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C Oxidase (c + oxidase)
Kinds of C Oxidase Terms modified by C Oxidase Selected AbstractsCaligus elongatus Nordmann genotypes on wild and farmed fishJOURNAL OF FISH DISEASES, Issue 2 2007Ų Ųines Abstract Two mitochondrial genotypes have been described for Caligus elongatus Nordmann in Norway. This article reports on the distribution of C. elongatus mitochondrial cytochrome C oxidase 1 genotypes from wild fish hosts from the SE Norwegian coast. For comparison, lice from areas with fish farming were included in the study. The genotype distribution of 841 lice from wild coastal (n = 535), wild North Sea pelagic (n = 26), farmed (n = 160) and wild hosts in areas of fish farming (n = 89) is presented. The genotype frequencies of C. elongatus on wild coastal hosts varied significantly between spring and autumn. Lice from these fish show a large proportion of genotype 1 lice in March,June every year. Genotype 2 lice were found more frequently in autumn. Genotype 1 was clearly associated with the lumpfish, Cyclopterus lumpus L. The genotype frequency appeared to be different in areas with aquaculture. Caligus elongatus from farmed fish and wild fish caught close to Atlantic salmon fish farms in Norway were predominantly genotype 1 in autumn. Genotypes of C. elongatus on the SE coast of Norway vary according to season and fish species. Factors involved in the encounter between fish and lice are important for the establishment of lice on their hosts. [source] Focused proteomics: Monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stainELECTROPHORESIS, Issue 15 2004James Murray Abstract We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro-Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post-translational modifications in control and patient samples using only small amounts of tissue. [source] Different pathophysiological mechanisms of intramitochondrial iron accumulation in acquired and congenital sideroblastic anemia caused by mitochondrial DNA deletionEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 2 2006Thomas Matthes Abstract:, Sideroblastic anemias (SA) are characterized by iron accumulation in the mitochondria of erythroblasts. Although we have evidence of mitochondrial gene alterations in sporadic congenital cases, the origin of acquired forms [refractory anemia with ring sideroblasts (RARS)], is still largely unknown. Here, we report the analysis of respiratory chain function in a patient with a large mitochondrial deletion and in patients with RARS. A young boy with SA showed symptoms typical of a mitochondrial disease with metabolic acidosis, muscle weakness and cerebral involvement. His bone marrow DNA was analyzed for the presence of mitochondrial deletions. We found a new mitochondrial (mt)DNA deletion spanning 3614 bp and including all the mt genes encoding complex IV, plus ATPase 6 and 8, and several transfer (t)RNAs. All tissues analyzed (liver, skeletal muscle, brain, pancreas) showed a heteroplasmic distribution of this mutant DNA. Bone marrow homogenates were obtained from five patients with RARS and from three patients with normal bone marrow and respiratory chain function assayed by spectrophotometric analysis. Cytochrome c oxidase (CCO) activity was greatly reduced in the patient's bone marrow. In contrast, CCO activity and global respiratory chain function were conserved in patients with RARS. We conclude that deficient CCO activity secondary to mtDNA deletions is related to intramitochondrial iron accumulation, as in our patient or in those with Pearson's syndrome, whereas other mechanisms, e.g. nuclear DNA mutations, have to be proposed to be involved in the acquired forms of SA. [source] Selective Long-Term Electrical Stimulation of Fast Glycolytic Fibres Increases Capillary Supply but not Oxidative Enzyme Activity in Rat Skeletal MusclesEXPERIMENTAL PHYSIOLOGY, Issue 5 2000S. Egginton Glycolytic fibres in rat extensor digitorum longus (EDL) and tibialis anterior (TA) were selectively activated, as demonstrated by glycogen depletion, by indirect electrical stimulation via electrodes implanted in the vicinity of the peroneal nerve using high frequency (40 Hz) trains (250 ms at 1 Hz) and low voltage (threshold of palpable contractions). This regime was applied 10 times per day, each bout being of 15 min duration with 60 min recovery, for 2 weeks. Cryostat sections of muscles were stained for alkaline phosphatase to depict capillaries, succinate dehydrogenase (SDH) to demonstrate oxidative fibres, and periodic acid-Schiff reagent (PAS) to verify glycogen depletion. Specific activity of hexokinase (HK), 6-phosphofructokinase, pyruvate kinase, glycogen phosphorylase and cytochrome c oxidase (COX) were estimated separately in homogenates of the EDL and the predominantly glycolytic cortex and oxidative core of the TA. Stimulation increased the activity of HK but not that of oxidative enzymes in fast muscles. Comparison of changes in oxidative capacity and capillary supply showed a dissociation in the predominantly glycolytic TA cortex. Here, COX was 3.9 ± 0.68 ,M min-1 (g wet wt)-1 in stimulated muscles compared with 3.7 ± 0.52 ,M min-1 (g wet wt)-1 in contralateral muscles (difference not significant), while the percentage of oxidative fibres (those positively stained for SDH) was also similar in stimulated (14.0 ± 2.8%) and contralateral (12.2 ± 1.9%) muscles. In contrast, the capillary to fibre ratio was significantly increased (2.01 ± 0.12 vs. 1.55 ± 0.04, P < 0.01). We conclude that capillary supply can be increased independently of oxidative capacity, possibly due to haemodynamic factors, and serves metabolite removal to a greater extent than substrate delivery. [source] Sulfide : quinone oxidoreductase (SQR) from the lugworm Arenicola marina shows cyanide- and thioredoxin-dependent activityFEBS JOURNAL, Issue 6 2008Ursula Theissen The lugworm Arenicola marina inhabits marine sediments in which sulfide concentrations can reach up to 2 mm. Although sulfide is a potent toxin for humans and most animals, because it inhibits mitochondrial cytochrome c oxidase at micromolar concentrations, A. marina can use electrons from sulfide for mitochondrial ATP production. In bacteria, electron transfer from sulfide to quinone is catalyzed by the membrane-bound flavoprotein sulfide : quinone oxidoreductase (SQR). A cDNA from A. marina was isolated and expressed in Saccharomyces cerevisiae, which lacks endogenous SQR. The heterologous enzyme was active in mitochondrial membranes. After affinity purification, Arenicola SQR isolated from yeast mitochondria reduced decyl-ubiquinone (Km = 6.4 ,m) after the addition of sulfide (Km = 23 ,m) only in the presence of cyanide (Km = 2.6 mm). The end product of the reaction was thiocyanate. When cyanide was substituted by Escherichia coli thioredoxin and sulfite, SQR exhibited one-tenth of the cyanide-dependent activity. Six amino acids known to be essential for bacterial SQR were exchanged by site-directed mutagenesis. None of the mutant enzymes was active after expression in yeast, implicating these amino acids in the catalytic mechanism of the eukaryotic enzyme. [source] Transcription of mammalian cytochrome c oxidase subunit IV-2 is controlled by a novel conserved oxygen responsive elementFEBS JOURNAL, Issue 21 2007Maik Hüttemann Subunit 4 of cytochrome c oxidase (CcO) is a nuclear-encoded regulatory subunit of the terminal complex of the mitochondrial electron transport chain. We have recently discovered an isoform of CcO 4 (CcO4-2) which is specific to lung and trachea, and is induced after birth. The role of CcO as the major cellular oxygen consumer, and the lung-specific expression of CcO4-2, led us to investigate CcO4-2 gene regulation. We cloned the CcO4-2 promoter regions of cow, rat and mouse and compared them with the human promoter. Promoter activity is localized within a 118-bp proximal region of the human promoter and is stimulated by hypoxia, reaching a maximum (threefold) under 4% oxygen compared with normoxia. CcO4-2 oxygen responsiveness was assigned by mutagenesis to a novel promoter element (5,-GGACGTTCCCACG-3,) that lies within a 24-bp region that is 79% conserved in all four species. This element is able to bind protein, and competition experiments revealed that, within the element, the four core bases 5,-TCNCA-3, are obligatory for transcription factor binding. CcO isolated from lung showed a 2.5-fold increased maximal turnover compared with liver CcO. We propose that CcO4-2 expression in highly oxygenated lung and trachea protects these tissues from oxidative damage by accelerating the last step in the electron transport chain, leading to a decrease in available electrons for free radical formation. [source] Probing the access of protons to the K pathway in the Paracoccus denitrificans cytochrome c oxidaseFEBS JOURNAL, Issue 2 2005Oliver-M. In recent studies on heme-copper oxidases a particular glutamate residue in subunit II has been suggested to constitute the entry point of the so-called K pathway. In contrast, mutations of this residue (E78II) in the Paracoccus denitrificans cytochrome c oxidase do not affect its catalytic activity at all (E78IIQ) or reduce it to about 50% (E78IIA); in the latter case, the mutation causes no drastic decrease in heme a3 reduction kinetics under anaerobic conditions, when compared to typical K pathway mutants. Moreover, both mutant enzymes retain full proton-pumping competence. While oxidized-minus-reduced Fourier-transform infrared difference spectroscopy demonstrates that E78II is indeed addressed by the redox state of the enzyme, absence of variations in the spectral range characteristic for protonated aspartic and glutamic acids at ,,1760 to 1710 cm,1 excludes the protonation of E78II in the course of the redox reaction in the studied pH range, although shifts of vibrational modes at 1570 and 1400 cm,1 reflect the reorganization of its deprotonated side chain at pH values greater than 4.8. We therefore conclude that protons do not enter the K channel via E78II in the Paracoccus enzyme. [source] The influence of temperature and osmolyte on the catalytic cycle of cytochrome c oxidaseFEBS JOURNAL, Issue 2 2003Jack A. Kornblatt The influence of temperature on cytochrome c oxidase (CCO) catalytic activity was studied in the temperature range 240,308 K. Temperatures below 273 K required the inclusion of the osmolyte ethylene glycol. For steady-state activity between 278 and 308 K the activation energy was 12 kcal·mol,1; the molecular activity or turnover number was 12 s,1 at 280 K in the absence of ethylene glycol. CCO activity was studied between 240 and 277 K in the presence of ethylene glycol. The activation energy was 30 kcal·mol,1; the molecular activity was 1 s,1 at 280 K. Ethylene glycol inhibits CCO by lowering the activity of water. The rate limitation in electron transfer (ET) was not associated with ET into the CCO as cytochrome a was predominantly reduced in the aerobic steady state. The activity of CCO in flash-induced oxidation experiments was studied in the low temperature range in the presence of ethylene glycol. Flash photolysis of the reduced CO complex in the presence of oxygen resulted in three discernable processes. At 273 K the rate constants were 1500 s,1, 150 s,1 and 30 s,1 and these dropped to 220 s,1, 27 s,1 and 3 s,1 at 240 K. The activation energies were 5 kcal·mol,1, 7 kcal·mol,1, and 8 kcal·mol,1, respectively. The fastest rate we ascribe to the oxidation of cytochrome a3, the intermediate rate to cytochrome a oxidation and the slowest rate to the re-reduction of cytochrome a followed by its oxidation. There are two comparisons that are important: (a) with vs. without ethylene glycol and (b) steady state vs. flash-induced oxidation. When one makes these two comparisons it is clear that the CCO only senses the presence of osmolyte during the reductive portion of the catalytic cycle. In the present work that would mean after a flash-induced oxidation and the start of the next reduction/oxidation cycle. [source] Purification and cDNA cloning of nitric oxide reductase cytochrome P450nor (CYP55A4) from Trichosporon cutaneumFEBS JOURNAL, Issue 11 2001Li Zhang Cytochrome P450nor is involved in fungal denitrification as nitric oxide (NO) reductase. Although the heme protein has been known to occur in restricted species of fungi that belong to ascomycotina, we have previously suggested that it would also occur in the yeast Trichosporon cutaneum, which is phylogenetically far from those P450nor-producing ascomycetous fungi. Here we isolated and characterized the heme protein from the basidiomycetous yeast T. cutaneum. P450nor of the yeast (TcP450nor) exhibited properties in terms of catalysis, absorption spectrum and molecular mass that are almost identical to those of its counterparts in ascomycetous fungi. We also isolated and sequenced its cDNA. The predicted primary structure of TcP450nor showed high sequence identities (around 65%) to those of other P450nors, indicating that they belong to the same family. TcP450nor protein cofractionated with cytochrome c oxidase by subcellular fractionation and its predicted primary structure contained an extension on its amino terminus that is characteristic of a mitochondrial-targeting signal, indicating that it is a mitochondrial protein like some of the isoforms of other fungi. On the other hand, TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells. The occurrence of P450nor across the subdivisions of eumycota suggests that P450nor and denitrification are distributed more universally among fungi than was previously thought. [source] Regulation of expression of terminal oxidases in Paracoccus denitrificansFEBS JOURNAL, Issue 8 2001Marijke F. Otten In order to study the induction of terminal oxidases in Paracoccus denitrificans, their promoters were fused to the lacZ reporter gene and analysed in the wild-type strain, in an FnrP-negative mutant, in a cytochrome bc1 -negative mutant, and in six single or double oxidase-negative mutant strains. The strains were grown under aerobic, semi-aerobic, and denitrifying conditions. The oxygen-sensing transcriptional-regulatory protein FnrP negatively regulated the activity of the qox promoter, which controls expression of the ba3 -type quinol oxidase, while it positively regulated the activity of the cco promoter, which controls expression of the cbb3 -type cytochrome c oxidase. The ctaDII and ctaC promoters, which control the expression of the aa3 -type cytochrome c oxidase subunits I and II, respectively, were not regulated by FnrP. The activities of the latter two promoters, however, did decrease with decreasing oxygen concentrations in the growth medium, suggesting that an additional oxygen-sensing mechanism exists that regulates transcription of ctaDII and ctaC. Apparently, the intracellular oxygen concentration (as sensed by FnrP) was not the only signal to which the oxidase promoters responded. At given extracellular oxygen status, both the qox and the cco promoters responded to mutations in terminal oxidase genes, whereas the ctaDII and ctaC promoters did not. The change of electron distribution through the respiratory network, resulting from elimination of one or more oxidase genes, may have changed intracellular signals that affect the activities of the qox and cco promoters. On the other hand, the re-routing of electron distribution in the respiratory mutants hardly affected the oxygen consumption rate as compared to that of the wild-type. This suggests that the mutants adapted their respiratory network in such a way that they were able to consume oxygen at a rate similar to that of the wild-type strain. [source] Photoinduced intracomplex electron transfer between cytochrome c oxidase and TUPS-modified cytochrome cFEBS JOURNAL, Issue 18 2000Alexander Kotlyar A novel method for initiating intramolecular electron transfer in cytochrome c oxidase is reported. The method is based upon photoreduction of cytochrome c labeled with thiouredopyrene-3,6,8-trisulfonate in complex with cytochrome oxidase. The thiouredopyrene-3,6,8-trisulfonate-labeled cytochrome c was prepared by incubating the thiol reactive form of the dye with yeast iso-1-cytochrome c, containing a single cysteine residue. Laser pulse excitation of a stoichiometrical complex between thiouredopyrene-3,6,8-trisulfonate-cytochrome c and bovine heart cytochrome oxidase at low ionic strength resulted in the reduction of cytochrome c by the excited form of thiouredopyrene-3,6,8-trisulfonate and subsequent intramolecular electron transfer from the reduced cytochrome c to cytochrome oxidase. The maximum efficiency by a single laser pulse resulted in the reduction of ,,17% of cytochrome a, and was achieved only at a 1 : 1 ratio of cytochrome c to cytochrome oxidase. At higher cytochrome c to cytochrome oxidase ratios the heme a reduction was strongly suppressed. [source] m.6267G>A: a recurrent mutation in the human mitochondrial DNA that reduces cytochrome c oxidase activity and is associated with tumors,HUMAN MUTATION, Issue 6 2006M. Esther Gallardo Abstract Complete sequencing of the mitochondrial genome of 13 cell lines derived from a variety of human cancers revealed nine novel mitochondrial DNA (mtDNA) variations. One of them, m.6267G>A, is a recurrent mutation that introduces the Ala122Thr substitution in the mitochondrially encoded cytochrome c oxidase I (MT-CO1): p.MT-CO1: Ala122Thr (GenBank: NP_536845.1). Biochemical analysis of the original cell lines and the transmitochondrial cybrids generated by transferring mitochondrial DNAs to a common nuclear background, indicate that cytochrome c oxidase (COX) activity, respiration, and growth in galactose are impaired by the m.6267G>A mutation. This mutation, found twice in the cancer cell lines included in this study, has been also encountered in one out of 63 breast cancer samples, one out of 64 colon cancer samples, one out of 260 prostate cancer samples, and in one out of 15 pancreatic cancer cell lines. In all instances the m.6267G>A mutation was associated to different mtDNA haplogroups. These findings, contrast with the extremely low frequency of the m.6267G>A mutation in the normal population (1:2264) and its apparent absence in other pathologies, strongly suggesting that the m.6267G>A missense mutation is a recurrent mutation specifically associated with cancer. Hum Mutat 27(6), 575,582, 2006. © 2006 Wiley-Liss, Inc. [source] Sequence and organization of the mitochondrial genome of the Chagas disease vector, Triatoma dimidiataINSECT MOLECULAR BIOLOGY, Issue 3 2001E. M. Dotson Abstract The 17 019 bp mitochondrial genome of Triatoma dimidiata is composed of thirteen protein coding sequences, twenty-two tRNAs, small and large ribosomal units, and a control region. The gene order and orientation are identical to that of Drosophila yakuba. The nucleotide composition is biased toward adenine and thymine (69.5% A + T). The 2.1 kb putative control region, known as the A + T rich region in most insects, has an A + T bias of 66%, but contains a 400 bp sequence that is 77.5% A + T and two other distinct regions: (1) one with a lower A + T bias (60.1%) and (2) a region of eight tandem repeat units. The identified 1.4 kb nuclear copy of mitochondrial sequences encompasses the string of Gs and the beginning of the cytochrome c oxidase 1 gene but lacks the 1.8 kb region spanning the eight tandem repeats and the 5, end of the NADH dehydrogenase subunit II gene. [source] Cytochrome c oxidase as the target of the heat shock protective effect in septic liverINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 5 2004Hsiang-Wen Chen Summary Liver function failure is one of the characteristics of critically ill, septic patients and is associated with worse outcome. Our previous studies have demonstrated that heat-shock response protects cells and tissue from subsequent insults and improves survival during sepsis. In this study, we have shown that mitochondrial cytochrome c oxidase (CCO) is one of the major sources of that protective effect. Experimental sepsis was induced by the cecal ligation and puncture (CLP) method. Heat-shock treatment was induced in rats by hyperthermia 24 h before CLP operation. The results showed that ATP content of the liver declined significantly, and the enzymatic activity of mitochondrial CCO was apparently suppressed during the late stages of sepsis. The mitochondrial ultrastructure of septic liver showed the deformity, mild swelling and inner membrane budding. Heat-shock treatment led to heat-shock protein 72 overexpression and prevented the downregulation of Grp75 during sepsis. On the contrary, the expression of the enzyme complex and its activity were preserved, associated with the minimization of ultrastructural deformities. In conclusion, the maintenance of mitochondrial function, especially the CCO, may be an important strategy in therapeutic interventions of a septic liver. [source] Cytochrome c oxidase biogenesis: New levels of regulationIUBMB LIFE, Issue 9 2008Flavia Fontanesi Abstract Eukaryotic cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is a multimeric enzyme of dual genetic origin, whose assembly is a complicated and highly regulated process. COX displays a concerted accumulation of its constitutive subunits. Data obtained from studies performed with yeast mutants indicate that most catalytic core unassembled subunits are posttranslationally degraded. Recent data obtained in the yeast Saccharomycescerevisiae have revealed another contribution to the stoichiometric accumulation of subunits during COX biogenesis targeting subunit 1 or Cox1p. Cox1p is a mitochondrially encoded catalytic subunit of COX which acts as a seed around which the full complex is assembled. A regulatory mechanism exists by which Cox1p synthesis is controlled by the availability of its assembly partners. The unique properties of this regulatory mechanism offer a means to catalyze multiple-subunit assembly. New levels of COX biogenesis regulation have been recently proposed. For example, COX assembly and stability of the fully assembled enzyme depend on the presence in the mitochondrial compartments of two partners of the oxidative phosphorylation system, the mobile electron carrier cytochrome c and the mitochondrial ATPase. The different mechanisms of regulation of COX assembly are reviewed and discussed. © 2008 IUBMB IUBMB Life, 60(9): 557,568, 2008 [source] Mitochondrial copper metabolism and delivery to cytochrome c oxidaseIUBMB LIFE, Issue 7 2008Darryl Horn Abstract Metals are essential elements of all living organisms. Among them, copper is required for a multiplicity of functions including mitochondrial oxidative phosphorylation and protection against oxidative stress. Here we will focus on describing the pathways involved in the delivery of copper to cytochrome c oxidase (COX), a mitochondrial metalloenzyme acting as the terminal enzyme of the mitochondrial respiratory chain. The catalytic core of COX is formed by three mitochondrially-encoded subunits and contains three copper atoms. Two copper atoms bound to subunit 2 constitute the CuA site, the primary acceptor of electrons from ferrocytochrome c. The third copper, CuB, is associated with the high-spin heme a3 group of subunit 1. Recent studies, mostly performed in the yeast Saccharomyces cerevisiae, have provided new clues about 1) the source of the copper used for COX metallation; 2) the roles of Sco1p and Cox11p, the proteins involved in the direct delivery of copper to the CuA and CuB sites, respectively; 3) the action mechanism of Cox17p, a copper chaperone that provides copper to Sco1p and Cox11p; 4) the existence of at least four Cox17p homologues carrying a similar twin CX9C domain suggestive of metal binding, Cox19p, Cox23p, Pet191p and Cmc1p, that could be part of the same pathway; and 5) the presence of a disulfide relay system in the intermembrane space of mitochondria that mediates import of proteins with conserved cysteines motifs such as the CX9C characteristic of Cox17p and its homologues. The different pathways are reviewed and discussed in the context of both mitochondrial COX assembly and copper homeostasis. © 2008 IUBMB IUBMB Life, 60(7): 421,429, 2008 [source] The role of mitochondria, cytochrome c and caspase-9 in embryonic lens fibre cell denucleationJOURNAL OF ANATOMY, Issue 2 2002E. J. Sanders Abstract During the differentiation of secondary lens fibre cells from the lens epithelium, the fibre cells lose all of their cytoplasmic organelles as well as their nuclei. The fibre cells, containing crystallins, which confer optical clarity, then persist in the adult lens. The process of denucleation of these cells has been likened to an apoptotic event which is not followed by the plasma membrane changes that are characteristic of apoptosis. We have examined the expression and subcellular translocation of molecules of the apoptotic cascade in differentiating lens epithelial cells in culture. In this culture system, the epithelial cells differentiate into lentoids composed of lens fibre cells. We find that caspase-9, which is expressed and activated before embryonic day 12 in intact lenses, is localized in the cytosol outside mitochondria in non-differentiating cultured cells. In lentoid cells, caspase-9 migrates into mitochondria after the latter undergo a membrane permeability transition that is characteristic of apoptotic cells. At the same time, caspase-9 co-localizes with cytochrome c in the cytosol. The cytochrome c is apparently released from the mitochondria in lentoid cells after the mitochondrial membrane permeability transition and during the period of nuclear shrinkage. Also during this time, the mitochondria aggregate around the degenerating nuclei. Cytochrome c disappears rapidly, while mitochondrial breakdown occurs approximately coincident with the disappearance of the nuclei, but mitochondrial remnants persist together with cytochrome c oxidase, which is a mitochondrial marker protein. Apaf-1, another cytosolic protein of the apoptotic cascade, also migrates to the permeabilized mitochondria and also co-localizes with caspase-9 and cytochrome c in the cytosol or mitochondria of denucleating cells, thus providing evidence for the formation of an ,apoptosome' in these cells, as in apoptotic cells. At no time did we observe the translocation of molecules between cytoplasmic compartments and the nucleus in differentiating lentoid cells. We suggest that the uncoupling of nuclear and membrane apoptotic events in these cells may be due to the early permeability changes in the mitochondria, resulting in the loss of mitochondrial signalling molecules, or to the failure of molecules to migrate to the nucleus in these cells, thus failing to activate nuclear-plasma membrane signalling pathways. [source] Charge parameterization of the metal centers in cytochrome c oxidaseJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 5 2008Mikael P. Johansson Abstract Reliable atomic point charges are of key importance for a correct description of the electrostatic interactions when performing classical, force field based simulations. Here, we present a systematic procedure for point charge derivation, based on quantum mechanical methodology suited for the systems at hand. A notable difference to previous procedures is to include an outer region around the actual system of interest. At the cost of increasing the system sizes, here up to 265 atoms, including the surroundings achieves near-neutrality for the systems as well as structural stability, important factors for reliable charge distributions. In addition, the common problem of converting between CH bonds and CC bonds at the border vanishes. We apply the procedure to the four redox-active metal centers of cytochrome c oxidase: CuA, haem a, haem a3, and CuB. Several relevant charge and ligand states are considered. Charges for two different force fields, CHARMM and AMBER, are presented. © 2007 Wiley Periodicals, Inc. J Comput Chem, 2008 [source] Mitochondrial dysfunction in a neural cell model of spinal muscular atrophyJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2009Gyula Acsadi Abstract Mutations of the survival motor neuron (SMN) gene in spinal muscular atrophy (SMA) lead to anterior horn cell death. The cause is unknown, but motor neurons depend substantially on mitochondrial oxidative phosphorylation (OxPhos) for normal function. Therefore, mitochondrial parameters were analyzed in an SMA cell culture model using small interfering RNA (siRNA) transfection that decreased Smn expression in NSC-34 cells to disease levels. Smn siRNA knock-down resulted in 35% and 66% reduced Smn protein levels 48 and 72 hr posttransfection, respectively. ATP levels were reduced by 14% and 26% at 48 and 72 hr posttransfection, respectively, suggesting decreased ATP production or increased energy demand in neural cells. Smn knock-down resulted in increased mitochondrial membrane potential and increased free radical production. Changes in activity of cytochrome c oxidase (CcO), a key OxPhos component, were observed at 72 hr with a 26% increase in oxygen consumption. This suggests a compensatory activation of the aerobic pathway, resulting in increased mitochondrial membrane potentials, a condition known to lead to the observed increase in free radical production. Further testing suggested that changes in ATP at 24 hr precede observable indices of cell injury at 48 hr. We propose that energy paucity and increased mitochondrial free radical production lead to accumulated cell damage and eventual cell death in Smn-depleted neural cells. Mitochondrial dysfunction may therefore be important in SMA pathology and may represent a new therapeutic target. © 2009 Wiley-Liss, Inc. [source] DNA barcodes to identify species and explore diversity in the Adelgidae (Insecta: Hemiptera: Aphidoidea)MOLECULAR ECOLOGY RESOURCES, Issue 2009R. G. FOOTTIT Abstract The Adelgidae are relatively small, cryptic insects, exhibiting complex life cycles with parthenogenetic reproduction. Due to these characteristics, the taxonomy of the group is problematic. Here, we test the effectiveness of the standard 658-bp barcode fragment from the 5,-end of the mitochondrial cytochrome c oxidase 1 gene (COI) in differentiating among 17 species of Adelgidae, in associating life-cycle stages, and in assessing patterns of geographical variation in selected species. Species of Adelgidae are well-differentiated by DNA barcodes, enabling the identification of different morphological forms, immature stages and individuals on different hosts and at different periods of the life cycle. DNA barcodes have uncovered cryptic diversity within taxa and, in other cases, a lack of sequence divergence in species pairs previously separated by life-cycle characteristics, indicating a need for further taxonomic analysis. [source] Species identification of aphids (Insecta: Hemiptera: Aphididae) through DNA barcodesMOLECULAR ECOLOGY RESOURCES, Issue 6 2008R. G. FOOTTIT Abstract A 658-bp fragment of mitochondrial DNA from the 5, region of the mitochondrial cytochrome c oxidase 1 (COI) gene has been adopted as the standard DNA barcode region for animal life. In this study, we test its effectiveness in the discrimination of over 300 species of aphids from more than 130 genera. Most (96%) species were well differentiated, and sequence variation within species was low, averaging just 0.2%. Despite the complex life cycles and parthenogenetic reproduction of aphids, DNA barcodes are an effective tool for identification. [source] Universal primer cocktails for fish DNA barcodingMOLECULAR ECOLOGY RESOURCES, Issue 4 2007NATALIA V. IVANOVA Abstract Reliable recovery of the 5, region of the cytochrome c oxidase 1 (COI) gene is critical for the ongoing effort to gather DNA barcodes for all fish species. In this study, we develop and test primer cocktails with a view towards increasing the efficiency of barcode recovery. Specifically, we evaluate the success of polymerase chain reaction amplification and the quality of resultant sequences using three primer cocktails on DNA extracts from representatives of 94 fish families. Our results show that M13-tailed primer cocktails are more effective than conventional degenerate primers, allowing barcode work on taxonomically diverse samples to be carried out in a high-throughput fashion. [source] Characterization of a Leishmania stage-specific mitochondrial membrane protein that enhances the activity of cytochrome c oxidase and its role in virulenceMOLECULAR MICROBIOLOGY, Issue 2 2010Ranadhir Dey Summary Leishmaniasis is caused by the dimorphic protozoan parasite Leishmania. Differentiation of the insect form, promastigotes, to the vertebrate form, amastigotes, and survival inside the vertebrate host accompanies a drastic metabolic shift. We describe a gene first identified in amastigotes that is essential for survival inside the host. Gene expression analysis identified a 27 kDa protein-encoding gene (Ldp27) that was more abundantly expressed in amastigotes and metacyclic promastigotes than in procyclic promastigotes. Immunofluorescence and biochemical analysis revealed that Ldp27 is a mitochondrial membrane protein. Co-immunoprecipitation using antibodies to the cytochrome c oxidase (COX) complex, present in the inner mitochondrial membrane, placed the p27 protein in the COX complex. Ldp27 gene-deleted parasites (Ldp27,/,) showed significantly less COX activity and ATP synthesis than wild type in intracellular amastigotes. Moreover, the Ldp27,/, parasites were less virulent both in human macrophages and in BALB/c mice. These results demonstrate that Ldp27 is an important component of an active COX complex enhancing oxidative phosphorylation specifically in infectious metacyclics and amastigotes and promoting parasite survival in the host. Thus, Ldp27 can be explored as a potential drug target and parasites devoid of the p27 gene could be considered as a live attenuated vaccine candidate against visceral leishmaniasis. [source] Cytochrome c oxidase of mammals contains a testes-specific isoform of subunit VIb,the counterpart to testes-specific cytochrome c?,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2003Maik Hüttemann Abstract Sperm motility is highly dependent on aerobic energy metabolism, of which the apparent rate-limiting step of the mitochondrial respiratory chain is catalyzed by cytochrome c oxidase (COX). COX is the only electron transport chain complex to display isoforms, consistent with its suggested rate-limiting role. Isoforms were previously described for four of the 13 subunits. We now report the discovery that COX subunit VIb displays a testes-specific isoform in human, bull, rat, and mouse (COX VIb-2). Analysis of a variety of rat and mouse tissues, including ovaries, demonstrates exclusive expression of VIb-2 in testes, whereas VIb-1 transcripts are absent in rodent testes, even at early developmental stages. In contrast, both isoforms are transcribed in human testes. In situ hybridizations with human, rat, and mouse testes sections reveal VIb-2 transcripts in all testicular cell types. Within the seminiferous tubules, VIb-1 shows stronger signals in the periphery than in the lumen. Previously, cytochrome c was the only component of the mitochondrial respiratory chain known to express a testes-specific isoform in mammals. COX subunit VIb connects the two COX monomers into the physiological dimeric form, and is the only COX subunit that, like cytochrome c, is solely located in the inter-membrane space. Significant differences between the isoform sequences, in particular changes in charged amino acids, suggest interactions with cytochrome c and sperm-specific energy requirements. Mol. Reprod. Dev. 66: 8,16, 2003. © 2003 Wiley-Liss, Inc. [source] Ophthalmoplegia due to mitochondrial DNA disease: The need for genetic diagnosisMUSCLE AND NERVE, Issue 1 2005Andrew M. Schaefer MB Abstract We describe a patient with chronic progressive external ophthalmoplegia (CPEO) who underwent muscle biopsy for suspected mitochondrial disease. In spite of normal histocytochemical cytochrome c oxidase (COX) activity and respiratory chain enzyme measurements in muscle, subsequent molecular genetic analysis revealed the presence of a single, large-scale deletion of mitochondrial DNA (mtDNA). The case serves to illustrate the importance of pursuing the proposed mitochondrial genetic abnormality, even in patients with normal biopsy findings. Muscle Nerve, 2005 [source] Molecular neuropathology of MELAS: level of heteroplasmy in individual neurones and evidence of extensive vascular involvementNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 4 2006J. Betts Mitochondrial DNA (mtDNA) disease is an important genetic cause of neurological disability. A variety of different clinical features are observed and one of the most common phenotypes is MELAS (Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis and Stroke-like episodes). The majority of patients with MELAS have the 3243A>G mtDNA mutation. The neuropathology is dominated by multifocal infarct-like lesions in the posterior cortex, thought to underlie the stroke-like episodes seen in patients. To investigate the relationship between mtDNA mutation load, mitochondrial dysfunction and neuropathological features in MELAS, we studied individual neurones from several brain regions of two individuals with the 3243A>G mutation using dual cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) histochemistry, and Polymerase Chain Reaction Restriction Fragment Lenght Polymorphism (PCR-RFLP) analysis. We found a low number of COX-deficient neurones in all brain regions. There appeared to be no correlation between the threshold level for the 3243A>G mutation to cause COX deficiency within single neurones and the degree of pathology in affected brain regions. The most severe COX deficiency associated with the highest proportion of mutated mtDNA was present in the walls of the leptomeningeal and cortical blood vessels in all brain regions. We conclude that vascular mitochondrial dysfunction is important in the pathogenesis of the stroke-like episodes in MELAS patients. As migraine is a commonly encountered feature in MELAS, we propose that coupling of the vascular mitochondrial dysfunction with cortical spreading depression (CSD) might underlie the selective distribution of ischaemic lesions in the posterior cortex in these patients. [source] Mitochondrial impacts of insecticidal formate esters in insecticide-resistant and insecticide-susceptible Drosophila melanogasterPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 6 2009Cheol Song Abstract BACKGROUND: Previous research on insecticidal formate esters in flies and mosquitoes has documented toxicity profiles, metabolism characteristics and neurological impacts. The research presented here investigated mitochondrial impacts of insecticidal formate esters and their hydrolyzed metabolite formic acid in the model dipteran insect Drosophila melanogaster Meig. These studies compared two Drosophila strains: an insecticide-susceptible strain (Canton-S) and a strain resistant by cytochrome P450 overexpression (Hikone-R). RESULTS: In initial studies investigating inhibition of mitochondrial cytochrome c oxidase, two proven insecticidal materials (hydramethylnon and sodium cyanide) caused significant inhibition. However, for insecticidal formate esters and formic acid, no significant inhibition was identified in either fly strain. Mitochondrial impacts of formate esters were then investigated further by tracking toxicant-induced cytochrome c release from mitochondria into the cytoplasm, a biomarker of apoptosis and neurological dysfunction. Formic acid and three positive control treatments (rotenone, antimycin A and sodium cyanide) induced cytochrome c release, verifying that formic acid is capable of causing mitochondrial disruption. However, when comparing formate ester hydrolysis and cytochrome c release between Drosophila strains, formic acid liberation was only weakly correlated with cytochrome c release in the susceptible Canton-S strain (r2 = 0.70). The resistant Hikone-R strain showed no correlation (r2 < 0.0001) between formate ester hydrolysis and cytochrome c release. CONCLUSION: The findings of this study provide confirmation of mitochondrial impacts by insecticidal formate esters and suggest links between mitochondrial disruption, respiratory inhibition, apoptosis and formate-ester-induced neurotoxicity. Copyright © 2009 Society of Chemical Industry [source] Design, synthesis, and characterization of a novel hemoproteinPROTEIN SCIENCE, Issue 2 2001Zhijin Xu Abstract Here we describe a synthetic protein (6H7H) designed to bind four heme groups via bis,histidine axial ligation. The hemes are designed to bind perpendicular to another in an orientation that mimics the relative geometry of the two heme a groups in the active site of cytochrome c oxidase. Our newly developed protein-design program, called CORE, was implemented in the design of this novel hemoprotein. Heme titration studies resolved four distinct KD values (KD1 = 80 nM, KD2 = 18 nM, KD3 , 3 mM, KD4 , 570 nM, with KD3 × K D4 = 1700); positive cooperativity in binding between the first and second heme, as well as substantial positive cooperativity between the third and forth heme, was observed. Chemical and thermal denaturation studies reveal a stable protein with native-like properties. Visible circular dichroism spectroscopy of holo-6H7H indicates excitonic coupling between heme groups. Further electrochemical and spectroscopic characterization of the holo-protein support a structure that is consistent with the predefined target structure. [source] Primary structure of a novel subunit in ba3 -cytochrome oxidase from thermus thermophilusPROTEIN SCIENCE, Issue 11 2000Tewfik Soulimane Abstract The ba3 -type cytochrome c oxidase from Thermus thermophilus is known as a two subunit enzyme. Deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix "subunit IIa" spanning the membrane. The hydrophobic N-terminally blocked protein was isolated in high yield using high-performance liquid chromatography. Its complete amino acid sequence was determined by a combination of automated Edman degradation of both the deformylated and the cyanogen bromide cleaved protein and automated C-terminal sequencing of the native protein. The molecular mass of 3,794 Da as determined by MALDI-MS and by ESI requires the N-terminal methionine to be formylated and is in good agreement with the value calculated from the formylmethionine containing sequence (3,766.5 Da + 28 Da = 3,794.5 Da). This subunit consits of 34 residues forming one helix across the membrane (Lys5-Ala34), which corresponds in space to the first transmembrane helix of subunit II of the cytochrome c oxidases from Paracoccus denitrificans and bovine heart, however, with opposite polarity. It is 35% identical to subunit IV of the ba3 -cytochrome oxidase from Natronobacterium pharaonis. The open reading frame encoding this new subunit IIa (cbaD) is located upstream of cbaB in the same operon as the genes for subunit I (cbaA) and subunit II (cbaB). [source] A comparative proteomic analysis for capsaicin-induced apoptosis between human hepatocarcinoma (HepG2) and human neuroblastoma (SK-N-SH) cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 22 2008Yu Mi Baek Abstract The endogenous ROS levels were increased during HepG2 apoptosis, whereas they were decreased during SK-N-SH apoptosis in response to capsaicin treatments. We used 2-DE-based proteomics to analyze the altered protein levels in both cells, with special attention on oxidative stress proteins before and after capsaicin treatments. The 2-DE analysis demonstrated that 23 proteins were increased and 26 proteins were decreased significantly (fold change>1.4) in capsaicin-treated apoptotic HepG2 and SK-N-SH cells, respectively. The distinct effect of capsaicin-induced apoptosis on the expression pattern of HepG2 proteins includes the downregulation of some antioxidant enzymes including aldose reductase (AR), catalase, enolase 1, peroxiredoxin 1, but upregulation of peroxiredoxin 6, cytochrome c oxidase, and SOD2. In contrast, most antioxidant enzymes were increased in SK-N-SH cells in response to capsaicin, where catalase might play a pivotal role in maintenance of low ROS levels in the course of apoptosis. The global gene expression for oxidative stress and antioxidant defense genes revealed that 84 gene expressions were not significantly different in HepG2 cells between control and capsaicin-treated cells. In contrast, a number of oxidative genes were downregulated in SK-N-SH cells, supporting the evidence of low ROS environment in apoptotic SK-N-SH cells after capsaicin treatment. It was concluded that the different relationship between endogenous ROS levels and apoptosis of two cancer cells presumably resulted from complicated expression patterns of many oxidative stress and antioxidant genes, rather than the individual role of some classical antioxidant enzymes such as SOD and catalase. [source] | |||||||||||||||||||||||||||||||