oxygen uptake rates were recorded by the high-resolution respirometer Oxygraph-2k.Plumbagin and menadione displayed highly cytotoxic activity on C6 cells (IC50 is 7.7 ± 0.28 μM and 9.6 ± 0.75 μM, respectively) and caused cell death by necrosis. Additionally, they increased the amount of intracellular ROS in a concentration-dependent manner. Moreover, even at very small concentrations (1-3 µM), these compounds significantly uncoupled mitochondrial oxidation from phosphorylation impairing energy production in cells. Lawsone had significantly lower viability decreasing and mitochondria-uncoupling effect, and exerted strong antioxidant activity.Plumbagin and menadione exhibit strong prooxidant, mitochondrial oxidative phosphorylation uncoupling and cytotoxic activity. In contrast, lawsone demonstrates a moderate effect on C6 cell viability and mitochondrial functions, and possesses strong antioxidant properties.unctions, and possesses strong antioxidant properties.)
Leyssens 1996 J Physiol + (1. As ATP has a higher affinity for Mg2+ t … 1. As ATP has a higher affinity for Mg2+ than ADP, the cytosolic magnesium concentration rises upon ATP hydrolysis. We have therefore used the Mg(2+)-sensitive fluorescent indicator Magnesium Green (MgG) to provide an index of changing ATP concentration in single rat cardiomyocytes in response to altered mitochondrial state. 2. In response to FCCP, [Mg2+]i rose towards a plateau coincident with the progression to rigor, which signals ATP depletion. Contamination of the MgG signal by changes in intracellular free Ca2+ concentration (the KD of MgG for Ca2+ is 4.7 microM) was excluded by simultaneous measurement of [Ca2+]i and [Mg2+]i in cells dual loaded with fura-2 and MgG. The response to FCCP was independent of external Mg2+, confirming an intracellular source for the rise in [Mg2+]i. 3. Simultaneous measurements of mitochondrial NAD(P)H autofluorescence and mitochondrial potential (delta psi m; .-1 fluorescence) and of autofluorescence and MgG allowed closer study of the relationship between [Mg2+]i and mitochondrial state. Oligomycin abolished the FCCP-induced rise in [Mg2+]i without altering the change in autofluorescence. Thus, the rise in [Mg2+]i in response to FCCP is consistent with the release of intracellular Mg2+ following ATP hydrolysis by the mitochondrial F1F0-ATPase. 4. The rise in [Mg2+]i was correlated with cell-attached recordings of ATP-sensitive K+ channel (KATP) activity. In response to FCCP, an increase in KATP channel activity was seen only as [Mg2+]i reached a plateau. In response to blockade of mitochondrial respiration and glycolysis with cyanide (CN-) and 2-deoxyglucose (DOG), [Mg2+]i rose more slowly but again KATP channel opening increased only when [Mg2+]i reached a plateau and the cells shortened. 5. Oligomycin decreased the rate of rise of [Mg2+]i delayed the onset of rigor and increased the rate of mitochondrial depolarization in response to CN-_DOG. Thus, with blockade of mitochondrial respiration delta psi m is maintained by the mitochondrial F1F0-ATPase at the expense of ATP reserves. 6. In response to CN-_DOG, the initial rise in [Mg2+]i was accompanied by a small rise in [Ca2+]i. After [Mg2+]i reached a plateau and rigor developed, [Ca2+]i rose progressively. On reperfusion, in hypercontracted cells, [Ca2+]i recovered before [Mg2+]i and [ca2+]i oscillations were sustained while [Mg2+]i decreased. Thus on reperfusion, full recovery of [ATP]i is slow, but the activation of contractile elements and the restoration of [Ca2+]i does not require the re-establishment of millimolar concentrations of ATP.hment of millimolar concentrations of ATP.)
Satoh 1995 Br J Pharmacol + (1. Effects of 3-hydroxy-3-methylglutaryl c … 1. Effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, pravastatin and simvastatin, on the myocardial level of coenzyme Q10, and on mitochondrial respiration were examined in dogs. 2. Either vehicle (control), pravastatin (4 mg kg-1 day-1), or simvastatin (2 mg kg-1 day-1) was administered orally for 3 weeks. First, the myocardial tissue level of coenzyme Q10 was determined in the 3 groups. Second, ischaemia was induced by ligating the left anterior descending coronary artery (LAD) in anaesthetized open chest dogs, pretreated with the inhibitors. After 30 min of ischaemia, nonischaemic and ischaemic myocardium were removed from the left circumflex and LAD regions, respectively, and immediately used for isolation of mitochondria. The mitochondrial respiration was determined by polarography, with glutamate and succinate used as substrates. 3. Simvastatin significantly decreased the myocardial level of coenzyme Q10, but pravastatin did not. 4. Ischaemia decreased the mitochondrial respiratory control index (RCI) in both groups. Significant differences in RCI between nonischaemic and ischaemic myocardium were observed in the control and simvastatin-treated groups. 5. Only in the simvastatin-treated group did ischaemia significantly decrease the ADP/O ratio, determined with succinate. 6. The present results indicate that simvastatin but not pravastatin may cause worsening of the myocardial mitochondrial respiration during ischaemia, probably because of reduction of the myocardial coenzyme Q10 level.tion of the myocardial coenzyme Q10 level.)
Chance 1962 J Biol Chem + (1. In succinate oxidation reactivated by a … 1. In succinate oxidation reactivated by a low concentration of adenosine triphosphate, addition of small amounts of adenosine diphosphatc will lead to reestablishment of the inhibited state of succinate oxidation after a short burst of respiration.2. The inhibited state is not relieved by either phosphate or phosphate acceptors. 3. The inhibition is closely correlated with a high degree of oxidation of mitochondrial reduced diphosphopyridine nucleotide, which occurs immediately on addition of adenosine diphosphate and is followed by the inhibition of succinate oxidation after the oxidation or rather small amounts of succinate. 4. Oxidation of more than approximately two-thirds of the total diphosphopyridinc nuclcotide (DPN) reducible by succinate and adenosine triphosphate eventually leads to inhibition of succinate oxidation. 5. Based on independent evidence for a compartmentation of mitochondrial pyridine nucleotide (approximately one-third available to DPN-linked substrates), it is proposed that inhibi- tion occurs when oxidation of DPN in the compartment available to malate causes oxaloacetate formation sufficient to inhibit succinate oxidation. 6. The general possibility of DPN control of malate oxidation is considered with respect to the whole question of oxaloacetate regulation of the citric acid cycle.etate regulation of the citric acid cycle.)
Hatefi 1962 J Biol Chem-XLII + (1. It has been shown that the electron tra … 1. It has been shown that the electron transfer system in beef heart mitochondria may be reconstituted either totally or in any desired sequential segment by appropriate combinations of two or more of the four primary complexes that have been isolated in highly purified form in this laboratory. 2. The four enzyme systems that collectively comprise the complete machinery for transfer of electrons from reduced diphosphopyridine nucleotide (DPNH; =NADH) and succinate to oxygen re: I, DPNH-coenzyme Q reductase; II, succinic-coenzyme Q reductase; III, QH2-cytochrome ''c'' reductase; and IV, cytochrome ''c'' reductase. The specific inhibitors of each complex have been studied. 3. By appropriate combinations of the primary complexes the following secondary activities have been reconstituted: V, DPNH-cytochrome ''c'' reductase; VI, succinic-cytochrome ''c'' reductase; VII, DPNH, succinic-cytochrome c reductase; VIII, DPNH oxidase; IX, succinic oxidase; and X, DPNH, succinic oxidase activity. The general oxidation-reduction properties of the reconstituted systems, both in the presence and the absence of the usual specific inhibitors of the electron transfer system, are essentially the same as those found for the same activities in the intact mitochondria and in the integrated particles derived therefrom. 4. The reconstituted activities are quite stable to repeated freezing, thawing, and storage at -2O °C, and for the most part, when once formed, are not dissociated by dilution of the mixture or by centrifugation. The evidence supporting the conclusion that reconstitution necessarily involves a particle-particle interaction is discussed.article-particle interaction is discussed.)
Opalka 2004 Br Poult Sci + (1. M. iliotibialis (MIT) and M. pectoralis … 1. M. iliotibialis (MIT) and M. pectoralis (MP) of the BUT Big 6 and Kelly BBB turkey were characterised with respect to physical properties, mitochondrial function, metabolic state, morphology and meat quality.2. Mitochondrial enzyme activity and respiration rates in MP declined with increasing age while glycolytic enzyme activity remained nearly constant.3. There were no major differences between BUT Big 6 and Kelly BBB with respect to histological, histochemical or biochemical variables. In spite of the greater adult weight of BUT Big 6, body proportion was equal in both strains.4. In agreement with the histochemical findings MIT showed higher oxidative capacities, while glycolytic enzyme activity was higher in MP.5. Pyruvate was the best substrate for oxidative phosphorylation in MIT, but not in MP. Pyruvate dehydrogenase (PDH) activity was up to 15 times less in MP and blood lactate was correlated with intramuscular pH.6. Turkey breast muscle was restricted in its ability to oxidise pyruvate, especially in those animals that tended to develop intramuscular acidosis post mortem.7. It is concluded that the ''in vivo'' metabolic environment results in acidosis and impaired meat quality, at least in turkey M. pectoralis.quality, at least in turkey M. pectoralis.)
Claude 1946 J Exp Med + (1. Materials and technical procedures invo … 1. Materials and technical procedures involved in the preparation of liver suspensions have been described and discussed. 2. Liver extracts prepared by the present method appear to contain almost exclusively elements of cytoplasmic origin and can be considered to represent, on a large scale, the cytoplasm of liver cells.large scale, the cytoplasm of liver cells.)
Wilson 1970 Biochim Biophys Acta + (1. Oxidation of NADH by fumarate coupled t … 1. Oxidation of NADH by fumarate coupled to synthesis of ATP was found to occur in cyanide-poisoned rat heart submitochondrial particles. The reaction was inhibited by amytal, thenoyltrifluoroacetone and malonate, indicating the involvement of a portion of the electron transfer chain.2. Cytochrome b became oxidized (while the other cytochromes remained reduced) during the oxidation of NADH by fumarate, suggesting that cytochrome b is part of the reaction pathway.3. Succinate was recovered as the reaction product and accounted for the NADH oxidized.4. The P/2e ratios indicate that one ATP was produced for each pair of electrons transferred to fumarate.5. The reaction was also demonstrated to be present in liver and gastrocnemius muscle of rat. The reaction rate in heart was 2.0 times that of gastrocnemius and 3.3 times that of liver. These differences are not related to the activities of NADH or succinate dehydrogenase.6. The ubiquitous nature of this reaction suggests that it could serve as an important physiological mechanism for generating extra glycolytic energy during periods of anoxia.lycolytic energy during periods of anoxia.)
Boveris 1973 Biochem J + (1. Pigeon heart mitochondria produce H(2)O … 1. Pigeon heart mitochondria produce H(2)O(2) at a maximal rate of about 20 nmol/min per mg of protein. 2. Succinate-glutamate and malate-glutamate are substrates which are able to support maximal H(2)O(2) production rates. With malate-glutamate, H(2)O(2) formation is sensitive to rotenone. Endogenous substrate, octanoate, stearoyl-CoA and palmitoyl-carnitine are by far less efficient substrates. 3. Antimycin A exerts a very pronounced effect in enhancing H(2)O(2) production in pigeon heart mitochondria; 0.26 nmol of antimycin A/mg of protein and the addition of an uncoupler are required for maximal H(2)O(2) formation. 4. In the presence of endogenous substrate and of antimycin A, ATP decreases and uncoupler restores the rates of H(2)O(2) formation. 5. Reincorporation of ubiquinone-10 and ubiquinone-3 to ubiquinone-depleted pigeon heart mitochondria gives a system in which H(2)O(2) production is linearly related to the incorporated ubiquinone. 6. The generation of H(2)O(2) by pigeon heart mitochondria in the presence of succinate-glutamate and in metabolic [[State 4]] has an optimum pH value of 7.5. In States 1 and 3u, and in the presence of antimycin A and uncoupler, the optimum pH value is shifted towards more alkaline values. 7. With increase of the partial pressure of O(2) to the hyperbaric region the formation of H(2)O(2) is markedly increased in pigeon heart mitochondria and in rat liver mitochondria. With rat liver mitochondria and succinate as substrate in State 4, an increase in the ''p''O(2) up to 1.97 MPa (19.5 atm) increases H(2)O(2) formation 10-15-fold. Similar ''p''O(2) profiles were observed when rat liver mitochondria were supplemented either with antimycin A or with antimycin A and uncoupler. No saturation of the system with O(2) was observed up to 1.97 MPa (19.5 atm). By increasing the ''p''O(2) to 1.97 MPa (19.5atm), H(2)O(2) formation in pigeon heart mitochondria with succinate as substrate increased fourfold in metabolic State 4, with antimycin A added the increase was threefold and with antimycin A and uncoupler it was 2.5-fold. In the last two saturation of the system with oxygen was observed, with an apparent ''K''(m) of about 71 kPa (0.7-0.8 atm) and a ''V''(max) of 12 and 20 nmol of H(2)O(2)/min per mg of protein. 8. It is postulated that in addition to the well-known flavin reaction, formation of H(2)O(2) may be due to interaction with an energy-dependent component of the respiratory chain at the cytochrome ''b'' level.atory chain at the cytochrome ''b'' level.)
Mitchell 1967 Biochem J + (1. Pulses of acidity of the outer aqueous … 1. Pulses of acidity of the outer aqueous phase of rat liver mitochondrial suspensions induced by pulses of respiration are due to the translocation of H(+) (or OH(-)) ions across the osmotic barrier (M phase) of the cristae membrane and cannot be attributed to the formation (with acid production) of a chemical intermediate that subsequently decomposes. 2. The effective quantity of protons translocated per bivalent reducing equivalent passing through the succinate-oxidizing and beta-hydroxybutyrate-oxidizing spans of the respiratory chain are very close to 4 and 6 respectively. These quotients are constant between pH5.5 and 8.5 and are independent of changes in the ionic composition of the mitochondrial suspension medium provided that the conditions permit the accurate experimental measurement of the proton translocation. 3. Apparent changes in the -->H(+)/O quotients may be induced by conditions preventing the occurrence of the usual backlash; these apparent changes of -->H(+)/O are attributable to a very fast electrically driven component of the decay of the acid pulses that is not included in the experimental extrapolations. 4. Apparent changes in the -->H(+)/O quotients may also be induced by the presence of anions, such as succinate, malonate and phosphate, or by cations such as Na(+). These apparent changes of -->H(+)/O are due to an increase in the rate of the pH-driven decay of the acid pulses. 5. The uncoupling agents, 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and gramicidin increase the effective proton conductance of the M phase and thus increase the rate of decay of the respiration-driven acid pulses, but do not change the initial -->H(+)/O quotients. The increase in effective proton conductance of the M phase caused by these uncouplers accounts quantitatively for their uncoupling action; and the fact that the initial -->H(+)/O quotients are unchanged shows that uncoupler-sensitive chemical intermediates do not exist between the respiratory-chain system and the effective proton-translocating mechanism. 6. Stoicheiometric acid-base changes associated with the activity of the regions of the respiratory chain on the oxygen side of the rotenone- and antimycin A-sensitive sites gives experimental support for a suggested configuration of loop 3.xperimental support for a suggested configuration of loop 3.)
Claude 1944 J Exp Med + (1. Rat tumor extracts, containing chiefly … 1. Rat tumor extracts, containing chiefly the cytoplasmic constituents of leukemic cells, were fractionated into three main portions, the different components separating in the centrifuge according to size. 2. Mitochondria were isolated by centrifugation at relatively low speed. Elementary composition of purified mitochondria was found to correspond to about 11.5 per cent nitrogen, 1.6 per cent phosphorus, and 27 per cent lipids. Phosphorus and nitrogen content of the lipid portion suggests that as much as 75 to 80 per cent of the lipids of mitochondria is represented by phospholipids. Tests for ribose nucleic acid were positive. 3. Microsomes were separated by means of centrifugation at 18,000 x g. A relation between the high phosphorus content of the microsomes and the marked basophilia of the cytoplasm of leukemic cells is suggested. 4. Phosphorus distribution in the tumor extract, and light absorption analysis of the third fraction, seem to demonstrate that nucleic acid was not present either in a free condition, or in the form of nucleoprotein of relatively low molecular weight. The nature of the results suggests that ribose nucleic acid occurs in the cytoplasm of leukemic cells only in association with formed elements of relatively large size, namely microsomes, and mitochondria.size, namely microsomes, and mitochondria.)
Ernster 1969 Eur J Biochem + (1. Submitochondrial particles were prepare … 1. Submitochondrial particles were prepared from beef‐heart mitochondria by sonication in the presence of EDTA. The particles were lyophilized and repeatedly extracted with pentane until no ubiquinone was found in the extract. Treatment of the ubiquinone‐depleted particles with pentane containing a suitable concentration of ubiquinone (ubiquinone‐50) and subsequent quick washing with ubiquinone‐free pentane resulted in a “re‐incorporation” of ubiquinone in an amount similar to that present in the original particles (3–6 nmoles/mg protein).2. The ubiquinone‐depleted particles exhibited very low or no succinate and NADH oxidase activities, which were restored upon the re‐incorporation of ubiquinone to the levels found in the lyophilized particles before extraction with pentane. Partial (about 50 %) extraction of ubiquinone resulted in markedly decreased succinate and NADH oxidase activities.3. Added cytochrome ''c'' did not replace ubiquinone in restoring the succinate or NADH oxidase activity of ubiquinone‐depleted particles. It stimulated the NADH oxidase, but not the succinate oxidase, activity of the “ubiquinone‐incorporated” particles, but the same stimulation occurred with the lyophilized particles before ubiquinone extraction. The normal, lyophilized, and “ubiquinone‐incorporated” particles contained equal amounts of both total and enzymatically reducible cytochromes.4. In the presence of KCN, NADH reduced the cytochromes, including cytochrome ''b'', only at insignificant rates in the ubiquinone‐depleted particles as compared to the normal and lyophilized preparations, and these rates were greatly stimulated upon the re‐incorporation of ubiquinone. Succinate caused a rapid partial (about 25 %) reduction of cytochrome ''b'', but not of the rest of the cytochromes, in the ubiquinone‐depleted particles. This reduction occurred also in the absence of KCN, and the fraction of cytochrome ''b'', so reduced was not reoxidized when succinate oxidation was inhibited by malonate. Evidence for the occurrence of such an enzymatically non‐oxidizable form of cytochrome ''b'' was also obtained in the normal, lyophilized and “ubiquinone‐incorporated” particles, but, in those cases, this cytochrome ''b'' was reduced by both succinate and NADH. In the presence of antimycin A, all cytochrome ''b'' in the ubiquinone‐depleted particles was rapidly reduced by succinate but not by NADH.5. The normal and lyophilized particles catalyzed a rotenone‐sensitive oxidation of NADH by fumarate. This reaction was completely absent from the ubiquinone‐depleted particles and was restored upon the re‐incorporation of ubiquinone.6. N,N,N′,N′‐Tetramethyl‐p‐phenylenediamine catalyzed an NADH and succinate oxidase activity in antimycin A‐inhibited particles. This NADH oxidase activity was partially sensitive to rotenone in the normal, lyophilized and “ubiquinone‐incorporated” particles, but completely rotenone‐insensitive in the ubiquinone‐depleted particles. All four types of particles were active in catalyzing the antimycin A‐sensitive oxidation of menadiol.7. It is concluded that uniquinone is essential for the interaction of succinate dehydrogenase, NADH dehydrogenase and cytochrome ''b'', and that this interaction is a requisite for the normal function of the respiratory chain. Functionally modified forms of cytochrome ''b'', arising as a consequence of structural damage or antimycin A treatment, are discussed in relation to existing information and proposals concerning the role of cytochrome ''b'' and ubiquinone in electron transport.'b'' and ubiquinone in electron transport.)
Henderson 1969 Biochem J + (1. The action of the antibiotics enniatin … 1. The action of the antibiotics enniatin A, valinomycin, the actin homologues, gramicidin, nigericin and dianemycin on mitochondria, erythrocytes and smectic mesophases of lecithin-dicetyl hydrogen phosphate was studied. 2. These antibiotics induced permeability to alkali-metal cations on all three membrane systems. 3. The ion specificity on each membrane system was the same. 4. Enniatin A, valinomycin and the actins did not induce permeability to protons, whereas nigericin and dianemycin rendered all three membrane systems freely permeable to protons. 5. Several differences were noted between permeability induced by nigericin and that induced by gramicidin. 6. The action of all these antibiotics on mitochondrial respiration could be accounted for by changes in passive ion permeability of the mitochondrial membrane similar to those induced in erythrocytes and phospholipid membranes, if it is assumed that a membrane potential is present in respiring mitochondria.tial is present in respiring mitochondria.)
Harafuji 1980 J Biochem + (1. The apparent binding constant (Kapp(Ca- … 1. The apparent binding constant (Kapp(Ca-G)) for GEDTA (ethylene glycol bis(β-aminoethyl ether)-N, N, N', N'-tetraacetic acid, EGTA) to calcium was determined under conditions of biological significance in the presence of various kinds of pH-buffering agents, using murexide or tetramethylmurexide as a Ca indicator.2. The value of Kapp(Ca-G) at pH 6.80 was 1.0×106M-1 at an ionic strength of 0.114 at 20°C, irrespective of the type of pH-buffering ions. This value is similar to that of Allen, Blinks and Prendergast (1977) (Science 196, 996-998), but still half that calculated from the results of Schwarzenbach, Senn and Anderegg (1957) (Helv. Chim. Acta 40, 1886-1900).3. The value of Kapp(Ca-G) varied according to the following equation as the ionic strength (I) was varied from 0.039 to 0.264:log Kapp(Ca-G)=6.460-[2_??_I/(1+_??_I)-0.4×I] (pH 6.80, 20°C)4. The discrepancy between the present results and previous ones (Ogawa, Y. (1968) J. Biochem. 64, 255-257) may have been due to inadequate regulation of the temperature of the reaction medium in the previous determinations, during which an increase in the temperature of the solution may have occurred.An increase of temperature causes a decrease in the pH of the solution in the presence of histidine, imidazole or Tris-maleate, but causes very little change of pH in the presence of phosphate or maleate.5. The association rate constant for GEDTA with calcium was determined by the stoppedflow method in solutions containing 100mM KCl and 20mM pH-buffering ions at 20°C: the values obtained were 1.4×106M-1s-1 in the presence of MOPS-KOH at pH 6.80; 3.0×106M-1s-1 with imidazole at pH 6.80; 1.0×106M-1s-1 with Tris-maleate at pH 6.80..0×106M-1s-1 with Tris-maleate at pH 6.80.)
Harris 1968 Biochem J + (1. The concentration-dependence of the int … 1. The concentration-dependence of the intramitochondrial accumulation of l-malate and succinate was measured and expressed in the form of adsorption isotherms. The accumulation, however, may arise because of an internal positive potential. 2. The competition for accumulation offered by some other anions, including phosphate, was measured and is expressed conventionally by additional terms in the adsorption equation. 3. The interactions between anions were also studied when one was acting as oxidized substrate. 4. In some examples there is a parallel between the effects of an added anion on both accumulation and oxidation; in other cases chemical participation of the added substance in metabolism is presumed to remove the correlation. 5. It is suggested that by combining kinetic data on penetration with stoicheiometric data on accumulation and specific reaction rates it may be possible to account for the rates of respiration obtained with intact mitochondria. 6. It is possible to show that there is a certain phosphate/substrate ratio for maximum phosphorylation rate with some substrates. This is to be expected when phosphate and substrate compete for accumulation.te and substrate compete for accumulation.)
Koenig 1969 Biochem J + (1. The effects of succinate oxidation on p … 1. The effects of succinate oxidation on pyruvate and also isocitrate oxidation by rat liver mitochondria were studied. 2. Succinate oxidation was without effect on pyruvate and isocitrate oxidation when respiration was maximally activated with ADP. 3. When respiration was partially inhibited by atractylate, succinate oxidation severely inhibited the oxidation of pyruvate and isocitrate. 4. This inhibitory effect of succinate was associated with a two- to three-fold increase in the reduction of mitochondrial NAD(+) but no change in the reduction of cytochrome b. 5. It is concluded that, in the partially energy-controlled state, respiration is more severely inhibited at the first phosphorylating site than at the other two. 6. The effects of succinate oxidation are compared with those of palmitoylcarnitine oxidation. It is concluded that a rapid flow of electrons directly into the respiratory chain at the level of cytochrome b is in itself inadequate to inhibit the oxidation of intramitochondrial NADH. 7. The effects of succinate oxidation on pyruvate oxidation were similar in rat heart and liver mitochondria.milar in rat heart and liver mitochondria.)
Hoek 1970 Biochim Biophys Acta + (1. The kinetics of the efflux of Pi and ma … 1. The kinetics of the efflux of Pi and malate as well as the relationship between Pi transport and intra- and extramitochondrial pH changes were studied in rat-liver mitochondria in the presence of rotenone and oligomycin at different pH's.2. At high pH a fast efflux of Pi from the mitochondria occurs in the first few seconds, followed by a slow re-entry of Pi into the mitochondria. Under the same conditions the exit of malate shows a time lag of 2–4 sec. The exit of malate coincides with the re-entry of Pi.3. In the presence of butylmalonate the exit of endogenous Pi is coupled with a concomitant alkalinization of the mitochondrial matrix space, as calculated from the distribution of 5,5-[14C]dimethyloxazolidine-2,4-dione.4. The stoicheiometry of the Pi-hydroxyl exchange was found to be 1:1.5. The kinetics of Pi transport are consistent with previous observations that there is a direct exchange between OH− and Pi, but not between OH− and malate. The equilibrium distribution of H2PO4− and OH− deviates from the Donnan distribution. This may be explained by assuming a pH-dependent binding of Pi in the mitochondria.pendent binding of Pi in the mitochondria.)
Claude 1945 J Exp Med + (1. The present paper constitutes a prelimi … 1. The present paper constitutes a preliminary study of the morphology of mitochondria by means of electron microscopy.2. The mitochondria that were the subject of this investigation were obtained from a lymphosarcoma of the rat. They were separated from the other components of the leukemic cells by a method of differential centrifugation, and thus made available for direct examination in the electron microscope.3. In the purified form the mitochondria appeared as spherical bodies, the majority of them varying in size approximately from 0.6 to 1.3 µ in diameter.4. Certain aspects of mitochondria in the electron microscope suggest that these elements are surrounded by a differentiated membrane. In some cases the limiting membrane seemed to be responsible for maintaining the general shape of the mitochondria, even when most of the mitochondrial substance had been lost.5. By means of the electron microscope, it is possible to distinguish small elements, 80 to 100 mµ in diameter, within the body of certain mitochondria. Further work is suggested to establish whether these small granules are normal constituents of mitochondria, and what relation may exist between them and ordinary microsomes.6. The nature of mitochondria as morphological units is discussed. Present evidence indicates that mitochondria constitute definite physical entities which can persist in the absence of the cytoplasm.n persist in the absence of the cytoplasm.)
Griffiths 1995 Biochem J + (1. The yield of mitochondria isolated from … 1. The yield of mitochondria isolated from perfused hearts subjected to 30 min ischaemia followed by 15 min reperfusion was significantly less than that for control hearts, and this was associated with a decrease in the rates of ADP-stimulated respiration. 2. The presence of 0.2 microM cyclosporin A (CsA) in the perfusion medium during ischaemia and reperfusion caused mitochondrial recovery to return to control values, but did not reverse the inhibition of respiration. 3. A technique has been devised to investigate whether the Ca(2+)-induced non-specific pore of the mitochondrial inner membrane opens during ischaemia and/or reperfusion of the isolated rat heart. The protocol involved loading the heart with 2-deoxy[3H]glucose ([3H]DOG), which will only enter mitochondria when the pore opens. Subsequent isolation of mitochondria demonstrated that [3H]DOG did not enter mitochondria during global isothermic ischaemia, but did enter during the reperfusion period. 4. The amount of [3H]DOG that entered mitochondria increased with the time of ischaemia, and reached a maximal value after 30-40 min of ischaemia. 5. CsA at 0.2 microM did not prevent [3H]DOG becoming associated with the mitochondria, but rather increased it; this was despite CsA having a protective effect on heart function similar to that shown previously [Griffiths and Halestrap (1993) J. Mol. Cell. Cardiol. 25, 1461-1469]. 6. The non-immunosuppressive CsA analogue [MeAla6]cyclosporin was shown to have a similar Ki to CsA on purified mitochondrial peptidyl-prolyl cis-trans-isomerase and mitochondrial pore opening, and also to have a similar protective effect against reperfusion injury. 7. Using isolated heart mitochondria, it was demonstrated that pore opening could become CsA-insensitive under conditions of adenine nucleotide depletion and high matrix [Ca2+] such as may occur during the initial phase of reperfusion. The apparent increase in mitochondrial [3H]DOG in the CsA-perfused hearts is explained by the ability of the drug to stabilize pore closure and so decrease the loss of [3H]DOG from the mitochondria during their preparation.the mitochondria during their preparation.)
Kearney 1960 J Biol Chem + (1. Unlike other known flavoproteins, in wh … 1. Unlike other known flavoproteins, in which the flavin is relatively loosely bound and is easily liberated by suitable methods of denaturation, in succinic dehydrogenase from beef heart the flavin component is so tightly held that neither treatment with strong acids nor thermal denaturation separates it from the protein.2. Extensive digestion of the purified dehydrogenase with suitable proteolytic enzymes liberates the flavin in an acid-soluble form, which is not, however, identical with known derivatives of riboflavin. The flavin appears in the digest in several chromatographically distinct forms, which may be separated from each other by purification on ion exchange resins or by chromatography on filter paper.3. The main flavin components have been extensively purified and degraded to the mononucleotide and dephosphorylated flavin levels. The dinucleotide contains 1 mole of 5’-adenylic acid, 2 atoms of phosphorus bound in pyrophosphate linkage and 1 mole of ribose. It differs from authentic flavin adenine dinucleotide (FAD) in numerous regards, including its inactivity in the n-amino acid oxidase test, shifted absorption spectrum, shifted pH-fluorescence curve, and in the presence of cationic group(s). After degradation to the mononucleotide and dephosphorylated flavin level, similar differences exist between the resulting compounds and authentic riboflavin 5’-phosphate and riboflavin, respectively. Irradiation in alkali degrades the flavin further, but the resulting compound is not identical with lumiflavin.4. These differences and the greater water solubility of the unphosphorylated compound as compared with riboflavin are best explained by the hypothesis that the flavin in the dehydrogenase is held to a peptide chain by a covalent linkage which survives proteolytic digestion. The compounds in the digest, therefore, would be peptides of FAD, representing fragments of the original enzyme.5. Evidence for the flavin peptide hypothesis has come from the finding that throughout very extensive purification by a variety of methods the flavin is always accompanied by peptide material. In the most purified fraction, believed to be free of contaminating peptides, alanine, serine, threonine, glutamic acid, and valine were present in molar ratio to the flavin and an additional mole of serine was present as N-terminal group. Similar amino acid compositions were found in 2 other samples, purified by different procedures.6. Evidence pertaining to the flavin peptide hypothesis and the possible structure of the flavin is discussed.ible structure of the flavin is discussed.)
Gnaiger 2013 Abstract MiP2013 + (10 years ago the uncoupling hypothesis was … 10 years ago the uncoupling hypothesis was presented for mitochondrial haplogroups of arctic populations suggesting that lower coupling of mitochondrial respiration to ATP production was selected for in favor of higher heat dissipation as an adaptation to cold climates [1,2]. Up to date no actual tests have been published to compare mitochondrial coupling in tissues obtained from human populations with regional mtDNA variations. Analysis of oxidative phosphorylation (OXPHOS) is a major component of mitochondrial phenotyping [3]. We studied mitochondrial coupling in small biopsies of arm and leg muscle of Inuit of the Thule and Dorset haplogroups in northern Greenland compared to Danes from western Europe haplogroups. Inuit had a higher capacity to oxidize fat substrate in leg and arm muscle, yet mitochondrial respiration compensating for proton leak was proportionate with OXPHOS capacity. Biochemical coupling efficiency was preserved across variations in muscle fiber type and uncoupling protein-3 content. After 42 days of skiing on the sea ice in northern Greenland, Danes demonstrated adaptive substrate control through an increase in fatty acid oxidation approaching the level of the Inuit, yet coupling control of oxidative phosphorylation was conserved. Our findings reveal that coupled ATP production is of primary evolutionary significance for muscle tissue independent of adaptations to the cold.ue independent of adaptations to the cold.)
ASMRM 2013 Seoul KR + (10th Conference of the Asian Society of Mitochondrial Research and Medicine - [http://asmrm2013.com/common_files/mess.asp ASMRM 2013], Seoul KR)
MiP2014 + (10th MiP''conference'': Joint IUBMB/MiP Symposium on Mitochondrial Physiology - a Point/Counterpoint Meeting, Obergurgl, Austria; with post-conference workshop '''[[MiPNet19.10 | 95th Oroboros O2k-Workshop]]'''.)
10th Conference of the International Coenzyme Q10 Association 2022 Hamburg DE + (10th Conference of the International Coenzyme Q10 Association, Hamburg, 2022)
10th European Algae Industry Summit 2020 Reykjavik IS + (10th European Algae Industry Summit, Reykjavik, Iceland, 2020)