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Hoeks 2008 FEBS Lett + (A high intake of dietary fat has been sugg … A high intake of dietary fat has been suggested to diminish mitochondrial functioning in skeletal muscle, possibly attributing to muscular fat accumulation. Here we show however, that an 8-week high-fat dietary intervention did not affect intrinsic functioning of rat skeletal muscle mitochondria assessed by respirometry, neither on a carbohydrate- nor on a lipid-substrate. Interestingly, PPARGC1A protein increased by approximately 2-fold upon high-fat feeding and we observed inconsistent results on different markers of mitochondrial density. Mitochondrial ROS production, assessed by electron spin resonance spectroscopy remained unaffected. Intramyocellular lipid levels increased significantly illustrating that a reduced innate mitochondrial function is not a prerequisite for intra-muscular fat accumulation.isite for intra-muscular fat accumulation.)
Park 2017 Metab Brain Dis + (A high-fat diet induces obesity in mice, l … A high-fat diet induces obesity in mice, leading to insulin resistance, decreased mitochondrial function, and increased apoptosis in the hippocampus, which eventually result in memory loss. The present study investigated the effect of physical exercise on memory, hippocampal mitochondrial function, and apoptosis in mice with in insulin resistance caused by obesity due to high-fat diet. Mice were randomly divided into four groups: control (CON), control and exercise (CON + EX), high fat diet (HFD), and high fat diet and exercise (HFD + EX). After receiving a high-fat (60%) diet for 20 weeks to induce obesity, the animals were subjected to an exercise routine 6 times per week, for 12 weeks. The exercise duration and intensity gradually increased over 4-week intervals. Hippocampal memory was examined using the step-down avoidance task. Mitochondrial function and apoptosis were also examined in the hippocampus and dentate gyrus. We found that obesity owing to a high-fat diet induced insulin resistance and caused a decrease in memory function. Insulin resistance also caused a decrease in mitochondrial function in the hippocampus by reducing Ca2+ retention and O2, respiration, increasing the levels of H2O2, and Cyp-D, and mPTP opening. In addition, apoptosis in the hippocampus increased owing to decreased expression of Bcl-2 and increased expression of Bax, cytochrome c, and caspase-3 and TUNEL-positive cells. In contrast, physical exercise led to reduced insulin resistance, improved mitochondrial function, and reduced apoptosis in the hippocampus. The results suggest that physiological stimulations such as exercise improve hippocampal function and suppress apoptosis, potentially preventing the memory loss associated with obesity-induced insulin resistance.potentially preventing the memory loss associated with obesity-induced insulin resistance.)
Kwon 2009 Thesis + (A high-fat diet leads to an accumulation o … A high-fat diet leads to an accumulation of lipid in skeletal muscle, and the development of both mitochondrial dysfunction and insulin resistance. Recently, our lab reported that lipid overload leads to elevated H2O2 emission from muscle mitochondria, and that mitochondrial-targeted scavenging of H2O2 completely prevents the development of high fat diet-induced insulin resistance. These findings raise the possibility that interventions which acutely restore cellular metabolic balance in muscle may also acutely restore insulin sensitivity. We hypothesized that mitochondrial function and insulin sensitivity can be restored in skeletal muscle of high-fat fed rats by creating an acute deficit in metabolic balance via 2 h low-intensity treadmill exercise or 16 h fasting. Male Sprague-Dawley rats (125-150g) were either maintained on a standard high carbohydrate- diet or fed a high-fat (60%) diet for 6 weeks and divided into three groups the day before the study: one group was maintained on the normal high-fat diet, another group was fasted overnight (16 h), and a third group completed a single 2 h bout of low-intensity treadmill exercise (10 m/min) and then were given normal overnight ad libitum access to the high-fat diet. Oral glucose tolerance tests were administrated to assess insulin action. Red gastrocnemius muscles were harvested and permeabilized fibers prepared for determination of mitochondrial respiratory function and H2O2 emission. A single 16 h fast significantly (P<0.05) improved insulin sensitivity in rats maintained on a high-fat diet (P<0.05). Oxygen consumption rate in permeabilized fibers in response to submaximal and maximal ADP concentration when supported exclusively with complex I substrates were not different among groups. However, when respiration was supported by fatty acids (palmitoylcarnitine plus malate, complex I + II substrates), high-fat diet plus exercise group showed higher (P<0.05) rates compared with high-fat diet group. There were no significant differences in H2O2 emission among the 4 groups. In conclusion, a single 16 h overnight fast is sufficient to restore insulin sensitivity in high fat diet-induced insulin resistant rats, providing evidence that insulin action in muscle is acutely sensitive to the metabolic state of cells. A single bout of low-intensity treadmill exercise in high-fat fed rats failed to restore insulin action but increased ADP-stimulated respiratory capacity, providing evidence of an as yet unidentified regulatory mechanism of the respiratory system. Somewhat surprisingly however, neither fasting nor exercise altered the H2O2 emitting potential in permeabilized fibers, suggesting that further work is required to better understand the factors influencing mitochondrial function and their potential link to insulin sensitivity.lized fibers, suggesting that further work is required to better understand the factors influencing mitochondrial function and their potential link to insulin sensitivity.)
Cavadas 2015 Hum Mutat + (A high-resolution mtDNA phylogenetic tree … A high-resolution mtDNA phylogenetic tree allowed us to look backward in time to investigate purifying selection. Purifying selection was very strong in the last 2,500 years, continuously eliminating pathogenic mutations back until the end of the Younger Dryas (∼11,000 years ago), when a large population expansion likely relaxed selection pressure. This was preceded by a phase of stable selection until another relaxation occurred in the out-of-Africa migration. Demography and selection are closely related: expansions led to relaxation of selection and higher pathogenicity mutations significantly decreased the growth of descendants. The only detectible positive selection was the recurrence of highly pathogenic nonsynonymous mutations (m.3394T>C-m.3397A>G-m.3398T>C) at interior branches of the tree, preventing the formation of a dinucleotide STR (TATATA) in the MT-ND1 gene. At the most recent time scale in 124 mother-children transmissions, purifying selection was detectable through the loss of mtDNA variants with high predicted pathogenicity. A few haplogroup-defining sites were also heteroplasmic, agreeing with a significant propensity in 349 positions in the phylogenetic tree to revert back to the ancestral variant. This nonrandom mutation property explains the observation of heteroplasmic mutations at some haplogroup-defining sites in sequencing datasets, which may not indicate poor quality as has been claimed.© 2015 WILEY PERIODICALS, INC.s has been claimed. © 2015 WILEY PERIODICALS, INC.)
Djafarzadeh 2017 J Vis Exp + (A high-resolution oxygraph is a device for … A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.ratory electron transport system capacity.)
Hatefi 1961 Biochim Biophys Acta + (A highly active DPNH-cytochrome c reductas … A highly active DPNH-cytochrome c reductase has been isolated from beef-heart mitochondria. The best preparations of the enzyme catalyze the reduction by DPNH of approx. 50–60 μmoles cytochrome c/min/mg protein at 38°. The enzymic activity is completely inhibited by Amytal, p-chloromercuriphenyl sulfonate, antimycin A, SN-5949 or 2-nonyl-4-hydroxyquinoline-N-oxide, and is stimulated by EDTA. The preparation contains DPNH flavoprotein, cytochromes b and c1, Coenzyme Q and non-heme iron and is essentially free of succinic-cytochrome c reductase as well as cytochrome oxidase activity.se as well as cytochrome oxidase activity.)
Kotarsky 2010 Mitochondrion + (A homozygous mutation in the complex III c … A homozygous mutation in the complex III chaperone BCS1L causes GRACILE syndrome (intrauterine growth restriction, aminoaciduria, cholestasis, hepatic iron overload, lactacidosis). In control and patient fibroblasts we localized BCS1L in inner mitochondrial membranes. In patient liver, kidney, and heart BCS1L and Rieske protein levels, as well as the amount and activity of complex III, were decreased. Major histopathology was found in kidney and liver with cirrhosis and iron deposition, but of iron-related proteins only ferritin levels were high. In placenta from a GRACILE fetus, the ferrooxidases ceruloplasmin and hephaestin were upregulated suggesting association between iron overload and placental dysfunction.n iron overload and placental dysfunction.)
Stodden 2020 Proc Natl Acad Sci U S A + (A key component of scientific communicatio … A key component of scientific communication is sufficient information for other researchers in the field to reproduce published findings. For computational and data-enabled research, this has often been interpreted to mean making available the raw data from which results were generated, the computer code that generated the findings, and any additional information needed such as workflows and input parameters. Many journals are revising author guidelines to include data and code availability. This work evaluates the effectiveness of journal policy that requires the data and code necessary for reproducibility be made available postpublication by the authors upon request. We assess the effectiveness of such a policy by (i) requesting data and code from authors and (ii) attempting replication of the published findings. We chose a random sample of 204 scientific papers published in the journal Science after the implementation of their policy in February 2011. We found that we were able to obtain artifacts from 44 % of our sample and were able to reproduce the findings for 26 %. We find this policy—author remission of data and code postpublication upon request—an improvement over no policy, but currently insufficient for reproducibility.urrently insufficient for reproducibility.)
Nissen 2017 Glia + (A key enzyme in brain glutamate homeostasi … A key enzyme in brain glutamate homeostasis is glutamate dehydrogenase (GDH) which links carbohydrate and amino acid metabolism mediating glutamate degradation to CO2 and expanding tricarboxylic acid (TCA) cycle capacity with intermediates, i.e. anaplerosis. Humans express two GDH isoforms, GDH1 and 2, whereas most other mammals express only GDH1. hGDH1 is widely expressed in human brain while hGDH2 is confined to astrocytes. The two isoforms display different enzymatic properties and the nature of these supports that hGDH2 expression in astrocytes potentially increases glutamate oxidation and supports the TCA cycle during energy-demanding processes such as high intensity glutamatergic signaling. However, little is known about how expression of hGDH2 affects the handling of glutamate and TCA cycle metabolism in astrocytes. Therefore, we cultured astrocytes from cerebral cortical tissue of hGDH2-expressing transgenic mice. We measured glutamate uptake and metabolism using [3 H]glutamate, while the effect on metabolic pathways of glutamate and glucose was evaluated by use of 13 C and 14 C substrates and analysis by mass spectrometry and determination of radioactively labeled metabolites including CO2 , respectively. We conclude that hGDH2 expression increases capacity for uptake and oxidative metabolism of glutamate, particularly during increased workload and aglycemia. Additionally, hGDH2 expression increased utilization of branched-chain amino acids (BCAA) during aglycemia and caused a general decrease in oxidative glucose metabolism. We speculate, that expression of hGDH2 allows astrocytes to spare glucose and utilize BCAAs during substrate shortages. These findings support the proposed role of hGDH2 in astrocytes as an important fail-safe during situations of intense glutamatergic activity.uations of intense glutamatergic activity.)
Andziak 2006 Aging Cell + (A key tenet of the oxidative stress theory … A key tenet of the oxidative stress theory of aging is that levels of accrued oxidative damage increase with age. Differences in damage generation and accumulation therefore may underlie the natural variation in species longevity. We compared age-related profiles of whole-organism lipid peroxidation (urinary isoprostanes) and liver lipid damage (malondialdehyde) in long living naked mole-rats [maximum lifespan (MLS) > 28.3 years] and shorter-living CB6F1 hybrid mice (MLS approximately 3.5 years). In addition, we compared age-associated changes in liver non-heme iron to assess how intracellular conditions, which may modulate oxidative processes, are affected by aging. Surprisingly, even at a young age, concentrations of both markers of lipid peroxidation, as well as of iron, were at least twofold (P < 0.005) greater in naked mole tats than in mice. This refutes the hypothesis that prolonged naked mole-rat longevity is due to superior protection against oxidative stress. The age-related profiles of all three parameters were distinctly species specific. Rates of lipid damage generation in mice were maintained throughout adulthood, while accrued damage in old animals was twice that of young mice. In naked mole-rats, urinary isoprostane excretion declined by half with age (P < 0.001), despite increases in tissue iron (P < 0.05). Contrary to the predictions of the oxidative stress theory, lipid damage levels did not change with age in mole-rats. These data suggest that the patterns of age-related changes in levels of markers of oxidative stress are species specific, and that the pronounced longevity of naked mole-rats is independent of oxidative stress parameters.le-rats is independent of oxidative stress parameters.)
Cottingham 1983 Biochim Biophys Acta + (A kinetic analysis of oxygen uptake was ca … A kinetic analysis of oxygen uptake was carried out in order to investigate the role of ubiquinone pool behaviour in plant mitochondria. The interaction of the external NADH dehydrogenase with either the cytochrome system or the cyanide-insensitive oxidase was examined under various conditions. The involvement of a ubiquinone pool can be deduced from the shape of the titration curve as the appropriate oxidase system is inhibited, by antimycin A for the cytochrome system and salicylhydroxamic acid for the cyanide-insensitive oxidase, at different activities of the NADH dehydrogenase. In the absence of a specific inhibitor, the turnover of the external NADH dehydrogenase was adjusted using a novel NADH-generating system involving the recycling of a low concentration of NAD+ by added glucose 6-phosphate dehydrogenase in the presence of substrate. The results show that ubiquinone pool behaviour is observed between the external NADH dehydrogenase and either the cytochrome b-c1 complex or the cyanide-insensitive oxidase. However, there is a substantial departure from pool behaviour during the simultaneous operation of both oxidases.e simultaneous operation of both oxidases.)
Laner 2017 Abstract EUROMIT2017 Cologne + (A lack of physical activity associates wit … A lack of physical activity associates with decreased mitochondrial capacity and is a major cause underlying metabolic dysregulation and preventable diseases in modern societies. In contrast, an active lifestyle supports enhanced mitochondrial capacities and reduces the risk of degenerative diseases. Despite this well-known relation between health and mitochondrial function, there is no regimented, quantitative system, or database organised to routinely test, compare and monitor mitochondrial capacities within individuals or populations. Every study of mitochondrial (mt) function and disease in human tissues and cells is faced with Evolution, Age, Gender, Lifestyle and Environment ([[EAGLE]]) as essential background conditions characterizing the individual patient, subject, study group, species, tissue or – to some extent - cell line. Only a large and well-coordinated network can manage to generate the necessary number of consistent data to address the complexity of EAGLE. Using [[high-resolution respirometry]], the [[K-Regio MitoFit]] and [[MitoEAGLE]] initiatives develop novel lab standards and diagnostic methods for monitoring of a mitochondrial fitness score. SOPs are elaborated for sample preparation, respiratory evaluation and data documentation. Fresh and cryopreserved cells obtained non-invasively from blood samples broaden the scope for respirometric mitochondrial diagnosis.for respirometric mitochondrial diagnosis.)
Gnaiger 2016 Abstract EBEC Riva del Garda 2016 + (A lack of physical activity associates wit … A lack of physical activity associates with decreased mitochondrial capacity and is a major cause underlying metabolic dysregulation and preventable diseases in modern societies. In contrast, an active lifestyle supports enhanced mitochondrial capacities and reduces the risk of degenerative diseases. Despite this well-known relation between health and mitochondrial function, there is no regimented, quantitative system, or database organised to routinely test, compare and monitor mitochondrial capacities within individuals or populations. Using high-resolution respirometry, the MitoFit and MitoEAGLE initiatives will develop novel lab standards and diagnostic methods for the monitoring of a mitochondrial fitness score. To this end, SOPs will be worked out regarding sample preparation, respiratory evaluation and data documentation. Fresh and cryopreserved cells obtained noninvasively from blood samples will serve as models, the latter allowing samples to be collected for later analysis, thereby broadening the scope for respirometric investigations.This approach will then be expanded to all sorts of human tissues and cells of interest and assess aspects relating to Evolution, Age, Gender, Lifestyle and Environment (EAGLE) as essential background conditions characterizing the individual patient, subject, study group, and/or species. The huge scope of this endeavour requires an international network of laboratories capable of generating the necessary number of consistent data to address the complexity of EAGLE. Coping with the mass of the expected data necessitates a dedicated MitoEAGLE knowledge management network developing harmonization protocols towards generating a rigorously monitored data repository on mitochondrial respiratory function. The resulting MitoEAGLE data management system will enable to interrelate results of a large number of studies, to interpret pathological phenotypes, and to set results into the multidimensional context of EAGLE.nto the multidimensional context of EAGLE.)
Lukasiak 2016 Eur J Pharmacol + (A large conductance potassium (BKCa) chann … A large conductance potassium (BKCa) channel opener, NS1619 (1,3-dihydro-1- [2-hydroxy-5-(trifluoromethyl) phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one), is well known for its protective effects against ischemia-reperfusion injury; however, the exact mode of its action remains unclear. The aim of this study was to characterize the effect of NS1619 on endothelial cells. The endothelial cell line EA.hy926, guinea pig hearts and submitochondrial particles isolated from the heart were used. In the isolated guinea pig hearts, which were perfused using the Langendorff technique, NS1619 caused a dose-dependent increase in coronary flow that was inhibited by L-NAME. In EA.hy926 cells, NS1619 also caused a dose-dependent increase in the intracellular calcium ion concentration [Ca(2+)]i, as measured using the FURA-2 fluorescent probe. Moreover, NS1619 decreased the oxygen consumption rate in EA.hy926 cells, as assessed using a Clark-type oxygen electrode. However, when NS1619 was applied in the presence of oligomycin, the oxygen consumption increased. NS1619 also decreased the mitochondrial membrane potential, as measured using a JC-1 fluorescent probe in the presence and absence of oligomycin. Additionally, the application of NS1619 to submitochondrial particles inhibited ATP synthase. In summary, NS1619 has pleiotropic actions on EA.hy926 cells and acts not only as an opener of the BKCa channel in EA.hy926 cells but also as an inhibitor of the respiratory chain component, sarcoplasmic reticulum ATPase, which leads to the release of Ca(2+) from the endoplasmic reticulum. Furthermore, NS1619 has the oligomycin-like property of inhibiting mitochondrial ATP synthase.Copyright © 2016 Elsevier B.V. All rights reserved. © 2016 Elsevier B.V. All rights reserved.)
Sperl 1994 J Inher Metab Dis + (A large number of enzyme systems are exami … A large number of enzyme systems are examined for the diagnosis of mitochondrial myopathies including the pyruvate dehidrogenase complex, tricarboxylic-acid-cycle enzymes and respiratory chain complexes. This investigation can be carried out in frozen tissue. For the study of oxidative phosphorilation in intact mitochondria, fresh muscle tissue is required, and isolation of mitochondria from large amounts of tissue (at least 500-1000 mg) is necessary. For ethical reason this imposes a serious limitation, especially in paediatric patients. Radiochemical measurements of oxidation rates in various substrates in 600 g supernatant from 100-300 mg amounts of muscle tissue has partly overcome this problem. (Bookelman ''et.al''., 1978). Owing to the low yield, the danger of selective isolation of different mitochondrial populations exists. In addition, since isolated mitochondria removed from their natural environment are more or less unstable, there is a possibility of artefacts. Recently, investigation of saponin-skinned muscle fibers by polarographic methods was reported for cardiac (Veksler ''et. al''., 1987) and human muscle tissue. In such permeabilized fibers, study of mitochondrial respiratory control is possible as in isolated mitochondria but without the disadvantages mentioned above (Letellier ''et.al''., 1992; Kunz ''et.al''., 1993).We investigated saponin-skinned muscle fibers in three patients suspected of a mitochondrial encephalo-myopathy. For our studies we used a specially developed respirometer with a sensitivity ten times higher than the established instruments (Kunz ''et.al''., 1993).ished instruments (Kunz ''et.al''., 1993).)
Lauterbach 2013 FEBS J + (A large number of industrially relevant en … A large number of industrially relevant enzymes depend upon nicotinamide cofactors, which are too expensive to be added in stoichiometric amounts. Existing NAD(P)H-recycling systems suffer from low activity, or the generation of side products. H₂-driven cofactor regeneration has the advantage of 100% atom efficiency and the use of H₂ as a cheap reducing agent, in a world where sustainable energy carriers are increasingly attractive. The state of development of H₂-driven cofactor-recycling systems and examples of their integration with enzyme reactions are summarized in this article. The O₂-tolerant NAD⁺-reducing hydrogenase from Ralstonia eutropha is a particularly attractive candidate for this approach, and we therefore discuss its catalytic properties that are relevant for technical applications.t are relevant for technical applications.)
Santoso 2019 Bioorg Med Chem + (A library of thirty-two quinolinequinones … A library of thirty-two quinolinequinones (QQs) with various amine substituents at the 6- and 7-positions were synthesised efficiently and in good yields for evaluation as potential anti-tuberculosis agents. ''Mycobacterium tuberculosis'' growth inhibition assays demonstrated that QQs bearing moderate length alkyl chains (i.e. heptylphenylamino- and octylamino-QQs), and aryl groups (i.e. phenylethylamino- and benzylamino-QQs) exhibited encouraging inhibitory activity, while QQ analogue 7-chloro-6-propargylamino-quinoline-5,8-dione (16b) had excellent inhibitory activity (MIC = 8 μM). The cLogP values and redox activities of the QQs were determined, and neither readout correlated with the anti-mycobacterial activities of the compounds. Notwithstanding, mode of action studies of 16b revealed that treatment of ''M. tuberculosis'' with this compound led to activation of NADH-dependent oxygen consumption suggesting a redox cycling mechanism. To this end, the promising anti-mycobacterial activity of several QQs and their ability to perturb oxygen management leading to an uncontrolled respiratory burst, as identified in this work and by others, demonstrates the merit of further optimising the anti-mycobacterial activity of this readily synthesised class of compound.Copyright © 2019. Published by Elsevier Ltd.right © 2019. Published by Elsevier Ltd.)
Antonenko 2011 J Biol Chem + (A limited decrease in mitochondrial membra … A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.vents further membrane potential decrease.)
Bergeson 1981 West J Med + (A major international movement is in progr … A major international movement is in progress to extend metrication using the International System of Units. Significantly involved is the field of medicine. Extensive changes adopted abroad now appear in foreign medical literature, and physicians in the United States commonly are unprepared to interpret medical information from abroad because the data are reported in unfamiliar terms. The system has broad immediate and future implications to American physicians.uture implications to American physicians.)
Fernandes 2012 Am J Hum Genet + (A major unanswered question regarding the … A major unanswered question regarding the dispersal of modern humans around the world concerns the geographical site of the first human steps outside of Africa. The "southern coastal route" model predicts that the early stages of the dispersal took place when people crossed the Red Sea to southern Arabia, but genetic evidence has hitherto been tenuous. We have addressed this question by analyzing the three minor west-Eurasian haplogroups, N1, N2, and X. These lineages branch directly from the first non-African founder node, the root of haplogroup N, and coalesce to the time of the first successful movement of modern humans out of Africa, ∼60 thousand years (ka) ago. We sequenced complete mtDNA genomes from 85 Southwest Asian samples carrying these haplogroups and compared them with a database of 300 European examples. The results show that these minor haplogroups have a relict distribution that suggests an ancient ancestry within the Arabian Peninsula, and they most likely spread from the Gulf Oasis region toward the Near East and Europe during the pluvial period 55-24 ka ago. This pattern suggests that Arabia was indeed the first staging post in the spread of modern humans around the world.Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.shed by Elsevier Inc. All rights reserved.)
Friedrich 2010 Abstract MiP2010 + (A medium of containing high levels of pota … A medium of containing high levels of potassium chloride (KCl) is commonly used when assessing respiratory function of isolated mitochondria from various tissues. However, the measured intracellular [K+] in kidney proximal tubular cells is about 60 mM and in cardiac myocytes approximately 130 mM. Therefore, the use of a similar media [K+] for all tissues seems unsupported. Here we investigated the effect of different [K+] on respiratory function in mitochondria isolated from kidney cortex and heart of healthy male Sprague-Dawley rats.Oxygen consumptions and the respiratory control ratios (RCR) were measured using respiratory medias containing [K+] of 15, 37, 81, 111 and 146 mM. In all measurements, the media contained (in mM): 1 EGTA, 20 HEPES, 5 MgCl2, 5 KPO4- and 1 g/l bovine serum albumin. pH was adjusted to 7.4 and the osmolarity to 330 mosm/kg HK2O using a 1:3 ratio of sucrose and mannitol.The RCR of kidney cortex mitochondria decreased when the [K+] was elevated compared to the media containing 15 mM K+ (5.2±0.2 vs. 2.5±0.2, 3.7±0.2, 3.9±0.2, 3.0±0.1, respectively). However, RCR of heart mitochondria was lowest at 37 mM (3.9±0.3) and was highest at 146 mM K+ (10.1±0.45). A two-way ANOVA showed that kidney cortex mitochondria have a different sensitivity towards K+ compared to heart mitochondria (interaction P<0.05, treatment P<0.05, group P<0.05). Glibenclamide (100 µM), an inhibitor of the ATP-sensitive K+ channel, increased RCR in kidney cortex mitochondria at 15 mM K+ (+32%), but significantly more at 146 mM K+ (+47%). Blockade of the voltage-gated K+ channel by 4-aminopyridine (4-AP, 1 mM) together with glibenclamide improved RCR by +73% at 146 mM K+. Neither of the applied K+-channel blockers had any effect on the RCR of heart mitochondria. Mitochondria swelling at increasing [K+] were observed in kidney cortex mitochondria, measured as loss of absorbance at 540 nm.Kidney cortex mitochondria in K+-based media are non-functional in [K+] ranging from 37-146 mM. Heart mitochondria do not display K+-sensitivity to the same degree, but rather increase respiratory function with increasing [K+]. Furthermore, we demonstrated that a tissue specific difference in mitochondria K+-channels may explain these differences. The present study therefore demonstrates the importance of choosing a correct ''in vitro media'' to ensure a high quality of mitochondria research.urthermore, we demonstrated that a tissue specific difference in mitochondria K+-channels may explain these differences. The present study therefore demonstrates the importance of choosing a correct ''in vitro media'' to ensure a high quality of mitochondria research.)
Lyon 2006 Anal Chem + (A method for low-level, low-potential elec … A method for low-level, low-potential electrochemical detection of hydrogen peroxide using a chemically activated redox mediator is presented. This method is unique in that it utilizes a mediator, Amplex Red, which is only redox-active when chemically oxidized by H2O2 in the presence of the enzyme horseradish peroxidase (HRP). When employed in concert with microelectrode square wave voltammetry to optimize sensing at ultralow concentrations (<1 microM), this method exhibits marked improvements in analytical sensitivity and detection limits (limit of detection as low as 8 pM) over existing protocols. Sensing schemes incorporating both freely diffusing and immobilized HRP are evaluated, and the resulting analytical sensitivities are 1.22 +/- 0.04 and (2.1 +/- 0.6) x 10(-1) microA/(microM mm2), respectively, for peroxide concentrations in the high picomolar to low micromolar range. A second linear region exists for lower peroxide concentrations. Furthermore, quantitative enzyme kinetics analysis using Michaelis-Menten parameters is possible through interpretation of data collected in this scheme. Km values for soluble and immobilized HRP were 84 +/- 13 and 504 +/- 19 microM, respectively. This method is amenable to any biological detection scheme that generates hydrogen peroxide as a reactive product.ates hydrogen peroxide as a reactive product.)
Cheng 2017 US Patent + (A method for treating a microbial infection in a subject includes administering to the subject a pharmaceutical composition which has a therapeutically effective amount of an antimicrobial peptide containing a derivative of P-113.)
Lee 2010 Curr Biol + (A mild inhibition of mitochondrial respira … A mild inhibition of mitochondrial respiration extends the life span of many organisms, including yeast, worms, flies, and mice, but the underlying mechanism is unknown. One environmental condition that reduces rates of respiration is hypoxia (low oxygen). Thus, it is possible that mechanisms that sense oxygen play a role in the longevity response to reduced respiration. The hypoxia-inducible factor HIF-1 is a highly conserved transcription factor that activates genes that promote survival during hypoxia. In this study, we show that inhibition of respiration in C. elegans can promote longevity by activating HIF-1. Through genome-wide screening, we found that RNA interference (RNAi) knockdown of many genes encoding respiratory-chain components induced hif-1-dependent transcription. Moreover, HIF-1 was required for the extended life spans of clk-1 and isp-1 mutants, which have reduced rates of respiration. Inhibiting respiration appears to activate HIF-1 by elevating the level of reactive oxygen species (ROS). We found that ROS are increased in respiration mutants and that mild increases in ROS can stimulate HIF-1 to activate gene expression and promote longevity. In this way, HIF-1 appears to link respiratory stress in the mitochondria to a nuclear transcriptional response that promotes longevity.iptional response that promotes longevity.)
Zelenka 2015 Oxid Med Cell Longev + (A moderate elevation of reactive oxygen sp … A moderate elevation of reactive oxygen species (ROS) production and a mild inhibition of mitochondrial respiratory chain have been associated with a health promotion and a lifespan extension in several animal models of aging. Here, we tested whether this phenomenon called mitohormesis could be mediated by L-lactate. The treatment with 5 mM L-lactate significantly increased H2O2 production and slightly inhibited the respiration in cultured skin fibroblasts and in isolated mitochondria. The L-lactate exposure was associated with oxidation of intracellular glutathione, phosphorylation of 5'AMP-activated protein kinase (AMPK), and induction of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) transcription. A replicative aging of fibroblasts (L0) with a constant (LC), or intermittent 5 mM L-lactate (LI) in media showed that the high-passage LI fibroblasts have higher respiration, lower H2O2 release, and lower secretion of L-lactate compared to L0 and LC. This protection against mitochondrial dysfunction in LI cells was associated with lower activity of mechanistic target of rapamycin complex 1 (mTORC1), less signs of cellular senescence, and increased autophagy compared to L0 and LC. In conclusion, we demonstrated that intermittent but not constant exposure to L-lactate triggers mitohormesis, prevents aging-associated mitochondrial dysfunction, and improves other markers of aging. prevents aging-associated mitochondrial dysfunction, and improves other markers of aging.)

(A moderate elevation of reactive oxygen species (ROS) production and a m)

Lee 2019 Nat Metab + (A moderate reduction of body temperature c … A moderate reduction of body temperature can induce a remarkable lifespan extension. Here we examine the link between cold temperature, germ line fitness and organismal longevity. We show that low temperature reduces age-associated exhaustion of germ stem cells (GSCs) in ''Caenorhabditis elegans'', a process modulated by thermosensory neurons. Notably, robust self-renewal of adult GSCs delays reproductive aging and is required for extended lifespan at cold temperatures. These cells release prostaglandin E2 (PGE2) to induce cbs-1 expression in the intestine, increasing somatic production of hydrogen sulfide (H2S), a gaseous signaling molecule that prolongs lifespan. Whereas loss of adult GSCs reduces intestinal cbs-1 expression and cold-induced longevity, application of exogenous PGE2 rescues these phenotypes. Importantly, tissue-specific intestinal overexpression of cbs-1 mimics cold-temperature conditions and extends longevity even at warm temperatures. Thus, our results indicate that GSCs communicate with somatic tissues to coordinate extended reproductive capacity with longevity.nded reproductive capacity with longevity.)
Heidler 2013 Abstract MiP2013 + (A morphological hallmark of the failing hu … A morphological hallmark of the failing human heart is a devastative autophagic degradation of cellular structures starting from the perinuclear region, proposed to actively shift the heart into a decompensated state [1]. We studied heart samples from different species, i.e. a mouse model of cardiac specific expression of MCP1 that autonomously develops heart failure [2], hibernating Syrian hamsters [3] and a pig model of mitochondrial dysfunction exposed to hyperbaric oxygen.Our data reveal an age-dependent increase of perinuclear degradation in mouse hearts that occurred prior to the onset of cardiac dysfunction. These center core-like lesions in the myofibrillar compartment are most likely the end-stage result of a vicious cycle that starts with a physiological response to lowered levels of cardiac workload. Accordingly we found that in hibernating Syrian hamsters under conditions of depressed metabolism interfibrillar mitochondria are reversibly silenced whilst subsarcolemmal mitochondria remain more active. Central remodeling of cardiomyocyte compartments is a phenomenon primarily known in the hibernating myocardium [4]. Here we show in pig hearts that the isolated impairment of the interfibrillary compartment can be fully re-activated upon treatment with hyperbaric oxygen.We conclude that differential compartment regulation by switching the activity status of mitochondrial sub-populations from on to off and vice versa might provide a hitherto unnoticed flexible on-demand plasticity in cardiomyocytes. Such alterations make proper myofibril contraction in the silenced compartment unlikely. Silenced mitochondria can be re-activated on demand. Only long-lasting mitochondrial silencing, e.g. upon chronic cardiac overload, might increase the risk of adverse cardiomyocyte remodeling. risk of adverse cardiomyocyte remodeling.)
Canton 1995 Biochem J + (A new criterion is utilized for the interp … A new criterion is utilized for the interpretation of flow-force relationships in rat liver mitochondria. The criterion is based on the view that the nature of the relationship between the H+/O ratio and the membrane potential can be inferred from the relationship between ohmic-uncoupler-induced extra respiration and the membrane potential. Thus a linear relationship between extra respiration and membrane potential indicates unequivocally the independence of the H+/O ratio from the membrane potential and the leak nature of the resting respiration [Brand, Chien, and Diolez (1994) Biochem. J. 297, 27-29]. On the other hand, a non-linear relationship indicates that the H+/O ratio is dependent on the membrane potential. The experimental assessment of this relationship in the presence of an additional ohmic leak, however, is rendered difficult by both the uncoupler-induced depression of membrane potential and the limited range of dependence of the H+/O ratio on the membrane potential. We have selected conditions, i.e. incubation of mitochondria at low temperatures, where the extent of non-linearity is markedly increased. It appears that the nature of the resting respiration of mitochondria in vitro is markedly dependent on the temperature: at low temperatures the percentage of resting respiration due to membrane leak decreases and that due to intrinsic uncoupling of the proton pumps increases. uncoupling of the proton pumps increases.)
Chinopoulos 2009 + (A novel method exploiting the differential … A novel method exploiting the differential affinity of ADP and ATP to Mg(2+) was developed to measure mitochondrial ADP-ATP exchange rate. The rate of ATP appearing in the medium after addition of ADP to energized mitochondria, is calculated from the measured rate of change in free extramitochondrial [Mg2+] reported by the membrane-impermeable 5K+ salt of the Mg2+-sensitive fluorescent indicator, Magnesium Green, using standard binding equations. The assay is designed such that the adenine nucleotide translocase (ANT) is the sole mediator of changes in [Mg2+] in the extramitochondrial volume, as a result of ADP-ATP exchange. We also provide data on the dependence of ATP efflux rate within the 6.8-7.8 matrix pH range as a function of membrane potential. Finally, by comparing the ATP-ADP steady-state exchange rate to the amount of the ANT in rat brain synaptic, brain nonsynaptic, heart and liver mitochondria, we provide molecular turnover numbers for the known ANT isotypes. and liver mitochondria, we provide molecular turnover numbers for the known ANT isotypes.)
Sjoevall 2015 US Patent + (A novel method useful in drug screening. T … A novel method useful in drug screening. The method is useful for testing effects of substances on the mitochondria, notably toxic or beneficial effects of drug substances or candidate drug substances. The method is based on measurement in live human mitochondria ''ex vivo'', but in a setting as near the ''in vivo'' situation as possible. The method is also useful for testing substances impact on the mitochondrial respiration. The method can be used to i) screening and selection of early or late stage drug candidates in cells derived from blood from healthy individuals or in so-called buffy coat, which is a concentrated solution of platelets and white blood cells, ii) testing a patient's sensitivity to a known mitochondrial toxicant, iii) analysing mitochondrial drug toxicity in clinical trials, and/or iv) analysing beneficial effects of drugs intended to improve mitochondrial function.ntended to improve mitochondrial function.)
Farrar 2015 US Patent + (A nucleic acid sequence encoding the yeast … A nucleic acid sequence encoding the yeast NDI1 protein of SEQ ID NO: 542 or a functional variant thereof having at least 90% sequence identity with SEQ ID NO: 2 is described. The nucleic acid sequence comprises at least 50 codons which are codon optimised compared with the sequence of yeast NDI1 gene of SEQ ID NO: 1. An immune optimised functional variant of the yeast NDI1 protein of SEQ ID NO: 542 having at least 90% sequence identity with SEQ ID NO: 542 is also described, and includes at least one amino acid change selected from the group consisting of L194F, K283E, K9R, S142N, L501M, L402I, A386S, S85K, F89H, L93M, K195E, L18M, K213E, K372E, L258F, K510E, L158M, R478Q, L482M. K372E, L258F, K510E, L158M, R478Q, L482M.)
Bentinger 2007 Mitochondrion + (A number of functions for coenzyme Q (CoQ) … A number of functions for coenzyme Q (CoQ) have been established during the years but its role as an effective antioxidant of the cellular membranes remains of dominating interest. This compound is our only endogenously synthesized lipid soluble antioxidant, present in all membranes and exceeding both in amount and efficiency that of other antioxidants. The protective effect is extended to lipids, proteins and DNA mainly because of its close localization to the oxidative events and the effective regeneration by continuous reduction at all locations. Its biosynthesis is influenced by nuclear receptors which may give the possibility, in the future, by using agonists or antagonists, of reestablishing the normal level in deficiencies caused by genetic mutations, aging or cardiomyopathy. An increase in CoQ concentration in specific cellular compartments in the presence of various types of oxidative stress appears to be of considerable interest.ss appears to be of considerable interest.)
Bastow 2016 J Cell Sci + (A number of genes have been linked to fami … A number of genes have been linked to familial forms of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Over 150 mutations within the gene encoding superoxide dismutase 1 (SOD1) have been implicated in ALS, but why such mutations lead to ALS-associated cellular dysfunction is unclear. In this study, we identify how ALS-linked SOD1 mutations lead to changes in the cellular health of the yeast ''Saccharomyces cerevisiae''. We find that it is not the accumulation of aggregates but the loss of Sod1 protein stability that drives cellular dysfunction. The toxic effect of Sod1 instability does not correlate with a loss of mitochondrial function or increased production of reactive oxygen species, but instead prevents acidification of the vacuole, perturbs metabolic regulation and promotes senescence. Central to the toxic gain-of-function seen with the SOD1 mutants examined was an inability to regulate amino acid biosynthesis. We also report that leucine supplementation results in an improvement in motor function in a ''Caenorhabditis elegans'' model of ALS. Our data suggest that metabolic dysfunction plays an important role in Sod1-mediated toxicity in both the yeast and worm models of ALS.© 2016. Published by The Company of Biologists Ltd.ublished by The Company of Biologists Ltd.)
Ruiz-Pesini 2004 Science + (A phylogenetic analysis of 1125 global hum … A phylogenetic analysis of 1125 global human mitochondrial DNA (mtDNA) sequences permitted positioning of all nucleotide substitutions according to their order of occurrence. The relative frequency and amino acid conservation of internal branch replacement mutations was found to increase from tropical Africa to temperate Europe and arctic northeastern Siberia. Particularly highly conserved amino acid substitutions were found at the roots of multiple mtDNA lineages from higher latitudes. These same lineages correlate with increased propensity for energy deficiency diseases as well as longevity. Thus, specific mtDNA replacement mutations permitted our ancestors to adapt to more northern climates, and these same variants are influencing our health today.variants are influencing our health today.)
Berg 2016 Science + (A preprint is a complete scientific manusc … A preprint is a complete scientific manuscript (often one also being submitted to a peer-reviewed journal) that is uploaded by the authors to a public server without formal review. After a brief inspection to ensure that the work is scientific in nature, the posted scientific manuscript can be viewed without charge on the Web.Conclusions: Preprints could play important roles in accelerating scientific progress; they could serve the needs and foster the careers of scientists; and, in cooperation with existing journals, they could enhance the current system for communicating results and ideas in the life sciences. However, preprints are relatively new to biology, and many questions remain unanswered. Will funding agencies encourage the use of preprint servers? Will all journals accept manuscripts for publication after they have been disseminated as preprints? Will the life sciences community find ways to make biology preprints easily discoverable? And will researchers themselves decide to submit, cite, and evaluate work presented in preprint form? The cooperative spirit displayed by the attendees at ASAPbio gives hope that these complex issues, as well as others that limit the communication of scientific ideas and results, can be addressed in a productive and thoughtful manner.sed in a productive and thoughtful manner.)
Committee 2018 COPE Discussion Document + (A preprint is a scholarly manuscript poste … A preprint is a scholarly manuscript posted by the author(s) in an openly accessible platform, usually before or in parallel with the peer review process. While the sharing of manuscripts via preprint platforms has been common in some disciplines (such as physics and mathematics) for many years, uptake in other disciplines traditionally had been low, possibly influenced by differences in research culture and strong opposition by some journal publishers [1]. The landscape has evolved rapidly in other fields in recent years, however, thanks to the launch of additional, discipline-specific preprint platforms and increased support by funders and initiatives such as ASAPBio[2, 3].ers and initiatives such as ASAPBio[2, 3].)
Gnaiger 1991 Soc Exp Biol Seminar Series + (A previous critique of the term facultative invertebrate anaerobiosis focused on the duration of anoxia. An addition, important, yet much neglected aspect is dicussed here, namely the extent and quantification of the 'anaerobic' condition.)
Podrabsky 2000 Am J Physiol Regul Integr Comp Physiol + (A previous phylogenetic analysis among 15 … A previous phylogenetic analysis among 15 taxa of the teleost fish ''Fundulus'' suggested that there should be thermal-adaptive differences in heart metabolism among populations. To test this hypothesis, the rate of oxygen consumption and the activities of all 11 glycolytic enzymes were measured in isolated heart ventricle from two populations of ''Fundulus heteroclitus''. Heart ventricular metabolism is greater in a northern population versus a southern population of these fish. Analysis of the amount of glycolytic enzymes indicates that 87% of the variation in cardiac metabolism within and between populations is explained by the variation in three enzymes (pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase). These enzymes are the same three enzymes that were predicted to be important based on previously determined phylogenetic patterns of expression. Our data indicate that near-equilibrium enzymes, as well as classically defined rate-limiting enzymes, can also influence metabolism.ng enzymes, can also influence metabolism.)
Gurakan 1990 Thermochim Acta + (A procedure has been developed for the pre … A procedure has been developed for the preparation of microbial biomass of standard, defined quality suitable for the determination of elemental composition and enthalpy of combustion. Furthermore methods for the determination of residual moisture and ash content of biomass samples have been established. The results indicate that samples should be prepared in a freeze-dried (lyophilized) state and that residual moisture content should be determined immediately prior to sample preparation for combustion calorimetry and elemental analysis. Results from such analysis should then be related to material which is first freeze- dried and subsequently oven dried (100 °C for 24 hours) as reference state. The method outlined here for microbial biomass should prove suitable for biological samples from a wide variety of sources including both pure proteins, fats etc. as well as cells and tissues.s, fats etc. as well as cells and tissues.)
Bradford 1976 Anal Biochem + (A protein determination method which invol … A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls. eliminated by the use of proper controls.)
Lam 1967 Arch Biochem Biophys + (A protein factor, designated as Factor B, … A protein factor, designated as Factor B, was extracted from lyophilized acetone-washed bovine heart mitochondria and purified by ammonium sulfate fractionation, and ion-exchange chromatography on DEAE-cellulose and CM-cellulose. Centrifugation in a sucrose density gradient showed that the activity of the purified factor was closely associated with a symmetrical protein peak comprising approximately 70% of the protein. Its molecular weight was estimated to be 32,000, using hemoglobin and cytochrome c as markers. Factor B produces several-fold stimulation of ATP-driven NAD reduction, and of net phosphorylation coupled to NADH or succinate oxidation in ammonia particles. The stimulation of ATP-driven NAD reduction activity exceeds that given by an optimal amount of oligomycin, and in the presence of a saturation level of Factor B, oligomycin stimulation disappears. Also, Factor B stimulation is evident in urea-depleted particles which have been supplemented by Factor A. These particles show no stimulation by oligomycin. The results suggest that Factor B may participate in the energy transfer reactions between the respiratory chain and the terminal step resulting in ATP synthesis. terminal step resulting in ATP synthesis.)
Wijermars 2016 Am J Transplant + (A recent seminal paper implicated ischemia … A recent seminal paper implicated ischemia-related succinate accumulation followed by succinate driven-reactive oxygen species formation as key driver of ischemia reperfusion injury. Although the data show that the mechanism is universal for all organs tested (kidney, liver, heart and brain), a remaining question is to what extend these observations for mouse translate to man. We here show that succinate accumulation is not a universal event during ischemia, and does not occur during renal graft procurement, in fact tissue succinate content progressively decreases with advancing graft ischemia time (p<0.007). Contrasting responses were also found with respect to mitochondrial susceptibility towards ischemia and reperfusion, with rodent mitochondria robustly resistant towards warm ischemia, but human and pig mitochondria being highly susceptible to warm ischemia (p<0.05). These observations suggest that succinate-driven reactive oxygen formation does not occur in the context of kidney transplantation. In fact absent allantoin release from the reperfused grafts suggests minimal oxidative stress during clinical reperfusion.This article is protected by copyright. All rights reserved. is protected by copyright. All rights reserved.)
Tomporowski 2003 Acta Psychol (Amst) + (A review was conducted of studies that ass … A review was conducted of studies that assessed the effects of acute bouts of physical activity on adults' cognitive performance. Three groups of studies were constituted on the basis of the type of exercise protocol employed. Each group was then evaluated in terms of information-processing theory. It was concluded that submaximal aerobic exercise performed for periods up to 60 min facilitate specific aspects of information processing; however, extended exercise that leads to dehydration compromises both information processing and memory functions. The selective effects of exercise on cognitive performance are explained in terms of Sanders' [Acta Psychol. 53 (1983) 61] cognitive-energetic model.. 53 (1983) 61] cognitive-energetic model.)
Meunier 1995 Biochemistry + (A screen has been performed of possible in … A screen has been performed of possible inhibitors of the quinol oxidation sites of the two terminal oxidases of ''Escherichia coli'', cytochromes bo and bd. Aurachin C and its analogues were found to be particularly effective inhibitors of both enzymes, whereas aurachin D and its analogues displayed a selectivity for inhibition of cytochrome bd. In addition, a tridecyl derivative of stigmatellin was found to inhibit cytochrome bo at concentrations which were without significant effect on cytochrome bd. Titration of membrane-bound cytochromes bo and bd with aurachin C gave an observed dissociation constant in the range of 10-8 M. A similar observed dissociation constant was determined for aurachin D inhibition of cytochrome bd. For both enzymes, their kinetic behavior during a series of substrate pulses indicates that it is reduction of the enzyme by quinol, and not reaction with oxygen, which is inhibited. It is concluded that the aurachins are powerful inhibitors of the quinol oxidation sites of bacterial cytochromes bo and bd. The effects of aurachin C on cytochrome bo were investigated in more detail. The number of inhibitor binding sites on the purified enzyme was determined by titration to be 0.6 per enzyme. At an inhibitorloxidase ratio of 1.0, electron donation into the enzyme from added quinol is extremely slow, making it very unlikely that there is more than one quinone-reactive site. Aurachin C caused a potent inhibition of electron donation from a pulse of quinol. In contrast, it was without effect on cyanide or carbon monoxide binding to the reduced enzyme, on cyanide binding to the oxidized enzyme, on the optical spectra of the heme groups, or on the kinetics of oxygen reduction after photolysis of carbon monoxide from the reduced enzyme. We conclude that binding of aurachin C specifically inhibits the quinol oxidation site and does not directly affect the properties of the binuclear center.irectly affect the properties of the binuclear center.)
Bers 1982 Am J Physiol + (A simple method for the accurate determina … A simple method for the accurate determination of free [Ca] in ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-buffered Ca solutions is described. This method is useful for calibration of Ca macro- and microelectrodes to low free [Ca] and should improve the reliability of calculated free [Ca] in more complex solutions. Briefly, free [Ca] in Ca-EGTA solutions is measured with a Ca electrode, bound Ca is calculated, and Scatchard and double-reciprocal plots are resolved for the total [EGTA] and the apparent Ca-EGTA association constant (K'Ca) in the solutions used. The free [Ca] is then recalculated using the determined parameters, giving a more accurate knowledge of the free [Ca] in these solutions and providing an accurate calibration curve for the Ca electrode. These solutions can then be used to calibrate other Ca electrodes (e.g., Ca microelectrodes) or the calibrated Ca electrode can be used to measure free [Ca] in solutions containing multiple metal ligands. This method allows determination of free [Ca], K'Ca, and total [EGTA] in the actual solutions used regardless of pH, temperature, or ionic strength. It does not require accurate knowledge of K'Ca or EGTA purity and circumvents many potential errors due to assumption of binding parameters. K'Ca was found to be 2.45 +/- 0.04 X 10(6) M-1 in 100 mM KCl, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 1 mM EGTA at pH 7.00 and 23 degrees C. Total [EGTA] varied with supplier but was always less than quoted. supplier but was always less than quoted.)
Stokich 2014 Cryobiology + (A simple method to cryogenically preserve … A simple method to cryogenically preserve hepatocyte monolayers is currently not available but such a technique would facilitate numerous applications in the field of biomedical engineering, cell line development, and drug screening. We investigated the effect of trehalose and dimethyl sulfoxide (Me2SO) in cryopreservation of human hepatocellular carcinoma (HepG2) cells in suspension and monolayer formats. HepG2 cell monolayers were incubated for 24 h at varying concentrations of trehalose (50-150 mM) prior to cryopreservation to identify the optimum concentration for such preincubation. When trehalose alone was used as the cryoprotective agent (CPA), cells in monolayer format did not survive freezing while cells in suspension demonstrated 14% viability 24 h after thawing. Only 6-13% of cells in monolayers survived freezing in cell culture medium supplemented with 10% Me2SO, but 42% of cells were recovered successfully if monolayers were preincubated with 100 mM trehalose prior to freezing in the Me2SO supplemented medium. Interestingly, for cells frozen in suspension in presence of 10% Me2SO, metabolic activity immediately following thawing did not change appreciably compared to unfrozen control cells. Finally, Raman spectroscopy techniques were employed to evaluate ice crystallization in the presence and absence of trehalose in freezing solutions without cells because crystallization may alter the extent of injury observed in cell monolayers. We speculate that biomimetic approaches of using protective sugars to preserve cells in monolayer format will facilitate the development of techniques for long-term preservation of human tissues and organs in the future.of human tissues and organs in the future.)
Small 1985 Biochem J + (A simple spectrophotometric assay was deve … A simple spectrophotometric assay was developed for peroxisomal fatty [[acyl-CoA oxidase]] activity. The assay, based on the H2O2-dependent oxidation of leuco-dichlorofluorescein catalysed by exogenous [[peroxidase]], is more sensitive than methods previously described. By using mouse liver samples, cofactor requirements were assessed and a linear relationship was demonstrated between dye oxidation and enzyme concentration. By using this assay on subcellular fractions, palmitoyl-CoA oxidase activity was localized for the first time in microperoxisomes of rat intestine. The assay was also adapted to measure D-amino acid oxidase activity, demonstrating the versatility of this method for measuring activity of other H2O2-producing oxidases.g activity of other H2O2-producing oxidases.)
Petrova 2014 Proc Chem + (A simple, accurate and rapid voltammetric … A simple, accurate and rapid voltammetric method has been developed for the quantitative determination of coenzyme Q10. Studies with direct current voltammetry were carried out using a glassy carbon electrode (GCE) in a phosphate buffer solution (pH 6.86). A well-defined oxidation peak of CoQ10 was obtained at -0.600 V vs Ag/AgCl. The magnitude of the oxidation peak current has been found to be related to the concentration of the coenzyme over the range of (2·10-5 to 2·10-4 M) (''r'' = 0.991). Antioxidant activity of CoQ10 was investigated.) (''r'' = 0.991). Antioxidant activity of CoQ10 was investigated.)
Lin 2012 FASEB J + (A single high-fat meal acutely increases s … A single high-fat meal acutely increases skeletal muscle mitochondrial H2O2 emitting potential (mEH2O2), shifts the intracellular redox environment to a more oxidized state, and increases circulating markers of oxidative stress. Bioenergetically, this implies an acute lipid load may elevate the reducing pressure/membrane potential ({Delta}{Psi}m) within mitochondria and, conversely, that even a mild increase in energy expenditure may be sufficient to prevent these effects. To test this hypothesis, male Sprague-Dawley rats received an oral lipid gavage (20% intralipid, 45 Kcal/kg lean body mass) or water followed either by 2h of rest or 1h of rest plus 1h of low intensity treadmill exercise (15 m/min, 0% grade). Permeabilized fiber bundles were prepared from red gastrocnemius muscle for testing mitochondrial function. In rats receiving lipid, {Delta}{Psi}m and mEH2O2 were higher (P<0.05) and calcium retention capacity (mCa2+RC, an index of resistance to mitochondrial permeability transition) was lower under state IV and/or "clamped" ADP-stimulated state III conditions. All three effects were prevented when lipid gavage was followed by low-intensity exercise. Respiratory capacity was unaffected by any of the interventions. These findings provide evidence that mitochondrial {Delta}{Psi}m, mEH2O2, and mCa2+RC are acutely affected by nutritional overload in skeletal muscle, but can be prevented by low intensity exercise. NIH DK073488ented by low intensity exercise. NIH DK073488)
Ortega 2017 Biol Reprod + (A single missense mutation at position 159 … A single missense mutation at position 159 of coenzyme Q9 (COQ9) (G→A; rs109301586) has been associated with genetic variation in fertility in Holstein cattle, with the A allele associated with higher fertility. COQ9 is involved in the synthesis of coenzyme COQ10, a component of the electron transport system of the mitochondria. Here we tested whether reproductive phenotype is associated with the mutation and evaluated functional consequences for cellular oxygen metabolism, body weight changes, and ovarian function. The mutation in COQ9 modifies predicted tertiary protein structure and affected mitochondrial respiration of peripheral blood mononuclear cells. The A allele was associated with low resting oxygen consumption and high electron transport system capacity. Phenotypic measurements for fertility were evaluated for up to five lactations in a population of 2273 Holstein cows. There were additive effects of the mutation (P < 0.05) in favor of the A allele for pregnancy rate, interval from calving to conception, and services per conception. There was no association of genotype with milk production or body weight changes ''postpartum''. The mutation in COQ9 affected ovarian function; the A allele was associated with increased mitochondrial DNA copy number in oocytes, and there were overdominance effects for COQ9 expression in oocytes, follicle number, and antimullerian hormone concentrations. Overall, results show how a gene involved in mitochondrial function is associated with overall fertility, possibly in part by affecting oocyte quality.possibly in part by affecting oocyte quality.)