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Insight into mammalian respiratory complexes defines the role of allosteric protein interactions in their proton-motive activity. In cytochrome c oxidase (CxIV) conformational change of subunit I, caused by O 2 binding to heme a 3 2+-Cu B + and reduction, and stereochemical transitions coupled to oxidation/reduction of heme a and Cu A , combined with electrostatic effects, determine the proton pumping activity. In ubiquinone-cytochrome c oxidoreductase (CxIII) conformational movement of Fe–S protein between cytochromes b and c 1 is the key element of the proton-motive activity. In NADH-ubiquinone oxidoreductase (CxI) ubiquinone binding and reduction result in conformational changes of subunits in the quinone reaction structure which initiate proton pumping. • The role of allosteric protein interactions in mammalian respiratory complexes. • Conformational changes and electrostatic effects determine proton pumping in CxIV. • Role of conformational movement of Fe–S protein in proton-motive activity of CxIII. • Redox coupled subunits conformational changes initiate proton pumping in CxI. [ABSTRACT FROM AUTHOR]

Seed germination is an energy demanding process that requires functional mitochondria upon imbibition. However, how mitochondria fine tune seed germination, especially in response to the dynamics of environmental temperature, remains largely unknown at the molecular level. Here, we report a mitochondrial matrix-localized heat shock protein GhHSP24.7, that regulates seed germination in a temperature-dependent manner. Suppression of GhHSP24.7 renders the seed insensitive to temperature changes and delays germination. We show that GhHSP24.7 competes with GhCCMH to bind to the maturation subunit protein GhCcmFc to form cytochrome C/C1 (CytC/C1) in the mitochondrial electron transport chain. GhHSP24.7 modulates CytC/C1 production to induce reactive oxygen species (ROS) generation, which consequently accelerates endosperm rupture and promotes seed germination. Overexpression of GhHSP24.7's homologous genes can accelerate seed germination in Arabidopsis and tomato, indicating its conserved function across plant species. Therefore, HSP24.7 is a critical factor that positively controls seed germination via temperature-dependent ROS generation. [ABSTRACT FROM AUTHOR]

Bemisia tabaci (Gennadius) is a species complex consisting of at least 40 cryptic species. Although the genetic diversity of B. tabaci has been studied in various regions, little is known about distribution in Bangladesh, which is covered by the Bengal delta, the largest delta on Earth. We conducted an extensive survey throughout the country and determined the nucleotide sequence of mitochondrial cytochrome c oxidase subunit 1 (COI) from 110 individuals. We then examined phylogenetic relationships. The results identified four cryptic species that expressed distinct interspecific variation but low intraspecific variation. Asia I was the most abundant, both Asia II 1 and Asia II 5 were moderately abundant, and Asia II 10 was found only in the central region. COI sequences of each cryptic species were distinctive and differentiated into many haplotypes. Our study provides important information to better understand the genetic diversity and geographic distribution of cryptic species in Bangladesh and nearby countries. [ABSTRACT FROM AUTHOR]

In mitochondria, copper is a Janus-faced trace element. While it is the essential cofactor of the mitochondrial cytochrome c oxidase, a surplus of copper can be highly detrimental to these organelles. On the one hand, mitochondria are strictly dependent on adequate copper supply for proper respiratory function, and the molecular mechanisms for metalation of the cytochrome c oxidase have been largely characterized. On the other hand, copper overload impairs mitochondria and uncertainties exist concerning the molecular mechanisms for mitochondrial metal uptake, storage and release. The latter issue is of fundamental importance in Wilson disease, a genetic disease characterized by dysfunctional copper excretion from the liver. Prime consequences of the progressive copper accumulation in hepatocytes are increasing mitochondrial biophysical and biochemical deficits. Focusing on this two-sided aspect of mitochondrial copper, we review mitochondrial copper homeostasis but also the impact of excessive mitochondrial copper in Wilson disease. [ABSTRACT FROM AUTHOR]

A study was conducted to investigate genetic diversity of Echinococcus isolates collected in Hungarian slaughterhouses between 2015 and 2018. Organs of 219 animals with suspected hydatidosis were collected during routine meat inspection and sent to our laboratory. Echinococcus infection was confirmed in 65 animals. These results indicate that prevalence data based on reporting of slaughterhouses are not reliable without the appropriate training of the people responsible for meat inspection. The genetic diversity was evaluated by the DNA sequence analysis of the cytochrome c oxydase subunit 1 (cox1) mitochondrial gene. Echinococcus intermedius (n = 31), Echinococcus granulosus s.s. (n = 2), and Echinococcus multilocularis (n = 3) was identified in swine. In cattle, only E. granulosus s.s. (n = 20) was detected. E. granulosus s.s. (n = 7) was the dominant species in sheep; nevertheless, E. intermedius was also identified in two animals. E. granulosus s.s. (n = 29) and E. intermedius (n = 33) were classified in 13 and three haplotypes, respectively. The genetic diversity and haplotype network of E. granulosus s.s. were similar to that observed in some other countries of Eastern Europe. The genetic diversity of E. intermedius was low with a single dominant haplotype. Cysts were fertile in nine sheep (100%), 22 swine (61%), and three cattle (15%) indicating that all three species play a role in some extent in the epidemiology of cystic echinococcosis in Hungary. Based on the number of animals killed in the slaughterhouses involved in the present study, the rate of infection was 0.013% in sheep, 0.007% in cattle, and 0.001% in swine. As animals with hydatidosis originated from family farms, control programs should mainly focus on these facilities. [ABSTRACT FROM AUTHOR]

The catalytic cycle of cytochrome c oxidase (COX) couples the reduction of oxygen to the translocation of protons across the inner mitochondrial membrane and involves several intermediate states of the heme a 3 -Cu B binuclear center with distinct absorbance properties. The absorbance maximum close to 605 nm observed during respiration is commonly assigned to the fully reduced species of hemes a or a 3 (R). However, by analyzing the absorbance of isolated enzyme and mitochondria in the Soret (420–450 nm), alpha (560–630 nm) and red (630–700 nm) spectral regions, we demonstrate that the Peroxy (P) and Ferryl (F) intermediates of the binuclear center are observed during respiration, while the R form is only detectable under nearly anoxic conditions in which electrons also accumulate in the higher extinction coefficient low spin a heme. This implies that a large fraction of COX (>50 %) is active, in contrast with assumptions that assign spectral changes only to R and/or reduced heme a. The concentration dependence of the COX chromophores and reduced c -type cytochromes on the transmembrane potential (ΔΨ m) was determined in isolated mitochondria during substrate or apyrase titration to hydrolyze ATP. The cytochrome c -type redox levels indicated that soluble cytochrome c is out of equilibrium with respect to both Complex III and COX. Thermodynamic analyses confirmed that reactions involving the chromophores we assign as the P and F species of COX are ΔΨ m -dependent, out of equilibrium, and therefore much slower than the ΔΨ m -insensitive oxidation of the R intermediate, which is undetectable due to rapid oxygen binding. • Reduction of mitochondrial cytochrome c oxidase hemes not detected during respiration. • Spectral fitting identifies absorbance peaks as species formed after oxygen binding. • Cytochrome c redox state shows electron transfer to oxygen is out of equilibrium. • Membrane potential slows conversion rate of intermediates relative to oxygen binding. [ABSTRACT FROM AUTHOR]

In Japan, Globodera pallida was first reported in Abashiri city, Hokkaido in 2015. Sequences of ribosomal DNA (rDNA) and mitochondrial cytochrome b gene (cytb) from the Japanese population of G. pallida were compared with those of overseas populations. The representative sequences for rDNA and for cytb from the Japanese population had 100% identity with the sequences from G. pallida populations of North America and Europe in GenBank. Phylogenetic analysis also showed that they clustered in the clade that included populations from Europe and North America. From these results, we concluded that the Japanese population of G. pallida is close to populations that have been reported in Europe and North America. Recent studies suggested that a few populations of G. pallida were introduced into Europe from South America and have spread to various places around the world, and according to our results, into Japan also. [ABSTRACT FROM AUTHOR]

Amyloid β (Aβ)1-42 is strongly associated with Alzheimer's disease (AD). The effects of Aβ1‑42 on astrocytes remain largely unknown. The present study focused on the effects of Aβ1‑42 on U87 human glioblastoma cells as astrocytes for in vitro investigation and mouse brains for in vivo investigation. The mechanism and regulation of mitochondria and cytochrome P450 reductase (CPR) were also investigated. As determined by MTT assays, low doses of Aβ1‑42 (<1 µM) marginally promoted astrocytosis compared with the 0 µM group within 24 h, however, after 48 h treatment these doses reduced cellular growth compared with the 0 µM group. Furthermore, Aβ1‑42 doses >5 µM inhibited the growth of U87 cells compared with the 0 µM group after 24 and 48 h treatment. Immunofluorescence analysis demonstrated that astrocytosis was also observed in early stage AD mice compared with wild‑type (WT) mice. In addition, concentrations of Aβ1‑42 were also significantly higher in early stage AD mice compared with WT mice, however, the levels were markedly lower compared with later stage AD mice, as determined by ELISA. In addition to increased levels of Aβ1‑42 in mice with later stage AD, reduced astrocyte staining was observed compared with WT mice. Western blotting indicated that the effect of Aβ1‑42 on U87 cell apoptosis may be regulated via Bcl‑2 and caspase‑3 located in mitochondria, whose functions, including adenosine triphosphate generation, electron transport chain and mitochondrial membrane potential, were inhibited by Aβ1‑42. During this process, the expression and activity of cytochrome P450 reductase was also downregulated. The current study provides novel insight into the effects of Aβ1‑42 on astrocytes and highlights a potential role for astrocytes in the protection against AD. [ABSTRACT FROM AUTHOR]

Inhibitors of the mitochondrial respiratory chain cytochrome bc1 complex, such as the antimalarial atovaquone and ELQ‐300, and many well‐studied compounds, are classified as either Qo or Qi site inhibitors based on their site of action. Here, we investigated the site of action of ELQ‐400 that showed an unusual behaviour, being effective against parasites resistant to the Qo site inhibitor atovaquone or to the Qi site inhibitor ELQ‐300. Analysis of yeast mutants and comparison with atovaquone and other ELQs strongly suggest that ELQ‐400 targets both Qo and Qi sites. Dual site inhibition would be particularly efficient as it would lower the risk of acquired resistance. However, such compounds are seldom found, which could be explained by structural and mechanistic differences between the sites. [ABSTRACT FROM AUTHOR]

Absorbance spectroscopy of intrinsic cardiac chromophores provides nondestructive assessment of cytosolic oxygenation and mitochondria redox state. Isolated perfused heart spectroscopy is usually conducted by collecting reflected light from the heart surface, which represents a combination of surface scattering events and light that traversed portions of the myocardium. Reflectance spectroscopy with complex surface scattering effects in the beating heart leads to difficulty in quantitating chromophore absorbance. In this study, surface scattering was minimized and transmural path length optimized by placing a light source within the left ventricular chamber while monitoring transmurally transmitted light at the epicardial surface. The customdesigned intrachamber light catheter was a flexible coaxial cable (2.42-Fr) terminated with an encapsulated side-firing LED of 1.8 x 0.8 mm, altogether similar in size to a Millar pressure catheter. The LED catheter had minimal impact on aortic flow and heart rate in Langendorff perfusion and did not impact stability of the left ventricule of the working heart. Changes in transmural absorbance spectra were deconvoluted using a library of chromophore reference spectra to quantify the relative contribution of specific chromophores to the changes in measured absorbance. This broad-band spectral deconvolution approach eliminated errors that may result from simple dualwavelength absorbance intensity. The myoglobin oxygenation level was only 82.2 ± 3.0%, whereas cytochrome c and cytochrome a + a3 were 13.3 ± 1.4% and 12.6 ± 2.2% reduced, respectively, in the Langendorff-perfused heart. The intracardiac illumination strategy permits transmural optical absorbance spectroscopy in perfused hearts, which provides a noninvasive real-time monitor of cytosolic oxygenation and mitochondria redox state. [ABSTRACT FROM AUTHOR]

In this work, we studied the protective effects of tamoxifen (TAM) on disulfiram (Dis)-induced mitochondrial membrane insult. The results indicate that TAM circumvents the inner membrane leakiness manifested as Ca2+ release, mitochondrial swelling, and collapse of the transmembrane electric gradient. Furthermore, it was found that TAM prevents inactivation of the mitochondrial enzyme aconitase and detachment of cytochrome c from the inner membrane. Interestingly, TAM also inhibited Dis-promoted generation of hydrogen peroxide. Given that TAM is an antioxidant molecule, it is plausible that its protection may be due to the inhibition of Dis-induced oxidative stress. [ABSTRACT FROM AUTHOR]

We propose a novel approach to assess simultaneously lipid composition in lipid droplets, the redox state of cytochromes, and the relative amount of [Fe-S] clusters in the electron transport chain in the mitochondria of skeletal myocytes by means of near-infrared Raman spectroscopy. Mitochondria dysfunction, disruption of oxidative phosphorylation, and change in lipid metabolism may be a consequence of the obesity-related insulin resistance and diabetes mellitus. Lipid metabolism affects the state of the mitochondrial electron transport chain although these relations are not well understood. We demonstrate the applicability of the proposed approach in a case study of myocytes of an obese patient before and after the gastric bypass surgery in comparison with a healthy lean donor. Ratios from chosen Raman peaks were calculated and compared between the different subjects. We show that the suggested technique allows to estimate qualitatively the relative amount of cholesterol and unsaturated lipids, ordering of lipid phase in lipid droplets, changes in the redox state of c-type and b-type cytochromes, and the relative amount of [Fe-S] clusters in the mitochondria of intact myocytes. The proposed approach opens perspectives to uncover relations between the functional state of mitochondrial cytochromes and lipid composition and to develop a monitoring tool once it is applied to a wide cohort of patients. Copyright © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Bonefish leptocephali of the genus Albula are difficult to identify to the species level due to morphological similarities between two different species present in the Northeastern Pacific Ocean, A. esuncula and A. gilberti. In this study, 22 bonefish leptocephali (premetamorphic and early metamorphic), collected from two locations in the southern Gulf of California were identified as Albula gilberti by comparing 459 bp of their mitochondrial cytochrome b gene sequences to those of four other species of bonefish. The characteristics of these A. gilberti leptocephali were compared to those of previously described bonefish leptocephali in the region. No distinctive morphological characteristics (meristic and pigmentation) were found that differentiate premetamorphic leptocephali of A. gilberti from those of other Albula species, making species identification by molecular-genetics a necessity. In early metamorphic leptocephali some differences in horizontal eye diameter-head length ratio, number of rays of pelvic and anal fins and myomere of pelvic-fin origin may help to differentiate A. gilberti from A. esuncula. [ABSTRACT FROM AUTHOR]

Mitochondria evolved as an endosymbiont providing the cell with a dizzying array of catabolic and anabolic processes essential for life. However, mitochondria have retained the ability to kill from within, and are widely considered the final executioners of programmed cell death. The groundbreaking discovery over 25 years ago that mitochondrial cytochrome c is released into the cytosol shone new and unexpected light onto this old organelle, revitalizing the field. The Bcl-2 family of proteins plays a central role in the maintenance of mitochondrial membrane integrity, but other factors are also involved in the cell death program. Indeed, contacts with the endoplasmic reticulum (ER), mitochondrial division and inner membrane cristae remodeling have emerged as key regulators of cytochrome c release. This review will focus on recent progress to define the functional contribution of the apoptotic ER/mitochondrial interface, which couples mitochondrial fission and cristae remodeling to calcium and lipid fluxes. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase (CcO) is the last electron acceptor in the respiratory chain. The CcOcore is formed by mitochondrial DNA-encoded Cox1, Cox2, and Cox3 subunits. Cox1 synthesis is highly regulated; for example, if CcO assembly is blocked, Cox1 synthesis decreases. Mss51 activates translation of COX1 mRNA and interacts with Cox1 protein in high-molecularweight complexes (COA complexes) to form the Cox1 intermediary assembly module. Thus, Mss51 coordinates both Cox1 synthesis and assembly. We previously reported that the last 15 residues of the Cox1 C terminus regulate Cox1 synthesis by modulating an interaction of Mss51 with Cox14, another component of the COA complexes. Here, using site-directed mutagenesis of the mitochondrial COX1 gene from Saccharomyces cerevisiae, we demonstrate that mutations P521A/P522A and V524E disrupt the regulatory role of the Cox1 C terminus. These mutations, as well as C terminus deletion (Cox1▵C15), reduced binding of Mss51 and Cox14 toCOAcomplexes. Mss51 was enriched in a translationally active form that maintains full Cox1 synthesis even if CcO assembly is blocked in these mutants. Moreover, Cox1 ▵C15, but not Cox1-P521A/P522A and Cox1-V524E, promoted formation of aberrant supercomplexes in CcOassembly mutants lacking Cox2 or Cox4 subunits. The aberrant supercomplex formation depended on the presence of cytochrome b and Cox3, supporting the idea that supercomplex assembly factors associate with Cox3 and demonstrating that supercomplexes can be formed even if CcO is inactive and not fully assembled. Our results indicate that the Cox1 C-terminal end is a key regulator of CcObiogenesis and that it is important for supercomplex formation/stability. [ABSTRACT FROM AUTHOR]

In the past, divergent results have been reported based on different methods and conditions used for enzymatic activity measurements of cytochrome c oxidase (CytOx). Here, we analyze in detail and show comparable and reproducible polarographic activity measurements of ATP-dependent inhibition of CytOx kinetics in intact and non-intact rat heart mitochondria and mitoplasts. We found that this mechanism is always present in isolated rat heart mitochondria and mitoplasts; however, it is measurable only at high ATP/ADP ratios using optimal protein concentrations. In the kinetics assay, measurement of this mechanism is independent of presence or absence of Tween-20 and the composition of measuring buffer. Furthermore, the effect of atractyloside on intact rat heart mitochondria confirms that (i) ATP inhibition occurs under uncoupled conditions [in the presence of carbonly cyanide m-chlorophenyl hydrazone (CCCP)] when the classical respiratory control is absent and (ii) high ATP/ADP ratios in the matrix as well as in the cytosolic space are required for full ATP inhibition of CytOx. Additionally, ATP inhibition measured in intact mitochondria extends in the presence of oligomycin, thus indicating further that the problem to measure the inhibitory effect of ATP on CytOx is apparently due to the lack of very high ATP/ADP ratios in isolated mitochondria. [ABSTRACT FROM AUTHOR]

There are large differences between flavonoids to protect against apoptosis, a process in which cytochrome c (Cyt c) plays a key role. In this work, we show that 7 of 13 flavonoids studied have a capacity to reduce Cyt c similar or higher than ascorbate, the flavonols quercetin, kaempferol and myricetin, flavanol epigallocatechin-gallate, anthocyanidins cyanidin and malvidin, and the flavone luteolin. In contrast, the kaempferol 3( O)- and 3,4′( O)-methylated forms, the flavanone naringenin, and also apigenin and chrysin, had a negligible reducing capacity. Equilibrium dialysis and quenching of 1,6-diphenyl-1,3,5-hexatriene fluorescence experiments showed that flavonoids did not interfere with Cyt c binding to cardiolipin (CL)/phosphatidylcholine (PC) vesicles. However, the CL-induced loss of Cyt c Soret band intensity was largely attenuated by flavonoids, pointing out a stabilizing action against Cyt c unfolding in the complex. Moreover, flavonoids that behave as Cyt c reductants also inhibited the pro-apoptotic CL-induced peroxidase activity of Cyt c, indicating that modulation of Cyt c signaling are probable mechanisms behind the protective biological activities of flavonoids. © 2016 BioFactors, 43(3):451-468, 2017 [ABSTRACT FROM AUTHOR]

The oleaginous yeast Debaryomyces hansenii is a good model to understand molecular mechanisms involved in halotolerance because of its impressive ability to survive under a wide range of salt concentrations. Several cellular adaptations are implicated in this response, including the presence of a cyanide-insensitive ubiquinol oxidase (Aox). This protein, which is present in several taxonomical orders, has been related to different stress responses. However, little is known about its role in mitochondria during transitions from low to high saline environments. In this report, we analyze the effects of Aox in shifts from low to high salt concentrations in the culture media. At early stages of a salt insult, we observed that this protein prevents the overflow of electrons on the mitochondrial respiratory chain, thus, decreasing the production of reactive oxygen species. Interestingly, in the presence of high osmolite concentrations, Aox activity is able to sustain a stable membrane potential when coupled to complex I, despite a compromised cytochrome pathway. Taken together, our results suggest that under high osmolarity conditions Aox plays a critical role regulating mitochondrial physiology. [ABSTRACT FROM AUTHOR]

Cell death represents the final outcome of several pathological conditions of the central nervous system and available evidence suggests that in both acute injuries and neurodegenerative diseases it is often associated with mitochondrial dysfunction. Thus, the possibility to prevent mitochondrial events involved in cell death might represent efficient tools to limit neuronal damage. In recent years, increased attention has been paid to the endogenous protein neuroglobin, since accumulating evidence showed that its high expression was associated with preserved mitochondrial function and to an increased survival of nerve cells in vitro and in vivo in a variety of experimental models of cell insult. The biological and structural features of neuroglobin and the mitochondria-related mechanisms of neuroglobin-induced neuroprotection will be here briefly discussed. In this respect, the inhibition of the intrinsic pathway of apoptosis emerges as a key neuroprotective effect induced by the protein. These findings could open the possibility to develop efficient neuroglobin-mediated therapeutic strategies aimed at minimizing the neuronal cell death occurring in impacting neurological pathologies like stroke and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

We here reported the mitochondrial (mt) genome of one of the heterotrophic microeukaryotes related to cryptophytes, Palpitomonas bilix. The P. bilix mt genome was found to be a linear molecule composed of "single copy region" (~16 kb) and repeat regions (~30 kb) arranged in an inverse manner at both ends of the genome. Linear mt genomes with large inverted repeats are known for three distantly related eukaryotes (including P. bilix), suggesting that this particular mt genome structure has emerged at least three times in the eukaryotic tree of life. The P. bilixmt genome contains 47 protein-coding genes including ccmA, ccmB, ccmC, and ccmF, which encode protein subunits involved in the system for cytochrome c maturation inherited from a bacterium (System I). We present data indicating that the phylogenetic relatives of P. bilix, namely, cryptophytes, goniomonads, and kathablepharids, utilize an alternative system for cytochrome c maturation, which has most likely emerged during the evolution of eukaryotes (System III). To explain the distribution of Systems I and III in P. bilix and its phylogenetic relatives, two scenarios are possible: (i) System I was replaced by System III on the branch leading to the common ancestor of cryptophytes, goniomonads, and kathablepharids, and (ii) the two systems co-existed in their common ancestor, and lost differentially among the four descendants. [ABSTRACT FROM AUTHOR]

The mitochondrial alternative oxidase pathway ( AOP) has been suggested to act as a sink for excess reducing power generated in the chloroplast under high-light ( HL) stress and thus may reduce photoinhibition. The aim of this study was to compare different species to investigate the in vivo regulation and role of AOP under HL stress., The in vivo activities of AOP (νalt) and the cytochrome oxidase pathway, chlorophyll fluorescence, metabolite profiles, alternative oxidase ( AOX) capacity and protein amount were determined in leaves of five C3 species under growth light and after HL treatment., Differences in respiration and metabolite levels were observed among species under growth light conditions. The HL response of νalt was highly species dependent, correlated with the AOP capacity and independent of AOX protein content. Nevertheless, significant correlations were observed between νalt, levels of key metabolites and photosynthetic parameters., The results show that the species-specific response of νalt is caused by the differential post-translational regulation of AOX. Significant correlations between respiration, metabolites and photosynthetic performance across species suggest that AOP may permit stress-related amino acid synthesis, whilst maintaining photosynthetic activity under HL stress. [ABSTRACT FROM AUTHOR]

Resting metabolic rate (RMR) in humans shows pronounced individual variations, but the underlying molecular mechanism remains elusive. Cytochrome c oxidase (COX) plays a key role in control of metabolic rate, and recent studies of the subunit 4 isoform 2 (COX IV-2) indicate involvement in the cellular response to hypoxia and oxidative stress. We evaluated whether the COX subunit IV isoform composition may explain the pronounced individual variations in resting metabolic rate (RMR). RMR was determined in healthy humans by indirect calorimetry and correlated to levels of COX IV-2 and COX IV-1 in vastus lateralis. Overexpression and knock down of the COX IV isoforms were performed in primary myotubes followed by evaluation of the cell respiration and production of reactive oxygen species. Here we show that COX IV-2 protein is constitutively expressed in human skeletal muscle and strongly correlated to RMR. Primary human myotubes overexpressing COX IV-2 displayed markedly (>60%) lower respiration, reduced (>50%) cellular H2O2 production, higher resistance toward both oxidative stress, and severe hypoxia compared with control cells. These results suggest an important role of isoform COX IV-2 in the control of energy expenditure, hypoxic tolerance, and mitochondrial ROS homeostasis in humans. [ABSTRACT FROM AUTHOR]

Mitochondrial holocytochrome c synthase (HCCS) is required for cytochrome c (cyt c) maturation and therefore respiration. HCCS efficiently attaches heme via two thioethers to CXXCH of mitochondrial but not bacterial cyt c even though they are functionally conserved. This inability is due to residues in the bacterial cyt c N terminus, but the molecular basis is unknown. Human cyts c with deletions of single residues in α helix-1, which mimic bacterial cyt c, are poorly matured by human HCCS. Focusing on ΔM13 cyt c, we co-purified this variant with HCCS, demonstrating that HCCS recognizes the bacterial- like cytochrome. Although an HCCS-WT cyt c complex contains two covalent links, HCCS-ΔM13 cyt c contains only one thioether attachment. Using multiple approaches, we show that the single attachment is to the second thiol of C15SQC18H, indicating thatα helix-1 is required for positioning the first cysteine for covalent attachment, whereas the histidine of CXXCH positions the second cysteine. Modeling of the N-terminal structure suggested that the serine residue (of CSQCH) would be anchored where the first cysteine should be inΔM13cyt c. An engineered cyt c with a CQCH motif in the ΔM13 background is matured at higher levels (2-3-fold), providing further evidence for α helix-1 positioning the first cysteine. Bacterial cyt c biogenesis pathways (Systems I and II) appear to recognize simply the CXXCH motif, not requiring α helix-1. Results here explain mechanistically how HCCS (System III) requires an extended region adjacent to CXXCH for maturation. [ABSTRACT FROM AUTHOR]

We tested the hypothesis that melatonin (Mel) enhances exogenous mitochondria (Mito) treatment against rodent hepatic ischemia-reperfusion (IR) injury. In vitro study utilized three groups of hepatocytes (i.e. nontreatment, menadione, and menadione-melatonin treatment, 4.0 × 105 each), while in vivo study used adult male Sprague Dawley rats (n = 40) equally divided into sham-control (SC), IR (60-min left-lobe ischemia + 72-hr reperfusion), IR-Mel (melatonin at 30 min/6/8 hr after reperfusion), IR-Mito (mitochondria 15,000 μg/rat 30 min after reperfusion), and IR-Mel-Mito. Following menadione treatment in vitro, oxidative stress (NOX-1/NOX-2/oxidized protein), apoptotic (cleaved caspase-3/PARP), DNA damage (γ-H2AX/CD90/XRCC1), mitochondria damage (cytosolic cytochrome c) biomarkers, and mitochondrial permeability transition were found to be lower, whereas mitochondrial cytochrome c were found to be higher in hepatocytes with melatonin treatment compared to those without (all P < 0.001). In vivo study demonstrated highest liver injury score and serum AST in IR group, but lowest in SC group and higher in IR-Mito group than that in groups IR-Mel and IR-Mel-Mito, and higher in IR-Mel group than that in IR-Mel-Mito group after 72-hr reperfusion (all P < 0.003). Protein expressions of inflammatory (TNF-α/NF-κB/IL-1β/MMP-9), oxidative stress (NOX-1/NOX-2/oxidized protein), apoptotic (caspase-3/PARP/Bax), and mitochondria damage (cytosolic cytochrome c) biomarkers displayed an identical pattern, whereas mitochondria integrity marker (mitochondrial cytochrome c) showed an opposite pattern compared to that of liver injury score (all P < 0.001) among five groups. Microscopically, expressions of apoptotic nuclei, inflammatory (MPO +/CD68 +/CD14 + cells), and DNA damage (γ-H2AX + cells) biomarkers exhibited an identical pattern compared to that of liver injury score (all P < 0.001) among five groups. Melatonin-supported mitochondria treatment offered an additional benefit of alleviating hepatic IR injury. [ABSTRACT FROM AUTHOR]

Purpose:Mitochondrial defects have been associated with a series of muscular diseases. Dysferlinopathy, however, has been rarely reported with mitochondrial dysfunction. Here we report a cohort of dysferlinopathy patients with mitochondrial abnormalities found in muscle.Methods: Clinical data and muscle pathologies of nine cases with dysferlinopathy were retrospectively studied. mtDNA copy number, protein levels and activities of mitochondrial enzyme complexes were assayed.Results: Nine patients were diagnosed as having dysferlinopathy byDYSFsequencing and quantification of dysferlin levels in muscle homogenates. Muscle biopsies exhibited dystrophic changes (n = 9), ragged-red fibers (n = 9) and cytochrome c oxidase-deficient fibers (n = 9). mtDNA copy number increased significantly in 56% (15/27) of fibers with mitochondrial histology. Protein levels of complex IV subunits II (n = 5), complex III subunit core 2 (n = 2) and complex I NDUFB1 (n = 1) decreased. Impaired activities of complexes I, III and IV were observed in 56%, 33% and 78% of subjects and the activities were reduced by 21%, 18% and 40%, respectively. Besides, loss activities of complexes I/IV and decreased ATP level were also found in fibroblasts from dysferlinopathy.Conclusion: Prominent mitochondrial abnormalities are common pathological findings in muscle from dysferlinopathy. Our data indicated that mitochondria may play a significant role in the progression of dysferlinopathy and also highlighted the potential of mitochondrial protective drugs in rescuing the symptoms of dysferlinopathy. [ABSTRACT FROM AUTHOR]

Background: Glutamate carboxypeptidase II (GCPII) inactivates the peptide co-transmitter N-acetylaspartylglutamate following synaptic release. Inhibition of GCPII elevates extracellular levels of the peptide, inhibits glutamate release and is neuroprotective in an animal model of traumatic brain injury. GCPII gene knockout mice were used to examine the cellular mechanisms underlying the neuroprotective efficacy of this transmitter system.Results: Following controlled cortical impact injury, GCPII knockout (KO) mice exhibited reduced TUNEL-positive nuclei in the contusion margin of the cerebral cortex relative to wild type mice. Impact injury reduced glutathione levels and superoxide dismutase and glutathione peroxidase activities and increased malondialdehyde. Each of these effects was moderated in KO mice relative to wild type. Similarly, the injury-induced increases in cleaved caspase-3, cytosolic cytochrome c levels and Bcl-2/Bax ratio observed in wild type mice were attenuated in the knockout mice.Conclusions: These data support the hypothesis that the neuroprotective efficacy of GCPII KO in traumatic brain injury is mediated via a reduction in oxidative stress. [ABSTRACT FROM AUTHOR]

The effect of NO between cytochromes b and c of the mitochondrial respiratory chain were studied using submitochondrial particles (SMP) from bovine heart and GSNO and SPER-NO as NO sources. Succinate-cytochrome c reductase (complex II-III) activity (222±4 nmol/min. mg protein) was inhibited by 51% in the presence of 500 μM GSNO and by 48% in the presence of 30 μM SPER-NO, in both cases at ~1.25 μM NO. Neither GSNO nor SPER-NO were able to inhibit succinate-Q reductase activity (complex II; 220±9 nmol/min. mg protein), showing that NO affects complex III. Complex II-III activity was decreased (36%) when SMP were incubated with l -arginine and mtNOS cofactors, indicating that this effect is also produced by endogenous NO. GSNO (500 μM) reduced cytochrome b 562 by 71%, in an [O 2 ] independent manner. Hyperbolic increases in O 2 •- (up to 1.3±0.1 nmol/min. mg protein) and H 2 O 2 (up to 0.64±0.05 nmol/min. mg protein) productions were observed with a maximal effect at 500 μM GSNO. The O 2 •- /H 2 O 2 ratio was 1.98 in accordance with the stoichiometry of the O 2 •- disproportionation. Moreover, H 2 O 2 production was increased by 72–74% when heart coupled mitochondria were exposed to 500 μM GSNO or 30 μM SPER-NO. SMP incubated in the presence of succinate showed an EPR signal ( g= 1.99) compatible with a stable semiquinone. This EPR signal was increased not only by antimycin but also by GSNO and SPER-NO. These signals were not modified under N 2 atmosphere, indicating that they are not a consequence to the effect of NOx species on complex III area. These results show that NO interacts with ubiquinone-cytochrome b area producing antimycin-like effects. This behaviour comprises the inhibition of electron transfer, the interruption of the oxidation of cytochromes b, and the enhancement of [UQH • ] ss which, in turn, leads to an increase in O 2 •- and H 2 O 2 mitochondrial production rates. [ABSTRACT FROM AUTHOR]

Despite lacking mitochondria and a known pathway for heme biosynthesis the micro-aerotolerant anaerobic protozoan parasite Giardia intestinalis encodes four members of the cytochrome b 5 family of electron transfer proteins, three of which are small, single-domain proteins. While these are similar in size and fold to their better-known mammalian counterparts the Giardia proteins have distinctly lower reduction potentials, ranging from −140 to −171 mV compared to +6 mV for the bovine microsomal protein. This difference is accounted for by a more polar heme environment in the Giardia proteins, as mutation of a conserved heme pocket tyrosine residue to phenylalanine in the Giardia cytochrome b 5 isotype-I (gCYTb5-I Y61F) raises its reduction potential by nearly 100 mV. All three isotypes have UV–visible spectra consistent with axial coordination of the heme by a pair of histidine residues, but electron paramagnetic spectroscopy indicates that the planes of their imidazole rings are nearly perpendicular rather than coplanar as observed in mammalian cytochrome b 5 , which may be due to geometrical constraints imposed by a one-residue shorter spacing between the ligand pair in the Giardia proteins. Although no function has yet to be ascribed to any Giardia cytochrome b 5 , the presence of similar sequences in many other eukaryotes indicates that these represent an under-characterized class of low reduction potential family members. [ABSTRACT FROM AUTHOR]

In Podospora anserina , the loss of function of the cytochrome segment of the mitochondrial respiratory chain is viable. This is due to the presence in this organism, as in most filamentous fungi, of an alternative respiratory oxidase (AOX) that provides a bypass to the cytochrome pathway. However mutants lacking a functional cytochrome pathway present multiple phenotypes including poorly colored thin mycelium and slow growth. In a large genetic screen based on the improvement of these phenotypes, we isolated a large number of independent suppressor mutations. Most of them led to the constitutive overexpression of the aox gene. In this study, we characterize a new suppressor mutation that does not affect the production of AOX. It is a loss-of-function mutation in the gene encoding the MED13 subunit of the kinase module of the Mediator complex. Inactivation of the cdk8 gene encoding another subunit of the same module also results in partial suppression of a cytochrome-deficient mutant. Analysis of strains lacking the MED13 or CDK8 subunits points to the importance of these subunits as regulators involved in diverse physiological processes such as growth, longevity and sexual development. Interestingly, transcriptional analyses indicate that in P. anserina , loss of the respiratory cytochrome pathway results in the up-regulation of glycolysis-related genes revealing a new type of retrograde regulation. The loss of MED13 augments the up-regulation of some of these genes. [ABSTRACT FROM AUTHOR]

This is the first study designed on mitochondrial Cytochrome b gene (Cyt-b) and Displacement (D) loop sequence diversity in two camel breeds (Mareecha and Bareela) of Pakistan to assess the status of genetic diversity and differentiation. Genomic DNA was extracted from total of 48 samples (Mareecha = 25 and Bareela = 23). The complete Cyto-b gene sequences (1140bp) analysis showed four parsimony informative sites were at position 137, 146, 337 and 806 and five haplotypes were constructed, indicating low Cyto-b gene genetic variability in selected two Pakistani camel breeds. Analysis of complete mt-DNA D-loop (1214 bp) sequences revealed eight different haplotypes resulting from 32 polymorphic sites (2.63% of all sites), of which 31 sites were singleton variable sites and one site was parsimony informative polymorphic site. The phylogenetic analysis showed two clades of camel (Camelus), dromedary and Bactrian populations that came out as distinct lineage and showed distinct genetic distance between them, and all Pakistani camel haplotypes were clustered with dromedary camels confirmed their genetic architecture as dromedary camels. Genetic variability assessment on camels is important to preserve this genetic resource and to develop future breeding plans to improve camel productivity. [ABSTRACT FROM AUTHOR]

Mutations in the MT-ATP6 gene are frequent causes of severe mitochondrial disorders. Typically, these are missense mutations, but another type is represented by the 9205delTA microdeletion, which removes the stop codon of the MT-ATP6 gene and affects the cleavage site in the MT-ATP8/MT-ATP6/MT-CO3 polycistronic transcript. This interferes with the processing of mRNAs for the Atp6 (Fo-a) subunit of ATP synthase and the Cox3 subunit of cytochrome c oxidase (COX). Two cases described so far presented with strikingly different clinical phenotypes - mild transient lactic acidosis or fatal encephalopathy. To gain more insight into the pathogenic mechanism, we prepared 9205delTA cybrids with mutation load ranging between 52 and 99% and investigated changes in the structure and function of ATP synthase and the COX. We found that 9205delTA mutation strongly reduces the levels of both Fo-a and Cox3 proteins. Lack of Fo-a alters the structure but not the content of ATP synthase, which assembles into a labile, ~60 kDa smaller, complex retaining ATP hydrolytic activity but which is unable to synthesize ATP. In contrast, lack of Cox3 limits the biosynthesis of COX but does not alter the structure of the enzyme. Consequently, the diminished mitochondrial content of COX and non-functional ATP synthase prevent most mitochondrial ATP production. The biochemical effects caused by the 9205delTA microdeletion displayed a pronounced threshold effect above ~90 % mutation heteroplasmy. We observed a linear relationship between the decrease in subunit Fo-a or Cox3 content and the functional presentation of the defect. Therefore we conclude that the threshold effect originated from a gene-protein level. [ABSTRACT FROM AUTHOR]

We have developed an aptameric nanosensor for fluorescence activation imaging of cytochrome c (Cyt c). Fluorescence imaging tools that enable visualization of key molecular players in apoptotic signaling are essential for cell biology and clinical theranostics. Cyt c is a major mediator in cell apoptosis. However, fluorescence imaging tools allowing direct visualization of Cyt c translocation in living cells have currently not been realized. We report for the first time the realization of a nanosensor tool that enables direct fluorescence activation imaging of Cyt c released from mitochondria in cell apoptosis. This strategy relies on spatially selective cytosolic delivery of a nanosensor constructed by assembly of a fluorophore-tagged DNA aptamer on PEGylated graphene nanosheets. The cytosolic release of Cyt c is able to dissociate the aptamer from graphene and trigger an activated fluorescence signal. The nanosensor is shown to exhibit high sensitivity and selectivity, rapid response, large signal-to-background ratio for in vitro, and intracellular detection of Cyt c. It also enables real-time visualization of the Cyt c release kinetics and direct identification of the regulators for apoptosis. The developed nanosensor may provide a very valuable tool for apoptotic studies and catalyze the fundamental interrogations of Cyt c-mediated biology. [ABSTRACT FROM AUTHOR]

Diverse signaling pathways are activated by perturbation of mitochondrial function under different growth conditions. Mitochondria have emerged as an important organelle for sensing and coping with stress in addition to being the sites of important metabolic pathways. Here, responses to moderate light and drought stress were examined in different Arabidopsis (Arabidopsis thaliana) mutant plants lacking a functional alternative oxidase (alternative oxidase1a [aox1a]), those with reduced cytochrome electron transport chain capacity (T3/T7 bacteriophage-type RNA polymerase, mitochondrial, and plastidial [rpoTmp]), and double mutants impaired in both pathways (aox1a:rpoTmp). Under conditions considered optimal for growth, transcriptomes of aox1a and rpoTmp were distinct. Under adverse growth conditions, however, transcriptome changes in aox1a and rpoTmp displayed a highly significant overlap and were indicative of a common mitochondrial stress response and downregulation of photosynthesis. This suggests that the role of mitochondria to support photosynthesis is provided through either the alternative pathway or the cytochrome pathway, and when either pathway is inhibited, such as under environmental stress, a common, dramatic, and succinct mitochondrial signal is activated to alter energy metabolism in both organelles. aox1a:rpoTmp double mutants grown under optimal conditions showed dramatic reductions in biomass production compared with aox1a and rpoTmp and a transcriptome that was distinct from aox1a or rpoTmp. Transcript data indicating activation of mitochondrial biogenesis in aox1a:rpoTmp were supported by a proteomic analysis of over 200 proteins. Under optimal conditions, aox1a: rpoTmp plants seemed to switch on many of the typical mitochondrial stress regulators. Under adverse conditions, aox1a:rpoTmp turned off these responses and displayed a biotic stress response. Taken together, these results highlight the diverse signaling pathways activated by the perturbation of mitochondrial function under different growth conditions. [ABSTRACT FROM AUTHOR]

The second mitochondria-derived activator of caspases (Smac/DIABLO), is a mitochondrial protein that is released along with cytochrome c during apoptosis and promotes caspase activity. It has been reported that c-Jun N-terminal kinase (JNK) is involved in the regulation of Smac release during apoptosis; however, the specific role of JNK is not well understood. The aim of the present study was to investigate whether JNK1 is activated during apoptosis induced by tumor necrosis factor-α (TNF-α) and whether the activation of JNK1 is functionally associated with Smac release from the mitochondria in HeLa cells. It was determined that during apoptotic progression induced by TNF-α, JNK1 is activated and translocated into the mitochondria. It was also shown that Smac release is markedly promoted by transient expression of JNK1, while it is suppressed by the expression of a dominant negative version of JNK1. Furthermore, expression of JNK1 also increases the levels of TNF-α-induced caspase-3 activity. To determine whether JNK1 activity is directly involved in the release of Smac, an in vitro phosphorylation assay was performed with the N-terminal 10-mer peptide fragment of Smac (pep0110). The results indicated that pep0110 is phosphorylated in dose and time-dependent manners by active JNK1, and that the phosphorylation of Smac at the N-terminal serine 6 residue is functionally linked to Smac release during TNF-α-induced apoptosis. In conclusion, this suggests that Smac is a major physiological substrate of JNK1 in the regulation of apoptosis. [ABSTRACT FROM AUTHOR]

Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-iso-form 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyrl activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity. [ABSTRACT FROM AUTHOR]

Coalescent theory provides powerful models for population genetic inference and is now increasingly important in estimates of divergence times and speciation research. We use molecular data and methods based on coalescent theory to investigate whether genetic evidence supports the hypothesis of A. pretrei and A. tucumana as separate species and whether genetic data allow us to assess which allopatric model seems to better explain the diversification process in these taxa. We sampled 13 A. tucumana from two provinces in northern Argentina and 28 A. pretrei from nine localities of Rio Grande do Sul, Brazil. A 491 bp segment of the mitochondrial gene cytochrome c oxidase I was evaluated using the haplotype network and phylogenetic methods. The divergence time and other demographic quantities were estimated using the isolation and migration model based on coalescent theory. The network and phylogenetic reconstructions showed similar results, supporting reciprocal monophyly for these two taxa. The divergence time of lineage separation was estimated to be approximately 1.3 million years ago, which corresponds to the lower Pleistocene. Our results enforce the current taxonomic status for these two Amazon species. They also support that A. pretrei and A. tucumana diverged with little or no gene flow approximately 1.3 million years ago, most likely after the establishment of a small population in the Southern Yungas forest by dispersion of a few founders from the A. pretrei ancestral population. This process may have been favored by habitat corridors formed in hot and humid periods of the Quaternary. Considering that these two species are considered threatened, the results were evaluated for their implications for the conservation of these two species. [ABSTRACT FROM AUTHOR]

Background Since the introduction of the array-CGH technique in the diagnostic workup of mental retardation, new recurrent copy number variations and novel microdeletion/microduplication syndromes were identified. These findings suggest that some genomic disorders have high penetrance but a wide range of phenotypic severity. Results We present the clinical and molecular description of four unrelated patients affected by neurodevelopmental disorders and overlapping 7q31.1 microdeletion/microduplication, identified by array-CGH and involving only part of the IMMP2L gene. Conclusion IMMP2L encodes an inner mitochondrial membrane protease-like protein, which is required for processing of cytochromes inside mitochondria. Numerous studies reported that this gene is implicated in behavioural disorders such as autistic spectrum disorders, attention-deficit hyperactivity disorders, and Gilles de la Tourette syndrome. We discuss the functions of the gene suggesting that IMMP2L may act as risk factor for neurological disease. [ABSTRACT FROM AUTHOR]

Mitochondrial respiratory chain complexes convert chemical energy into a membrane potential by connecting electron transport with charge separation. Electron transport relies on redox cofactors that occupy strategic positions in the complexes. How these redox cofactors are assembled into the complexes is not known. Cytochrome b, a central catalytic subunit of complex III, contains two heme fos. Here, we unravel the sequence of events in the mitochondrial inner membrane by which cytochrome b is hemylated. Heme incorporation occurs in a strict sequential process that involves interactions of the newly synthesized cytochrome b with assembly factors and structural complex III subunits. These interactions are functionally connected to cofactor acquisition that triggers the progression of cytochrome b through successive assembly intermediates. Failure to he-mylate cytochrome b sequesters the Cbp3-Cbp6 complex in early assembly intermediates, thereby causing a reduction in cytochrome b synthesis via a feedback loop that senses hemylation of cytochrome b. [ABSTRACT FROM AUTHOR]

At the onset of apoptosis, the peroxidation of cardiolipin at the inner mitochondrial membrane by cytochrome c requires an open coordination site on the heme. We report a 1.45-Å resolution structure of yeast iso-1-cytochrome c with the Met80 heme ligand swung out of the heme crevice and replaced by a water molecule. This conformational change requires modest adjustments to the main chain of the heme crevice loop and is facilitated by a trimethyllysine 72-to-alanine mutation. This mutation also enhances the peroxidase activity of iso-1-cytochrome c. The structure shows a buried water channel capable of facilitating peroxide access to the active site and of moving protons produced during peroxidase activity to the protein surface. Alternate positions of the side chain of Arg38 appear to mediate opening and closing of the buried water channel. In addition, two buried water molecules can adopt alternate positions that change the network of hydrogen bonds in the buried water channel. Taken together, these observations suggest that low and high proton conductivity states may mediate peroxidase function. Comparison of yeast and mammalian cytochrome c sequences, in the context of the steric factors that permit opening of the heme crevice, suggests that higher organisms have evolved to inhibit peroxidase activity, providing a more stringent barrier to the onset of apoptosis. [ABSTRACT FROM AUTHOR]

Highlights: [•] First study to genetically characterise E. setonicis and E. quokka. [•] Characterisation at the 18S rRNA gene. [•] Characterisation at the mitochondrial cytochrome oxidase gene (COl). [•] Morphological characterisation. [•] Insights into Eimeria phylogeny. [ABSTRACT FROM AUTHOR]

We compared the capacity of rat liver and heart mitochondria to remove exogenously produced HO, determining their ability to decrease fluorescence generated by HO detector system. In the absence of substrates, liver and heart mitochondria removed HO at similar rates. Respiratory substrate addition increased removal rates, indicating a respiration-dependent process. Moreover, the rates were higher with pyruvate/malate than with succinate and in heart than in liver mitochondria. Generally, the changes in HO removal rates mirrored those of HO release rates excluding the possibility that endogenous and exogenous HO competed for the removing system. This idea was supported by the observation that the heaviest of three liver mitochondrial fractions exhibited the highest rates of both HO release and removal. Pharmacological inhibition showed tissue-linked differences in antioxidant enzyme contribution to HO removal which were consistent with the differences in antioxidant system activities. The enzymatic processes accounted only in part for net HO removal and the non-enzymatic ones participated to HO scavenging to a degree that was higher for heart than for liver mitochondria. The idea that non-enzymatic scavenging was due in great part to hemoproteins action was consistent with observation that the concentration of cytochromes, in particular cytochrome c, was higher in heart mitochondria. Indirect support was also obtained by a technique of enhanced luminescence, utilizing the capacity of cytochrome c/HO to catalyze the luminol oxidation, which showed that luminescence response to an oxidative challenge was higher in heart mitochondria. [ABSTRACT FROM AUTHOR]

Background: Apaf1 (apoptotic protease activating factor 1) is the central component of the apoptosome, a multiprotein complex that activates procaspase-9 after cytochrome c release from the mitochondria in the intrinsic pathway of apoptosis. Other cellular roles, including a pro-survival role, have also been described for Apaf1, while the relative contribution of each function to cell death, but also to cell homeostatic conditions, remain to be clarified. Methodology and Principal Findings: Here we examined the response to apoptosis induction of available embryonic fibroblasts from Apaf1 knockout mice (MEFS KO Apaf1). In the absence of Apaf1, cells showed mitochondria with an altered morphology that affects cytochrome c release and basal metabolic status. Conclusions: We analysed mitochondrial features and cell death response to etoposide and ABT-737 in two different Apaf1-deficient MEFS, which differ in the immortalisation protocol. Unexpectedly, MEFS KO Apaf1 immortalised with the SV40 antigen (SV40IM-MEFS Apaf1) and those which spontaneously immortalised (SIM-MEFS Apaf1) respond differently to apoptotic stimuli, but both presented relevant differences at the mitochondria when compared to MEFS WT, indicating a role for Apaf1 at the mitochondria. [ABSTRACT FROM AUTHOR]

Abstract: Apart from complex I (NADH:ubiquinone oxidoreductase) the mitochondrial cytochrome bc 1 complex (complex III; ubiquinol:cytochrome c oxidoreductase) has been identified as the main producer of superoxide and derived reactive oxygen species (ROS) within the mitochondrial respiratory chain. Mitochondrial ROS are generally linked to oxidative stress, aging and other pathophysiological settings like in neurodegenerative diseases. However, ROS produced at the ubiquinol oxidation center (center P, Qo site) of complex III seem to have additional physiological functions as signaling molecules during cellular processes like the adaptation to hypoxia. The molecular mechanism of superoxide production that is mechanistically linked to the electron bifurcation during ubiquinol oxidation is still a matter of debate. Some insight comes from extensive kinetic studies with mutated complexes from yeast and bacterial cytochrome bc 1 complexes. This review is intended to bridge the gap between those mechanistic studies and investigations on complex III ROS in cellular signal transduction and highlights factors that impact superoxide generation. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes. [Copyright &y& Elsevier]

We developed a Raman spectroscopy-based approach for simultaneous study of redox changes in c-and b-type cytochromes and for a semiquantitative estimation of the amount of oxygenated myoglobin in a perfused rat heart. Excitation at 532 nm was used to obtain Raman scattering of the myocardial surface of the isolated heart at normal and hypoxic conditions. Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb). The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling mitochondrial respiration. Conversely, transient hypoxia causes the reversible increase in the intensity of peaks assigned to cytochromes c and c1, reflecting electron stacking proximal to cytochrome oxidase due to the lack of terminal electron acceptor O2. Intensities of peaks assigned to oxy- and deoxyhemoglobin were used for the semiquantitative estimation of oMb deoxygenation that was found to be of approximately 50 under hypoxia conditions. [ABSTRACT FROM AUTHOR]

Mammalian cells typically contain thousands of copies of mitochondrial DNA assembled into hundreds of nucleolds. Here we analyzed the dynamic features of nucleoids in terms of mitochondrial membrane dynamics involving balanced fusion and fission. In mitochondrial fission GTPase dynamin-related protein (Drpl)deficient cells, nucleoids were enlarged by their clustering within hyperfused mitochondria. In normal cells, mitochondrial fission often occurred adjacent to nucleoids, since localization of Mff and Drpl is dependent on the nucleoids. Thus, mitochondrial fission adjacent to nucleoids should prevent their clustering by maintaining small and fragmented nucleoids. The enhanced clustering of nucleoids resulted in the formation of highly stacked cristae structures in enlarged bulb-like mitochondria (mito-bulbs). Enclosure of proapoptotic factor cytochrome c, but not of Smac/DIABLO, into the highly stacked cristae suppressed its release from mitochondria under apoptotic stimuli. In the absence of nucleoids, Drpl deficiency failed to form mito-buibs and to protect against apoptosis. Thus, mitochondrial dynamics by fission and fusion play a critical role in controlling mitochondrial nucleoid structures, contributing to cristae reformation and the proapoptotic status of mitochondria. [ABSTRACT FROM AUTHOR]

The modification particularities of the structural and functional state of the inner mitochondrial membrane of the rat liver and myocardium were observed in conditions of artificial hypobiosis, which was created using hypoxic and hypercapnic gas medium with a body temperature reduction. Under the artificial hypobiosis the activity of NAD·H-generating enzymes of the Krebs cycle of the liver mitochondria decreases. The established changes of the enzymes activity and cytochromes content of the inner mitochondrial membrane indicate the decrease of the oxidative activity of a respiratory chain, that can be limited on a terminal (cytochrome c oxidase) site and leads to the decrease (by 49% at an average) of the H+-ATPase activity of the liver mitochondria. Under the artificial hypobiosis the detected increase of the succinate-KoQ-oxidoreductase activi ty (by 65% at average) causes the maintaining of the functional activity of a mitochondrial respiratory chain, considering the high (relative to control) cytochrome c oxidase and H+-ATPase activities of the mitochondria of the rats' myocardium. The structural changes of the inner mitochondrial membrane of the liver and myocardium in experimental conditions are accompanied by the increase of hydrophobicity of tryptophan residues microenvironment and the intramolecular modifications of protein molecules. [ABSTRACT FROM AUTHOR]

Within the species-rich European harvestman genus Trogulus Latreille, 1802, the Balkan Trogulus torosus species-group as defined by Schönhofer and Martens is revised. The group is remarkable because it includes the world's largest Opiliones species, Trogulus torosus Simon, 1885, and Trogulus ozimeci sp. nov. is the first member of the family showing obvious adaption to subterranean life. According to nuclear 28S and mitochondrial cytochrome b gene data, the T. torosus species-group and the Trogulus hirtus species-group form a monophyletic unit. Only the former is treated here as a paraphyletic group. Despite this paraphyly, the T. torosus species-group members share a number of morphological, morphometric, geographical, and ecological traits and are treated here as a unit for revisional work. Morphometric data are shown to be not only useful for species discrimination but also for species-group delineation. Within the T. torosus species-group six species are recognized: Trogulus banaticus Avram, 1971, Trogulus setosissimus Roewer, 1940, and T. torosus are re-described; Trogulus megaligrava sp. nov., T. ozimeci sp. nov., and Trogulus tenuitarsus sp. nov. are described as new. Unusually within Trogulus, strong affiliations with subterranean habitats exist and are discussed. © 2013 The Linnean Society of London [ABSTRACT FROM AUTHOR]

Circulating blood platelets contain small numbers of fully functional mitochondria. Accumulating evidence demonstrates that these mitochondria regulate the pro-thrombotic function of platelets through not only energy generation, but also redox signalling and the initiation of apoptosis. Beyond its regulation of haemostasis, platelet mitochondrial function has also traditionally been used to identify and study mitochondrial dysfunction in human disease, owing to the easy accessibility of platelets compared with other metabolically active tissues. In the present article, we provide a brief overview of what is currently known about the function of mitochondria in platelets and review how platelet mitochondria have been used to study mitochondrial function in human disease. [ABSTRACT FROM AUTHOR]