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Sample records for brucei mitochondrial membranes

  1. Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo-ATPase.

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    Anthonius A Eze

    2016-08-01

    Full Text Available Isometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b. gambiense and T. b. rhodesiense. Resistance to isometamidium is a growing concern, as is cross-resistance to the diamidine drugs diminazene and pentamidine.Two isometamidium resistant Trypanosoma brucei clones were generated (ISMR1 and ISMR15, being 7270- and 16,000-fold resistant to isometamidium, respectively, which retained their ability to grow in vitro and establish an infection in mice. Considerable cross-resistance was shown to ethidium bromide and diminazene, with minor cross-resistance to pentamidine. The mitochondrial membrane potentials of both resistant cell lines were significantly reduced compared to the wild type. The net uptake rate of isometamidium was reduced 2-3-fold but isometamidium efflux was similar in wild-type and resistant lines. Fluorescence microscopy and PCR analysis revealed that ISMR1 and ISMR15 had completely lost their kinetoplast DNA (kDNA and both lines carried a mutation in the nuclearly encoded γ subunit gene of F1 ATPase, truncating the protein by 22 amino acids. The mutation compensated for the loss of the kinetoplast in bloodstream forms, allowing near-normal growth, and conferred considerable resistance to isometamidium and ethidium as well as significant resistance to diminazene and pentamidine, when expressed in wild type trypanosomes. Subsequent exposure to either isometamidium or ethidium led to rapid loss of kDNA and a further increase in isometamidium resistance.Sub-lethal exposure to isometamidium gives rise to viable but highly resistant trypanosomes that, depending on sub-species, are infective to humans and cross-resistant to at least some diamidine drugs. The crucial mutation is in the F1 ATPase γ subunit, which

  2. Non-cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei

    NARCIS (Netherlands)

    Bienen, E. J.; Maturi, R. K.; Pollakis, G.; Clarkson, A. B.

    1993-01-01

    The life cycle of Trypanosoma brucei brucei involves a series of differentiation steps characterized by marked changes in mitochondrial development and function. The bloodstream forms of this parasite completely lack cytochromes and have not been considered to have any Krebs cycle function. It has

  3. Trypanosoma brucei mitochondrial respiratome: Composition and organization in procyclic form

    KAUST Repository

    Acestor, Nathalie

    2011-05-24

    The mitochondrial respiratory chain is comprised of four different protein complexes (I-IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by F oF 1-ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. An Atypical Mitochondrial Carrier That Mediates Drug Action in Trypanosoma brucei.

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    Juan P de Macêdo

    2015-05-01

    Full Text Available Elucidating the mechanism of action of trypanocidal compounds is an important step in the development of more efficient drugs against Trypanosoma brucei. In a screening approach using an RNAi library in T. brucei bloodstream forms, we identified a member of the mitochondrial carrier family, TbMCP14, as a prime candidate mediating the action of a group of anti-parasitic choline analogs. Depletion of TbMCP14 by inducible RNAi in both bloodstream and procyclic forms increased resistance of parasites towards the compounds by 7-fold and 3-fold, respectively, compared to uninduced cells. In addition, down-regulation of TbMCP14 protected bloodstream form mitochondria from a drug-induced decrease in mitochondrial membrane potential. Conversely, over-expression of the carrier in procyclic forms increased parasite susceptibility more than 13-fold. Metabolomic analyses of parasites over-expressing TbMCP14 showed increased levels of the proline metabolite, pyrroline-5-carboxylate, suggesting a possible involvement of TbMCP14 in energy production. The generation of TbMCP14 knock-out parasites showed that the carrier is not essential for survival of T. brucei bloodstream forms, but reduced parasite proliferation under standard culture conditions. In contrast, depletion of TbMCP14 in procyclic forms resulted in growth arrest, followed by parasite death. The time point at which parasite proliferation stopped was dependent on the major energy source, i.e. glucose versus proline, in the culture medium. Together with our findings that proline-dependent ATP production in crude mitochondria from TbMCP14-depleted trypanosomes was reduced compared to control mitochondria, the study demonstrates that TbMCP14 is involved in energy production in T. brucei. Since TbMCP14 belongs to a trypanosomatid-specific clade of mitochondrial carrier family proteins showing very poor similarity to mitochondrial carriers of mammals, it may represent an interesting target for drug

  5. Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei

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    Abdulsalam A.M. Alkhaldi

    2016-04-01

    Full Text Available Lipophilic bisphosphonium salts are among the most promising antiprotozoal leads currently under investigation. As part of their preclinical evaluation we here report on their mode of action against African trypanosomes, the etiological agents of sleeping sickness. The bisphosphonium compounds CD38 and AHI-9 exhibited rapid inhibition of Trypanosoma brucei growth, apparently the result of cell cycle arrest that blocked the replication of mitochondrial DNA, contained in the kinetoplast, thereby preventing the initiation of S-phase. Incubation with either compound led to a rapid reduction in mitochondrial membrane potential, and ATP levels decreased by approximately 50% within 1 h. Between 4 and 8 h, cellular calcium levels increased, consistent with release from the depolarized mitochondria. Within the mitochondria, the Succinate Dehydrogenase complex (SDH was investigated as a target for bisphosphonium salts, but while its subunit 1 (SDH1 was present at low levels in the bloodstream form trypanosomes, the assembled complex was hardly detectable. RNAi knockdown of the SDH1 subunit produced no growth phenotype, either in bloodstream or in the procyclic (insect forms and we conclude that in trypanosomes SDH is not the target for bisphosphonium salts. Instead, the compounds inhibited ATP production in intact mitochondria, as well as the purified F1 ATPase, to a level that was similar to 1 mM azide. Co-incubation with azide and bisphosphonium compounds did not inhibit ATPase activity more than either product alone. The results show that, in T. brucei, bisphosphonium compounds do not principally act on succinate dehydrogenase but on the mitochondrial FoF1 ATPase.

  6. Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei

    Science.gov (United States)

    Alkhaldi, Abdulsalam A.M.; Martinek, Jan; Panicucci, Brian; Dardonville, Christophe; Zíková, Alena; de Koning, Harry P.

    2015-01-01

    Lipophilic bisphosphonium salts are among the most promising antiprotozoal leads currently under investigation. As part of their preclinical evaluation we here report on their mode of action against African trypanosomes, the etiological agents of sleeping sickness. The bisphosphonium compounds CD38 and AHI-9 exhibited rapid inhibition of Trypanosoma brucei growth, apparently the result of cell cycle arrest that blocked the replication of mitochondrial DNA, contained in the kinetoplast, thereby preventing the initiation of S-phase. Incubation with either compound led to a rapid reduction in mitochondrial membrane potential, and ATP levels decreased by approximately 50% within 1 h. Between 4 and 8 h, cellular calcium levels increased, consistent with release from the depolarized mitochondria. Within the mitochondria, the Succinate Dehydrogenase complex (SDH) was investigated as a target for bisphosphonium salts, but while its subunit 1 (SDH1) was present at low levels in the bloodstream form trypanosomes, the assembled complex was hardly detectable. RNAi knockdown of the SDH1 subunit produced no growth phenotype, either in bloodstream or in the procyclic (insect) forms and we conclude that in trypanosomes SDH is not the target for bisphosphonium salts. Instead, the compounds inhibited ATP production in intact mitochondria, as well as the purified F1 ATPase, to a level that was similar to 1 mM azide. Co-incubation with azide and bisphosphonium compounds did not inhibit ATPase activity more than either product alone. The results show that, in T. brucei, bisphosphonium compounds do not principally act on succinate dehydrogenase but on the mitochondrial FoF1 ATPase. PMID:27054061

  7. A Flagellar Pocket Membrane Fraction from Trypanosoma brucei rhodesiense: Immunogold Localization and Nonvariant Immunoprotection

    Science.gov (United States)

    1988-01-01

    membrane components. specific glycoproteins from Trypanosoma equiperdum variants. Exp. Parasitol. 64:1-11. FEBS Lett. 82:93-96. 20. Melton, D. W., D. S...Fraction from Trypanosoma brucei rhodesiense: Immunogold Localization and Nonvariant Immunoprotection JOHN G. OLENICK,* RUTH WOLFF,’ ROBERT K. NAUMAN...obtained for each of three distinct variable antigenic types (VATs) of a serodeme of Trypanosoma brucei rhodesiense Wellcome strain. Products of

  8. Tail characteristics of Trypanosoma brucei mitochondrial transcripts are developmentally altered in a transcript-specific manner.

    Science.gov (United States)

    Gazestani, Vahid H; Hampton, Marshall; Shaw, Aubie K; Salavati, Reza; Zimmer, Sara L

    2018-02-01

    The intricate life cycle of Trypanosoma brucei requires extensive regulation of gene expression levels of the mtRNAs for adaptation. Post-transcriptional gene regulatory programs, including unencoded mtRNA 3' tail additions, potentially play major roles in this adaptation process. Intriguingly, T. brucei mitochondrial transcripts possess two distinct unencoded 3' tails, each with a differing functional role; i.e., while one type is implicated in RNA stability (in-tails), the other type appears associated with translation (ex-tails). We examined the degree to which tail characteristics differ among cytochrome c oxidase subunits I and III (CO1 and CO3), and NADH dehydrogenase subunit 1 (ND1) transcripts, and to what extent these characteristics differ developmentally. We found that CO1, CO3 and ND1 transcripts possess longer in-tails in the mammalian life stage. By mathematically modelling states of in-tail and ex-tail addition, we determined that the typical length at which an in-tail is extended to become an ex-tail differs by transcript and, in the case of ND1, by life stage. To the best of our knowledge, we provide the first evidence that developmental differences exist in tail length distributions of mtRNAs, underscoring the potential involvement of in-tail and ex-tail populations in mitochondrial post-transcriptional regulation mechanisms. Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  9. Apocytochrome b and other mitochondrial DNA sequences are differentially expressed during the life cycle of Trypanosoma brucei.

    OpenAIRE

    Feagin, J E; Jasmer, D P; Stuart, K

    1985-01-01

    Cytochromes and Krebs cycle enzymes are not detected in bloodstream forms of Trypanosoma brucei but are present in procyclic forms. We have analyzed transcription of mitochondrial sequences which contain the apocytochrome b gene and several other open reading frames (ORFs). Multiple transcripts map to individual DNA sequences located on both DNA strands. Larger low abundance transcripts map to multiple ORFs and may be precursor RNAs. Small abundant transcripts map to G + C rich sequences that...

  10. Mitochondrial fusion through membrane automata.

    Science.gov (United States)

    Giannakis, Konstantinos; Andronikos, Theodore

    2015-01-01

    Studies have shown that malfunctions in mitochondrial processes can be blamed for diseases. However, the mechanism behind these operations is yet not sufficiently clear. In this work we present a novel approach to describe a biomolecular model for mitochondrial fusion using notions from the membrane computing. We use a case study defined in BioAmbient calculus and we show how to translate it in terms of a P automata variant. We combine brane calculi with (mem)brane automata to produce a new scheme capable of describing simple, realistic models. We propose the further use of similar methods and the test of other biomolecular models with the same behaviour.

  11. Formation and Regulation of Mitochondrial Membranes

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    Laila Cigana Schenkel

    2014-01-01

    Full Text Available Mitochondrial membrane phospholipids are essential for the mitochondrial architecture, the activity of respiratory proteins, and the transport of proteins into the mitochondria. The accumulation of phospholipids within mitochondria depends on a coordinate synthesis, degradation, and trafficking of phospholipids between the endoplasmic reticulum (ER and mitochondria as well as intramitochondrial lipid trafficking. Several studies highlight the contribution of dietary fatty acids to the remodeling of phospholipids and mitochondrial membrane homeostasis. Understanding the role of phospholipids in the mitochondrial membrane and their metabolism will shed light on the molecular mechanisms involved in the regulation of mitochondrial function and in the mitochondrial-related diseases.

  12. Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane

    NARCIS (Netherlands)

    Bohnert, Maria; Wenz, Lena-Sophie; Zerbes, Ralf M.; Horvath, Susanne E.; Stroud, David A.; von der Malsburg, Karina; Mueller, Judith M.; Oeljeklaus, Silke; Perschil, Inge; Warscheid, Bettina; Chacinska, Agnieszka; Veenhuis, Marten; van der Klei, Ida J.; Daum, Guenther; Wiedemann, Nils; Becker, Thomas; Pfanner, Nikolaus; van der Laan, Martin

    2012-01-01

    Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of

  13. Proteins and lipids of glycosomal membranes from Leishmania tarentolae and Trypanosoma brucei [v1; ref status: indexed, http://f1000r.es/x1

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    Claudia Colasante

    2013-01-01

    Full Text Available In kinetoplastid protists, several metabolic pathways, including glycolysis and purine salvage, are located in glycosomes, which are microbodies that are evolutionarily related to peroxisomes. With the exception of some potential transporters for fatty acids, and one member of the mitochondrial carrier protein family, proteins that transport metabolites across the glycosomal membrane have yet to be identified. We show here that the phosphatidylcholine species composition of Trypanosoma brucei glycosomal membranes resembles that of other cellular membranes, which means that glycosomal membranes are expected to be impermeable to small hydrophilic molecules unless transport is facilitated by specialized membrane proteins. Further, we identified 464 proteins in a glycosomal membrane preparation from Leishmania tarentolae. The proteins included approximately 40 glycosomal matrix proteins, and homologues of peroxisomal membrane proteins - PEX11, GIM5A and GIM5B; PXMP4, PEX2 and PEX16 - as well as the transporters GAT1 and GAT3. There were 27 other proteins that could not be unambiguously assigned to other compartments, and that had predicted trans-membrane domains. However, no clear candidates for transport of the major substrates and intermediates of energy metabolism were found. We suggest that, instead, these metabolites are transported via pores formed by the known glycosomal membrane proteins.

  14. The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase of Trypanosomatidae and the glycocomal redox balance of insect stages of Trypanosoma brucei and Leishmania spp.

    NARCIS (Netherlands)

    Guerra, D.G.; Decottignies, A.; Bakker, B.M.; Michels, P.A.M.

    2006-01-01

    The genes for the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase were identified in Trypanosoma brucei and Leishmania major genomes. We have expressed the L. major gene in Saccharomyces cerevisiae and confirmed the subcellular localization and activity of the produced enzyme. Using

  15. Propofol affinity to mitochondrial membranes does not alter mitochondrial function.

    Science.gov (United States)

    Félix, Luís M; Correia, Fernando; Pinto, Pedro A; Campos, Sónia P; Fernandes, Telma; Videira, Romeu; Oliveira, M M; Peixoto, Francisco P; Antunes, Luís M

    2017-05-15

    The molecular mechanisms of hepatotoxicity after propofol anaesthesia have not been fully elucidated, although there is a relation with mitochondrial dysfunction. The action of propofol on mitochondrial hepatic functions in a rat model was evaluated by infusion for 4h with 25 and 62.5mg/kg/h propofol or 3.125ml/kg/h (vehicle). Liver mitochondrial respiratory rates were evaluated as well as mitochondrial transmembrane potential (ΔΨ), calcium fluxes, mitochondrial enzymatic activities (Complex I-V) and oxidative stress biomarkers (superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase, lipid peroxidation and the oxidised/reduced glutathione ratio). Biophysical interactions with membrane models were also performed. The mitochondrial transmembrane potential was decreased and the opening time of the mitochondrial permeability transition pore was slightly reduced for the highest dose. The activity of complex II was stimulated by propofol, which also causes fluctuations on some respiratory parameters, whereas the antioxidant system was affected in a nonspecific manner. Fluorescence quenching studies suggested that propofol is preferably located in deeper regions of the bilayer and has a high affinity to mitochondrial membranes. It is suggested that propofol interacts with liver mitochondrial membranes with mild modification in mitochondrial function. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Variation of G-rich mitochondrial transcripts among stocks of Trypanosoma brucei.

    Science.gov (United States)

    Jasmer, D P; Feagin, J E; Payne, M; Stuart, K

    1987-01-15

    We have compared maxicircle transcripts from eight stocks of subspecies of Trypanosoma brucei. Transcripts from the rRNA and protein genes have a constant size among stocks and exhibit only minor variation in abundance. In contrast, four of the G+C rich sequences encode multiple transcripts that very markedly in size or abundance. Maxicircle nucleotide sequence comparison of three stocks shows very limited sequence divergence suggesting that sequence divergence may not explain the transcript variability. These results suggest that the G-rich transcripts do not encode proteins and that their variability among stocks may result from posttranscriptional processing events.

  17. Dual functions of α-ketoglutarate dehydrogenase E2 in the Krebs cycle and mitochondrial DNA inheritance in Trypanosoma brucei.

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    Sykes, Steven E; Hajduk, Stephen L

    2013-01-01

    The dihydrolipoyl succinyltransferase (E2) of the multisubunit α-ketoglutarate dehydrogenase complex (α-KD) is an essential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally regulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. We found that despite the absence of a functional α-KD, bloodstream form (BF) trypanosomes express α-KDE2, which localized to the mitochondrial matrix and inner membrane. Furthermore, α-KDE2 fractionated with the mitochondrial genome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for α-KDE2 in kDNA maintenance was revealed in α-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showed pronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation of cells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochondrial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that α-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribution of kDNA networks to daughter cells at cytokinesis.

  18. Wafer-scale Mitochondrial Membrane Potential Assays

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    Lim, Tae-Sun; Davila, Antonio; Zand, Katayoun; Douglas, Wallace C.; Burke, Peter J.

    2012-01-01

    It has been reported that mitochondrial metabolic and biophysical parameters are associated with degenerative diseases and the aging process. To evaluate these biochemical parameters, current technology requires several hundred milligrams of isolated mitochondria for functional assays. Here, we demonstrate manufacturable wafer-scale mitochondrial functional assay lab-on-a-chip devices, which require mitochondrial protein quantities three orders of magnitude less than current assays, integrated onto 4” standard silicon wafer with new fabrication processes and materials. Membrane potential changes of isolated mitochondria from various well-established cell lines such as human HeLa cell line (Heb7A), human osteosarcoma cell line (143b) and mouse skeletal muscle tissue were investigated and compared. This second generation integrated lab-on-a-chip system developed here shows enhanced structural durability and reproducibility while increasing the sensitivity to changes in mitochondrial membrane potential by an order of magnitude as compared to first generation technologies. We envision this system to be a great candidate to substitute current mitochondrial assay systems. PMID:22627274

  19. INFECTED WITH TRYPANOSOMA BRUCEI BRUCEI

    African Journals Online (AJOL)

    v brucei infection where for example tant components of extra-cellular the parasite infectivity potential and toxicological effects have been sues in animal. They are ... homeostasis (1,2, 3). With a unique ability to main- tain the cellular contents and ions stable concentrations, Trypano- soma b. brucei, to which humans.

  20. RNA interference analyses suggest a transcript-specific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in Trypanosoma brucei

    NARCIS (Netherlands)

    Vondrusková, Eva; van den Burg, Janny; Zíková, Alena; Ernst, Nancy Lewis; Stuart, Kenneth; Benne, Rob; Lukes, Julius

    2005-01-01

    Mitochondrial RNA-binding proteins MRP1 and MRP2 occur in a heteromeric complex that appears to play a role in U-insertion/deletion editing in trypanosomes. Reduction in the levels of MRP1 (gBP21) and/or MRP2 (gBP25) mRNA by RNA interference in procyclic Trypanosoma brucei resulted in severe growth

  1. Identification of a bacterial-like HslVU protease in the mitochondria of Trypanosoma brucei and its role in mitochondrial DNA replication.

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    Ziyin Li

    2008-04-01

    Full Text Available ATP-dependent protease complexes are present in all living organisms, including the 26S proteasome in eukaryotes, Archaea, and Actinomycetales, and the HslVU protease in eubacteria. The structure of HslVU protease resembles that of the 26S proteasome, and the simultaneous presence of both proteases in one organism was deemed unlikely. However, HslVU homologs have been identified recently in some primordial eukaryotes, though their potential function remains elusive. We characterized the HslVU homolog from Trypanosoma brucei, a eukaryotic protozoan parasite and the causative agent of human sleeping sickness. TbHslVU has ATP-dependent peptidase activity and, like its bacterial counterpart, has essential lysine and N-terminal threonines in the catalytic subunit. By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA. RNAi of TbHslVU dramatically affects the kDNA by causing over-replication of the minicircle DNA. This leads to defects in kDNA segregation and, subsequently, to continuous network growth to an enormous size. Multiple discrete foci of nicked/gapped minicircles are formed on the periphery of kDNA disc, suggesting a failure in repairing the gaps in the minicircles for kDNA segregation. TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote. It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms.

  2. Malleable mitochondrion of Trypanosoma brucei.

    Science.gov (United States)

    Verner, Zdeněk; Basu, Somsuvro; Benz, Corinna; Dixit, Sameer; Dobáková, Eva; Faktorová, Drahomíra; Hashimi, Hassan; Horáková, Eva; Huang, Zhenqiu; Paris, Zdeněk; Peña-Diaz, Priscila; Ridlon, Lucie; Týč, Jiří; Wildridge, David; Zíková, Alena; Lukeš, Julius

    2015-01-01

    The importance of mitochondria for a typical aerobic eukaryotic cell is undeniable, as the list of necessary mitochondrial processes is steadily growing. Here, we summarize the current knowledge of mitochondrial biology of an early-branching parasitic protist, Trypanosoma brucei, a causative agent of serious human and cattle diseases. We present a comprehensive survey of its mitochondrial pathways including kinetoplast DNA replication and maintenance, gene expression, protein and metabolite import, major metabolic pathways, Fe-S cluster synthesis, ion homeostasis, organellar dynamics, and other processes. As we describe in this chapter, the single mitochondrion of T. brucei is everything but simple and as such rivals mitochondria of multicellular organisms. Copyright © 2015. Published by Elsevier Inc.

  3. Mitochondrial DNA mutations provoke dominant inhibition of mitochondrial inner membrane fusion.

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    Cécile Sauvanet

    Full Text Available Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA. We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial ATP6 gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP or to maternally inherited Leigh Syndrome (MILS in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from

  4. TMEM65 is a mitochondrial inner-membrane protein

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    Naotaka Nishimura

    2014-04-01

    Full Text Available It has been reported that the expression of TMEM65 is regulated by steroid receptor RNA activator (SRA. To date, however, the localization and function of TMEM65 remained unknown. We analyzed the intracellular localization of TMEM65. Immunoblot and immunostaining analysis revealed mitochondrial localization of TMEM65. Alkali extraction analysis and digitonin extraction test using isolated mitochondria revealed that TMEM65 is an integral membrane protein that localizes to the inner-membrane of mitochondria. Analysis using deletion mutants of TMEM65 suggested that the N-terminal region (1–20 of this protein is sufficient for mitochondrial targeting and that this mitochondrial targeting signal (MTS is cleaved between the amino acid positions 35 and 64, which contain a putative recognition site of matrix processing protease (MPP. Together, these results suggest that TMEM65 is imported into the mitochondria, integrated into mitochondrial inner-membrane, and processed into its mature form by an MPP.

  5. Development of a no-wash assay for mitochondrial membrane potential using the styryl dye DASPEI

    DEFF Research Database (Denmark)

    Reveles Jensen, Kristian; Rekling, Jens C

    2010-01-01

    Mitochondrial dysfunction is a hallmark of several diseases and may also result from drugs with unwanted side effects on mitochondrial biochemistry. The mitochondrial membrane potential is a good indicator of mitochondrial function. Here, the authors have developed a no-wash mitochondrial membrane...

  6. The nucleotide sequence of the variable region in Trypanosoma brucei completes the sequence analysis of the maxicircle component of mitochondrial kinetoplast DNA

    NARCIS (Netherlands)

    Sloof, P.; de Haan, A.; Eier, W.; van Iersel, M.; Boel, E.; van Steeg, H.; Benne, R.

    1992-01-01

    The nucleotide sequence of two non-contiguous DNA fragments of 4.0 and 2.2 kb, respectively, of the kinetoplast maxicircle of Trypanosoma brucei brucei EATRO strain 427 has been determined, completing the sequence analysis of the so-called variable region (see also de Vries et al., 1988, Mol.

  7. Mechanisms of ER Stress-Mediated Mitochondrial Membrane Permeabilization.

    LENUS (Irish Health Repository)

    Gupta, Sanjeev

    2010-01-01

    During apoptosis, the process of mitochondrial outer membrane permeabilization (MOMP) represents a point-of-no-return as it commits the cell to death. Here we have assessed the role of caspases, Bcl-2 family members and the mitochondrial permeability transition pore on ER stress-induced MOMP and subsequent cell death. Induction of ER stress leads to upregulation of several genes such as Grp78, Edem1, Erp72, Atf4, Wars, Herp, p58ipk, and ERdj4 and leads to caspase activation, release of mitochondrial intermembrane proteins and dissipation of mitochondrial transmembrane potential (DeltaPsim). Mouse embryonic fibroblasts (MEFs) from caspase-9, -2 and, -3 knock-out mice were resistant to ER stress-induced apoptosis which correlated with decreased processing of pro-caspase-3 and -9. Furthermore, pretreatment of cells with caspase inhibitors (Boc-D.fmk and DEVD.fmk) attenuated ER stress-induced loss of DeltaPsim. However, only deficiency of caspase-9 and -2 could prevent ER stress-mediated loss of DeltaPsim. Bcl-2 overexpression or pretreatment of cells with the cell permeable BH4 domain (BH4-Tat) or the mitochondrial permeability transition pore inhibitors, bongkrekic acid or cyclosporine A, attenuated the ER stress-induced loss of DeltaPsim. These data suggest a role for caspase-9 and -2, Bcl-2 family members and the mitochondrial permeability transition pore in loss of mitochondrial membrane potential during ER stress-induced apoptosis.

  8. Structural and Functional Association of Trypanosoma brucei MIX Protein with Cytochrome c Oxidase Complex ▿ †

    Science.gov (United States)

    Zíková, Alena; Panigrahi, Aswini K.; Uboldi, Alessandro D.; Dalley, Rachel A.; Handman, Emanuela; Stuart, Kenneth

    2008-01-01

    A mitochondrial inner membrane protein, designated MIX, seems to be essential for cell viability. The deletion of both alleles was not possible, and the deletion of a single allele led to a loss of virulence and aberrant mitochondrial segregation and cell division in Leishmania major. However, the mechanism by which MIX exerts its effect has not been determined. We show here that MIX is also expressed in the mitochondrion of Trypanosoma brucei, and using RNA interference, we found that its loss leads to a phenotype that is similar to that described for Leishmania. The loss of MIX also had a major effect on cytochrome c oxidase activity, on the mitochondrial membrane potential, and on the production of mitochondrial ATP by oxidative phosphorylation. Using a tandem affinity purification tag, we found that MIX is associated with a multiprotein complex that contains subunits of the mitochondrial cytochrome c oxidase complex (respiratory complex IV), the composition of which was characterized in detail. The specific function of MIX is unknown, but it appears to be important for the function of complex IV and for mitochondrial segregation and cell division in T. brucei. PMID:18776036

  9. Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty.

    Directory of Open Access Journals (Sweden)

    Jason Carnes

    2015-01-01

    Full Text Available Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA. In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.

  10. Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty.

    Science.gov (United States)

    Carnes, Jason; Anupama, Atashi; Balmer, Oliver; Jackson, Andrew; Lewis, Michael; Brown, Rob; Cestari, Igor; Desquesnes, Marc; Gendrin, Claire; Hertz-Fowler, Christiane; Imamura, Hideo; Ivens, Alasdair; Kořený, Luděk; Lai, De-Hua; MacLeod, Annette; McDermott, Suzanne M; Merritt, Chris; Monnerat, Severine; Moon, Wonjong; Myler, Peter; Phan, Isabelle; Ramasamy, Gowthaman; Sivam, Dhileep; Lun, Zhao-Rong; Lukeš, Julius; Stuart, Ken; Schnaufer, Achim

    2015-01-01

    Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.

  11. Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy.

    Science.gov (United States)

    McKenzie, Matthew; Lim, Sze C; Duchen, Michael R

    2017-01-24

    Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca 2+ ) buffers to tightly regulate intracellular Ca 2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (ΔΨm) to sequester Ca 2+ . The influx of Ca 2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains ΔΨm, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix. We describe here a method for simultaneously measuring mitochondria Ca 2+ uptake and ΔΨm in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca 2+ can be measured using the fluorescent Ca 2+ indicator Fluo-4, AM, with measurement of ΔΨm using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca 2+ and ΔΨm simultaneously. Using the sequential addition of Ca 2+ aliquots, mitochondrial Ca 2+ uptake can be monitored, and the concentration at which Ca 2+ induces mitochondrial membrane permeability transition and the loss of ΔΨm determined.

  12. Alkaloids Induce Programmed Cell Death in Bloodstream Forms of Trypanosomes (Trypanosoma b. brucei

    Directory of Open Access Journals (Sweden)

    Michael Wink

    2008-10-01

    Full Text Available The potential induction of a programmed cell death (PCD in Trypanosoma b. brucei by 55 alkaloids of the quinoline, quinolizidine, isoquinoline, indole, terpene, tropane, steroid, and piperidine type was studied by measuring DNA fragmentation and changes in mitochondrial membrane potential. For comparison, the induction of apoptosis by the same alkaloids in human leukemia cells (Jurkat APO-S was tested. Several alkaloids of the isoquinoline, quinoline, indole and steroidal type (berberine, chelerythrine, emetine, sanguinarine, quinine, ajmalicine, ergotamine, harmine, vinblastine, vincristine, colchicine, chaconine, demissidine and veratridine induced programmed cell death, whereas quinolizidine, tropane, terpene and piperidine alkaloids were mostly inactive. Effective PCD induction (EC50 below 10 µM was caused in T. brucei by chelerythrine, emetine, sanguinarine, and chaconine. The active alkaloids can be characterized by their general property to inhibit protein biosynthesis, to intercalate DNA, to disturb membrane fluidity or to inhibit microtubule formation.

  13. Modulation of myometrium mitochondrial membrane potential by calmodulin antagonists

    Directory of Open Access Journals (Sweden)

    S. G. Shlykov

    2014-02-01

    Full Text Available Influence of calmodulin antagonists on mitochondrial membrane potential was investigated using­ a flow cytometry method, confocal microscopy and fluorescent potential-sensitive probes TMRM and MTG. Influence of different concentrations of calmodulin antagonists on mitochondrial membrane potential was studied using flow cytometry method and a fraction of myometrium mitochondria of unpregnant rats. It was shown that 1-10 µМ calmidazolium gradually reduced mitochondria membrane potential. At the same time 10-100 µМ trifluope­razine influenced as follows: 10 µМ – increased polarization, while 100 µМ – caused almost complete depolarization of mitochondrial membranes. In experiments which were conducted with the use of confocal microscopy method and myometrium cells it was shown, that MTG addition to the incubation medium­ led to the appearance of fluorescence signal in a green range. Addition of the second probe (ТМRM resulted in the appearance of fluorescent signal in a red range. Mitochondrial membrane depolarization by 1µМ СССР or 10 mМ NaN3 was accompanied by the decline of “red” fluo­rescence intensity, “green” fluorescence was kept. The 10-15 minute incubation of myometrium cells in the presen­ce 10 µМ calmidazolium or 100 µМ trifluoperazine was accompanied by almost complete decrease of the TMRM fluorescent signal. Thus, with the use of potential-sensitive fluorescent probes TMRM and MTG it was shown, that calmodulin antagonists modulate mitochondrial membrane potential of myometrium cells.

  14. Disaccharide modulation of the mitochondrial membrane fluidity changes induced by the membrane potential.

    Science.gov (United States)

    Ricchelli, F; Camerin, M; Beghetto, C; Crisma, M; Moretto, V; Gobbo, S; Salvato, B; Salet, C; Moreno, G

    2001-02-01

    The influence of the medium composition on the dynamic properties of mitochondrial membranes on depolarization was studied by following the fluorescence anisotropy changes of mitochondria-bound 1,6-diphenyl-1,3,5-hexatriene (DPH) and hematoporphyrin (HP) as reporters, respectively, of lipid and protein regions. On collapse of the potential, the membrane fluidity increased in NaCl-, KCl-, and monosaccharide-based media and decreased in disaccharides. Infrared spectroscopy experiments suggested that disaccharides likely change water's structure and association on the membrane surface. These results indicate that disaccharides induce membrane perturbation, which may interfere in the study of structure-function correlation in biological membranes.

  15. What happens when Trypanosoma brucei leaves Africa.

    Science.gov (United States)

    Jensen, Robert E; Simpson, Larry; Englund, Paul T

    2008-10-01

    Julius Lukes and co-workers evaluated the evolutionary origin of Trypanosoma equiperdum and Trypanosoma evansi, parasites that cause horse and camel diseases. Although similar to T. brucei, the sleeping-sickness parasite, these trypanosomes do not cycle through the tsetse fly and have been able to spread beyond Africa. Transmission occurs sexually, or via blood-sucking flies or vampire bats. They concluded that these parasites, which resemble yeast petite mutants, are T. brucei sub-species, which have evolved recently through changes in mitochondrial DNA.

  16. Trypanosoma brucei TBRGG1, a mitochondrial oligo(U)-binding protein that co-localizes with an in vitro RNA editing activity

    NARCIS (Netherlands)

    Vanhamme, L.; Perez-Morga, D.; Marchal, C.; Speijer, D.; Lambert, L.; Geuskens, M.; Alexandre, S.; Ismaïli, N.; Göringer, U.; Benne, R.; Pays, E.

    1998-01-01

    We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their

  17. On-line measurements of oscillating mitochondrial membrane potential in glucose-fermenting Saccharomyces cerevisiae.

    Science.gov (United States)

    Andersen, Ann Zahle; Poulsen, Allan K; Brasen, Jens Christian; Olsen, Lars Folke

    2007-09-01

    We employed the fluorescent cyanine dye DiOC(2)(3) to measure membrane potential in semi-anaerobic yeast cells under conditions where glycolysis was oscillating. Oscillations in glycolysis were studied by means of the naturally abundant nicotinamide adenine dinucleotide (NADH). We found that the mitochondrial membrane potential was oscillating, and that these oscillations displayed the same frequency and duration as the NADH oscillations. It was confirmed that DiOC(2)(3) localizes itself in the mitochondrial membrane and thus reports qualitative changes solely in mitochondrial membrane potential. Our studies showed that glycolytic oscillations perturb the mitochondrial membrane potential and that the mitochondria do not have any controlling effect on the dynamics of glycolysis under these conditions. Depolarization of the mitochondrial membrane by addition of FCCP quenched mitochondrial membrane potential oscillations and delocalized DiOC(2)(3), while glycolysis continued to oscillate unaffected. Copyright (c) 2007 John Wiley & Sons, Ltd.

  18. Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal membrane origins.

    Science.gov (United States)

    Mellgren, Ronald L

    2008-04-24

    HEK293 cell detergent-resistant membranes (DRMs) isolated by the standard homogenization protocol employing a Teflon pestle homogenizer yielded a prominent opaque band at approximately 16% sucrose upon density gradient ultracentrifugation. In contrast, cell disruption using a ground glass tissue homogenizer generated three distinct DRM populations migrating at approximately 10%, 14%, and 20% sucrose, named DRM subfractions A, B, and C, respectively. Separation of the DRM subfractions by mechanical disruption suggested that they are physically associated within the cellular environment, but can be dissociated by shear forces generated during vigorous homogenization. All three DRM subfractions possessed cholesterol and ganglioside GM1, but differed in protein composition. Subfraction A was enriched in flotillin-1 and contained little caveolin-1. In contrast, subfractions B and C were enriched in caveolin-1. Subfraction C contained several mitochondrial membrane proteins, including mitofilin and porins. Only subfraction B appeared to contain significant amounts of plasma membrane-associated proteins, as revealed by cell surface labeling studies. A similar distribution of DRM subfractions, as assessed by separation of flotillin-1 and caveolin-1 immunoreactivities, was observed in CHO cells, in 3T3-L1 adipocytes, and in HEK293 cells lysed in detergent-free carbonate. Teflon pestle homogenization of HEK293 cells in the presence of the actin-disrupting agent latrunculin B generated DRM subfractions A-C. The microtubule-disrupting agent vinblastine did not facilitate DRM subfraction separation, and DRMs prepared from fibroblasts of vimentin-null mice were present as a single major band on sucrose gradients, unless pre-treated with latrunculin B. These results suggest that the DRM subfractions are interconnected by the actin cytoskeleton, and not by microtubes or vimentin intermediate filaments. The subfractions described may prove useful in studying discrete protein

  19. Calcium flux across plant mitochondrial membranes: possible molecular players

    Directory of Open Access Journals (Sweden)

    Luca eCarraretto

    2016-03-01

    Full Text Available Plants, being sessile organisms, have evolved the ability to integrate external stimuli into metabolic and developmental signals. A wide variety of signals, including abiotic, biotic, and developmental stimuli, were observed to evoke specific spatio-temporal Ca2+ transients which are further transduced by Ca2+ sensor proteins into a transcriptional and metabolic response. Most of the research on Ca2+ signaling in plants has been focused on the transport mechanisms for Ca2+ across the plasma- and the vacuolar membranes as well as on the components involved in decoding of cytoplasmic Ca2+ signals, but how intracellular organelles such as mitochondria are involved in the process of Ca2+ signaling is just emerging. The combination of the molecular players and the elicitors of Ca2+ signaling in mitochondria together with newly generated detection systems for measuring organellar Ca2+ concentrations in plants has started to provide fruitful grounds for further discoveries. In the present review we give an updated overview of the currently identified/hypothesized pathways, such as voltage-dependent anion channels, homologs of the mammalian mitochondrial uniporter, LETM1, a plant glutamate receptor family member, adenine nucleotide/phosphate carriers and the permeability transition pore, that may contribute to the transport of Ca2+ across the outer and inner mitochondrial membranes in plants. We briefly discuss the relevance of the mitochondrial Ca2+ homeostasis for ensuring optimal bioenergetic performance of this organelle.

  20. Polyethylenimine-mediated impairment of mitochondrial membrane potential, respiration and membrane integrity

    DEFF Research Database (Denmark)

    Larsen, Anna Karina; Malinska, Dominika; Koszela-Piotrowska, Izabela

    2012-01-01

    -dependent manner can affect functions (membrane potential, swelling and respiration) and ultrastructural integrity of freshly isolated rat liver mitochondria. The threshold concentration for detection of PEI-mediated impairment of rat liver mitochondrial functions is 3 µg/mL, however, lower PEI levels still exert...

  1. Non-radioactive method for labelling Trypanosoma brucei brucei ...

    African Journals Online (AJOL)

    It was observed that (i) the biological activities of the T.brucei brucei like the motility, viability, and growth were not affected adversely by the biotinylation; (ii) as little as 100ug Sulfo-NHS-LC-Biotin was enough to label about 6x106 Trypanosoma Brucei Brucei; (iii) high temperature appears to have adverse effect on the ...

  2. Combinations of alkaloids affecting different molecular targets with the saponin digitonin can synergistically enhance trypanocidal activity against Trypanosoma brucei brucei

    National Research Council Canada - National Science Library

    Krstin, Sonja; Peixoto, Herbenya Silva; Wink, Michael

    2015-01-01

    .... In this study, the potential synergism of mutual combinations of bioactive alkaloids and alkaloids with a membrane-active steroidal saponin, digitonin, was explored with regard to their effect on T. b. brucei...

  3. TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions.

    Science.gov (United States)

    Martínez-Reyes, Inmaculada; Diebold, Lauren P; Kong, Hyewon; Schieber, Michael; Huang, He; Hensley, Christopher T; Mehta, Manan M; Wang, Tianyuan; Santos, Janine H; Woychik, Richard; Dufour, Eric; Spelbrink, Johannes N; Weinberg, Samuel E; Zhao, Yingming; DeBerardinis, Ralph J; Chandel, Navdeep S

    2016-01-21

    Mitochondrial metabolism is necessary for the maintenance of oxidative TCA cycle function and mitochondrial membrane potential. Previous attempts to decipher whether mitochondria are necessary for biological outcomes have been hampered by genetic and pharmacologic methods that simultaneously disrupt multiple functions linked to mitochondrial metabolism. Here, we report that inducible depletion of mitochondrial DNA (ρ(ο) cells) diminished respiration, oxidative TCA cycle function, and the mitochondrial membrane potential, resulting in diminished cell proliferation, hypoxic activation of HIF-1, and specific histone acetylation marks. Genetic reconstitution only of the oxidative TCA cycle function specifically in these inducible ρ(ο) cells restored metabolites, resulting in re-establishment of histone acetylation. In contrast, genetic reconstitution of the mitochondrial membrane potential restored ROS, which were necessary for hypoxic activation of HIF-1 and cell proliferation. These results indicate that distinct mitochondrial functions associated with respiration are necessary for cell proliferation, epigenetics, and HIF-1 activation. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. On-line measurements of oscillating mitochondrial membrane potential in glucose-fermenting Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Andersen, Ann Zahle; Poulsen, Allan K; Brasen, Jens Christian

    2007-01-01

    We employed the fluorescent cyanine dye DiOC(2)(3) to measure membrane potential in semi-anaerobic yeast cells under conditions where glycolysis was oscillating. Oscillations in glycolysis were studied by means of the naturally abundant nicotinamide adenine dinucleotide (NADH). We found...... studies showed that glycolytic oscillations perturb the mitochondrial membrane potential and that the mitochondria do not have any controlling effect on the dynamics of glycolysis under these conditions. Depolarization of the mitochondrial membrane by addition of FCCP quenched mitochondrial membrane...... potential oscillations and delocalized DiOC(2)(3), while glycolysis continued to oscillate unaffected....

  5. A Protein Complex Map of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Vahid H Gazestani

    2016-03-01

    Full Text Available The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org.

  6. Photodynamic action of chlorin e6 on thymocyte plasmatic and mitochondrial membrane potentials

    Science.gov (United States)

    Gyulkhandanyan, Grigor V.

    2005-08-01

    Transmembrane potentials appear to be cell state sensitive characteristics and can give information about cell damage initial stage. Photodynamic action of the photosensitizer chlorin e6 on plasmatic and mitochondrial membrane potentials of the rat thymus lymphocytes was studied using voltage-sensitive dye rhodamine 6G. It has been revealed that mitochondrial membrane potential is more sensitive characteristic of membrane disfunction than plasmatic one at the cell photodamage.

  7. Regulation of glycolytic oscillations by mitochondrial and plasma membrane H+-ATPases

    DEFF Research Database (Denmark)

    Olsen, Lars Folke; Andersen, Ann Zahle; Lunding, Anita

    2009-01-01

    We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3,3'-diethylo......We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3......,3'-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H(+)-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol...... in a number of different situations (changing pH or the presence and absence of inhibitors). Finally, the intracellular pH was determined and shown to oscillate. The results support earlier work suggesting that the coupling between glycolysis and mitochondrial membrane potential is mediated by the ADP...

  8. Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry.

    Science.gov (United States)

    Kanno, Chihiro; Kang, Sung-Sik; Kitade, Yasuyuki; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

    2016-08-01

    The present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen-thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE-PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE-PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE-PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE-PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously.

  9. The Possible Role of Nonbilayer Structures in Regulating ATP Synthase Activity in Mitochondrial Membranes.

    Science.gov (United States)

    Gasanov, S E; Kim, A A; Dagda, R K

    2016-07-01

    The effects of temperature and of the membrane-active protein CTII on the formation of nonbilayer structures in mitochondrial membranes were studied by (31)P-NMR. Increasing the temperature of isolated mitochondrial fractions correlated with an increase in ATP synthase activity and the formation of nonbilayer packed phospholipids with immobilized molecular mobility. Computer modeling was employed for analyzing the interaction of mitochondrial membrane phospholipids with the molecular surface of CTII, which behaves like a dicyclohexylcarbodiimide-binding protein (DCCD-BP) of the F0 group in a lipid phase. Overall, our studies suggest that proton permeability toroidal pores formed in mitochondrial membranes consist of immobilized nonbilayer-packed phospholipids formed via interactions with DCCD-BP. Our studies support the existence of a proton transport along a concentration gradient mediated via transit toroidal permeability pores which induce conformational changes necessary for mediating the catalytic activity of ATP synthase in the subunits of the F0-F1 complex.

  10. Cholesterol and Peroxidized Cardiolipin in Mitochondrial Membrane Properties, Permeabilization and Cell Death

    Science.gov (United States)

    Montero, Joan; Mari, Montserrat; Colell, Anna; Morales, Albert; Basañez, Gorka; Garcia-Ruiz, Carmen; Fernández-Checa, Jose C.

    2010-01-01

    Mitochondria are known to actively regulate cell death with the final phenotype of demise being determined by the metabolic and energetic status of the cell. Mitochondrial membrane permeabilization (MMP) is a critical event in cell death, as it regulates the degree of mitochondrial dysfunction and the release of intermembrane proteins that function in the activation and assembly of caspases. In addition to the crucial role of proapoptotic members of the Bcl-2 family, the lipid composition of the mitochondrial membranes is increasingly recognized to modulate MMP and hence cell death. The unphysiological accumulation of cholesterol in mitochondrial membranes regulates their physical properties, faciliating or impairing MMP during Bax and death ligand-induced cell death depending on the level of mitochondrial GSH (mGSH), which in turn regulates the oxidation status of cardiolipin. Cholesterol-mediated mGSH depletion stimulates TNF-induced reactive oxygen species and subsequent cardiolipin peroxidation, which destabilizes the lipid bilayer and potentiates Bax-induced membrane permeabilization. These data suggest that the balance of mitochondrial cholesterol to peroxidized cardiolipin regulates mitochondrial membrane properties and permeabilization, emerging as a rheostat in cell death. PMID:20153716

  11. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

    Science.gov (United States)

    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

    Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. © FASEB.

  12. Mitochondrial membrane studies using impedance spectroscopy with parallel pH monitoring.

    Directory of Open Access Journals (Sweden)

    Divya Padmaraj

    Full Text Available A biological microelectromechanical system (BioMEMS device was designed to study complementary mitochondrial parameters important in mitochondrial dysfunction studies. Mitochondrial dysfunction has been linked to many diseases, including diabetes, obesity, heart failure and aging, as these organelles play a critical role in energy generation, cell signaling and apoptosis. The synthesis of ATP is driven by the electrical potential across the inner mitochondrial membrane and by the pH difference due to proton flux across it. We have developed a tool to study the ionic activity of the mitochondria in parallel with dielectric measurements (impedance spectroscopy to gain a better understanding of the properties of the mitochondrial membrane. This BioMEMS chip includes: 1 electrodes for impedance studies of mitochondria designed as two- and four-probe structures for optimized operation over a wide frequency range and 2 ion-sensitive field effect transistors for proton studies of the electron transport chain and for possible monitoring other ions such as sodium, potassium and calcium. We have used uncouplers to depolarize the mitochondrial membrane and disrupt the ionic balance. Dielectric spectroscopy responded with a corresponding increase in impedance values pointing at changes in mitochondrial membrane potential. An electrical model was used to describe mitochondrial sample's complex impedance frequency dependencies and the contribution of the membrane to overall impedance changes. The results prove that dielectric spectroscopy can be used as a tool for membrane potential studies. It can be concluded that studies of the electrochemical parameters associated with mitochondrial bioenergetics may render significant information on various abnormalities attributable to these organelles.

  13. Rapeseed oil-rich diet alters hepatic mitochondrial membrane lipid composition and disrupts bioenergetics.

    Science.gov (United States)

    Monteiro, João P; Pereira, Cláudia V; Silva, Ana M; Maciel, Elisabete; Baldeiras, Inês; Peixoto, Francisco; Domingues, Maria R; Jurado, Amália S; Oliveira, Paulo J

    2013-12-01

    Diet is directly related with physiological alterations occurring at a cell and subcellular level. However, the role of diet manipulation on mitochondrial physiology is still largely unexplored. Aiming at correlating diet with alterations of mitochondrial membrane composition and bioenergetics, Wistar-Han male rats were fed for 11, 22 and 33 days with a rapeseed oil-based diet and mitochondrial bioenergetics, and membrane composition were compared at each time point with a standard diet group. Considerable differences were noticed in mitochondrial membrane lipid composition, namely in terms of fatty acyl chains and relative proportions of phospholipid classes, the modified diet inducing a decrease in the saturated to unsaturated molar ratio and an increase in the phosphatidylcholine to phosphatidylethanolamine molar ratio. Mass spectrometry lipid analysis showed significant differences in the major species of cardiolipin, with an apparent increased incorporation of oleic acid as a result of exposure to the modified diet. Rats fed the modified diet during 22 days showed decreased hepatic mitochondrial state 3 respiration and were more susceptible to Ca(2+)-induced transition pore opening. Rapeseed oil-enriched diet also appeared to promote a decrease in hydroperoxide production by the respiratory chain, although a simultaneous decrease in vitamin E content was detected. In conclusion, our data indicate that the rapeseed oil diet causes negative alterations on hepatic mitochondrial bioenergetics, which may result from membrane remodeling. Such alterations may have an impact not only on energy supply to the cell, but also on drug-induced hepatic mitochondrial liabilities.

  14. Preliminary crystallographic studies of yeast mitochondrial peripheral membrane protein Tim44p

    Energy Technology Data Exchange (ETDEWEB)

    Josyula, Ratnakar [Department of Cell Biology, Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States); Jin, Zhongmin [SER-CAT, APS, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439 (United States); McCombs, Deborah; DeLucas, Lawrence [Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States); Sha, Bingdong, E-mail: bdsha@uab.edu [Department of Cell Biology, Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States)

    2006-02-01

    Tim44p is an essential mitochondrial peripheral membrane protein. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, the yeast Tim44p has been crystallized. Protein translocations across mitochondrial membranes play critical roles in mitochondrion biogenesis. Protein transport from the cell cytosol to the mitochondrial matrix is carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tim44p is an essential mitochondrial peripheral membrane protein and a major component of the TIM23 translocon. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, the yeast Tim44p was crystallized. The crystals diffract to 3.2 Å using a synchrotron X-ray source and belong to space group P6{sub 3}22, with unit-cell parameters a = 124.25, c = 77.83 Å. There is one Tim44p molecule in one asymmetric unit, which corresponds to a solvent content of approximately 43%. Structure determination by MAD methods is under way.

  15. Astrocytic mitochondrial membrane hyperpolarization following extended oxygen and glucose deprivation.

    Directory of Open Access Journals (Sweden)

    Andrej Korenić

    Full Text Available Astrocytes can tolerate longer periods of oxygen and glucose deprivation (OGD as compared to neurons. The reasons for this reduced vulnerability are not well understood. Particularly, changes in mitochondrial membrane potential (Δψ(m in astrocytes, an indicator of the cellular redox state, have not been investigated during reperfusion after extended OGD exposure. Here, we subjected primary mouse astrocytes to glucose deprivation (GD, OGD and combinations of both conditions varying in duration and sequence. Changes in Δψ(m, visualized by change in the fluorescence of JC-1, were investigated within one hour after reconstitution of oxygen and glucose supply, intended to model in vivo reperfusion. In all experiments, astrocytes showed resilience to extended periods of OGD, which had little effect on Δψ(m during reperfusion, whereas GD caused a robust Δψ(m negativation. In case no Δψ(m negativation was observed after OGD, subsequent chemical oxygen deprivation (OD induced by sodium azide caused depolarization, which, however, was significantly delayed as compared to normoxic group. When GD preceded OD for 12 h, Δψ(m hyperpolarization was induced by both GD and subsequent OD, but significant interaction between these conditions was not detected. However, when GD was extended to 48 h preceding OGD, hyperpolarization enhanced during reperfusion. This implicates synergistic effects of both conditions in that sequence. These findings provide novel information regarding the role of the two main substrates of electron transport chain (glucose and oxygen and their hyperpolarizing effect on Δψ(m during substrate deprivation, thus shedding new light on mechanisms of astrocyte resilience to prolonged ischemic injury.

  16. Integrity of the plasma membrane, the acrosomal membrane, and the mitochondrial membrane potential of sperm in Nelore bulls from puberty to sexual maturity

    Directory of Open Access Journals (Sweden)

    L.S.L.S. Reis

    2016-06-01

    Full Text Available ABSTRACT This study evaluated the plasma membrane integrity, acrosomal membrane integrity, and mitochondrial membrane potential of Nelore bull sperm from early puberty to early sexual maturity and their associations with sperm motility and vigor, the mass motility of the spermatozoa (wave motion, scrotal circumference, and testosterone. Sixty Nelore bulls aged 18 to 19 months were divided into four lots (n=15 bulls/lot and evaluated over 280 days. Semen samples, collected every 56 days by electroejaculation, were evaluated soon after collection for motility, vigor and wave motion under an optical microscope. Sperm membrane integrity, acrosomal integrity, and mitochondrial activity were evaluated under a fluorescent microscope using probe association (FITC-PSA, PI, JC-1, H342. The sperm were classified into eight integrity categories depending on whether they exhibited intact or damaged membranes, an intact or damaged acrosomal membrane, and high or low mitochondrial potential. The results show that bulls have a low amount of sperm with intact membranes at puberty, and the sperm show low motility, vigor, and wave motion; however, in bulls at early sexual maturity, the integrity of the sperm membrane increased significantly. The rate of sperm membrane damage was negatively correlated with motility, vigor, wave motion, and testosterone in the bulls, and a positive correlation existed between sperm plasma membrane integrity and scrotal circumference. The integrity of the acrosomal membrane was not influenced by puberty. During puberty and into early sexual maturity, bulls show low sperm mitochondrial potential, but when bulls reached sexual maturity, high membrane integrity with high mitochondrial potential was evident.

  17. Interaction of ADP, atractyloside, and gummiferin on the ADP translocase of the inner mitochondrial membrane

    Energy Technology Data Exchange (ETDEWEB)

    Vignais, P.V.; Vignais, P.M.; Defaye, G.; Lauquin, G.; Doussiere, J.; Chabert, J.; Brandolin, G.

    1972-05-01

    From international conference on mechanism in bioenergetica; Bari, Italy (1 May 1972). Two specific inhibitors of the adenine nucleotide translocation, gummiferin (GUM), identified to 4-carboxyatractyloside and atractyloside (ATR), were labeled with /sup 35/S and their binding properties to whole mitochondria and inner mitochondrial membrane vesicles used to monitor changes of membrane conformation induced by ADP. (auth)

  18. In vitro induction of rat liver mitochondrial membrane permeability ...

    African Journals Online (AJOL)

    Alteration of mitochondrial functions such as permeability transition (PT), a process associated with the uncoupling of oxidative phosphorylation, has been found to play a vital role in the apoptotic process induced by certain anti-cancer agents. When triggered, PT facilitates the release of mitochondrial apoptogenic proteins ...

  19. Taxonomy Icon Data: Trypanosoma brucei [Taxonomy Icon

    Lifescience Database Archive (English)

    Full Text Available Trypanosoma brucei Trypanosoma brucei Trypanosoma_brucei_L.png Trypanosoma_brucei_NL.png Trypanosoma_bruce...i_S.png Trypanosoma_brucei_NS.png http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Trypanosoma+bruce...i&t=L http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Trypanosoma+brucei&t=NL http://bioscie...ncedbc.jp/taxonomy_icon/icon.cgi?i=Trypanosoma+brucei&t=S http://biosciencedbc.jp.../taxonomy_icon/icon.cgi?i=Trypanosoma+brucei&t=NS http://togodb.biosciencedbc.jp/togodb/view/taxonomy_icon_comment_en?species_id=121 ...

  20. Palmitoleic acid induces the cardiac mitochondrial membrane permeability transition despite the presence of L-carnitine.

    Science.gov (United States)

    Oyanagi, Eri; Uchida, Masataka; Miyakawa, Takeshi; Miyachi, Motohiko; Yamaguchi, Hidetaka; Nagami, Kuniatsu; Utsumi, Kozo; Yano, Hiromi

    Although palmitoleic acid (C16:1) is associated with arrhythmias, and increases in an age-dependent matter, the effects of L-carnitine, which is essential for the transport of long-chain fatty acids into the mitochondria, are unclear. It has been postulated that L-carnitine may attenuate palmitate (C16:0)-induced mitochondrial dysfunction and the apoptosis of cardiomyocytes. The aim of this study was to elucidate the activity of L-carnitine in the prevention of the palmitoleic acid-induced mitochondrial membrane permeability transition and cytochrome c release using isolated cardiac mitochondria from rats. Palmitoleoyl-CoA-induced mitochondrial respiration was not accelerated by L-carnitine treatment, and this respiration was slightly inhibited by oligomycin, which is an inhibitor of ATP synthase. Despite pretreatment with L-carnitine, the mitochondrial membrane potential decreased and mitochondrial swelling was induced by palmitoleoyl-CoA. In the presence of a combination of L-carnitine and tiron, a free radical scavenger, there was attenuated mitochondrial swelling and cytochrome c release following palmitoleoyl-CoA treatment. We concluded that palmitoleic acid, but not palmitate, induces the cardiac mitochondrial membrane permeability transition despite the presence of L-carnitine. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Loss of prohibitin membrane scaffolds impairs mitochondrial architecture and leads to tau hyperphosphorylation and neurodegeneration.

    Directory of Open Access Journals (Sweden)

    Carsten Merkwirth

    Full Text Available Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegeneration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds. In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the dynamin-like GTPase OPA1 (optic atrophy 1, which mediates mitochondrial inner membrane fusion and cristae morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus, demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1, affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability, mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins. Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for

  2. A nontoxic, photostable and high signal-to-noise ratio mitochondrial probe with mitochondrial membrane potential and viscosity detectivity

    Science.gov (United States)

    Chen, Yanan; Qi, Jianguo; Huang, Jing; Zhou, Xiaomin; Niu, Linqiang; Yan, Zhijie; Wang, Jianhong

    2018-01-01

    Herein, we reported a yellow emission probe 1-methyl-4-(6-morpholino-1, 3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl) pyridin-1-ium iodide which could specifically stain mitochondria in living immortalized and normal cells. In comparison to the common mitochondria tracker (Mitotracker Deep Red, MTDR), this probe was nontoxic, photostable and ultrahigh signal-to-noise ratio, which could real-time monitor mitochondria for a long time. Moreover, this probe also showed high sensitivity towards mitochondrial membrane potential and intramitochondrial viscosity change. Consequently, this probe was used for imaging mitochondria, detecting changes in mitochondrial membrane potential and intramitochondrial viscosity in physiological and pathological processes.

  3. A nontoxic, photostable and high signal-to-noise ratio mitochondrial probe with mitochondrial membrane potential and viscosity detectivity.

    Science.gov (United States)

    Chen, Yanan; Qi, Jianguo; Huang, Jing; Zhou, Xiaomin; Niu, Linqiang; Yan, Zhijie; Wang, Jianhong

    2018-01-15

    Herein, we reported a yellow emission probe 1-methyl-4-(6-morpholino-1, 3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl) pyridin-1-ium iodide which could specifically stain mitochondria in living immortalized and normal cells. In comparison to the common mitochondria tracker (Mitotracker Deep Red, MTDR), this probe was nontoxic, photostable and ultrahigh signal-to-noise ratio, which could real-time monitor mitochondria for a long time. Moreover, this probe also showed high sensitivity towards mitochondrial membrane potential and intramitochondrial viscosity change. Consequently, this probe was used for imaging mitochondria, detecting changes in mitochondrial membrane potential and intramitochondrial viscosity in physiological and pathological processes. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Uncoupling proteins 2 and 3 alter mitochondrial membrane ...

    African Journals Online (AJOL)

    Saccharomyces cerevisiae). Total phospholipids were extracted and their fatty acyl composition was analysed. Expression of hUCP2 and hUCP3 significantly altered the mitochondrial phospholipid fatty acyl composition. Palmitoleoyl groups (16:1(n-7)) ...

  5. Hepatic response in Trypanosomia brucei brucei infected-rats ...

    African Journals Online (AJOL)

    Ethanol leaf extract of Tithonia diversifolia was investigated for its hepato-curative activity in Trypanosoma brucei brucei infected-rats. The phytochemical compositions of the plant extract were also evaluated. The leaf extract was administered 14 days post-infection at dose of 200 and 400 mg/kg orally once daily.

  6. Mitochondrial DNA damage associated with lipid peroxidation of the mitochondrial membrane induced by Fe2+-citrate

    Directory of Open Access Journals (Sweden)

    Andréa M. Almeida

    2006-09-01

    Full Text Available Iron imbalance/accumulation has been implicated in oxidative injury associated with many degenerative diseases such as hereditary hemochromatosis, beta-thalassemia, and Friedreich's ataxia. Mitochondria are particularly sensitive to iron-induced oxidative stress - high loads of iron cause extensive lipid peroxidation and membrane permeabilization in isolated mitochondria. Here we detected and characterized mitochondrial DNA damage in isolated rat liver mitochondria exposed to a Fe2+-citrate complex, a small molecular weight complex. Intense DNA fragmentation was induced after the incubation of mitochondria with the iron complex. The detection of 3' phosphoglycolate ends at the mtDNA strand breaks by a 32P-postlabeling assay, suggested the involvement of hydroxyl radical in the DNA fragmentation induced by Fe2+-citrate. Increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine also suggested that Fe2+-citrate-induced oxidative stress causes mitochondrial DNA damage. In conclusion, our results show that iron-mediated lipid peroxidation was associated with intense mtDNA damage derived from the direct attack of reactive oxygen species.Desequilíbrio/acúmulo de ferro tem sido implicado em injúria oxidativa associada a diversas doenças degenerativas tais como, hemocromatose hereditária, beta-talassemia e ataxia de Friedreich. As mitocôndrias são particularmente sensíveis a estresse oxidativo induzido por ferro - um carregamento alto de ferro em mitocôndrias isoladas pode causar uma extensiva peroxidação lipídica e a permeabilização de membrana. Nesse estudo, nós detectamos e caracterizamos danos do DNA mitocondrial em mitocôndrias isoladas de fígado de rato, expostas ao complexo Fe2+-citrato, um dos complexos de baixo peso molecular. A intensa fragmentação do DNA foi induzida após a incubação das mitocôndrias com o complexo de ferro. A detecção de finais 3' de fosfoglicolato nas quebras de fitas de DNA mitocondrial pelo ensaio 32

  7. Effect of hydrogen peroxide and hypochlorite on mitochondrial membrane potential in permeabilized rat heart cells

    Energy Technology Data Exchange (ETDEWEB)

    Konno, N.; Kako, K.J. (Univ. of Ottawa, Ontario (Canada))

    1991-03-15

    The chemiosmotic theory states that the proton electrochemical potential gradient across the membrane drives mitochondrial energy transduction. Mitochondria can take up Ca accumulated in the cytosol. Therefore, oxidant-induced ATP depletion and Ca overload in the cell may be the result of mitochondrial dysfunction. Consequently, the authors measured membrane potential of mitochondria in situ in isolated rat heart myocytes with {sup 3}H-triphenylmethylphosphonium. This was followed by permeabilization using digitonin and rapid centrifugation using density gradient of bromododecane. They found that the membrane potentials, 118 mV with isolated and 161 mV with in situ mitochondria, were relatively well maintained under oxidant stress. High concentrations of oxidants reduced also the cellular ATP level, whereas the matrix volume was not significantly changed. The H{sub 2}O{sub 2} effect on the mitochondrial membrane potential was more pronounced when the extra-mitochondrial free Ca concentration was increased in permeabilized myocytes. These results support the view that heart mitochondria are equipped with well developed defense mechanisms against oxidants and thus the electrochemical gradient of inner membrane is affected only by a relatively large concentration of H{sub 2}O{sub 2} and HOCl.

  8. Formation of Mitochondrial Outer Membrane Derived Protrusions and Vesicles in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Akihiro Yamashita

    Full Text Available Mitochondria are dynamic organelles that have inner and outer membranes. In plants, the inner membrane has been well studied but relatively little is known about the outer membrane. Here we report that Arabidopsis cells have mitochondrial outer membrane-derived structures, some of which protrude from the main body of mitochondria (mitochondrial outer-membrane protrusions; MOPs, while others form vesicle-like structures without a matrix marker. The latter vesicle-like structures are similar to some mammalian MDVs (mitochondrial-derived vesicles. Live imaging demonstrated that a plant MDV budded off from the tip of a MOP. MDVs were also observed in the drp3a drp3b double mutant, indicating that they could be formed without the mitochondrial fission factors DRP3A and DRP3B. Double staining studies showed that the MDVs were not peroxisomes, endosomes, Golgi apparatus or trans-Golgi network (TGN. The numbers of MDVs and MOPs increased in senescent leaves and after dark treatment. Together, these results suggest that MDVs and MOPs are related to leaf senescence.

  9. Apoptosis and mitochondrial membrane potential changes of T ...

    African Journals Online (AJOL)

    Background: Down's syndrome (DS) patients present a high risk of developing alterations of the immune system. The increased susceptibility to bacterial and viral infection, hematologic malignancies and autoimmune disease, suggest that immunodeficiency is an integral part of DS. Little is known about the mitochondrial ...

  10. SUMOylation in Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Cornelia Andrea Klein

    2013-10-01

    Full Text Available Small ubiquitin like modifier (SUMO proteins are involved in many processes in eukaryotes. We here show that Trypanosoma brucei SUMO (Tb927.5.3210 modifies many proteins. The levels of SUMOylation were unaffected by temperature changes but were increased by severe oxidative stress. We obtained evidence that trypanosome homologues of the SUMO conjugating enzyme Ubc9 (Tb927.2.2460 and the SUMO-specific protease SENP (Tb927.9.2220 are involved in SUMOylation and SUMO removal, respectively.

  11. Rat liver mitochondrial membrane characteristics and mitochondrial functions are more profoundly altered by dietary lipid quantity than by dietary lipid quality: effect of different nutritional lipid patterns.

    Science.gov (United States)

    Aoun, Manar; Feillet-Coudray, Christine; Fouret, Gilles; Chabi, Béatrice; Crouzier, David; Ferreri, Carla; Chatgilialoglu, Chryssostomos; Wrutniak-Cabello, Chantal; Cristol, Jean Paul; Carbonneau, Marie-Annette; Coudray, Charles

    2012-03-01

    Dietary lipids are known to affect the composition of the biological membrane and functions that are involved in cell death and survival. The mitochondrial respiratory chain enzymes are membrane protein complexes whose function depends on the composition and fluidity of the mitochondrial membrane lipid. The present study aimed at investigating the impact of different nutritional patterns of dietary lipids on liver mitochondrial functions. A total of forty-eight Wistar male rats were divided into six groups and fed for 12 weeks with a basal diet, lard diet or fish oil diet, containing either 50 or 300 g lipid/kg. The 30 % lipid intake increased liver NEFA, TAG and cholesterol levels, increased mitochondrial NEFA and TAG, and decreased phospholipid (PL) levels. SFA, PUFA and unsaturation index (UI) increased, whereas MUFA and trans-fatty acids (FA) decreased in the mitochondrial membrane PL in 30 % fat diet-fed rats compared with 5 % lipid diet-fed rats. PL UI increased with fish oil diet v. basal and lard-rich diets, and PL trans-FA increased with lard diet v. basal and fish oil diets. The 30 % lipid diet intake increased mitochondrial membrane potential, membrane fluidity, mitochondrial respiration and complex V activity, and decreased complex III and IV activities. With regard to lipid quality effects, β-oxidation decreased with the intake of basal or fish oil diets compared with that of the lard diet. The intake of a fish oil diet decreased complex III and IV activities compared with both the basal and lard diets. In conclusion, the characteristics and mitochondrial functions of the rat liver mitochondrial membrane are more profoundly altered by the quantity of dietary lipid than by its quality, which may have profound impacts on the pathogenesis and development of non-alcoholic fatty liver disease.

  12. Mechanism of Trypanosoma brucei gambiense resistance to human serum

    DEFF Research Database (Denmark)

    Uzureau, Pierrick; Uzureau, Sophie; Lecordier, Laurence

    2013-01-01

    The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease......GP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According...

  13. Heinrich Wieland--prize lecture. Transport of proteins across mitochondrial membranes.

    Science.gov (United States)

    Neupert, W

    1994-03-01

    The vast majority of proteins comprising the mitochondrion are encoded by nuclear genes, synthesized on ribosomes in the cytosol, and translocated into the various mitochondrial subcompartments. During this process proteins must cross the lipid membranes of the mitochondrion without interfering with the integrity or functions of the organelle. In recent years an approach combining biochemical, molecular, genetic, and morphological methodology has provided insights into various aspects of this complex process of intracellular protein sorting. In particular, a greater understanding of the molecular specificity and mechanism of targeting of mitochondrial preproteins has been reached, as a protein complex of the outer membrane which facilitates recognition and initial membrane insertion has been identified and characterized. Furthermore, pathways and components involved in the translocation of pre-proteins across the two mitochondrial membranes are being dissected and defined. The energetics of translocation and the processes of unfolding and folding of proteins during transmembrane transfer are closely linked to the function of a host of proteins known as heat-shock proteins or molecular chaperones, present both outside and inside the mitochondrion. In addition, the analysis of the process of folding of polypeptides in the mitochondrial matrix has allowed novel and unexpected insights into general pathways of protein folding assisted by folding factors. Pathways of sorting of proteins to the four different mitochondrial subcompartments--the outer membrane (OM), intermembrane space, inner membrane (IM) and matrix--are only partly understood and reveal an amazing complexity and variation. Many additional protein factors are involved in these latter processes, a few of which have been analyzed, such as cytochrome c heme lyase and cytochrome c1 heme lyase, enzymes that catalyze the covalent addition of the heme group to cytochrome c and c1 preproteins, and the

  14. Yeast Mitochondrial Interactosome Model: Metabolon Membrane Proteins Complex Involved in the Channeling of ADP/ATP

    Directory of Open Access Journals (Sweden)

    Benjamin Clémençon

    2012-02-01

    Full Text Available The existence of a mitochondrial interactosome (MI has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp and inorganic phosphate (PiC carriers as well as the VDAC (or mitochondrial porin catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F0, F1 under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003.

  15. Mitochondrial protein import in trypanosomes: Expect the unexpected.

    Science.gov (United States)

    Harsman, Anke; Schneider, André

    2017-02-01

    Mitochondria have many different functions, the most important one of which is oxidative phosphorylation. They originated from an endosymbiotic event between a bacterium and an archaeal host cell. It was the evolution of a protein import system that marked the boundary between the endosymbiotic ancestor of the mitochondrion and a true organelle that is under the control of the nucleus. In present day mitochondria more than 95% of all proteins are imported from the cytosol in a proces mediated by hetero-oligomeric protein complexes in the outer and inner mitochondrial membranes. In this review we compare mitochondrial protein import in the best studied model system yeast and the parasitic protozoan Trypanosoma brucei. The 2 organisms are phylogenetically only remotely related. Despite the fact that mitochondrial protein import has the same function in both species, only very few subunits of their import machineries are conserved. Moreover, while yeast has 2 inner membrane protein translocases, one specialized for presequence-containing and one for mitochondrial carrier proteins, T. brucei has a single inner membrane translocase only, that mediates import of both types of substrates. The evolutionary implications of these findings are discussed. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia.

    Science.gov (United States)

    Cooper, Helen M; Yang, Yang; Ylikallio, Emil; Khairullin, Rafil; Woldegebriel, Rosa; Lin, Kai-Lan; Euro, Liliya; Palin, Eino; Wolf, Alexander; Trokovic, Ras; Isohanni, Pirjo; Kaakkola, Seppo; Auranen, Mari; Lönnqvist, Tuula; Wanrooij, Sjoerd; Tyynismaa, Henna

    2017-04-15

    De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity. © The Author 2017. Published by Oxford University Press.

  17. Role of cardiolipins in the inner mitochondrial membrane: insight gained through atom-scale simulations

    DEFF Research Database (Denmark)

    Róg, Tomasz; Martinez-Seara, Hector; Munck, Nana

    2009-01-01

    , the exceptional nature of cardiolipins is characterized by their small charged head group connected to typically four hydrocarbon chains. In this work, we present atomic-scale molecular dynamics simulations of the inner mitochondrial membrane modeled as a mixture of cardiolipins (CLs), phosphatidylcholines (PCs...

  18. Clearing the outer mitochondrial membrane from harmful proteins via lipid droplets

    Czech Academy of Sciences Publication Activity Database

    Bischof, J.; Salzmann, M.; Streubel, M.K.; Hašek, Jiří; Geltinger, F.; Duschl, J.; Bresgen, N.; Briza, P.; Hašková, Danuša; Lejsková, Renata; Sopjani, M.; Richter, K.; Rinnerthaler, M.

    2017-01-01

    Roč. 3, March 20 (2017), č. článku 17016. E-ISSN 2058-7716 R&D Projects: GA ČR(CZ) GA16-05497S; GA MŠk(CZ) 7AMB16AT006 Institutional support: RVO:61388971 Keywords : mitochondrial membrane * harmful proteins * lipid droplets Subject RIV: EE - Microbiology, Virology

  19. Synchronism in mitochondrial ROS flashes, membrane depolarization and calcium sparks in human carcinoma cells.

    Science.gov (United States)

    Kuznetsov, Andrey V; Javadov, Sabzali; Saks, Valdur; Margreiter, Raimund; Grimm, Michael

    2017-06-01

    Mitochondria are major producers of reactive oxygen species (ROS) in many cells including cancer cells. However, complex interrelationships between mitochondrial ROS (mitoROS), mitochondrial membrane potential (ΔΨm) and Ca2+ are not completely understood. Using human carcinoma cells, we further highlight biphasic ROS dynamics: - gradual mitoROS increase followed by mitoROS flash. Also, we demonstrate heterogeneity in rates of mitoROS generation and flash initiation time. Comparing mitochondrial and near-extra-mitochondrial signals, we show that mechanisms of mitoROS flashes in single mitochondria, linked to mitochondrial permeability transition pore opening (ΔΨm collapse) and calcium sparks, may involve flash triggering by certain levels of external ROS released from the same mitochondria. In addition, mitochondria-mitochondria interactions can produce wave propagations of mitoROS flashes and ΔΨm collapses in cancer cells similar to phenomena of ROS-induced ROS release (RIRR). Our data suggest that in cancer cells RIRR, activation of mitoROS flashes and mitochondrial depolarization may involve participation of extramitochondrial-ROS produced either by individual mitochondria and/or by neighboring mitochondria. This could represent general mechanisms in ROS-ROS signaling with suggested role in both mitochondrial and cellular physiology and signaling. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Immunocytochemical localization of the translocase of the outer mitochondrial membrane (Tom20) in the human cochlea.

    Science.gov (United States)

    Balaker, Ashley E; Ishiyama, Paul; Lopez, Ivan A; Ishiyama, Gail; Ishiyama, Akira

    2013-02-01

    Mitochondrial degeneration in the inner ear is likely a contributing factor in age-related hearing loss and other otopathologies such as Meniere's disease. Most mitochondrial proteins are synthesized in the cytosol and imported through the mitochondrial membranes by translocators. The translocase of the outer membrane (Tom) is the universal entry gate for all proteins that are imported into mitochondria. Altered function of the translocator could alter protein transport into the mitochondria, and disrupt function. In this study, we determined the immunolocalization of Tom20, a major mitochondrial protein import receptor, in microdissected human cochlea frozen sections obtained from postmortem autopsy and celloidin-embedded archival specimens. We used affinity purified rabbit polyclonal antibodies against Tom20. We also determined the Tom20 immunolocalization in the mouse inner ear. In the human and mouse cochlea, Tom20 was ubiquitously distributed in the organ of Corti, allowing well-delineated visualization of inner and outer hair cells. Tom20 immunoreactivity localized in the cytoplasm of spiral ganglia neurons. In the inner ear of aged subjects with Meniere's disease, there was decreased expression of Tom20. These results suggest that Tom20 can be used in the inner ear as a marker for mitochondrial protein import. Copyright © 2012 Wiley Periodicals, Inc.

  1. A Farnesylated Coxiella burnetii Effector Forms a Multimeric Complex at the Mitochondrial Outer Membrane during Infection.

    Science.gov (United States)

    Fielden, Laura F; Moffatt, Jennifer H; Kang, Yilin; Baker, Michael J; Khoo, Chen Ai; Roy, Craig R; Stojanovski, Diana; Newton, Hayley J

    2017-05-01

    Coxiella burnetii, the causative agent of Q fever, establishes a unique lysosome-derived intracellular niche termed the Coxiella-containing vacuole (CCV). The Dot/Icm-type IVB secretion system is essential for the biogenesis of the CCV and the intracellular replication of Coxiella Effector proteins, translocated into the host cell through this apparatus, act to modulate host trafficking and signaling processes to facilitate CCV development. Here we investigated the role of CBU0077, a conserved Coxiella effector that had previously been observed to localize to lysosomal membranes. CBU0077 was dispensable for the intracellular replication of Coxiella in HeLa and THP-1 cells and did not appear to participate in CCV biogenesis. Intriguingly, native and epitope-tagged CBU0077 produced by Coxiella displayed specific punctate localization at host cell mitochondria. As such, we designated CBU0077 MceA (mitochondrial Coxiellaeffector protein A). Analysis of ectopically expressed MceA truncations revealed that the capacity to traffic to mitochondria is encoded within the first 84 amino acids of this protein. MceA is farnesylated by the host cell; however, this does not impact mitochondrial localization. Examination of mitochondria isolated from infected cells revealed that MceA is specifically integrated into the mitochondrial outer membrane and forms a complex of approximately 120 kDa. Engineering Coxiella to express either MceA tagged with 3×FLAG or MceA tagged with 2×hemagglutinin allowed us to perform immunoprecipitation experiments that showed that MceA forms a homo-oligomeric species at the mitochondrial outer membrane during infection. This research reveals that mitochondria are a bona fide target of Coxiella effectors and MceA is a complex-forming effector at the mitochondrial outer membrane during Coxiella infection. Copyright © 2017 American Society for Microbiology.

  2. Carnitine/acylcarnitine translocase and carnitine palmitoyltransferase 2 form a complex in the inner mitochondrial membrane.

    Science.gov (United States)

    Console, Lara; Giangregorio, Nicola; Indiveri, Cesare; Tonazzi, Annamaria

    2014-09-01

    Carnitine/acylcarnitine translocase and carnitine palmitoyltransferase 2 are members of the carnitine system, which are responsible of the regulation of the mitochondrial CoA/acyl-CoA ratio and of supplying substrates for the ß-oxidation to mitochondria. This study, using cross-Linking reagent, Blue native electrophoresis and immunoprecipitation followed by detection with immunoblotting, shows conclusive evidence about the interaction between carnitine palmitoyltransferase 2 and carnitine/acylcarnitine translocase supporting the channeling of acylcarnitines and carnitine at level of the inner mitochondrial membrane.

  3. Regulation of Mitochondrial Dynamics and Autophagy by the Mitochondria-Associated Membrane.

    Science.gov (United States)

    Tagaya, Mitsuo; Arasaki, Kohei

    2017-01-01

    Mitochondria are powerhouses and central to metabolism in cells. They are highly dynamic organelles that continuously fuse, divide, and move along the cytoskeleton to form the mitochondrial network. The fusion and fission are catalyzed by four dynamin-related GTPases in mammals that are controlled by a variety of protein-protein interactions and posttranslational modifications. Mitochondrial dynamics and metabolism are linked and regulate each other. Starvation induces mitochondrial elongation, which enables the mitochondria to produce energy more efficiently and to escape from autophagic degradation. Damaged portions of mitochondria are removed from the healthy parts by division, and subsequently degraded via a specific mode of autophagy termed mitophagy. Recent studies shed light on the contribution of the endoplasmic reticulum to mitochondrial dynamics and the cooperation of the two organelles for the progression of autophagy including mitophagy. A subdomain of the endoplasmic reticulum apposed to mitochondria is called the mitochondria-associated membrane (MAM), which comprises a unique set of proteins that interact with mitochondrial proteins. Here we review our current understanding of the molecular mechanisms of mitochondrial dynamics and mitochondria-related processes in the context of the interaction with the endoplasmic reticulum.

  4. Alterations in Lipid Levels of Mitochondrial Membranes Induced by Amyloid-ß: A Protective Role of Melatonin

    Directory of Open Access Journals (Sweden)

    Sergio A. Rosales-Corral

    2012-01-01

    Full Text Available Alzheimer pathogenesis involves mitochondrial dysfunction, which is closely related to amyloid-ß (Aß generation, abnormal tau phosphorylation, oxidative stress, and apoptosis. Alterations in membranal components, including cholesterol and fatty acids, their characteristics, disposition, and distribution along the membranes, have been studied as evidence of cell membrane alterations in AD brain. The majority of these studies have been focused on the cytoplasmic membrane; meanwhile the mitochondrial membranes have been less explored. In this work, we studied lipids and mitochondrial membranes in vivo, following intracerebral injection of fibrillar amyloid-ß (Aß. The purpose was to determine how Aß may be responsible for beginning of a vicious cycle where oxidative stress and alterations in cholesterol, lipids and fatty acids, feed back on each other to cause mitochondrial dysfunction. We observed changes in mitochondrial membrane lipids, and fatty acids, following intracerebral injection of fibrillar Aß in aged Wistar rats. Melatonin, a well-known antioxidant and neuroimmunomodulator indoleamine, reversed some of these alterations and protected mitochondrial membranes from obvious damage. Additionally, melatonin increased the levels of linolenic and n-3 eicosapentaenoic acid, in the same site where amyloid ß was injected, favoring an endogenous anti-inflammatory pathway.

  5. Adaptations of Trypanosoma brucei to gradual loss of kinetoplast DNA: Trypanosoma equiperdum and Trypanosoma evansi are petite mutants of T. brucei.

    Science.gov (United States)

    Lai, De-Hua; Hashimi, Hassan; Lun, Zhao-Rong; Ayala, Francisco J; Lukes, Julius

    2008-02-12

    Trypanosoma brucei is a kinetoplastid flagellate, the agent of human sleeping sickness and ruminant nagana in Africa. Kinetoplastid flagellates contain their eponym kinetoplast DNA (kDNA), consisting of two types of interlocked circular DNA molecules: scores of maxicircles and thousands of minicircles. Maxicircles have typical mitochondrial genes, most of which are translatable only after RNA editing. Minicircles encode guide RNAs, required for decrypting the maxicircle transcripts. The life cycle of T. brucei involves a bloodstream stage (BS) in vertebrates and a procyclic stage (PS) in the tsetse fly vector. Partial [dyskinetoplastidy (Dk)] or total [akinetoplastidy (Ak)] loss of kDNA locks the trypanosome in the BS form. Transmission between vertebrates becomes mechanical without PS and tsetse mediation, allowing the parasite to spread outside the African tsetse belt. Trypanosoma equiperdum and Trypanosoma evansi are agents of dourine and surra, diseases of horses, camels, and water buffaloes. We have characterized representative strains of T. equiperdum and T. evansi by numerous molecular and classical parasitological approaches. We show that both species are actually strains of T. brucei, which lost part (Dk) or all (Ak) of their kDNA. These trypanosomes are not monophyletic clades and do not qualify for species status. They should be considered two subspecies, respectively T. brucei equiperdum and T. brucei evansi, which spontaneously arose recently. Dk/Ak trypanosomes may potentially emerge repeatedly from T. brucei.

  6. Impact of Storage and Purification on Mitochondrial Membrane Potential of Boar Spermatozoa

    Directory of Open Access Journals (Sweden)

    Aristotelis G. Lymberopoulos

    2013-05-01

    Full Text Available This study aimed to evaluate the effect of semen purification and storage on sperm mitochondrial membrane potential (ΔΨm. Gel-free whole ejaculates were collected from five proven fertile Large White boars aged two to three years. Aliquots of fresh semen were split, diluted in one step with commercial extenders and incubated at 37oC for 5-10 minutes. Semen was cooled to 18oC and packaged in 15-ml sterile propylene tubes. After 4-10 hours post-semen collection, stored semen was purified by colloidal centrifugation. After 48 hours post-semen collection, stored semen was incubated at 37oC and evaluated after 45 minutes for motility, velocity and sperm ΔΨm. Samples were stained with 2.99 μM JC-1 and 2.32 μM EH-1 and assessed by Fluorescence microscopy. After centrifugation a significant improvement of motility (P<0.035, and velocity (P<0.012 was noticed. The percentage of spermatozoa with intact plasma membrane and high/low mitochondrial membrane potential was statistical higher after centrifugation and storage at 18°C for 48 hours. In conclusion, colloidal purification of boar semen can improve sperm quality and  mitochondrial membrane potential.

  7. Outer Mitochondrial Membrane Localization of Apoptosis-Inducing Factor: Mechanistic Implications for Release

    Directory of Open Access Journals (Sweden)

    Seong-Woon Yu

    2009-10-01

    Full Text Available Poly(ADP-ribose polymerase-1-dependent cell death (known as parthanatos plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor, but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.

  8. Variations in mitochondrial membrane potential correlate with malic acid production by natural isolates of Saccharomyces cerevisiae sake strains

    National Research Council Canada - National Science Library

    Oba, Takahiro; Kusumoto, Kenichi; Kichise, Yuki; Izumoto, Eiji; Nakayama, Shunichi; Tashiro, Kosuke; Kuhara, Satoru; Kitagaki, Hiroshi

    2014-01-01

    .... In the present study, we isolated naturally occurring strains of yeast from sake mash that produce high levels of malic acid and demonstrate that variations in mitochondrial membrane potential...

  9. Voltage-dependent anion channels: the wizard of the mitochondrial outer membrane.

    Science.gov (United States)

    Mertins, Barbara; Psakis, Georgios; Essen, Lars-Oliver

    2014-12-01

    Voltage dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane. Although they are essential in metabolite exchange, cell defense and apoptosis, the molecular mechanism of these VDAC-mediated processes remains elusive. Here we review recent progress in terms of VDACs' structure and regulation, with a special focus on the molecular aspects of gating and the interaction with effector proteins.

  10. Practical techniques for bovine sperm simultaneous fluorimetric assessment of plasma, acrosomal and mitochondrial membranes.

    Science.gov (United States)

    Celeghini, E C C; de Arruda, R P; de Andrade, A F C; Nascimento, J; Raphael, C F

    2007-10-01

    This experiment was performed to develop and validate practical techniques for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function in bovine spermatozoa using associations of fluorescent probes. Four protocols of fluorescent probes association were defined: protocol 1: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and rhodamine 123; protocol 2: PI, FITC-PSA and MitoTracker Green FM (MITO); protocol 3: PI, Hoechst 33342 (H342), FITC-PSA and CMXRos; and protocol 4: PI, H342, FITC-PSA and JC-1. Three ejaculates from each of the four bulls (n = 12) were utilized, showing sperm motility >/=80% and abnormal morphology membrane integrity (R(2) = 0.95, 0.93 and 0.92, respectively), acrosome integrity (R(2) = 0.95, 0.92 and 0.91, respectively) and mitochondrial function (R(2) = 0.84, 0.93 and R(2) = 0.93, respectively). These techniques are efficient for the simultaneous integrity evaluation of plasma and acrosomal membranes and mitochondrial function in bovine spermatozoa. However, JC-1 has an advantage over MITO and CMXRos, as it separates two cell populations with high and low mitochondrial membrane potential.

  11. Apricot melanoidins prevent oxidative endothelial cell death by counteracting mitochondrial oxidation and membrane depolarization.

    Directory of Open Access Journals (Sweden)

    Annalisa Cossu

    Full Text Available The cardiovascular benefits associated with diets rich in fruit and vegetables are thought to be due to phytochemicals contained in fresh plant material. However, whether processed plant foods provide the same benefits as unprocessed ones is an open question. Melanoidins from heat-processed apricots were isolated and their presence confirmed by colorimetric analysis and browning index. Oxidative injury of endothelial cells (ECs is the key step for the onset and progression of cardiovascular diseases (CVD, therefore the potential protective effect of apricot melanoidins on hydrogen peroxide-induced oxidative mitochondrial damage and cell death was explored in human ECs. The redox state of cytoplasmic and mitochondrial compartments was detected by using the redox-sensitive, fluorescent protein (roGFP, while the mitochondrial membrane potential (MMP was assessed with the fluorescent dye, JC-1. ECs exposure to hydrogen peroxide, dose-dependently induced mitochondrial and cytoplasmic oxidation. Additionally detected hydrogen peroxide-induced phenomena were MMP dissipation and ECs death. Pretreatment of ECs with apricot melanoidins, significantly counteracted and ultimately abolished hydrogen peroxide-induced intracellular oxidation, mitochondrial depolarization and cell death. In this regard, our current results clearly indicate that melanoidins derived from heat-processed apricots, protect human ECs against oxidative stress.

  12. A collection of yeast mutants selectively resistant to ionophores acting on mitochondrial inner membrane.

    Science.gov (United States)

    Petrezselyova, Silvia; Lalakova, Jana; Abelovska, Lenka; Klobucnikova, Vlasta; Tomaska, Lubomir

    2008-03-01

    Valinomycin and nigericin are potassium ionophores acting selectively on the mitochondrial inner membrane of Saccharomyces cerevisiae [Kovac, L., Bohmerova, E., Butko, P., 1982a. Ionophores and intact cells. I. Valinomycin and nigericin act preferentially on mitochondria and not on the plasma membrane of Saccharomyces cerevisiae. Biochim. Biophys. Acta 721, 341-348]. However, the molecular mechanism of their action is not understood. Here we show that their selective effect on mitochondrial membranes is not caused by the pleiotropic drug resistance system. To identify the molecular components mediating the action of ionophores we isolated several mutants specifically resistant to valinomycin and/or nigericin. In contrast to the parental strain, these mutants do not form respiratory-deficient cells in the presence of ionophores. Moreover, all mutants harbor extensively fragmented mitochondria and these morphological defects can be alleviated by the ionophores. Interestingly, we observed that these mitochondrial defects may be accompanied by changes in vacuolar dynamics. Our results demonstrate that the classical genetic approach can provide a starting point for the analysis of components involved in the action of ionophores on mitochondria-related processes in eukaryotic cell.

  13. Trypanosoma brucei TbIF1 inhibits the essential F1-ATPase in the infectious form of the parasite.

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    Brian Panicucci

    2017-04-01

    Full Text Available The mitochondrial (mt FoF1-ATP synthase of the digenetic parasite, Trypanosoma brucei, generates ATP during the insect procyclic form (PF, but becomes a perpetual consumer of ATP in the mammalian bloodstream form (BF, which lacks a canonical respiratory chain. This unconventional dependence on FoF1-ATPase is required to maintain the essential mt membrane potential (Δψm. Normally, ATP hydrolysis by this rotary molecular motor is restricted to when eukaryotic cells experience sporadic hypoxic conditions, during which this compulsory function quickly depletes the cellular ATP pool. To protect against this cellular treason, the highly conserved inhibitory factor 1 (IF1 binds the enzyme in a manner that solely inhibits the hydrolytic activity. Intriguingly, we were able to identify the IF1 homolog in T. brucei (TbIF1, but determined that its expression in the mitochondrion is tightly regulated throughout the life cycle as it is only detected in PF cells. TbIF1 appears to primarily function as an emergency brake in PF cells, where it prevented the restoration of the Δψm by FoF1-ATPase when respiration was chemically inhibited. In vitro, TbIF1 overexpression specifically inhibits the hydrolytic activity but not the synthetic capability of the FoF1-ATP synthase in PF mitochondria. Furthermore, low μM amounts of recombinant TbIF1 achieve the same inhibition of total mt ATPase activity as the FoF1-ATPase specific inhibitors, azide and oligomycin. Therefore, even minimal ectopic expression of TbIF1 in BF cells proved lethal as the indispensable Δψm collapsed due to inhibited FoF1-ATPase. In summary, we provide evidence that T. brucei harbors a natural and potent unidirectional inhibitor of the vital FoF1-ATPase activity that can be exploited for future structure-based drug design.

  14. High-confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope-tag organelle enrichment and SILAC proteomics.

    Science.gov (United States)

    Güther, Maria Lucia S; Urbaniak, Michael D; Tavendale, Amy; Prescott, Alan; Ferguson, Michael A J

    2014-06-06

    The glycosome of the pathogenic African trypanosome Trypanosoma brucei is a specialized peroxisome that contains most of the enzymes of glycolysis and several other metabolic and catabolic pathways. The contents and transporters of this membrane-bounded organelle are of considerable interest as potential drug targets. Here we use epitope tagging, magnetic bead enrichment, and SILAC quantitative proteomics to determine a high-confidence glycosome proteome for the procyclic life cycle stage of the parasite using isotope ratios to discriminate glycosomal from mitochondrial and other contaminating proteins. The data confirm the presence of several previously demonstrated and suggested pathways in the organelle and identify previously unanticipated activities, such as protein phosphatases. The implications of the findings are discussed.

  15. Potential-dependent membrane permeabilization and mitochondrial aggregation caused by anticancer polyarginine-KLA peptides.

    Science.gov (United States)

    Lemeshko, Victor V

    2010-01-15

    The anticancer activity of the polycationic peptide (KLAKLAK)(2), as a possible mitochondria-damaging agent, named KLA (l-form) or kla (d-form), has been increased by the fusion with hepta-arginine cell delivery vectors r7 and R7 (peptides r7-kla and R7-KLA, respectively), as shown in the literature. We demonstrated that 3.6muM r7-kla or R7-KLA, but not kla, caused significant permeabilization of the inner and the outer membranes of energized rat liver mitochondria. In addition, r7-kla or R7-KLA induced mitochondrial aggregation, thus causing the inhibition of metabolic activity. Potential-dependent mechanism of permeabilization of the inner mitochondrial membrane by these peptides was also observed for the plasma membrane of red blood cells. The obtained results suggest that polyarginine cell delivery vectors of anticancer polycationic peptides not only increase their direct potential-dependent permeabilization of biological membranes, but also create the capacity to cause aggregation of mitochondria, as a new mechanism of cytotoxic action of these peptides. Copyright (c) 2009 Elsevier Inc. All rights reserved.

  16. TSPO, a Mitochondrial Outer Membrane Protein, Controls Ethanol-Related Behaviors in Drosophila

    Science.gov (United States)

    Lin, Ran; Rittenhouse, Danielle; Sweeney, Katelyn; Potluri, Prasanth; Wallace, Douglas C.

    2015-01-01

    The heavy consumption of ethanol can lead to alcohol use disorders (AUDs) which impact patients, their families, and societies. Yet the genetic and physiological factors that predispose humans to AUDs remain unclear. One hypothesis is that alterations in mitochondrial function modulate neuronal sensitivity to ethanol exposure. Using Drosophila genetics we report that inactivation of the mitochondrial outer membrane translocator protein 18kDa (TSPO), also known as the peripheral benzodiazepine receptor, affects ethanol sedation and tolerance in male flies. Knockdown of dTSPO in adult male neurons results in increased sensitivity to ethanol sedation, and this effect requires the dTSPO depletion-mediated increase in reactive oxygen species (ROS) production and inhibition of caspase activity in fly heads. Systemic loss of dTSPO in male flies blocks the development of tolerance to repeated ethanol exposures, an effect that is not seen when dTSPO is only inactivated in neurons. Female flies are naturally more sensitive to ethanol than males, and female fly heads have strikingly lower levels of dTSPO mRNA than males. Hence, mitochondrial TSPO function plays an important role in ethanol sensitivity and tolerance. Since a large array of benzodiazepine analogues have been developed that interact with the peripheral benzodiazepine receptor, the mitochondrial TSPO might provide an important new target for treating AUDs. PMID:26241038

  17. TSPO, a Mitochondrial Outer Membrane Protein, Controls Ethanol-Related Behaviors in Drosophila.

    Directory of Open Access Journals (Sweden)

    Ran Lin

    2015-08-01

    Full Text Available The heavy consumption of ethanol can lead to alcohol use disorders (AUDs which impact patients, their families, and societies. Yet the genetic and physiological factors that predispose humans to AUDs remain unclear. One hypothesis is that alterations in mitochondrial function modulate neuronal sensitivity to ethanol exposure. Using Drosophila genetics we report that inactivation of the mitochondrial outer membrane translocator protein 18kDa (TSPO, also known as the peripheral benzodiazepine receptor, affects ethanol sedation and tolerance in male flies. Knockdown of dTSPO in adult male neurons results in increased sensitivity to ethanol sedation, and this effect requires the dTSPO depletion-mediated increase in reactive oxygen species (ROS production and inhibition of caspase activity in fly heads. Systemic loss of dTSPO in male flies blocks the development of tolerance to repeated ethanol exposures, an effect that is not seen when dTSPO is only inactivated in neurons. Female flies are naturally more sensitive to ethanol than males, and female fly heads have strikingly lower levels of dTSPO mRNA than males. Hence, mitochondrial TSPO function plays an important role in ethanol sensitivity and tolerance. Since a large array of benzodiazepine analogues have been developed that interact with the peripheral benzodiazepine receptor, the mitochondrial TSPO might provide an important new target for treating AUDs.

  18. Toxins in botanical dietary supplements: blue cohosh components disrupt cellular respiration and mitochondrial membrane potential.

    Science.gov (United States)

    Datta, Sandipan; Mahdi, Fakhri; Ali, Zulfiqar; Jekabsons, Mika B; Khan, Ikhlas A; Nagle, Dale G; Zhou, Yu-Dong

    2014-01-24

    Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA "black box" warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3), exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage.

  19. Toxins in Botanical Dietary Supplements: Blue Cohosh Components Disrupt Cellular Respiration and Mitochondrial Membrane Potential

    Science.gov (United States)

    Datta, Sandipan; Mahdi, Fakhri; Ali, Zulfiqar; Jekabsons, Mika B.; Khan, Ikhlas A.; Nagle, Dale G.; Zhou, Yu-Dong

    2014-01-01

    Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA “Black Box” warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3) exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage. PMID:24328138

  20. Role of charge screening and delocalization for lipophilic cation permeability of model and mitochondrial membranes.

    Science.gov (United States)

    Trendeleva, Tatiana A; Sukhanova, Evgenia I; Rogov, Anton G; Zvyagilskaya, Renata A; Seveina, Inna I; Ilyasova, Tatiana M; Cherepanov, Dmitry A; Skulachev, Vladimir P

    2013-09-01

    The effects of the mitochondria-targeted lipophilic cation dodecyltriphenylphosphonium (C12TPP, the charge is delocalized and screened by bulky hydrophobic residues) and those of lipophilic cations decyltriethylammonium bromide and cetyltrimethylammonium bromide (C10TEA and C16TMA, the charges are localized and screened by less bulky residues) on bilayer planar phospholipid membranes and tightly-coupled mitochondria from the yeast Yarrowia lipolytica have been compared. In planar membranes, C12TPP was found to generate a diffusion potential as if it easily penetrates these membranes. In the presence of palmitate, C12TPP induced H(+) permeability like plastoquinonyl decyltriphenilphosphonium that facilitates transfer of fatty acid anions (Severin et al., PNAS, 2010, 107, 663-668). C12TPP was shown to stimulate State 4 respiration of mitochondria and caused a mitochondrial membrane depolarization with a half-maximal effect at 6μM. Besides, C12TPP profoundly potentiated the uncoupling effect of endogenous or added fatty acids. C10TEA and C16TMA inhibited State 4 respiration and decreased the membrane potential, though at much higher concentrations than C12TPP, and they did not promote the uncoupling action of fatty acids. These relationships were modeled by molecular dynamics. They can be explained by different membrane permeabilities for studied cations, which in turn are due to different availabilities of the positive charge in these cations to water dipoles. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. The Involvement of Mitochondrial Membrane Potential in Cross-Resistance Between Radiation and Docetaxel

    Energy Technology Data Exchange (ETDEWEB)

    Kuwahara, Yoshikazu [Department of Radiation Biology and Medicine, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, Sendai (Japan); Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai (Japan); Roudkenar, Mehryar Habibi; Suzuki, Masatoshi; Urushihara, Yusuke; Fukumoto, Motoi [Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai (Japan); Saito, Yohei [Department of Radiopharmacy, Tohoku Medical and Pharmaceutical University, Sendai (Japan); Fukumoto, Manabu, E-mail: manabu.fukumoto.a8@tohoku.ac.jp [Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai (Japan); Department of Molecular Pathology, Tokyo Medical University, Tokyo (Japan)

    2016-11-01

    Purpose: To understand the molecular mechanisms underlying cancer cell radioresistance, clinically relevant radioresistant (CRR) cells that continue to proliferate during exposure to 2 Gy/day X-rays for more than 30 days were established. A modified high-density survival assay for anticancer drug screening revealed that CRR cells were resistant to an antimicrotubule agent, docetaxel (DTX). The involvement of reactive oxygen species (ROS) from mitochondria (mtROS) in the cross-resistance to X-rays and DTX was studied. Methods and Materials: Sensitivity to anticancer agents was determined by a modified high-density cell survival or water-soluble tetrazolium salt assay. DTX-induced mtROS generation was determined by MitoSOX red staining. JC-1 staining was used to visualize mitochondrial membrane potential. DTX-induced DNA double-strand breaks were determined by γ-H2AX staining. To obtain mitochondrial DNA-lacking (ρ{sup 0}) cells, the cells were cultured for 3 to 4 weeks in medium containing ethidium bromide. Results: Treatment with DTX increased mtROS in parental cells but not in CRR cells. DTX induced DNA double-strand breaks in parental cells. The mitochondrial membrane potential of CRR cells was lower in CRR cells than in parental cells. Depletion of mtDNA induced DTX resistance in parental cells. Treatment with dimethyl sulfoxide also induced DTX resistance in parental cells. Conclusions: The mitochondrial dysfunction observed in CRR cells contributes to X-ray and DTX cross-resistance. The activation of oxidative phosphorylation in CRR cells may represent an effective approach to overcome radioresistant cancers. In general, the overexpression of β-tubulin or multidrug efflux pumps is thought to be involved in DTX resistance. In the present study, we discovered another DTX resistant mechanism by investigating CRR cells.

  2. A novel algorithm identifies stress-induced alterations in mitochondrial connectivity and inner membrane structure from confocal images.

    Directory of Open Access Journals (Sweden)

    Mathieu Ouellet

    2017-06-01

    Full Text Available Mitochondria exist as a highly interconnected network that is exquisitely sensitive to variations in nutrient availability, as well as a large array of cellular stresses. Changes in length and connectivity of this network, as well as alterations in the mitochondrial inner membrane (cristae, regulate cell fate by controlling metabolism, proliferation, differentiation, and cell death. Given the key roles of mitochondrial dynamics, the process by which mitochondria constantly fuse and fragment, the measure of mitochondrial length and connectivity provides crucial information on the health and activity of various cell populations. However, despite the importance of accurately measuring mitochondrial networks, the tools required to rapidly and accurately provide this information are lacking. Here, we developed a novel probabilistic approach to automatically measure mitochondrial length distribution and connectivity from confocal images. This method accurately identified mitochondrial changes caused by starvation or the inhibition of mitochondrial function. In addition, we successfully used the algorithm to measure changes in mitochondrial inner membrane/matrix occurring in response to Complex III inhibitors. As cristae rearrangements play a critical role in metabolic regulation and cell survival, this provides a rapid method to screen for proteins or compounds affecting this process. The algorithm will thus provide a robust tool to dissect the molecular mechanisms underlying the key roles of mitochondria in the regulation of cell fate.

  3. Peroxynitrite-mediated nitrosative stress decreases motility and mitochondrial membrane potential in human spermatozoa.

    Science.gov (United States)

    Uribe, P; Boguen, R; Treulen, F; Sánchez, R; Villegas, J V

    2015-03-01

    Nitrosative stress is produced by high levels of reactive nitrogen species (RNS). The RNS include peroxynitrite, a highly reactive free radical produced from a diffusion-controlled reaction between nitric oxide and superoxide anion. Peroxynitrite causes nitration and oxidation of lipids, proteins and DNA, and is thus considered an important pathogenic mechanism in various diseases. Although high levels of peroxynitrite are associated with astenozoospermia, few reports exist regarding the in vitro effect of high levels of this RNS on human sperm. The aim of this study was to evaluate the in vitro effect of nitrosative stress caused by peroxynitrite on the viability, motility and mitochondrial membrane potential of human spermatozoa. To do this, human spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a molecule that generates peroxynitrite. Incubations were done at 37°C for up to 4 h with SIN-1 concentrations between 0.2 and 1.0 mmol/l. Generation of peroxynitrite was confirmed using dihydrorhodamine 123 (DHR) by spectrophotometry and flow cytometry. Sperm viability was assessed by propidium iodide staining; sperm motility was analyzed by CASA, and the state of mitochondrial membrane potential (ΔΨm) by JC-1 staining. Viability and ΔΨm were measured by flow cytometry. The results showed an increase in DHR oxidation, demonstrating the generation of peroxynitrite through SIN-1. Peroxynitrite decreased progressive and total motility, as well as some sperm kinetic parameters. Mitochondrial membrane potential also decreased. These alterations occurred with no decrease in sperm viability. In conclusion, peroxynitrite-induced nitrosative stress impairs vital functions in the male gamete, possibly contributing to male infertility. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  4. Evaluation of sperm superoxide anion production and Mitochondrial Membrane Potential: flowcytometry in rats with experimental varicocele

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    Jafari A

    2009-05-01

    Full Text Available "n Normal 0 false false false EN-GB X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Varicocele is a major cause of male infertility, but its pathophysiology is unclear. Recent studies declare that fertile varicocele people with normal semen analysis are also at risk of loss of infertility. The exact mechanism by which varicocele damages spermatogenesis is still unknown. Some studies have reported increased Reactive Oxygen Species (ROS is a major factor in semen of men with varicocele. The aim of this study was to investigate whether the source of elevated ROS is intracellular or not. In addition, we studied Mitochondrial Membrane Potential (MMP, viability, antioxidant activity, sperm count and motility in these rats."n"n Methods: The study group consisted of 28 male rats divided in four groups: control, sham, varicocele 1, varicocele 2, Experimental varicocele was established by partial ligation of the left renal vein in last two groups. Animals were sacrificed two and six months after surgery and dilation of the internal spermatic veins was observed. Then, superoxide anion production and Mitochondrial Membrane Potential were evaluated by Flow cytometry sperm characteristics were evaluated by Flow cytometry. Sperm superoxide anion production was assessed by the dihydroethidium and

  5. Variations in mitochondrial membrane potential correlate with malic acid production by natural isolates of Saccharomyces cerevisiae sake strains.

    Science.gov (United States)

    Oba, Takahiro; Kusumoto, Kenichi; Kichise, Yuki; Izumoto, Eiji; Nakayama, Shunichi; Tashiro, Kosuke; Kuhara, Satoru; Kitagaki, Hiroshi

    2014-08-01

    Research on the relationship between mitochondrial membrane potential and fermentation profile is being intensely pursued because of the potential for developing advanced fermentation technologies. In the present study, we isolated naturally occurring strains of yeast from sake mash that produce high levels of malic acid and demonstrate that variations in mitochondrial membrane potential correlate with malic acid production. To define the underlying biochemical mechanism, we determined the activities of enzymes required for malic acid synthesis and found that pyruvate carboxylase and malate dehydrogenase activities in strains that produce high levels of malic acid were elevated compared with the standard sake strain K901. These results inspired us to hypothesize that decreased mitochondrial membrane potential was responsible for increased malic acid synthesis, and we present data supporting this hypothesis. Thus, the mitochondrial membrane potential of high malic acid producers was lower compared with standard strains. We conclude that mitochondrial membrane potential correlates with malic acid production. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  6. Depolarization of mitochondrial membrane potential is the initial event in non-nucleoside reverse transcriptase inhibitor efavirenz induced cytotoxicity.

    Science.gov (United States)

    Ganta, Krishna Kumar; Mandal, Anirban; Chaubey, Binay

    2017-02-01

    Efavirenz is a non-nucleoside reverse transcriptase inhibitor (NNRTI) and an active constituent of the highly active antiretroviral therapy regime. It has significantly contributed in control and management of human immunodeficiency virus propagation. However, EFV administration has also led to severe adverse effects, several reports highlighted the role of EFV in mitochondrial dysfunction and toxicity but the molecular mechanism has been poorly understood. In present study, human hepatoma cells Huh 7.5 were treated with clinically relevant concentrations of EFV and parameters like cytotoxicity, mitochondrial transmembrane potential, mitochondrial morphology, cytochrome c release, mitochondria-mediated apoptosis, mtDNA and mtRNA levels and EFV distribution into mitochondrial compartment were evaluated to understand sequence of events leading to cell death in EFV-treated cells. EFV at its clinically relevant concentration was significantly toxic after 48 and 72 h of treatments. EFV-mediated toxicity is initiated with the permeabilization of mitochondrial outer membrane and change in mitochondrial membrane potential (Δψm) which triggers a series of events like cytochrome c release, alteration in mitochondrial morphology and mitochondria-mediated apoptosis. Total mitochondrial content is reduced after 48 h of EFV treatment at IC50 concentration which is also reflected in reduced mitochondrial DNA and RNA levels. After detecting EFV in mitochondrial compartment after 12 h of incubation with EFV, we hypothesize that EFV being a lipophilic molecule is internalized into the mitochondrial compartment causing depolarization of Δψm which subsequently leads to a cascade of events causing cell death.

  7. Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids.

    Science.gov (United States)

    Raza Shaikh, Saame; Brown, David A

    2013-01-01

    Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid-protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Phytochemicals prevent mitochondrial membrane permeabilization and protect SH-SY5Y cells against apoptosis induced by PK11195, a ligand for outer membrane translocator protein.

    Science.gov (United States)

    Wu, Yuqiu; Shamoto-Nagai, Masayo; Maruyama, Wakako; Osawa, Toshihiko; Naoi, Makoto

    2017-01-01

    Epidemiological studies present the beneficial effects of dietary habits on prevention of aging-associated decline of brain function. Phytochemicals, the second metabolites of food, protect neuronal cells from cell death in cellular models of neurodegenerative disorders, and the neuroprotective activity has been ascribed to the anti-oxidant and anti-inflammatory functions. In this paper, the cellular mechanism of neuroprotection by phytochemicals was investigated, using the cellular model of mitochondrial apoptosis induced by PK11195, a ligand of outer membrane translocator protein, in SH-SY5Y cells. PK11195 induced mitochondrial membrane permeabilization with rapid transit production of superoxide (superoxide flashes) and calcium release from mitochondria, and activated apoptosis signal pathway. Study on the structure-activity relationship of astaxanthin, ferulic acid derivatives, and sesame lignans revealed that these phytochemicals inhibited mitochondrial membrane permeabilization and protected cells from apoptosis. Ferulic acid derivatives and sesame lignans inhibited or enhanced the mitochondrial pore formation and cell death by PK11195 according to their amphiphilic properties, not directly depending on the antioxidant activity. Regulation of pore formation at mitochondrial membrane is discussed as a novel mechanism behind neuroprotective activity of phytochemicals in aging and age-associated neurodegenerative disorders, and also behind dual functions of phytochemicals in neuronal and cancer cells.

  9. Fatty acids composition of inner mitochondrial membrane of rat cardiomyocytes and hepatocytes during hypoxia-hypercapnia

    Directory of Open Access Journals (Sweden)

    S. V. Khyzhnyak

    2016-06-01

    Full Text Available We studied the influence of hypoxic-hypercapnic environment under the effect of hypothermia (artificial hibernation on fatty acids spectrum of inner mitochondrial membrane (IMM lipids of rat cardiomyocytes and hepatocytes. Specific for cellular organelles redistribution of IMM fatty acids was determined. It led to the reduction of total amount of saturated fatty acids (SFAs and increase of unsaturated fatty acids (UFAs in cardiomyocytes and to the increase of SFAs and decrease of UFAs in hepatocytes. The decrease in the content of oleic acid and increased content of arachidonic and docosahexaenoic acids in IMM were shown. This may be due to their role in the regulatory systems during hibernation, as well as following exit therefrom. It is assumed that artificial hibernation state is characterized by the stress reaction leading to optimal readjustment of fatty acids composition of membrane lipids, which supports functional activity of mitochondria in hepatocytes and cardiomyocytes.

  10. Eukaryote-wide sequence analysis of mitochondrial β-barrel outer membrane proteins

    Directory of Open Access Journals (Sweden)

    Fujita Naoya

    2011-01-01

    Full Text Available Abstract Background The outer membranes of mitochondria are thought to be homologous to the outer membranes of Gram negative bacteria, which contain 100's of distinct families of β-barrel membrane proteins (BOMPs often forming channels for transport of nutrients or drugs. However, only four families of mitochondrial BOMPs (MBOMPs have been confirmed to date. Although estimates as high as 100 have been made in the past, the number of yet undiscovered MBOMPs is an open question. Fortunately, the recent discovery of a membrane integration signal (the β-signal for MBOMPs gave us an opportunity to look for undiscovered MBOMPs. Results We present the results of a comprehensive survey of eukaryotic protein sequences intended to identify new MBOMPs. Our search employs recent results on β-signals as well as structural information and a novel BOMP predictor trained on both bacterial and mitochondrial BOMPs. Our principal finding is circumstantial evidence suggesting that few MBOMPs remain to be discovered, if one assumes that, like known MBOMPs, novel MBOMPs will be monomeric and β-signal dependent. In addition to this, our analysis of MBOMP homologs reveals some exceptions to the current model of the β-signal, but confirms its consistent presence in the C-terminal region of MBOMP proteins. We also report a β-signal independent search for MBOMPs against the yeast and Arabidopsis proteomes. We find no good candidates MBOMPs in yeast but the Arabidopsis results are less conclusive. Conclusions Our results suggest there are no remaining MBOMPs left to discover in yeast; and if one assumes all MBOMPs are β-signal dependent, few MBOMP families remain undiscovered in any sequenced organism.

  11. Interaction of fullerene nanoparticles with biomembranes: from the partition in lipid membranes to effects on mitochondrial bioenergetics.

    Science.gov (United States)

    Santos, Sandra M; Dinis, Augusto M; Peixoto, Francisco; Ferreira, Lino; Jurado, Amália S; Videira, Romeu A

    2014-03-01

    Partition and localization of C60 and its derivative C60(OH)18-22 in lipid membranes and their impact on mitochondrial activity were studied, attempting to correlate those events with fullerene characteristics (size, surface chemistry, and surface charge). Fluorescence quenching studies suggested that C60(OH)18-22 preferentially populated the outer regions of the bilayer, whereas C60 preferred to localize in deeper regions of the bilayer. Partition coefficient values indicated that C60 exhibited higher affinity for dipalmitoylphosphatidylcholine and mitochondrial membranes than C60(OH)18-22. Both fullerenes affected the mitochondrial function, but the inhibitory effects promoted by C60 were more pronounced than those induced by C60(OH)18-22 (up to 20 nmol/mg of mitochondrial protein). State 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations are inhibited by both fullerenes when glutamate/malate or succinate was used as substrate. Phosphorylation system and electron transport chain of mitochondria are affected by both fullerenes, but only C60 increased the inner mitochondrial membrane permeability to protons, suggesting perturbations in the structure and dynamics of that membrane. At concentrations of C60(OH)18-22 above 20 nmol/mg of mitochondrial protein, the activity of FoF1-ATP synthase was also decreased. The evaluation of transmembrane potential showed that the mitochondria phosphorylation cycle decreased upon adenosine diphosphate addition with increasing fullerenes concentration and the time of the repolarization phase increased as a function of C60(OH)18-22 concentration. Our results suggest that the balance between hydrophilicity and hydrophobicity resulting from the surface chemistry of fullerene nanoparticles, rather than the cluster size or the surface charge acquired by fullerenes in water, influences their membrane interactions and consequently their effects on mitochondrial bioenergetics.

  12. Effects of Insecticides on the Fluidity of Mitochondrial Membranes of the Diamondback Moth, Plutella xylostella, Resistant and Susceptible to Avermectin

    Science.gov (United States)

    Hu, J.; Liang, P.; Shi, X.; Gao, X.

    2008-01-01

    The effects of various insecticides on the fluidity of mitochondrial membranes and cross-resistance were investigated in the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) using strains that were both resistant and susceptible to avermectin. The resistant strain of P. xylostella, AV-R, developed 1078-fold resistance to avermetins with a high level of cross-resistance to the analogs of avermectins, ivermectin and emamectin benzoate. It had more than 1000 times greater resistance when compared with the avermectin-susceptible strain, XH-S. Mitochondrial membrane fluidity was measured by detecting fluorescence polarization using DPH (1,6-Diphenyl -1,3,5-hexatriene) as the fluorescence probe. Abamectin, emamectin benzoate, ivermectin, cypermethrin and fenvalerate decreased the fluidity of mitochondrial membranes in the XH-S strain at 25°C. However, fipronil and acephate did not change the fluidity of mitochondrial membrane when the concentration of these insecticides was 1×10-4 mol/L. Membrane fluidity increased as the temperature increased. The thermotropic effect on the polarization value of DPH increased as the insecticide concentration was increased. There was a significant difference of mitochondrial membrane fluidity between both XH-S and AV-R when temperature was less than 25°C and no difference was observed when the temperature was more than 25°C. The low-dose abamectin (0.11 mg/L) in vivo treatment caused a significant change of membrane fluidity in the XH-S strain and no change in the AV-R strain. However, a high-dose abamectin (11.86 mg/L) resulted in 100% mortality of the XH-S strain. In vivo treatment may cause a significant change of membrane fluidity in the AV-R strain PMID:20345311

  13. Lipid, membrane, and mitochondrial characteristics of Ustilago maydis following exposure to ergosterol biosynthesis inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Waterfield, W.F. III

    1986-01-01

    Pencoazole at 0.5 ..mu..g/ml inhibited ergosterol biosynthesis in U. maydis. Polar lipids of sporidia grown with 0.5 ..mu..g/ml penconazole for 7.5 or 22 hr or 1.0 ..mu..g/ml fenarimol for 7.5 hr contained more 18:2 than 18:1 fatty acids. There was usually more 18:1 than 18:2 fatty acids in polar lipids of untreated sporidia but this ratio was influenced by culture cell density. The high 18:2 to 18:1 ratio in the polar lipids from penconazole grown cells was unaffected by cell density. There was an increase in free fatty acids and these were enriched with 18:2 members in cells grown with 0.5 ..mu..g/ml penconazole for 22 hr. Unsaturation of triglycerides fatty acids did not differ appreciably from that of untreated sporidia. Untreated WT U. maydis protoplasts lysed more slowly in 0.3 M sorbitol than those prepared from WT sporidia grown for 16 hr with 1.0 ..mu..g/ml penconazole or 2.0 ..mu..g/ml fenarimol or from untreated erg-40 sporidia. Protoplasts were more permeable to crystal violet than were those from untreated WT sporidia. Mitochondria from untreated WT sporidia oxidizing pyruvate plus malate or succinate yielded higher ADP/O rations than mitochondria from erg-40 or penconazole grown WT sporidia. The mitochondrial ATPase of control cells had a Km of 0.8 mM ATP whereas the mitochondrial ATPase of penconazole grown WT and erg-40 had a Km value of 3.7 and 3.2 mM ATP, respectively. When the mitochondrial catalytic subunit of the ATPase from these mitochondria were solubilized, the Km did not differ. These studies suggest that changes in sterols and membrane fatty acids resulting from treatments with EBI fungicides cause increased membrane fluidity which affects membrane stability, permeability and activity of the mitochondrial ATPase.

  14. Membrane association of mitochondrial DNA facilitates base excision repair in mammalian mitochondria.

    Science.gov (United States)

    Boesch, Pierre; Ibrahim, Noha; Dietrich, André; Lightowlers, Robert N

    2010-03-01

    Mitochondrial DNA encodes a set of 13 polypeptides and is subjected to constant oxidative stress due to ROS production within the organelle. It has been shown that DNA repair in the mitochondrion proceeds through both short- and long-patch base excision repair (BER). In the present article, we have used the natural competence of mammalian mitochondria to import DNA and study the sub-mitochondrial localization of the repair system in organello. Results demonstrate that sequences corresponding to the mtDNA non-coding region interact with the inner membrane in a rapid and saturable fashion. We show that uracil containing import substrates are taken into the mitochondrion and are used as templates for damage driven DNA synthesis. After further sub-fractionation, we show that the length of the repair synthesis patch differs in the soluble and the particulate fraction. Bona fide long patch BER synthesis occurs on the DNA associated with the particulate fraction, whereas a nick driven DNA synthesis occurs when the uracil containing DNA accesses the soluble fraction. Our results suggest that coordinate interactions of the different partners needed for BER is only found at sites where the DNA is associated with the membrane.

  15. Ethanol Influences on Bax Associations with Mitochondrial Membrane Proteins in Neonatal Rat Cerebellum

    Science.gov (United States)

    Heaton, Marieta Barrow; Siler-Marsiglio, Kendra; Paiva, Michael; Kotler, Alexandra; Rogozinski, Jonathan; Kubovec, Stacey; Coursen, Mary; Madorsky, Vladimir

    2012-01-01

    These studies investigated interactions taking place at the mitochondrial membrane in neonatal rat cerebellum following ethanol exposure, and focused on interactions between pro-apoptotic Bax and proteins of the permeability transition pore (PTP), voltage-dependent anion channel (VDAC), and adenine nucleotide translocator (ANT), of the outer and inner mitochondrial membranes, respectively. Cultured cerebellar granule cells were used to assess the role of these interactions in ethanol neurotoxicity. Analyses were made at the age of maximal cerebellar ethanol vulnerability (P4), compared to the later age of relative resistance (P7), to determine whether differential ethanol sensitivity was mirrored by differences in these molecular interactions. We found that following ethanol exposure, Bax pro-apoptotic associations with both VDAC and ANT were increased, particularly at the age of greater ethanol sensitivity, and these interactions were sustained at this age for at least two hours post-exposure. Since Bax:VDAC interactions disrupt protective VDAC interactions with mitochondrial hexokinase (HXK), we also assessed VDAC:HXK associations following ethanol treatment, and found such interactions were altered by ethanol treatment, but only at two-hours post-exposure, and only in the P4, ethanol-sensitive cerebellum. Ethanol neurotoxicity in cultured neuronal preparations was abolished by pharmacological inhibition of both VDAC and ANT interactions with Bax, but not by a Bax channel blocker. Therefore, we conclude that at this age, within the constraints of our experimental model, a primary mode of Bax-induced initiation of the apoptosis cascade following ethanol insult involves interactions with proteins of the PTP complex, and not channel formation independent of PTP constituents. PMID:22767450

  16. Cockayne syndrome group B protein promotes mitochondrial DNA stability by supporting the DNA repair association with the mitochondrial membrane

    National Research Council Canada - National Science Library

    Aamann, Maria D; Sorensen, Martin M; Hvitby, Christina; Berquist, Brian R; Muftuoglu, Meltem; Tian, Jingyan; de Souza-Pinto, Nadja C; Scheibye-Knudsen, Morten; Wilson, 3rd, David M; Stevnsner, Tinna; Bohr, Vilhelm A

    2010-01-01

    Cockayne syndrome (CS) is a human premature aging disorder associated with severe developmental deficiencies and neurodegeneration, and phenotypically it resembles some mitochondrial DNA (mtDNA) diseases...

  17. Role of Pterocarpus santalinus against mitochondrial dysfunction and membrane lipid changes induced by ulcerogens in rat gastric mucosa.

    Science.gov (United States)

    Narayan, Shoba; Devi, R S; Devi, C S Shyamala

    2007-11-20

    Free radicals produced by ulcerogenic agents affect the TCA cycle enzymes located in the outer membrane of the mitochondria. Upon induction with ulcerogens, peroxidation of membrane lipids bring about alterations in the mitochondrial enzyme activity. This indicates an increase in the permeability levels of the mitochondrial membrane. The ability of PSE to scavenge the reactive oxygen species results in restoration of activities of TCA cycle enzymes. NSAIDs interfere with the mitochondrial beta-oxidation of fatty acids in vitro and in vivo, resulting in uncoupling of mitochondrial oxidative phosphorylation process. This usually results in diminished cellular ATP production. The recovery of gastric mucosal barrier function through maintenance of energy metabolism results in maintenance of ATP levels, as observed in this study upon treatment with PSE. Membrane integrity altered by peroxidation is known to have a modified fatty acid composition, a disruption of permeability, a decrease in electrical resistance, and increase in flip-flopping between monolayers and inactivated cross-linked proteins. The severe depletion of arachidonic acid in ulcer induced groups was prevented upon treatment with PSE. The acid inhibitory property of the herbal extract enables the maintenance of GL activity upon treatment with PSE. The ability to prevent membrane peroxidation has been traced to the presence of active constituents in the PSE. In essence, PSE has been found to prevent mitochondrial dysfunction, provide mitochondrial cell integrity, through the maintenance of lipid bilayer by its ability to provide a hydrophobic character to the gastric mucosa, further indicating its ability to reverse the action of NSAIDs and mast cell degranulators in gastric mucosa.

  18. L-carnitine is essential to beta-oxidation of quarried fatty acid from mitochondrial membrane by PLA(2).

    Science.gov (United States)

    Yano, Hiromi; Oyanagi, Eri; Kato, Yasuko; Samejima, Yoshiyuki; Sasaki, Junzo; Utsumi, Kozo

    2010-09-01

    Mitochondrial beta-oxidation is an important system involved in the energy production of various cells. In this system, the function of L-carnitine is essential for the uptake of fatty acids to mitochondria. However, it is unclear whether or not endogenous respiration, ADP-induced O(2) consumption without substrates, is caused by L-carnitine treatment. In this study, we investigated whether L-carnitine is essential to the beta-oxidation of quarried fatty acids from the mitochondrial membrane by phospholipase A(2) (PLA(2)) using isolated mitochondria from the liver of rats. Intact mitochondria were incubated in a medium containing Pi, CoA and L-carnitine. The effect of L-carnitine treatment on ADP-induced mitochondrial respiration was observed without exogenous respiratory substrate. Increase in mitochondrial respiration was induced by treatment with L-carnitine in a concentration-dependent manner. Treatment with rotenone, a complex I blocker, completely inhibited ADP-induced oxygen consumption even in the presence of L-carnitine. Moreover, the L-carnitine dependent ADP-induced mitochondrial oxygen consumption did not increase when PLA(2) inhibitors were treated before ADP treatment. The L-carnitine-dependent ADP-induced oxygen consumption did contribute to ATP productions but not heat generation via an uncoupling system. These results suggest that L-carnitine might be essential to the beta-oxidation of quarried fatty acids from the mitochondrial membrane by PLA(2).

  19. Supplementation of T3 Recovers Hypothyroid Rat Liver Cells from Oxidatively Damaged Inner Mitochondrial Membrane Leading to Apoptosis

    Science.gov (United States)

    Mukherjee, Sutapa; Samanta, Luna; Roy, Anita; Bhanja, Shravani; Chainy, Gagan B. N.

    2014-01-01

    Hypothyroidism is a growing medical concern. There are conflicting reports regarding the mechanism of oxidative stress in hypothyroidism. Mitochondrial oxidative stress is pivotal to thyroid dysfunction. The present study aimed to delineate the effects of hepatic inner mitochondrial membrane dysfunction as a consequence of 6-n-propyl-2-thiouracil-induced hypothyroidism in rats. Increased oxidative stress predominance in the submitochondrial particles (SMP) and altered antioxidant defenses in the mitochondrial matrix fraction correlated with hepatocyte apoptosis. In order to check whether the effects caused by hypothyroidism are reversed by T3, the above parameters were evaluated in a subset of T3-treated hypothyroid rats. Complex I activity was inhibited in hypothyroid SMP, whereas T3 supplementation upregulated electron transport chain complexes. Higher mitochondrial H2O2 levels in hypothyroidism due to reduced matrix GPx activity culminated in severe oxidative damage to membrane lipids. SMP and matrix proteins were stabilised in hypothyroidism but exhibited increased carbonylation after T3 administration. Glutathione content was higher in both. Hepatocyte apoptosis was evident in hypothyroid liver sections; T3 administration, on the other hand, exerted antiapoptotic and proproliferative effects. Hence, thyroid hormone level critically regulates functional integrity of hepatic mitochondria; hypothyroidism injures mitochondrial membrane lipids leading to hepatocyte apoptosis, which is substantially recovered upon T3 supplementation. PMID:24987693

  20. Supplementation of T3 Recovers Hypothyroid Rat Liver Cells from Oxidatively Damaged Inner Mitochondrial Membrane Leading to Apoptosis

    Directory of Open Access Journals (Sweden)

    Sutapa Mukherjee

    2014-01-01

    Full Text Available Hypothyroidism is a growing medical concern. There are conflicting reports regarding the mechanism of oxidative stress in hypothyroidism. Mitochondrial oxidative stress is pivotal to thyroid dysfunction. The present study aimed to delineate the effects of hepatic inner mitochondrial membrane dysfunction as a consequence of 6-n-propyl-2-thiouracil-induced hypothyroidism in rats. Increased oxidative stress predominance in the submitochondrial particles (SMP and altered antioxidant defenses in the mitochondrial matrix fraction correlated with hepatocyte apoptosis. In order to check whether the effects caused by hypothyroidism are reversed by T3, the above parameters were evaluated in a subset of T3-treated hypothyroid rats. Complex I activity was inhibited in hypothyroid SMP, whereas T3 supplementation upregulated electron transport chain complexes. Higher mitochondrial H2O2 levels in hypothyroidism due to reduced matrix GPx activity culminated in severe oxidative damage to membrane lipids. SMP and matrix proteins were stabilised in hypothyroidism but exhibited increased carbonylation after T3 administration. Glutathione content was higher in both. Hepatocyte apoptosis was evident in hypothyroid liver sections; T3 administration, on the other hand, exerted antiapoptotic and proproliferative effects. Hence, thyroid hormone level critically regulates functional integrity of hepatic mitochondria; hypothyroidism injures mitochondrial membrane lipids leading to hepatocyte apoptosis, which is substantially recovered upon T3 supplementation.

  1. Immunoaffinity purification and characterization of mitochondrial membrane-bound D-3-hydroxybutyrate dehydrogenase from Jaculus orientalis

    Directory of Open Access Journals (Sweden)

    Cherkaoui-Malki Mustapha

    2008-09-01

    Full Text Available Abstract Background The interconversion of two important energy metabolites, 3-hydroxybutyrate and acetoacetate (the major ketone bodies, is catalyzed by D-3-hydroxybutyrate dehydrogenase (BDH1: EC 1.1.1.30, a NAD+-dependent enzyme. The eukaryotic enzyme is bound to the mitochondrial inner membrane and harbors a unique lecithin-dependent activity. Here, we report an advanced purification method of the mammalian BDH applied to the liver enzyme from jerboa (Jaculus orientalis, a hibernating rodent adapted to extreme diet and environmental conditions. Results Purifying BDH from jerboa liver overcomes its low specific activity in mitochondria for further biochemical characterization of the enzyme. This new procedure is based on the use of polyclonal antibodies raised against BDH from bacterial Pseudomonas aeruginosa. This study improves the procedure for purification of both soluble microbial and mammalian membrane-bound BDH. Even though the Jaculus orientalis genome has not yet been sequenced, for the first time a D-3-hydroxybutyrate dehydrogenase cDNA from jerboa was cloned and sequenced. Conclusion This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain. In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal. Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction.

  2. Naja naja oxiana Cobra Venom Cytotoxins CTI and CTII Disrupt Mitochondrial Membrane Integrity: Implications for Basic Three-Fingered Cytotoxins.

    Directory of Open Access Journals (Sweden)

    Sardar E Gasanov

    Full Text Available Cobra venom cytotoxins are basic three-fingered, amphipathic, non-enzymatic proteins that constitute a major fraction of cobra venom. While cytotoxins cause mitochondrial dysfunction in different cell types, the mechanisms by which cytotoxins bind to mitochondria remain unknown. We analyzed the abilities of CTI and CTII, S-type and P-type cytotoxins from Naja naja oxiana respectively, to associate with isolated mitochondrial fractions or with model membranes that simulate the mitochondrial lipid environment by using a myriad of biophysical techniques. Phosphorus-31 nuclear magnetic resonance (31P-NMR spectroscopy data suggest that both cytotoxins bind to isolated mitochondrial fractions and promote the formation of aberrant non-bilayer structures. We then hypothesized that CTI and CTII bind to cardiolipin (CL to disrupt mitochondrial membranes. Collectively, 31P-NMR, electron paramagnetic resonance (EPR, proton NMR (1H-NMR, deuterium NMR (2H-NMR spectroscopy, differential scanning calorimetry, and erythrosine phosphorescence assays suggest that CTI and CTII bind to CL to generate non-bilayer structures and promote the permeabilization, dehydration and fusion of large unilamellar phosphatidylcholine (PC liposomes enriched with CL. On the other hand, CTII but not CTI caused biophysical alterations of large unilamellar PC liposomes enriched with phosphatidylserine (PS. Mechanistically, single molecule docking simulations identified putative CL, PS and PC binding sites in CTI and CTII. While the predicted binding sites for PS and PC share a high number of interactive amino acid residues in CTI and CTII, the CL biding sites in CTII and CTI are more divergent as it contains additional interactive amino acid residues. Overall, our data suggest that cytotoxins physically associate with mitochondrial membranes by binding to CL to disrupt mitochondrial structural integrity.

  3. Naja naja oxiana Cobra Venom Cytotoxins CTI and CTII Disrupt Mitochondrial Membrane Integrity: Implications for Basic Three-Fingered Cytotoxins.

    Science.gov (United States)

    Gasanov, Sardar E; Shrivastava, Indira H; Israilov, Firuz S; Kim, Aleksandr A; Rylova, Kamila A; Zhang, Boris; Dagda, Ruben K

    2015-01-01

    Cobra venom cytotoxins are basic three-fingered, amphipathic, non-enzymatic proteins that constitute a major fraction of cobra venom. While cytotoxins cause mitochondrial dysfunction in different cell types, the mechanisms by which cytotoxins bind to mitochondria remain unknown. We analyzed the abilities of CTI and CTII, S-type and P-type cytotoxins from Naja naja oxiana respectively, to associate with isolated mitochondrial fractions or with model membranes that simulate the mitochondrial lipid environment by using a myriad of biophysical techniques. Phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy data suggest that both cytotoxins bind to isolated mitochondrial fractions and promote the formation of aberrant non-bilayer structures. We then hypothesized that CTI and CTII bind to cardiolipin (CL) to disrupt mitochondrial membranes. Collectively, 31P-NMR, electron paramagnetic resonance (EPR), proton NMR (1H-NMR), deuterium NMR (2H-NMR) spectroscopy, differential scanning calorimetry, and erythrosine phosphorescence assays suggest that CTI and CTII bind to CL to generate non-bilayer structures and promote the permeabilization, dehydration and fusion of large unilamellar phosphatidylcholine (PC) liposomes enriched with CL. On the other hand, CTII but not CTI caused biophysical alterations of large unilamellar PC liposomes enriched with phosphatidylserine (PS). Mechanistically, single molecule docking simulations identified putative CL, PS and PC binding sites in CTI and CTII. While the predicted binding sites for PS and PC share a high number of interactive amino acid residues in CTI and CTII, the CL biding sites in CTII and CTI are more divergent as it contains additional interactive amino acid residues. Overall, our data suggest that cytotoxins physically associate with mitochondrial membranes by binding to CL to disrupt mitochondrial structural integrity.

  4. Naja naja oxiana Cobra Venom Cytotoxins CTI and CTII Disrupt Mitochondrial Membrane Integrity: Implications for Basic Three-Fingered Cytotoxins

    Science.gov (United States)

    Gasanov, Sardar E.; Shrivastava, Indira H.; Israilov, Firuz S.; Kim, Aleksandr A.; Rylova, Kamila A.; Zhang, Boris; Dagda, Ruben K.

    2015-01-01

    Cobra venom cytotoxins are basic three-fingered, amphipathic, non-enzymatic proteins that constitute a major fraction of cobra venom. While cytotoxins cause mitochondrial dysfunction in different cell types, the mechanisms by which cytotoxins bind to mitochondria remain unknown. We analyzed the abilities of CTI and CTII, S-type and P-type cytotoxins from Naja naja oxiana respectively, to associate with isolated mitochondrial fractions or with model membranes that simulate the mitochondrial lipid environment by using a myriad of biophysical techniques. Phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy data suggest that both cytotoxins bind to isolated mitochondrial fractions and promote the formation of aberrant non-bilayer structures. We then hypothesized that CTI and CTII bind to cardiolipin (CL) to disrupt mitochondrial membranes. Collectively, 31P-NMR, electron paramagnetic resonance (EPR), proton NMR (1H-NMR), deuterium NMR (2H-NMR) spectroscopy, differential scanning calorimetry, and erythrosine phosphorescence assays suggest that CTI and CTII bind to CL to generate non-bilayer structures and promote the permeabilization, dehydration and fusion of large unilamellar phosphatidylcholine (PC) liposomes enriched with CL. On the other hand, CTII but not CTI caused biophysical alterations of large unilamellar PC liposomes enriched with phosphatidylserine (PS). Mechanistically, single molecule docking simulations identified putative CL, PS and PC binding sites in CTI and CTII. While the predicted binding sites for PS and PC share a high number of interactive amino acid residues in CTI and CTII, the CL biding sites in CTII and CTI are more divergent as it contains additional interactive amino acid residues. Overall, our data suggest that cytotoxins physically associate with mitochondrial membranes by binding to CL to disrupt mitochondrial structural integrity. PMID:26091109

  5. Antioxidant activity of capsaicin on radiation-induced oxidation of murine hepatic mitochondrial membrane preparation

    Directory of Open Access Journals (Sweden)

    Gangabhagirathi R

    2015-06-01

    Full Text Available Ramachandran Gangabhagirathi,1 Ravi Joshi,2 1Bioorganic Division, 2Radiation and Photochemistry Division, Bhabha Atomic Research Center, Trombay, Mumbai, India Abstract: Capsaicin is the major capsaicinoid in chili peppers and is widely used as a spice. It is also used for topical applications in cases of peripheral neuropathy. The present study deals with its role in modulation of gamma radiation-induced damages of the biochemical constituents of rat liver mitochondrial membrane (RLM preparation. The extent of lipid hydroperoxide formation, depletion in protein thiols, and formation of protein carbonyls have been biochemically assessed in the presence of varying concentrations of capsaicin in RLM. Decrease in the activities of the important antioxidant enzyme superoxide dismutase, which is involved in the scavenging of free radicals, and the mitochondrial marker enzyme succinate dehydrogenase have been also looked into. Capsaicin has been found to efficiently inhibit radiation-induced biochemical alterations, namely lipid peroxidation and protein oxidation. It also significantly prevented radiation-induced loss in the activity of antioxidant enzyme and the important endogenous antioxidant glutathione. The study suggests that capsaicin can act as an antioxidant and radioprotector in physiological systems. Keywords: capsaicin, gamma radiation, radioprotection, lipid peroxidation, protein oxidation, enzyme activity

  6. Peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands

    Energy Technology Data Exchange (ETDEWEB)

    Snyder, S.H.; Verma, A.; Trifiletti, R.R.

    1987-10-01

    The peripheral-type benzodiazepine receptor is a site identified by its nanomolar affinity for (/sup 3/H)diazepam, similar to the affinity of diazepam for the central-type benzodiazepine receptor in the brain. The peripheral type benzodiazepine receptor occurs in many peripheral tissues but has discrete localizations as indicated by autoradiographic studies showing uniquely high densities of the receptors in the adrenal cortex and in Leydig cells of the testes. Subcellular localization studies reveal a selective association of the receptors with the outer membrane of mitochondria. Photoaffinity labeling of the mitochondrial receptor with (/sup 3/H)flunitrazepam reveals two discrete labeled protein bands of 30 and 35 kDa, respectively. The 35-kDa band appears to be identical with the voltage-dependent anion channel protein porin. Fractionation of numerous peripheral tissues reveals a single principal endogenous ligand for the receptor, consisting of porphyrins, which display nanomolar affinity. Interactions of porphyrins with the mitochondrial receptor may clarify its physiological role and account for many pharmacological actions of benzodiazepines.

  7. Apoptotic Bax at Oxidatively Stressed Mitochondrial Membranes: Lipid Dynamics and Permeabilization.

    Science.gov (United States)

    Dingeldein, Artur Peter Günther; Pokorná, Šárka; Lidman, Martin; Sparrman, Tobias; Šachl, Radek; Hof, Martin; Gröbner, Gerhard

    2017-05-23

    Mitochondria are crucial compartments of eukaryotic cells because they function as the cellular power plant and play a central role in the early stages of programmed cell death (apoptosis). To avoid undesired cell death, this apoptotic pathway is tightly regulated by members of the Bcl-2 protein family, which interact on the external surface of the mitochondria, i.e., the mitochondrial outer membrane (MOM), and modulate its permeability to apoptotic factors, controlling their release into the cytosol. A growing body of evidence suggests that the MOM lipids play active roles in this permeabilization process. In particular, oxidized phospholipids (OxPls) formed under intracellular stress seem to directly induce apoptotic activity at the MOM. Here we show that the process of MOM pore formation is sensitive to the type of OxPls species that are generated. We created MOM-mimicking liposome systems, which resemble the cellular situation before apoptosis and upon triggering of oxidative stress conditions. These vesicles were studied using 31 P solid-state magic-angle-spinning nuclear magnetic resonance spectroscopy and differential scanning calorimetry, together with dye leakage assays. Direct polarization and cross-polarization nuclear magnetic resonance experiments enabled us to probe the heterogeneity of these membranes and their associated molecular dynamics. The addition of apoptotic Bax protein to OxPls-containing vesicles drastically changed the membranes' dynamic behavior, almost completely negating the previously observed effect of temperature on the lipids' molecular dynamics and inducing an ordering effect that led to more cooperative membrane melting. Our results support the hypothesis that the mitochondrion-specific lipid cardiolipin functions as a first contact site for Bax during its translocation to the MOM in the onset of apoptosis. In addition, dye leakage assays revealed that different OxPls species in the MOM-mimicking vesicles can have opposing

  8. Cyclosporin A does not protect the disruption of the inner mitochondrial membrane potential induced by potassium ionophores in intact K562 cells.

    Science.gov (United States)

    Marques-Santos, Luis F; Coqueiro, Vivian M; Rumjanek, Vivian M

    2006-03-01

    Mitochondrial dysfunction has been widely associated with programmed cell death. Studies of intact cells are important for the understanding of the process of cell death and its relation to mitochondrial physiology. Using cytofluorometric approaches we studied the mitochondrial behavior in an erythroleukemic cell line. The effects of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), potassium exchanger (nigericin), potassium ionophore (valinomycin), Na+K+-ATPase inhibitor (ouabain) and mitochondrial permeability transition pore inhibitor (cyclosporin A) were evaluated. Cyclosporin A (CSA) was very effective in attenuating the disruption of inner mitochondrial membrane potential induced by CCCP. However, CSA failed to protect the loss of inner mitochondrial membrane potential induced by potassium intracellular flux manipulation. Our findings suggest that mitochondrial cyclophilin is not involved in the cell events mediated by deregulation of potassium flux, underlining the need for further studies in intact tumor cells for a better understanding of the involvement of mitochondria physiology in cell death events.

  9. Contribution of glucose transport to the control of the glycolytic flux in Trypanosoma brucei.

    NARCIS (Netherlands)

    Bakker, B.M.; Walsh, M.C.; ter Kuile, B.; Mensonides, F.I.C.; Michels, P.A.M.; Opperdoes, F.R.; Westerhoff, H.V.

    1999-01-01

    The rate of glucose transport across the plasma membrane of the bloodstream form of Trypanosoma brucei was modulated by titration of the hexose transporter with the inhibitor phloretin, and the effect on the glycolytic flux was measured. A rapid glucose uptake assay was developed to measure the

  10. Redox-active nanoceria depolarize mitochondrial membrane of human colon cancer cells

    Science.gov (United States)

    Jana, Saikat Kumar; Banerjee, Priyanka; Das, Soumen; Seal, Sudipta; Chaudhury, Koel

    2014-06-01

    Nanotherapeutics is emerging as a promising option to the various limitations and side effects associated with conventional chemotherapy. The present study investigates the cytotoxic effect of redox-active cerium oxide nanoparticles (nanoceria) on human colorectal adenocarcinoma-derived cell line (HCT 15). Exposure of these cells to nanoceria for 24 h with concentration ranging between 10 and 100 μM resulted in a significant reduction of cell viability in a dose-dependent manner. Further, at a concentration of 10 µM, nanoceria exhibited time-dependent cytotoxic effect when exposed to the cells for 24, 48, and 72 h. Upon treatment of the cells with nanoceria, reactive oxygen species (ROS) and lipid peroxidation which are indicators of oxidative stress and cytotoxicity increased significantly, in a dose-dependent manner. Nanoceria was also found to depolarize the mitochondrial membrane, thereby collapsing the membrane potential and leading to initiation of apoptosis. Scanning electron microscopic study of nanoceria-treated HCT 15 cells showed morphological changes and loss of filopodia and lamellipodia, indicating arrest of metastatic spread. Summarizing, when cultured HCT 15 cells are exposed to nanoceria, a dose-dependent cytotoxic effect mediated by ROS generation is observed.

  11. Ligand tunnels in T. brucei and human CYP51: Insights for parasite-specific drug design.

    Science.gov (United States)

    Yu, Xiaofeng; Nandekar, Prajwal; Mustafa, Ghulam; Cojocaru, Vlad; Lepesheva, Galina I; Wade, Rebecca C

    2016-01-01

    Cytochrome P450 sterol 14α-demethylase (CYP51) is an essential enzyme for sterol biosynthesis and a target for anti-parasitic drug design. However, the design of parasite-specific drugs that inhibit parasitic CYP51 without severe side effects remains challenging. The active site of CYP51 is situated in the interior of the protein. Here, we characterize the potential ligand egress routes and mechanisms in Trypanosoma brucei and human CYP51 enzymes. We performed Random Acceleration Molecular Dynamics simulations of the egress of four different ligands from the active site of models of soluble and membrane-bound T. brucei CYP51 and of soluble human CYP51. In the simulations, tunnel 2f, which leads to the membrane, was found to be the predominant ligand egress tunnel for all the ligands studied. Tunnels S, 1 and W, which lead to the cytosol, were also used in T. brucei CYP51, whereas tunnel 1 was the only other tunnel used significantly in human CYP51. The common tunnels found previously in other CYPs were barely used. The ligand egress times were shorter for human than T. brucei CYP51, suggesting lower barriers to ligand passage. Two gating residues, F105 and M460, in T. brucei CYP51 that modulate the opening of tunnels 2f and S were identified. Although the main egress tunnel was the same, differences in the tunnel-lining residues, ligand passage and tunnel usage were found between T. brucei and human CYP51s. The results provide a basis for the design of selective anti-parasitic agents targeting the ligand tunnels. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. [Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane Na+, K+-ATPase].

    Science.gov (United States)

    Danylovych, H V; Danylovych, Iu V; Kolomiiets', O V; Kosterin, S O; Rodik, R V; Cherenok, S O; Kal'chenko, V I; Chunikhin, O Iu; Horchev, V F; Karakhim, S O

    2012-01-01

    The influence of supramolecular macrocyclic compounds--calix[4]arenes C-97, C-99, C-107, which are ouabainomymetic high affinity inhibitors of Na+, K(+)-ATPase, on the polarization level of plasmic and mitochondrial membranes of rat uterine smooth muscle cells was investigated. The influence of these compounds on the myocytes characteristic size was studied. By using a confocal microscopy and specific for mitochondrial MitoTracker Orange CM-H2TMRos dye it was proved that the potential-sensitive fluorescent probe DiOC6(3) interacts with mitochondria. Artificial potential collapse of plasmic membrane in this case was modeled by myocytes preincubation with ouabain (1 mM). Further experiments performed using the method of flow cytometry with DiOC6(3) have shown that the compounds C-97, C-99 and C-107 at concentration 50-100 nM caused depolarization of the plasma membrane (at the level of 30% relative to control values) in conditions of artificial collapse of mitochondrial potential by myocytes preincubation in the presence of 5 mM of sodium azide. Under artificial sarcolemma depolarization by ouabain, calixarenes C-97, C-99 and C-107 at 100 nM concentrations caused a transient increase of mitochondrial membrane potential, that is 40% of the control level and lasted about 5 minutes. Calixarenes C-99 and C-107 caused a significant increase in fluorescence of myocytes in these conditions, which was confirmed by confocal microscopy too. It was proved by photon correlation spectroscopy method that the C-99 and C-107 caused an increase of characteristic size of myocytes.

  13. Cockayne syndrome group B protein promotes mitochondrial DNA stability by supporting the DNA repair association with the mitochondrial membrane

    DEFF Research Database (Denmark)

    Aamann, Maria Diget; Sorensen, Martin M; Hvitby, Christina Poulsen

    2010-01-01

    Cockayne syndrome (CS) is a human premature aging disorder associated with severe developmental deficiencies and neurodegeneration, and phenotypically it resembles some mitochondrial DNA (mtDNA) diseases. Most patients belong to complementation group B, and the CS group B (CSB) protein plays a role...

  14. [Changes in mitochondrial membrane potentials and its exponential relation with phosphatidylserine translocation in the plasma membrane as markers in the initial events of apoptosis: evaluation in different spermatic fractions].

    Science.gov (United States)

    Barroso Villa, Gerardo; Karchmer Krivitzky, Samuel; Castelazo Morales, Ernesto; Carballo Mondragón, Esperanza; Kably Ambe, Alberto

    2002-04-01

    To determine the integrity of the plasmatic membrane through phosphatidylserine (PS) translocation in two spermatic fractions and their correlation with the spermatic mitochondrial membrane potential. The analysis of both spermatic fractions was carried out through a discontinuous gradient separation with Percoli, in order to obtain two samples with high and low mobility (90-40%). Twelve patients were recruited for the initial evaluation of seminal parameters. Mitochondrial membrane integrity was determined using a second antibody (Mitosensor), and was analyzed by fluorescence microscopy, evaluating an average of 200 cells. A 450-490 nm excitation filter was used for this analysis. Cytoplasmatic assessment was carried out by anexine V bonding to PS, in order to determine the initial events of cellular death. Non parameter tests were used in order to determine the differences between mitochondrial potentials and plasmatic membrane processes. Linear correlation tests were used for the anexine V and Mitosensor ratios. Due to the study's design, some differences were observed regarding the displacement parameters and the presence of apoptosis, both, in the plasmatic membrane and in mitochondrial membrane potentials. A positive correlation between both, mitochondrial and cytoplasmic membrane functions was also found. This is the first study performing a comparative analysis between mitochondrial membrane function and cytoplasmatic PS expression as early cellular death markers. The male infertility population is probably associated with an increase in this kind of apoptosis processes.

  15. Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.

    Science.gov (United States)

    Lee, Sukyeong; Augustin, Steffen; Tatsuta, Takashi; Gerdes, Florian; Langer, Thomas; Tsai, Francis T F

    2011-02-11

    FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.

  16. Inactivation of cytochrome c oxidase activity in mitochondrial membranes during redox cycling of doxorubicin.

    Science.gov (United States)

    Demant, E J

    1991-02-15

    Interactions of doxorubicin (DX) with the cardiolipin-dependent cytochrome c oxidase have been examined by using pig heart submitochondrial particles (SMP). A progressive and irreversible loss of oxidase activity is demonstrated in 2 hr incubations of the SMP with 10-100 microM DX in air-equilibrated medium with excess NADH to support redox-cycling of the drug. This oxidative mechanism for oxidase inactivation occurs in connection with a peroxidation process in the bulk membrane lipid, and is independent on turnover of the enzyme. It is related in a complex manner to the electron flux in the respiratory chain with antioxidant properties, and is maximal at the high reduction level of respiratory chain Complex I obtained in the presence of rotenone. Reduction of DX per se plays a minor role, and trace concentrations of chelatable metal ions (iron) are required to catalyse the reaction. Iron in the iron storage protein ferritin is released by DX, and at physiological low O2 concentrations ([O2] less than 20 microM), this iron is a better promoter of oxidase inactivation than is endogenous iron in the SMP. Kinetic analysis of inactivation data indicates the interaction of DX with low affinity (Km 35-55 microM) binding sites in the SMP membranes. Overall, the results point to the possible role of ferritin-iron in the mechanism of DX mitochondrial toxicity and argue against site specific effects of the DX-reduction/oxidation cycle on the cytochrome c oxidase or on its essential phospholipid (cardiolipin) environment.

  17. Microcystic cyanobacteria causes mitochondrial membrane potential alteration and reactive oxygen species formation in primary cultured rat hepatocytes.

    OpenAIRE

    Ding, W X; Shen, H M; Shen, Y; Zhu, H G; Ong, C N

    1998-01-01

    Cyanobacteria contamination of water has become a growing public health problem worldwide. Microcystis aeruginosa is one of the most common toxic cyanobacteria. It is capable of producing microcystins, a group of cyclic heptapeptide compounds with potent hepatotoxicity and tumor promotion activity. The present study investigated the effect of microcystic cyanobacteria on primary cultured rat hepatocytes by examining mitochondrial membrane potential (MMP) changes and intracellular reactive oxy...

  18. Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver.

    Science.gov (United States)

    Theurey, Pierre; Tubbs, Emily; Vial, Guillaume; Jacquemetton, Julien; Bendridi, Nadia; Chauvin, Marie-Agnès; Alam, Muhammad Rizwan; Le Romancer, Muriel; Vidal, Hubert; Rieusset, Jennifer

    2016-04-01

    Mitochondria-associated endoplasmic reticulum membranes (MAM) play a key role in mitochondrial dynamics and function and in hepatic insulin action. Whereas mitochondria are important regulators of energy metabolism, the nutritional regulation of MAM in the liver and its role in the adaptation of mitochondria physiology to nutrient availability are unknown. In this study, we found that the fasted to postprandial transition reduced the number of endoplasmic reticulum-mitochondria contact points in mouse liver. Screening of potential hormonal/metabolic signals revealed glucose as the main nutritional regulator of hepatic MAM integrity both in vitro and in vivo Glucose reduced organelle interactions through the pentose phosphate-protein phosphatase 2A (PP-PP2A) pathway, induced mitochondria fission, and impaired respiration. Blocking MAM reduction counteracted glucose-induced mitochondrial alterations. Furthermore, disruption of MAM integrity mimicked effects of glucose on mitochondria dynamics and function. This glucose-sensing system is deficient in the liver of insulin-resistant ob/ob and cyclophilin D-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission, and altered mitochondrial respiration. These data indicate that MAM contribute to the hepatic glucose-sensing system, allowing regulation of mitochondria dynamics and function during nutritional transition. Chronic disruption of MAM may participate in hepatic mitochondrial dysfunction associated with insulin resistance. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

  19. Improvement of the energy metabolism of recombinant CHO cells by cell sorting for reduced mitochondrial membrane potential.

    Science.gov (United States)

    Hinterkörner, Georg; Brugger, Gudrun; Müller, Dethardt; Hesse, Friedemann; Kunert, Renate; Katinger, Hermann; Borth, Nicole

    2007-05-10

    One of the major problems in process performance of mammalian cell cultures is the production of lactic acid. Cell specific glucose uptake rates usually correlate to glucose concentration and approximately 80% of the metabolised glucose is converted into lactic acid. As the mitochondrial membrane potential was shown to correlate to cell specific glucose uptake rates, we used Rhodamine 123, a lipophilic cationic dye for cell sorting to improve the energy metabolism of existing production cell lines. Two recombinant CHO cell lines with known differences in lactic acid production rate were used to evaluate Rhodamine 123 staining as a descriptor for glucose uptake rates and to determine whether it is possible to isolate subclones with altered metabolic properties. Such subclones would exhibit an improved process performance, and in addition could be used as models for genomic and metabolic studies. From the cell line with high lactate production, a subclone sorted for reduced mitochondrial membrane potential was found to have a lower specific lactate formation rate compared to the parental cell line in batch cultures. In addition, the glucose consumption rate was also reduced, while both the growth rate and the final cell concentration were increased. A subclone sorted for high mitochondrial membrane potential, on the other hand, had a higher glucose consumption rate, a higher lactate production rate and reduced growth. The potential of using flow cytometric cell sorting methods based on physiological activity for cell line optimisation is discussed.

  20. Genome-Wide Screens in Saccharomyces cerevisiae Highlight a Role for Cardiolipin in Biogenesis of Mitochondrial Outer Membrane Multispan Proteins.

    Science.gov (United States)

    Sauerwald, Julia; Jores, Tobias; Eisenberg-Bord, Michal; Chuartzman, Silvia Gabriela; Schuldiner, Maya; Rapaport, Doron

    2015-09-01

    A special group of mitochondrial outer membrane (MOM) proteins spans the membrane several times via multiple helical segments. Such multispan proteins are synthesized on cytosolic ribosomes before their targeting to mitochondria and insertion into the MOM. Previous work recognized the import receptor Tom70 and the mitochondrial import (MIM) complex, both residents of the MOM, as required for optimal biogenesis of these proteins. However, their involvement is not sufficient to explain either the entire import pathway or its regulation. To identify additional factors that are involved in the biogenesis of MOM multispan proteins, we performed complementary high-throughput visual and growth screens in Saccharomyces cerevisiae. Cardiolipin (CL) synthase (Crd1) appeared as a candidate in both screens. Our results indeed demonstrate lower steady-state levels of the multispan proteins Ugo1, Scm4, and Om14 in mitochondria from crd1Δ cells. Importantly, MOM single-span proteins were not affected by this mutation. Furthermore, organelles lacking Crd1 had a lower in vitro capacity to import newly synthesized Ugo1 and Scm4 molecules. Crd1, which is located in the mitochondrial inner membrane, condenses phosphatidylglycerol together with CDP-diacylglycerol to obtain de novo synthesized CL molecules. Hence, our findings suggest that CL is an important component in the biogenesis of MOM multispan proteins. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Helicobacter pylori VacA toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane.

    Directory of Open Access Journals (Sweden)

    Grazyna Domańska

    2010-04-01

    Full Text Available The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% beta-strands, similar to pore-forming beta-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylaminobenzoic acid (NPPB, a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.

  2. Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Alloatti, Andres [Instituto de Biologia Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Santa Fe (Argentina); Gupta, Shreedhara; Gualdron-Lopez, Melisa; Nguewa, Paul A. [Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Universite Catholique de Louvain, Brussels (Belgium); Altabe, Silvia G. [Instituto de Biologia Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Santa Fe (Argentina); Deumer, Gladys; Wallemacq, Pierre [Department of Clinical Chemistry, Cliniques Universitaires Saint-Luc, LTAP, Universite Catholique de Louvain, Brussels (Belgium); Michels, Paul A.M. [Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Universite Catholique de Louvain, Brussels (Belgium); Uttaro, Antonio D., E-mail: toniuttaro@yahoo.com.ar [Instituto de Biologia Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Santa Fe (Argentina)

    2011-08-26

    Highlights: {yields} Inhibiting {Delta}9 desaturase drastically changes T. brucei's fatty-acid composition. {yields} Isoxyl specifically inhibits the {Delta}9 desaturase causing a growth arrest. {yields} RNA interference of desaturase expression causes a similar effect. {yields} Feeding T. brucei-infected mice with Isoxyl decreases the parasitemia. {yields} 70% of Isoxyl-treated mice survived the trypanosome infection. -- Abstract: Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes named desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei's survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC{sub 50}) of PCF was 1.0 {+-} 0.2 {mu}M for Isoxyl and 5 {+-} 2 {mu}M for 10-TS, whereas BSF appeared more susceptible with EC{sub 50} values 0.10 {+-} 0.03 {mu}M (Isoxyl) and 1.0 {+-} 0.6 {mu}M (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents' survival.

  3. Rab23 is a flagellar protein in Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Field Mark C

    2011-06-01

    Full Text Available Abstract Background Rab small GTPases are important mediators of membrane transport, and orthologues frequently retain similar locations and functions, even between highly divergent taxa. In metazoan organisms Rab23 is an important negative regulator of Sonic hedgehog signaling and is crucial for correct development and differentiation of cellular lineages by virtue of an involvement in ciliary recycling. Previously, we reported that Trypanosoma brucei Rab23 localized to the nuclear envelope 1, which is clearly inconsistent with the mammalian location and function. As T. brucei is unicellular the potential that Rab23 has no role in cell signaling was possible. Here we sought to further investigate the role(s of Rab23 in T. brucei to determine if Rab23 was an example of a Rab protein with divergent function in distinct taxa. Methods/major findings The taxonomic distribution of Rab23 was examined and compared with the presence of flagella/cilia in representative taxa. Despite evidence for considerable secondary loss, we found a clear correlation between a conventional flagellar structure and the presence of a Rab23 orthologue in the genome. By epitope-tagging, Rab23 was localized and found to be present at the flagellum throughout the cell cycle. However, RNAi knockdown did not result in a flagellar defect, suggesting that Rab23 is not required for construction or maintenance of the flagellum. Conclusions The location of Rab23 at the flagellum is conserved between mammals and trypanosomes and the Rab23 gene is restricted to flagellated organisms. These data may suggest the presence of a Rab23-mediated signaling mechanism in trypanosomes.

  4. Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation.

    Science.gov (United States)

    van Weelden, Susanne W H; Fast, Beate; Vogt, Achim; van der Meer, Pieter; Saas, Joachim; van Hellemond, Jaap J; Tielens, Aloysius G M; Boshart, Michael

    2003-04-11

    The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitro differentiation from wild type and mutant (Delta aco::NEO/Delta aco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO(2). Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.

  5. Microcystic cyanobacteria causes mitochondrial membrane potential alteration and reactive oxygen species formation in primary cultured rat hepatocytes.

    Science.gov (United States)

    Ding, W X; Shen, H M; Shen, Y; Zhu, H G; Ong, C N

    1998-07-01

    Cyanobacteria contamination of water has become a growing public health problem worldwide. Microcystis aeruginosa is one of the most common toxic cyanobacteria. It is capable of producing microcystins, a group of cyclic heptapeptide compounds with potent hepatotoxicity and tumor promotion activity. The present study investigated the effect of microcystic cyanobacteria on primary cultured rat hepatocytes by examining mitochondrial membrane potential (MMP) changes and intracellular reactive oxygen species (ROS) formation in cells treated with lyophilized freshwater microcystic cyanobacteria extract (MCE). Rhodamine 123 (Rh-123) was used as a fluorescent probe for changes in mitochondrial fluorescence intensity. The mitochondrial Rh-123 fluorescence intensity in MCE-treated hepatocytes, examined using a laser confocal microscope, responded in a dose- and time-dependent manner. The results thus indicate that the alteration of MMP might be an important event in the hepatotoxicity caused by cyanobacteria. Moreover, the parallel increase of ROS formation detected using another fluorescent probe, 2',7'-dichlorofluorescin diacetate also suggests the involvement of oxidative stress in the hepatotoxicity caused by cyanobacteria. The fact that MMP changes precede other cytotoxic parameters such as nuclear staining by propidium iodide and cell morphological changes suggests that mitochondrial damage is closely associated with MCE-induced cell injury in cultured rat hepatocytes.

  6. Evidence of proteolipid domain formation in an inner mitochondrial membrane mimicking model

    DEFF Research Database (Denmark)

    Cheniour, Mouhedine; Brewer, Jonathan R.; Bagatolli, Luis

    2017-01-01

    Background Mitochondrial creatine kinase (mtCK) is highly abundant in mitochondria; its quantity is equimolecular to the Adenylic Nucleotide Translocator and represents 1% of the mitochondrial proteins. It is a multitask protein localized in the mitochondria intermembrane space where it binds...

  7. Zinc and calcium alter the relationship between mitochondrial respiration, ROS and membrane potential in rainbow trout (Oncorhynchus mykiss) liver mitochondria.

    Science.gov (United States)

    Sharaf, Mahmoud S; Stevens, Don; Kamunde, Collins

    2017-08-01

    At excess levels, zinc (Zn) disrupts mitochondrial functional integrity and induces oxidative stress in aquatic organisms. Although much is known about the modulation of Zn toxicity by calcium (Ca) in fish, their interactions at the mitochondrial level have scarcely been investigated. Here we assessed the individual and combined effects of Zn and Ca on the relationship between mitochondrial respiration, ROS and membrane potential (ΔΨmt) in rainbow trout liver mitochondria. We tested if cation uptake through the mitochondrial calcium uniporter (MCU) is a prerequisite for Zn- and/or Ca-induced alteration of mitochondrial function. Furthermore, using our recently developed real-time multi-parametric method, we investigated the changes in respiration, ΔΨmt, and reactive oxygen species (ROS, as hydrogen peroxide (H2O2)) release associated with Ca-induced mitochondrial depolarization imposed by transient and permanent openings of the mitochondrial permeability transition pore (mPTP). We found that independent of the MCU, Zn precipitated an immediate depolarization of the ΔΨmt that was associated with relatively slow enhancement of H2O2 release, inhibition of respiration and reversal of the positive correlation between ROS and ΔΨmt. In contrast, an equitoxic dose of Ca caused transient depolarization, and stimulation of both respiration and H2O2 release, effects that were completely abolished when the MCU was blocked. Contrary to our expectation that mitochondrial transition ROS Spike (mTRS) would be sensitive to both Zn and Ca, only Ca suppressed it. Moreover, Zn and Ca in combination immediately depolarized the ΔΨmt, and caused transient and sustained stimulation of respiration and H2O2 release, respectively. Lastly, we uncovered and characterized an mPTP-independent Ca-induced depolarization spike that was associated with exposure to moderately elevated levels of Ca. Importantly, we showed the stimulation of ROS release associated with highly elevated but not

  8. Thermal acclimation in American alligators: Effects of temperature regime on growth rate, mitochondrial function, and membrane composition.

    Science.gov (United States)

    Price, Edwin R; Sirsat, Tushar S; Sirsat, Sarah K G; Kang, Gurdeep; Keereetaweep, Jantana; Aziz, Mina; Chapman, Kent D; Dzialowski, Edward M

    2017-08-01

    We investigated the ability of juvenile American alligators (Alligator mississippiensis) to acclimate to temperature with respect to growth rate. We hypothesized that alligators would acclimate to cold temperature by increasing the metabolic capacity of skeletal muscles and the heart. Additionally, we hypothesized that lipid membranes in the thigh muscle and liver would respond to low temperature, either to maintain fluidity (via increased unsaturation) or to maintain enzyme reaction rates (via increased docosahexaenoic acid). Alligators were assigned to one of 3 temperature regimes beginning at 9 mo of age: constant warm (30°C), constant cold (20°C), and daily cycling for 12h at each temperature. Growth rate over the following 7 mo was highest in the cycling group, which we suggest occurred via high digestive function or feeding activity during warm periods and energy-saving during cold periods. The warm group also grew faster than the cold group. Heart and liver masses were proportional to body mass, while kidney was proportionately larger in the cold group compared to the warm animals. Whole-animal metabolic rate was higher in the warm and cycling groups compared to the cold group - even when controlling for body mass - when assayed at 30°C, but not at 20°C. Mitochondrial oxidative phosphorylation capacity in permeabilized fibers of thigh muscle and heart did not differ among treatments. Membrane fatty acid composition of the brain was largely unaffected by temperature treatment, but adjustments were made in the phospholipid headgroup composition that are consistent with homeoviscous adaptation. Thigh muscle cell membranes had elevated polyunsaturated fatty acids in the cold group relative to the cycling group, but this was not the case for thigh muscle mitochondrial membranes. Liver mitochondria from cold alligators had elevated docosahexaenoic acid, which might be important for maintenance of reaction rates of membrane-bound enzymes. Copyright © 2016

  9. Comparative kinetics of damage to the plasma and mitochondrial membranes by intra-cellularly synthesized and externally-provided photosensitizers using multi-color FACS.

    Science.gov (United States)

    Haupt, Sara; Malik, Zvi; Ehrenberg, Benjamin

    2014-01-01

    Photodynamic therapy (PDT) of cancer involves inflicting lethal damage to the cells of malignant tumors, primarily by singlet oxygen that is generated following light-absorption in a photosensitizer molecule. Dysfunction of cells is manifested in many ways, including peroxidation of cellular components, membrane rupture, depolarization of electric potentials, termination of mitochondrial activity, onset of apoptosis and necrosis and eventually cell lysis. These events do not necessarily occur in linear fashion and different types of damage to cell components occur, most probably, in parallel. In this report we measured the relative rates of damage to two cellular membranes: the plasma membrane and the mitochondrial membrane. We employed photosensitizers of diverse hydrophobicities and used different incubation procedures, which lead to their different intra-cellular localizations. We monitored the damage that was inflicted on these membranes, by employing optical probes of membrane integrity, in a multi-color FACS experiment. The potentiometric indicator JC-1 monitored the electric cross-membrane potential of the mitochondria and the fluorometric indicator Draq7 monitored the rupture of the plasma membrane. We show that the electric depolarization of the mitochondrial membrane and the damage to the enveloping plasma membrane proceed with different kinetics that reflect the molecular character and intracellular location of the sensitizer: PpIX that is synthesized in the cells from ALA causes rapid mitochondrial damage and very slow damage to the plasma membrane, while externally added PpIX has an opposite effect. The hydrophilic sensitizer HypS4 can be taken up by the cells by different incubation conditions, and these affect its intracellular location, and as a consequence either the plasma membrane or the mitochondria is damaged first. A similar correlation was found for additional extracellularly-provided photosensitizers HP and PpIX.

  10. Correlation between fluidising effects on phospholipid membranes and mitochondrial respiration of propofol and p-nitrosophenol homologues.

    Science.gov (United States)

    Momo, Federico; Fabris, Sabrina; Wisniewska, Anna; Fiore, Cristina; Bindoli, Alberto; Scutari, Guido; Stevanato, Roberto

    2003-03-25

    Nitrosopropofol (2-6-diisopropyl-4-nitrosophenol) has dramatic consequences for respiration, ATP synthesis and the transmembrane potential of isolated rat liver mitochondria at concentrations at which propofol (2-6-diisopropylphenol) does not cause any apparent effects. These results correlate well with the observation that nitrosopropofol is also a stronger perturbing agent of phospholipid membranes. In this paper we verify the possible biological activity of different phenols and nitrosophenols on mitochondrial respiration. We then discuss their interactions with phospholipid liposomes, studied with differential scanning calorimetry, spin labelling techniques and UV-Vis spectrophotometry, in order to obtain information on drug distribution and the modifications they impose on lipid bilayer. The results of the experiments performed on mitochondria and model membranes prove an interesting correlation between the effects of the molecules on both systems.

  11. Mitochondrial Membrane Potential and Nuclear and Gene Expression Changes During Human Disc Cell Apoptosis: In Vitro and In Vivo Annulus Findings.

    Science.gov (United States)

    Gruber, Helen E; Hoelscher, Gretchen L; Bethea, Synthia; Hanley, Edward N

    2015-06-15

    A study using cultured human annulus cells and human annular tissue. To further explore and define mitochondrial mechanisms related to disc cell apoptosis in vitro and in vivo. Mitochondrial-dependent intrinsic signaling pathways are a well-recognized component of apoptosis (programmed cell death). Disc cell apoptosis is important because it is a major mechanism by which cell numbers decrease during disc degeneration. Our objective was to further explore and define mitochondrial mechanisms related to disc cell apoptosis. High-content screening techniques were used to study nuclear morphology and mitochondrial membrane potentials in cultured annulus cells. Gene expression in annulus tissue was studied with microarray analysis. Cultured cells showed significantly increased nuclear size (an indicator of apoptosis) with increasing Thompson grade (P potential (which results from the difference in electrical potential generated by the electrochemical gradient across the inner membrane of the mitochondrion) versus Thompson grade was identified in cultured human annulus cells in control conditions (r = 0.356, P potential was identified versus nontreated cells. Gene expression patterns in more degenerated Thompson grade III, IV, and V discs versus healthier grade I and II discs showed significant upregulation of a number of genes with well-recognized apoptosis roles in mitochondrial potential decline (ITM2B, beta-2-microglobulin, and cathepsin B, DAP, GAS1, and PDCD5) and TNF-α associations (cathepsin B, RAC1, and PPT1). Data presented here show the in vivo expression of apoptosis-related genes associated with the loss of mitochondrial membrane integrity and decreased mitochondrial membrane potential with increasing Thompson scores. These data, which mimic our novel, direct cell-based in vitro findings, stress the importance of mitochondrial changes related to apoptosis and TNF-α during human disc degeneration. N/A.

  12. Interaction between Trypanosoma brucei and Haemonchus ...

    African Journals Online (AJOL)

    In order to investigate the immunomodulatory influence of concurrent T. brucei and H. contortus infection in West African Dwarf (WAD) goats, 28 infected and 7 uninfected (control) of 8-9 months old male WAD goats were studied. The infected goats were separated into resistant (Class 1) and susceptible (Class 2) Faecal ...

  13. Haematology of experimental Trypanosoma brucei rhodesiense ...

    African Journals Online (AJOL)

    Haematological aberrations associated with human infective trypanosomes were investigated in the vervet monkey model of the Rhodesian sleeping sickness. Four monkeys were infected intravenously with 104 Trypanosoma brucei rhodesiense and monitored for changes in the blood profile using a haematological ...

  14. Characterization of Trypanosoma brucei gambiense stocks isolated ...

    African Journals Online (AJOL)

    Characterization of Trypanosoma brucei gambiense stocks isolated from humans by RAPD fingerprinting in Côte d'Ivoire: another evidence for multiple infections. ... a given isoenzyme profile (zymodeme), confirming that this fingerprinting method has a higher discriminative power (faster molecular clock) than isoenzymes.

  15. Wild chimpanzees are infected by Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Milan Jirků

    2015-12-01

    Finally, we demonstrated that the mandrill serum was able to efficiently lyse T. b. brucei and T. b. rhodesiense, and to some extent T. b. gambiense, while the chimpanzee serum failed to lyse any of these subspecies.

  16. Evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential, membrane integrity, sperm motility and in vitro fertilization in bovine epididymal sperm.

    Science.gov (United States)

    Nichi, M; Rijsselaere, T; Losano, Jda; Angrimani, Dsr; Kawai, Gkv; Goovaerts, Igf; Van Soom, A; Barnabe, V H; De Clercq, Jbp; Bols, Pej

    2017-04-01

    The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post-mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty-six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2-3 hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer-assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre-freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post-thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures. © 2016 Blackwell Verlag GmbH.

  17. Genetic control of resistance to Trypanosoma brucei brucei infection in mice.

    Directory of Open Access Journals (Sweden)

    Matyáš Síma

    2011-06-01

    Full Text Available Trypanosoma brucei brucei infects livestock, with severe effects in horses and dogs. Mouse strains differ greatly in susceptibility to this parasite. However, no genes controlling these differences were mapped.We studied the genetic control of survival after T. b. brucei infection using recombinant congenic (RC strains, which have a high mapping power. Each RC strain of BALB/c-c-STS/A (CcS/Dem series contains a different random subset of 12.5% genes from the parental "donor" strain STS/A and 87.5% genes from the "background" strain BALB/c. Although BALB/c and STS/A mice are similarly susceptible to T. b. brucei, the RC strain CcS-11 is more susceptible than either of them. We analyzed genetics of survival in T. b. brucei-infected F(2 hybrids between BALB/c and CcS-11. CcS-11 strain carries STS-derived segments on eight chromosomes. They were genotyped in the F(2 hybrid mice and their linkage with survival was tested by analysis of variance.We mapped four Tbbr (Trypanosoma brucei brucei response loci that influence survival after T. b. brucei infection. Tbbr1 (chromosome 3 and Tbbr2 (chromosome 12 have effects on survival independent of inter-genic interactions (main effects. Tbbr3 (chromosome 7 influences survival in interaction with Tbbr4 (chromosome 19. Tbbr2 is located on a segment 2.15 Mb short that contains only 26 genes.This study presents the first identification of chromosomal loci controlling susceptibility to T. b. brucei infection. While mapping in F(2 hybrids of inbred strains usually has a precision of 40-80 Mb, in RC strains we mapped Tbbr2 to a 2.15 Mb segment containing only 26 genes, which will enable an effective search for the candidate gene. Definition of susceptibility genes will improve the understanding of pathways and genetic diversity underlying the disease and may result in new strategies to overcome the active subversion of the immune system by T. b. brucei.

  18. Sensitivity of mitochondrial DNA depleted ρ0 cells to H2O2 depends on the plasma membrane status.

    Science.gov (United States)

    Tomita, Kazuo; Kuwahara, Yoshikazu; Takashi, Yuko; Tsukahara, Takao; Kurimasa, Akihiro; Fukumoto, Manabu; Nishitani, Yoshihiro; Sato, Tomoaki

    2017-08-19

    To clarify the relationship between mitochondrial DNA (mtDNA)-depleted ρ0 cells and the cellular sensitivity to hydrogen peroxide (H2O2), we established HeLa and SAS ρ0 cell lines and investigated their survival rate in H2O2, radical scavenging enzymes, plasma membrane potential status, and chronological change in intracellular H2O2 amount under the existence of extracellular hydrogen peroxide compared with the parental cells. The results revealed that ρ0 cells had higher sensitivity to H2O2 than their parental cells, even though the catalase activity of ρ0 cells was up-regulated, and the membrane potential of the ρ0 cells was lower than their parental cells. Furthermore, the internal H2O2 amount significantly increased only in ρ0 cells after 50 μM H2O2 treatment for 1 h. These results suggest that plasma membrane status of ρ0 cells may cause degradation, and the change could lead to enhanced membrane permeability to H2O2. As a consequence, ρ0 cells have a higher H2O2 sensitivity than the parental cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related

    Energy Technology Data Exchange (ETDEWEB)

    Berk, P.D.; Potter, B.J.; Sorrentino, D.; Zhou, S.L.; Isola, L.M.; Stump, D.; Kiang, C.L.; Thung, S. (Mount Sinai School of Medicine, New York, NY (USA)); Wada, H.; Horio, Y. (Univ. of Osaka (Japan))

    1990-05-01

    The hepatic plasma membrane fatty acid-binding protein (h-FABP{sub PM}) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate. Sinusoidally enriched liver plasma membranes and purified h-FABP{sub PM} have GOT enzymatic activity. Monospecific rabbit antiserum against h-FABP{sub PM} reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of ({sup 3}H)oleate but not that of ({sup 35}S)sulfobromophthalein or ({sup 14}C)taurocholate. The inhibition of oleate uptake produced by anti-h-FABP{sub PM} can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABP{sub PM} and mGOT are closely related.

  20. Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes.

    Science.gov (United States)

    Dean, Samuel; Gould, Matthew K; Dewar, Caroline E; Schnaufer, Achim C

    2013-09-03

    Viability of the tsetse fly-transmitted African trypanosome Trypanosoma brucei depends on maintenance and expression of its kinetoplast (kDNA), the mitochondrial genome of this parasite and a putative target for veterinary and human antitrypanosomatid drugs. However, the closely related animal pathogens T. evansi and T. equiperdum are transmitted independently of tsetse flies and survive without a functional kinetoplast for reasons that have remained unclear. Here, we provide definitive evidence that single amino acid changes in the nuclearly encoded F1FO-ATPase subunit γ can compensate for complete physical loss of kDNA in these parasites. Our results provide insight into the molecular mechanism of compensation for kDNA loss by showing FO-independent generation of the mitochondrial membrane potential with increased dependence on the ADP/ATP carrier. Our findings also suggest that, in the pathogenic bloodstream stage of T. brucei, the huge and energetically demanding apparatus required for kDNA maintenance and expression serves the production of a single F1FO-ATPase subunit. These results have important implications for drug discovery and our understanding of the evolution of these parasites.

  1. Palmitoylation of the immunity related GTPase, Irgm1: impact on membrane localization and ability to promote mitochondrial fission.

    Directory of Open Access Journals (Sweden)

    Stanley C Henry

    Full Text Available The Immunity-Related GTPases (IRG are a family of large GTPases that mediate innate immune responses. Irgm1 is particularly critical for immunity to bacteria and protozoa, and for inflammatory homeostasis in the intestine. Although precise functions for Irgm1 have not been identified, prior studies have suggested roles in autophagy/mitophagy, phagosome remodeling, cell motility, and regulating the activity of other IRG proteins. These functions ostensibly hinge on the ability of Irgm1 to localize to intracellular membranes, such as those of the Golgi apparatus and mitochondria. Previously, it has been shown that an amphipathic helix, the αK helix, in the C-terminal portion of the protein partially mediates membrane binding. However, in absence of αK, there is still substantial binding of Irgm1 to cellular membranes, suggesting the presence of other membrane binding motifs. In the current work, an additional membrane localization motif was found in the form of palmitoylation at a cluster of cysteines near the αK. An Irgm1 mutant possessing alanine to cysteine substitutions at these amino acids demonstrated little residual palmitoylation, yet it displayed only a small decrease in localization to the Golgi and mitochondria. In contrast, a mutant containing the palmitoylation mutations in combination with mutations disrupting the amphipathic character of the αK displayed a complete loss of apparent localization to the Golgi and mitochondria, as well as an overall loss of association with cellular membranes in general. Additionally, Irgm1 was found to promote mitochondrial fission, and this function was undermined in Irgm1 mutants lacking the palmitoylation domain, and to a greater extent in those lacking the αK, or the αK and palmitoylation domains combined. Our data suggest that palmitoylation together with the αK helix firmly anchor Irgm1 in the Golgi and mitochondria, thus facilitating function of the protein.

  2. Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

    DEFF Research Database (Denmark)

    Pszon-Bartosz, Kamila Justyna; Hansen, Jesper S.; Stibius, Karin B.

    2011-01-01

    establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein...... in a bilayer array with a total membrane area of 2mm2 within 20min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications....

  3. Trypanosoma brucei: two steps to spread out from Africa.

    Science.gov (United States)

    Lun, Zhao-Rong; Lai, De-Hua; Li, Feng-Jun; Lukes, Julius; Ayala, Francisco J

    2010-09-01

    Trypanosoma brucei equiperdum and Trypanosoma brucei evansi are typically considered separate species, although a recent study suggested that these organisms can be classified as subspecies of Trypanosoma brucei, which we also favor. Here we present a scenario that attempts to explain the continuing evolution of the dyskinetoplastic and akinetoplastic strains, as a consequence of loss of selective pressure(s) leading to the loss of kinetoplast DNA. Copyright 2010 Elsevier Ltd. All rights reserved.

  4. Identification of Trypanosoma evansi, Trypanosoma equiperdum and Trypanosoma brucei brucei using repetitive DNA probes.

    Science.gov (United States)

    Zhang, Z Q; Baltz, T

    1994-06-01

    The phylogenetic relatedness of 15 stocks of Trypanosoma evansi, three stocks of Trypanosoma equiperdum and one stock of Trypanosoma brucei brucei was determined using Southern blot analysis of restriction enzyme digested DNA, probed with two repetitive DNA sequences from T. b. brucei. A dendrogram derived by cluster analysis of restriction fragment length polymorphism (RFLP) revealed three groups of related stocks. Group 1 included 14 stocks of T. evansi and one stock of T. equiperdum. Group 2 included two stocks of T. equiperdum and one stock of T. evansi. Group 3 included the one stock of T. brucei brucei. Group 2 is more closely related to Group 3 than Group 1, by analysis of the banding patterns. Further analysis of the T. evansi in Group 1 revealed that the patterns of isolates from different provinces in China were identical, but differed from T. evansi isolated from Africa, South America and the Philippines. These results provide insight into the origins of T. evansi and suggest that RFLP may be a useful means of distinguishing closely related trypanosomes.

  5. Demonstration of an intramitochondrial invertase activity and the corresponding sugar transporters of the inner mitochondrial membrane in Jerusalem artichoke (Helianthus tuberosus L.) tubers.

    Science.gov (United States)

    Szarka, András; Horemans, Nele; Passarella, Salvatore; Tarcsay, Akos; Orsi, Ferenc; Salgó, András; Bánhegyi, Gábor

    2008-10-01

    Genetic evidences indicate that alkaline/neutral invertases are present in plant cell organelles, and they might have a novel physiological function in mitochondria. The present study demonstrates an invertase activity in the mitochondrial matrix of Helianthus tuberosus tubers. The pH optimum, the kinetic parameters and the inhibitor profile of the invertase activity indicated that it belongs to the neutral invertases. In accordance with this topology, transport activities responsible for the mediation of influx/efflux of substrate/products were studied in the inner mitochondrial membrane. The transport of sucrose, glucose and fructose was shown to be bidirectional, saturable and independent of the mitochondrial respiration and membrane potential. Sucrose transport was insensitive to the inhibitors of the proton-sucrose symporters. The different kinetic parameters and inhibitors as well as the absence of cross-inhibition suggest that sucrose, glucose and fructose transport are mediated by separate transporters in the inner mitochondrial membrane. The mitochondrial invertase system composed by an enzyme activity in the matrix and the corresponding sugar transporters might have a role in both osmoregulation and intermediary metabolism.

  6. Detection of Trypanosoma brucei gambiense and T. b. rhodesiense ...

    African Journals Online (AJOL)

    Detection of Trypanosoma brucei gambiense and T. b. rhodesiense in Glossina fuscipes fuscipes ( Diptera: Glossinidae ) and Stomoxys flies using the polymerase chain reaction (PCR) technique in southern Sudan.

  7. Improved glycaemic control decreases inner mitochondrial membrane leak in type 2 diabetes

    DEFF Research Database (Denmark)

    Rabøl, R; Højberg, P M V; Almdal, T

    2009-01-01

    AIM: Several mechanisms have been targeted as culprits of weight gain during antihyperglycaemic treatment in type 2 diabetes (T2DM). These include reductions in glucosuria, increased food intake from fear of hypoglycaemia, the anabolic effect of insulin, decreased metabolic rate and increased...... efficiency in fuel usage. The purpose of the study was to test the hypothesis that mitochondrial efficiency increases as a result of insulin treatment in patients with type 2 diabetes. METHODS: We included ten patients with T2DM (eight males) on oral antidiabetic treatment, median age: 51.5 years (range: 39......-67) and body mass index (BMI): 30.1 +/- 1.2 kg/m2 (mean +/- s.e.). Muscle biopsies from m. vastus lateralis and m. deltoideus were obtained before and after seven weeks of intensive insulin treatment, and mitochondrial respiration was measured using high-resolution respirometry. State 3 respiration...

  8. Investigation of nitrosactive compounds influence on polarization of the mitochondrial inner membrane in the rat uterus myocytes using potential sensitive fluorescent probe DіOC(6(3

    Directory of Open Access Journals (Sweden)

    Yu. V. Danylovych

    2014-02-01

    Full Text Available The effect of nitrosactive compounds (sodium nitroprusside and sodium nitrite on the polarization level of the uterus myocytes inner mitochondrial membrane using the confocal laser microscopy and fluorescent probe potentialsensitive DiOC6(3 (3,3′-dihexyloxacarbocyanine was ivestigated. Colocalisation of mitochondrial membranes specific fluorescent probes (MitoTracker Orange CM H2TMRos­, 10 – nonyl acridine orange and DiOC6(3 was demon­strated. It was shown that sodium nitroprusside at 0.1 mM concentration caused a moderate decrease in mitochondrial transmembrane potential. That observation was confirmed by flow cytometry. Action efficiency of sodium nitrite in a similar concentration was significantly lower than that of sodium nitroprusside. It is shown that it was sodium nitroprusside which caused a slight swelling of the mitochondria. A possible protecting role of nitric oxi­de as to mitochondria was discussed.

  9. Cytosolic peroxidases protect the lysosome of bloodstream African trypanosomes from iron-mediated membrane damage.

    Directory of Open Access Journals (Sweden)

    Corinna Hiller

    2014-04-01

    Full Text Available African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I-II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I-II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective

  10. Anaerobic respiration sustains mitochondrial membrane potential in a prolyl hydroxylase pathway-activated cancer cell line in a hypoxic microenvironment.

    Science.gov (United States)

    Takahashi, Eiji; Sato, Michihiko

    2014-02-15

    To elucidate how tumor cells produce energy in oxygen-depleted microenvironments, we studied the possibility of mitochondrial electron transport without oxygen. We produced well-controlled oxygen gradients (ΔO2) in monolayer-cultured cells. We then visualized oxygen levels and mitochondrial membrane potential (ΔΦm) in individual cells by using the red shift of green fluorescent protein (GFP) fluorescence and a cationic fluorescent dye, respectively. In this two-dimensional tissue model, ΔΦm was abolished in cells >500 μm from the oxygen source [the anoxic front (AF)], indicating limitations in diffusional oxygen delivery. This result perfectly matched GFP-determined ΔO2. In cells pretreated with dimethyloxaloylglycine (DMOG), a prolyl hydroxylase domain-containing protein (PHD) inhibitor, the AF was expanded to 1,500-2,000 μm from the source. In these cells, tissue ΔO2 was substantially decreased, indicating that PHD pathway activation suppressed mitochondrial respiration. The expansion of the AF and the reduction of ΔO2 were much more prominent in a cancer cell line (Hep3B) than in the equivalent fibroblast-like cell line (COS-7). Hence, the results indicate that PHD pathway-activated cells can sustain ΔΦm, despite significantly decreased electron flux to complex IV. Complex II inhibition abolished the effect of DMOG in expanding the AF, although tissue ΔO2 remained shallow. Separate experiments demonstrated that complex II plays a substantial role in sustaining ΔΦm in DMOG-pretreated Hep3B cells with complex III inhibition. From these results, we conclude that PHD pathway activation can sustain ΔΦm in an otherwise anoxic microenvironment by decreasing tissue ΔO2 while activating oxygen-independent electron transport in mitochondria.

  11. Isolation and characterization of a Ca/sup 2 +/ carrier candidate from calf heart inner mitochondrial membrane

    Energy Technology Data Exchange (ETDEWEB)

    Jeng, A.Y.

    1979-01-01

    A protein was isolated from calf heart inner mitochondrial membrane with the aid of an electron paramagnetic resonance assay based on the relative binding properties of Ca/sup 2 +/, Mn/sup 2 +/, and Mg/sup 2 +/ to the protein. Partial delipidation of the protein was performed by using either the organic solvent extraction procedure or the silicic acid column chromatography. Control experiments indicated that the Ca/sup 2 +/ transport properties of the isolated protein were not due to the contaminating phospholipids. A complete delipidation procedure was developd by using Sephadex LH-20 column chromatography. Further characterization of the physical and chemical properties of the delipidated protein showed that delipidated protein becomes more hydrophobic in the presence of Ca/sup 2 +/ and alkaline pH in the organic solvent extraction experiments. Two possible models of calciphorin-mediated Ca/sup 2 +/ transport in mitochondria are proposed. (PCS)

  12. Membrane trafficking and mitochondrial abnormalities precede subunit c deposition in a cerebellar cell model of juvenile neuronal ceroid lipofuscinosis

    Directory of Open Access Journals (Sweden)

    Cattaneo Elena

    2004-12-01

    Full Text Available Abstract Background JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a ~1 kb CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Δex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology. Results To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbCln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Δex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced. Conclusions These findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival.

  13. Functional and dynamic state of inner mitochondrial membrane of sarcoma 37 in mice under administration of sodium dichloroacetate

    Directory of Open Access Journals (Sweden)

    S. V. Khyzhnyak

    2014-12-01

    Full Text Available The activity of enzymes of the respiratory chain and structural-dynamic properties of the inner mitochondrial membrane (IMM of sarcoma 37 (S37 in mice under sodium dichloroacetate (SDA administration in a daily dose of 86 mg/kg of body weight starting from the 2nd day after tumor transplantation were investigated. The dynamic and structural state of the IMM components was determined using the fluorescent probes. With S37 growth the intensification of glycolytic metabolism occurred on the background of suppressed functional capacity of mitochondrial respiratory chain enzymes. The changes of conformational properties of protein molecules and the increase of IMM lipid phase microviscosity were shown. The administration of SDA promotes the decrease of lactate content and the increase of pyruvate dehydrogenase activity in S37. This was accompanied by further suppression of the functional activity of the respiratory chain complexes and Н+-АТРase coupled with conformational modification of protein molecules and changes of the structural orderliness of the IMM lipid phase, possibly due to intensification of reactive oxygen species generation.

  14. Continuous renal replacement therapy (CRRT) attenuates myocardial inflammation and mitochondrial injury induced by venovenous extracorporeal membrane oxygenation (VV ECMO) in a healthy piglet model.

    Science.gov (United States)

    Shen, Juanhong; Yu, Wenkui; Chen, Qiyi; Shi, Jialiang; Hu, Yimin; Zhang, Juanjuan; Gao, Tao; Xi, Fengchan; He, Changsheng; Gong, Jianfeng; Li, Ning; Li, Jieshou

    2013-10-01

    In this study, we investigated the myocardial inflammation and mitochondrial function during venovenous extracorporeal membrane oxygenation (VV ECMO) and further evaluated the effects of continuous renal replacement therapy (CRRT) on them. Eighteen piglets were assigned to the control group, ECMO group, and ECMO+CRRT group. Myocardial inflammation was assessed by the activity of myeloperoxidase (MPO), myocardial concentrations, and mRNA expression of TNF-α, IL-1β, and IL-6; mitochondrial function was assessed by activities of mitochondrial complexes I-V. VV ECMO elicited a general activation of serum and myocardial inflammation and significantly decreased the activities of mitochondrial complexes I and IV. After being combined with CRRT, serum and myocardial concentrations of IL-1β and IL-6, myocardial mRNA expression of IL-6, and the activity of MPO were decreased significantly; the activities of mitochondrial complexes were increased. We conclude that myocardial inflammation was activated during ECMO therapy, inducing mitochondrial injury; moreover, CRRT reduced myocardial inflammation and partially ameliorated mitochondrial function.

  15. Effect of narcotics on membrane-bound mitochondrial processes in fish

    DEFF Research Database (Denmark)

    Vergauwen, Lucia; Nørgaard Schmidt, Stine; Michiels, Ellen

    Around 70% of industrial chemicals are hydrophobic compounds which are assumed to elicit toxicity through narcosis by accumulating in membranes and disrupting membrane integrity and function. Although narcosis has been recognized as an important toxicity mechanism for decades, ecotoxicological...... research has been mostly limited to the development of quantitative structure activity relations (QSARs) to predict toxicity, resulting in insufficient understanding of the exact mechanisms involved. In this study we investigate specific aspects of the mechanism of narcosis in fish using both alternative...

  16. Utilization of fluorescent probe association for simultaneous assessment of plasmatic, acrosomal, and mitochondrial membranes of rooster spermatozoa

    Directory of Open Access Journals (Sweden)

    ECC Celeghini

    2007-09-01

    Full Text Available This experiment was designed with the objective of developing a simple, practical, and high repeatability technique for the simultaneous evaluation of the integrity of the plasmatic and acrosomal membranes, as well as funcional mitochondria of domestic fowl spermatozoa using an association of fluorescent probes. Four ejaculates (motility > 80% and abnormal morphology < 10% from each of six Ross male broiler breeder (n=24 were diluted in TALP sperm medium (25x10(6 spermatozoa/mL and split into two aliquots, and one of these aliquots was flash frozen in liquid nitrogen and thawed to damage all cellular membranes. Three treatments were prepared from these aliquots, with the following ratios of Fresh semen:Flash frozen semen: 100:0 (T100, 50:50 (T50, and 0:100 (T0. A 150-µL aliquot of diluted semen was placed in a microcentrifuge tube with the addition of 2-µL PI, 2-µL MITO, and 50-µL FITC-PSA, and incubated at 38.5º C/8 min in the dark. An 8-µL sample was placed on a slide, coverslipped, and examined by epifluorescence microscopy. Each sample had 200 cells counted and classified based on the fluorescence emitted by each probe. By regression analysis, plasma membrane integrity, as detected by PI, was determined as: v=4.17+0.82X (R²=0.95. Acrosome integrity, as detected by FITC-PSA, generated the equation: v=4.19+0.84X (R²=0.96. Functional mitochondria was estimated by the equation v=3.20+0.83X (R²=0.96. This is an efficient technique to simultaneously evaluate plasmatic, acrosomal, and mitochondrial membranes in fowl sperm. It is suggested that its application in flow cytometry systems allows this methodology to be applied in large scale.

  17. Atypical composition and structure of the mitochondrial dimeric ATP synthase from Euglena gracilis.

    Science.gov (United States)

    Yadav, K N Sathish; Miranda-Astudillo, Héctor V; Colina-Tenorio, Lilia; Bouillenne, Fabrice; Degand, Hervé; Morsomme, Pierre; González-Halphen, Diego; Boekema, Egbert J; Cardol, Pierre

    2017-04-01

    Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Here we studied the mitochondrial F1FO-ATP synthase (or Complex V) from the photosynthetic eukaryote E. gracilis in detail. The enzyme was purified by a two-step chromatographic procedure and its subunit composition was resolved by a three-dimensional gel electrophoresis (BN/SDS/SDS). Twenty-two different subunits were identified by mass-spectrometry analyses among which the canonical α, β, γ, δ, ε, and OSCP subunits, and at least seven subunits previously found in Trypanosoma. The ADP/ATP carrier was also associated to the ATP synthase into a dimeric ATP synthasome. Single-particle analysis by transmission electron microscopy of the dimeric ATP synthase indicated that the structures of both the catalytic and central rotor parts are conserved while other structural features are original. These new features include a large membrane-spanning region joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring, the latter having not been reported for any mitochondrial F-ATPase. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. (Berenil(B)) in the Treatment of Experimental Trypanosoma brucei ...

    African Journals Online (AJOL)

    Dr Olaleye

    Animal welfare was observed in accordance with. International guidelines for the use of laboratory animals for biomedical research (EC, 1996). Trypanosomes: Trypanosoma brucei brucei (Federe strain) obtained from NITR, Vom, was used and the parasites were maintained by serial passages in rats. One millilitre of.

  19. The Effect of Garlic Extracts on Experimental Trypanosoma brucei ...

    African Journals Online (AJOL)

    The anti-trypanosomal effect of aqueous and methanolic extracts of garlic were studied in Trypanosoma brucei brucei infected rabbits. With the establishment of infection, parasitaemia, anaemia, leucopenia, neutropenia and lymphocytosis developed. There was decrease in total serum protein, albumin and increase in ...

  20. Pathogenicity of Trypanosoma brucei in African giant rats ...

    African Journals Online (AJOL)

    Pathogenicity of Trypanosoma brucei in African giant rats ( Cricetomys gambianus , Water House) ... The course of trypanosomosis was investigated over a period of two weeks in six African giant rats (Cricetomys gambianus) experimentally infected with Trypanosoma brucei. Six other rats served as uninfected control.

  1. Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons.

    Science.gov (United States)

    Ludtmann, Marthe H R; Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Preza, Elisavet; Carro, Eva; Houlden, Henry; Gandhi, Sonia; Wray, Selina; Abramov, Andrey Y

    2017-05-26

    Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Elevated hydrostatic pressures induce apoptosis and oxidative stress through mitochondrial membrane depolarization in PC12 neuronal cells: A cell culture model of glaucoma.

    Science.gov (United States)

    Tök, Levent; Nazıroğlu, Mustafa; Uğuz, Abdülhadi Cihangir; Tök, Ozlem

    2014-10-01

    Despite the importance of oxidative stress and apoptosis through mitochondrial depolarization in neurodegenerative diseases, their roles in etiology of glaucoma are poorly understood. We aimed to investigate whether oxidative stress and apoptosis formation are altered in rat pheochromocytoma-derived cell line-12 (PC12) neuronal cell cultures exposed to elevated different hydrostatic pressures as a cell culture model of glaucoma. Cultured PC12 cells were subjected to 0, 15 and 70 mmHg hydrostatic pressure for 1 and 24 h. Then, the following values were analyzed: (a) cell viability; (b) lipid peroxidation and intracellular reactive oxygen species production; (c) mitochondrial membrane depolarization; (d) cell apoptosis; (e) caspase-3 and caspase-9 activities; (f) reduced glutathione (GSH) and glutathione peroxidase (GSH-Px). The hydrostatic pressures (15 and 70 mmHg) increased oxidative cell damage through a decrease of GSH and GSH-Px values, and increasing mitochondrial membrane potential. Additionally, 70 mmHg hydrostatic pressure for 24 h indicated highest apoptotic effects, as demonstrated by plate reader analyses of apoptosis, caspase-3 and -9 values. The present data indicated oxidative stress, apoptosis and mitochondrial changes in PC12 cell line during different hydrostatic pressure as a cell culture model of glaucoma. This findings support the view that mitochondrial oxidative injury contributes early to glaucomatous optic neuropathy.

  3. A Mitochondrial Membrane Exopolyphosphatase Is Modulated by, and Plays a Role in, the Energy Metabolism of Hard Tick Rhipicephalus (Boophilus microplus Embryos

    Directory of Open Access Journals (Sweden)

    Carlos Logullo

    2011-06-01

    Full Text Available The physiological roles of polyphosphates (polyP recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (Pi and this activation was much greater using polyP3 than polyP15. After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP3 was 10 times stronger than that for polyP15. Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 µg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5’-triphosphate synthesis in hard tick embryos.

  4. Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

    Directory of Open Access Journals (Sweden)

    S Andrea Moreno

    2015-06-01

    Full Text Available Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF of T. b. brucei: (i fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme in glycosomes, (ii enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes and a GAPDH isoenzyme in the cytosol, (iii malate dehydrogenase in cytosol and (iv glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

  5. Apoptotic Bax at Oxidatively Stressed Mitochondrial Membranes: Lipid Dynamics and Permeabilization

    Czech Academy of Sciences Publication Activity Database

    Dilgendein, A. P.; Pokorná, Šárka; Lidman, M.; Sparrman, T.; Šachl, Radek; Hof, Martin; Gröbner, G.

    2017-01-01

    Roč. 112, č. 10 (2017), s. 2147-2158 ISSN 0006-3495 R&D Projects: GA ČR(CZ) GBP208/12/G016 Institutional support: RVO:61388955 Keywords : LOW-FREQUENCY MOTION * OXIDIZED PHOSPHOLIPIDS * BILAYER-MEMBRANES Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 3.656, year: 2016

  6. In vitro and in vivo activation of mitochondrial membrane permeability transition pore using triiodothyronine

    Czech Academy of Sciences Publication Activity Database

    Endlicher, R.; Drahota, Zdeněk; Červinková, Z.

    2016-01-01

    Roč. 65, č. 2 (2016), s. 321-331 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GB14-36804G Institutional support: RVO:67985823 Keywords : rat liver mitochondria * membrane permeability transition pore * thyroid hormones Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.461, year: 2016

  7. Elevated mRNA-levels of distinct mitochondrial and plasma membrane Ca2+ transporters in individual hypoglossal motor neurons of endstage SOD1 transgenic mice.

    Directory of Open Access Journals (Sweden)

    Tobias eMühling

    2014-11-01

    Full Text Available Disturbances in Ca2+ homeostasis and mitochondrial dysfunction have emerged as major pathogenic features in familial and sporadic forms of Amyotrophic Lateral Sclerosis (ALS, a fatal degenerative motor neuron disease. However, the distinct molecular ALS-pathology remains unclear. Recently, an activity-dependent Ca2+ homeostasis deficit, selectively in highly vulnerable cholinergic motor neurons in the hypoglossal nucleus (hMNs from a common ALS mouse model, endstage superoxide dismutase SOD1G93A transgenic mice, was described. This functional deficit was defined by a reduced hMN mitochondrial Ca2+ uptake capacity and elevated Ca2+ extrusion across the plasma membrane. To address the underlying molecular mechanisms, here we quantified mRNA-levels of respective potential mitochondrial and plasma membrane Ca2+ transporters in individual, choline-acetyltransferase (ChAT positive hMNs from wildtype (WT and endstage SOD1G93A mice, by combining UV laser microdissection with RT-qPCR techniques, and specific data normalization. As ChAT cDNA levels as well as cDNA and genomic DNA levels of the mitochondrially encoded NADH dehydrogenase ND1 were not different between hMNs from WT and endstage SOD1G93A mice, these genes were used to normalize hMN-specific mRNA-levels of plasma membrane and mitochondrial Ca2+ transporters, respectively. We detected about 2-fold higher levels of the mitochondrial Ca2+ transporters MCU/MICU1, Letm1 and UCP2 in remaining hMNs from endstage SOD1G93A mice. These higher expression-levels of mitochondrial Ca2+ transporters in individual hMNs were not associated with a respective increase in number of mitochondrial genomes, as evident from hMN specific ND1 DNA quantification. Normalized mRNA-levels for the plasma membrane Na2+/Ca2+exchanger NCX1 was also about 2-fold higher in hMNs from SOD1G93A mice. Thus, pharmacological stimulation of Ca2+ transporters in highly vulnerable hMNs might offer a novel neuroprotective strategy for ALS.

  8. Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function.

    Science.gov (United States)

    Johnston, S D; Satake, N; Zee, Y; López-Fernández, C; Holt, W V; Gosálvez, J

    2012-06-01

    This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (r=0.573; Pkoala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo- and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (r=0.878; Pkoala SDF, the mechanisms resulting in relaxed chromatin require further study. A lack of correlation between the percentage of sperm with relaxed chromatin and SDF suggests that the timing of these pathologies are asynchronous. We propose an integrative model of cryo-induced osmotic injury that involves a combination of structural damage (rupture of membrane) and oxidative stress that first leads to the reduction of MMP and the relaxation of chromatin, which is then ultimately followed by an increase in DNA fragmentation.

  9. Quantum squeezed light for probing mitochondrial membranes and study of neuroprotectants.

    Energy Technology Data Exchange (ETDEWEB)

    Gourley, Paul Lee; Copeland, Robert Guild; McDonald, Anthony Eugene; Hendricks, Judy K.; Naviaux, Robert K. (University of California, San Diego, CA)

    2005-01-01

    We report a new nanolaser technique for measuring characteristics of human mitochondria. Because mitochondria are so small, it has been difficult to study large populations using standard light microscope or flow cytometry techniques. We recently discovered a nano-optical transduction method for high-speed analysis of submicron organelles that is well suited to mitochondrial studies. This ultrasensitive detection technique uses nano-squeezing of light into photon modes imposed by the ultrasmall organelle dimensions in a semiconductor biocavity laser. In this paper, we use the method to study the lasing spectra of normal and diseased mitochondria. We find that the diseased mitochondria exhibit larger physical diameter and standard deviation. This morphological differences are also revealed in the lasing spectra. The diseased specimens have a larger spectral linewidth than the normal, and have more variability in their statistical distributions.

  10. Mitochondrial membranes with mono- and divalent salt: changes induced by salt ions on structure and dynamics

    DEFF Research Database (Denmark)

    Pöyry, Sanja; Róg, Tomasz; Karttunen, Mikko

    2009-01-01

    We employ atomistic simulations to consider how mono- (NaCl) and divalent (CaCl(2)) salt affects properties of inner and outer membranes of mitochondria. We find that the influence of salt on structural properties is rather minute, only weakly affecting lipid packing, conformational ordering......, and membrane electrostatic potential. The changes induced by salt are more prominent in dynamical properties related to ion binding and formation of ion-lipid complexes and lipid aggregates, as rotational diffusion of lipids is slowed down by ions, especially in the case of CaCl(2). In the same spirit, lateral...... diffusion of lipids is slowed down rather considerably for increasing concentration of CaCl(2). Both findings for dynamic properties can be traced to the binding of ions with lipid head groups and the related changes in interaction patterns in the headgroup region, where the binding of Na(+) and Ca(2+) ions...

  11. Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes

    Science.gov (United States)

    Gan, Zhuohui; Audi, Said H.; Bongard, Robert D.; Gauthier, Kathryn M.

    2011-01-01

    Our goal was to quantify mitochondrial and plasma potential (Δψm and Δψp) based on the disposition of rhodamine 123 (R123) or tetramethylrhodamine ethyl ester (TMRE) in the medium surrounding pulmonary endothelial cells. Dyes were added to the medium, and their concentrations in extracellular medium ([Re]) were measured over time. R123 [Re] fell from 10 nM to 6.6 ± 0.1 (SE) nM over 120 min. TMRE [Re] fell from 20 nM to a steady state of 4.9 ± 0.4 nM after ∼30 min. Protonophore or high K+ concentration ([K+]), used to manipulate contributions of membrane potentials, attenuated decreases in [Re], and P-glycoprotein (Pgp) inhibition had the opposite effect, demonstrating the qualitative impact of these processes on [Re]. A kinetic model incorporating a modified Goldman-Hodgkin-Katz model was fit to [Re] vs. time data for R123 and TMRE, respectively, under various conditions to obtain (means ± 95% confidence intervals) Δψm (−130 ± 7 and −133 ± 4 mV), Δψp (−36 ± 4 and −49 ± 4 mV), and a Pgp activity parameter (KPgp, 25 ± 5 and 51 ± 11 μl/min). The higher membrane permeability of TMRE also allowed application of steady-state analysis to obtain Δψm (−124 ± 6 mV). The consistency of kinetic parameter values obtained from R123 and TMRE data demonstrates the utility of this experimental and theoretical approach for quantifying intact cell Δψm and Δψp. Finally, steady-state analysis revealed that although room air- and hyperoxia-exposed (95% O2 for 48 h) cells have equivalent resting Δψm, hyperoxic cell Δψm was more sensitive to depolarization with protonophore, consistent with previous observations of pulmonary endothelial hyperoxia-induced mitochondrial dysfunction. PMID:21239539

  12. Shifting the paradigm: the putative mitochondrial protein ABCB6 resides in the lysosomes of cells and in the plasma membrane of erythrocytes.

    Directory of Open Access Journals (Sweden)

    Katalin Kiss

    Full Text Available ABCB6, a member of the adenosine triphosphate-binding cassette (ABC transporter family, has been proposed to be responsible for the mitochondrial uptake of porphyrins. Here we show that ABCB6 is a glycoprotein present in the membrane of mature erythrocytes and in exosomes released from reticulocytes during the final steps of erythroid maturation. Consistent with its presence in exosomes, endogenous ABCB6 is localized to the endo/lysosomal compartment, and is absent from the mitochondria of cells. Knock-down studies demonstrate that ABCB6 function is not required for de novo heme biosynthesis in differentiating K562 cells, excluding this ABC transporter as a key regulator of porphyrin synthesis. We confirm the mitochondrial localization of ABCB7, ABCB8 and ABCB10, suggesting that only three ABC transporters should be classified as mitochondrial proteins. Taken together, our results challenge the current paradigm linking the expression and function of ABCB6 to mitochondria.

  13. Influence of mitochondrial membrane potential of spermatozoa on in vitro fertilisation outcome.

    Science.gov (United States)

    Marchetti, P; Ballot, C; Jouy, N; Thomas, P; Marchetti, C

    2012-04-01

    To determine whether the outcome of in vitro fertilisation (IVF) is influenced by the percentage of spermatozoa with functional mitochondria, a total of 91 random couples undergoing IVF were included. Mitochondrial function was determined by flow cytometry and expressed as percentage of spermatozoa. Conventional sperm parameters were studied by light microscopy. Reproductive outcome parameters were fertilisation rate, embryo quality and clinical pregnancy. It was found that the fertilisation rate was correlated with the percentage of spermatozoa (r = 0.24, P = 0.01) as well as with the percentage of highly motile spermatozoa. However, we did not find any relationship between the percentage of spermatozoa and embryo quality. Nevertheless, no patient who exhibited less than 64% of spermatozoa achieved pregnancy. It is concluded that determination of Δψ(m) provides accurate information to guide physicians to identify male patients for whom IVF will be unlikely to result in pregnancy. Therefore, we suggest that the percentage of spermatozoa may contribute to identify the most appropriate treatment for an individual patient. © 2011 Blackwell Verlag GmbH.

  14. Inhibition of the iron-catalysed formation of hydroxyl radicals by nitrosouracil derivatives: protection of mitochondrial membranes against lipid peroxidation.

    Science.gov (United States)

    Rabion, A; Verlhac, J B; Fraisse, L; Roche, B; Seris, J L

    1993-01-01

    A new series of metal ligands containing the 1,3-dimethyl-6-amino-5- nitrosouracil moiety has been synthesized and they have been studied as potential inhibitors of iron-dependent lipid peroxidation. For this purpose, these new derivatives have been tested in the Fenton induced deoxyribose degradation assay, which allows a quantitative measurement of their inhibitory effect towards hydroxyl radical generation. When iron(II) is complexed by these ligands, a strong inhibition of deoxyribose degradation is observed, especially in the case of tris-[2-(1,3-dimethyl-5-nitrosouracil-6-yl)aminoethyl] amine (5). This inhibitory effect is clearly related to a specific complexation of iron(II) and is not due to the direct scavenging of hydroxyl radical by the ligand. Inhibition of the iron mediated Fenton reaction presumably results from inactivation of the reactivity of the metal center towards hydrogen peroxide. These derivatives, as well as long alkyl chain substituted nitrosouracils were evaluated in the protection of biological membranes against lipid peroxidation (induced by iron(II)/dihydroxyfumaric acid and determined with the 2-thiobarbituric acid test). Ligand 5 inhibited lipid peroxidation at a rate similar to Desferal (desferrioxamine B) and slightly higher than bathophenanthroline sulphonate (BPS), which are respectively good iron(III) and iron(II) chelators. When covalently bound with a long alkyl chain, the increase of lipophilic character of the ligand allows its location near the mitochondrial membrane, where lipid peroxidation occurs. Lower concentrations (IC50 = 4 microM) are then necessary to inhibit lipid peroxidation. This IC50 concentration should be compared to those obtained for Trolox (IC50 = 3 microM) or the 21-aminosteroid U74500A (IC50 = 1 microM) described previously.

  15. Crystallization of Mitochondrial Respiratory Complex II fromChicken Heart: A Membrane-Protein Complex Diffracting to 2.0Angstrom

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Li-shar; Borders, Toni M.; Shen, John T.; Wang, Chung-Jen; Berry, Edward A.

    2004-12-17

    Procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory Complex II. The crystals have the potential to diffract to at least 2.0 Angstrom with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites.

  16. Aging Yeast Cells Undergo a Sharp Entry into Senescence Unrelated to the Loss of Mitochondrial Membrane Potential

    Directory of Open Access Journals (Sweden)

    Steffen Fehrmann

    2013-12-01

    Full Text Available In budding yeast, a mother cell can produce a finite number of daughter cells before it stops dividing and dies. Such entry into senescence is thought to result from a progressive decline in physiological function, including a loss of mitochondrial membrane potential (ΔΨ. Here, we developed a microfluidic device to monitor the dynamics of cell division and ΔΨ in real time at single-cell resolution. We show that cells do not enter senescence gradually but rather undergo an abrupt transition to a slowly dividing state. Moreover, we demonstrate that the decline in ΔΨ, which is observed only in a fraction of cells, is not responsible for entry into senescence. Rather, the loss of ΔΨ is an age-independent and heritable process that leads to clonal senescence and is therefore incompatible with daughter cell rejuvenation. These results emphasize the importance of quantitative single-cell measurements to decipher the causes of cellular aging.

  17. Low intensity ultrasound induces apoptosis via MPT channel on mitochondrial membrane: Target for regulating cancer therapy or not?

    Science.gov (United States)

    Feng, Yi; Wan, Mingxi

    2017-03-01

    To discuss how the mitochondrion is involved in low intensity ultrasound induced apoptosis, HepG2 cells were irradiated by low intensity focused ultrasound (ISPTA = 3W/cm2, 1 min) and then cultured from 3-12 h post irradiation in the study. The morphological alteration was examined by light and fluorescent microscopy respectively. Cell viability and apoptosis were examined by trypan blue staining and flow cytometry with double staining of FITC-labelled Annexin-V/PI. Key proteins responded to irradiation were screened out by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and shotgun proteomic methods with Agilent 1100 HPLC-Chip-MS technology. Representative apoptotic morphological characteristics and increased percentage of apoptotic cells were achieved. Six important proteins (4 up-regulated and 2 down-regulated) were selected and analyzed. It revealed low intensity focused ultrasound could induce apoptosis in HepG2 cells and the US-induced apoptosis was mitochondria-dependent and caspases-dependent. Moreover, mitochondrial membrane permeability transition (MPT) is related to ultrasound induced apoptosis, but VDAC may be not the main MPT channel. Understanding it could help to assist the cancer therapy by regulating the MPT as the target.

  18. Achiral Mannich-Base Curcumin Analogs Induce Unfolded Protein Response and Mitochondrial Membrane Depolarization in PANC-1 Cells

    Directory of Open Access Journals (Sweden)

    Gábor J. Szebeni

    2017-10-01

    Full Text Available Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549, hepatocellular carcinoma (HepG2 and pancreatic cancer cell line (PANC-1. Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G0/G1 cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER and the up-regulation of ER stress-related unfolded protein response (UPR genes: HSPA5, ATF4, XBP1, and DDIT3. The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.

  19. ATG24 Represses Autophagy and Differentiation and Is Essential for Homeostasy of the Flagellar Pocket in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Ana Brennand

    Full Text Available We have previously identified homologs for nearly half of the approximately 30 known yeast Atg's in the genome database of the human sleeping sickness parasite Trypanosoma brucei. So far, only a few of these homologs have their role in autophagy experimentally confirmed. Among the candidates was the ortholog of Atg24 that is involved in pexophagy in yeast. In T. brucei, the peroxisome-like organelles named glycosomes harbor core metabolic processes, especially glycolysis. In the autotrophic yeast, autophagy is essential for adaptation to different nutritional environments by participating in the renewal of the peroxisome population. We hypothesized that autophagic turnover of the parasite's glycosomes plays a role in differentiation during its life cycle, which demands adaptation to different host environments and associated dramatic changes in nutritional conditions. We therefore characterized T. brucei ATG24, the T. brucei ortholog of yeast Atg24 and mammalian SNX4, and found it to have a regulatory role in autophagy and differentiation as well as endocytic trafficking. ATG24 partially localized on endocytic membranes where it was recruited via PI3-kinase III/VPS34. ATG24 silencing severely impaired receptor-mediated endocytosis of transferrin, but not adsorptive uptake of a lectin, and caused a major enlargement of the flagellar pocket. ATG24 silencing approximately doubled the number of autophagosomes, suggesting a role in repressing autophagy, and strongly accelerated differentiation, in accordance with a role of autophagy in parasite differentiation. Overexpression of the two isoforms of T. brucei ATG8 fused to GFP slowed down differentiation, possibly by a dominant-negative effect. This was overcome by ATG24 depletion, further supporting its regulatory role.

  20. Blockade of Electron Transport at the Onset of Reperfusion Decreases Cardiac Injury in Aged Hearts by Protecting the Inner Mitochondrial Membrane

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    Qun Chen

    2012-01-01

    Full Text Available Myocardial injury is increased in the aged heart following ischemia-reperfusion (ISC-REP compared to adult hearts. Intervention at REP with ischemic postconditioning decreases injury in the adult heart by attenuating mitochondrial driven cell injury. Unfortunately, postconditioning is ineffective in aged hearts. Blockade of electron transport at the onset of REP with the reversible inhibitor amobarbital (AMO decreases injury in adult hearts. We tested if AMO treatment at REP protects the aged heart via preservation of mitochondrial integrity. Buffer-perfused elderly Fischer 344 24 mo. rat hearts underwent 25 min global ISC and 30 min REP. AMO (2.5 mM or vehicle was given for 3 min at the onset of REP. Subsarcolemmal (SSM and interfibrillar (IFM mitochondria were isolated after REP. Oxidative phosphorylation (OXPHOS and mitochondrial inner membrane potential were measured. AMO treatment at REP decreased cardiac injury. Compared to untreated ISC-REP, AMO improved inner membrane potential in SSM and IFM during REP, indicating preserved inner membrane integrity. Thus, direct pharmacologic modulation of electron transport at REP protects mitochondria and decreases cardiac injury in the aged heart, even when signaling-induced pathways of postconditioning that are upstream of mitochondria are ineffective.

  1. CHARACTERIZATION AND ANTIPARASITIC ACTIVITY OF BENZOPHENONE THIOSEMICARBAZONES ON Trypanosoma brucei brucei

    Directory of Open Access Journals (Sweden)

    Georges C. Accrombessi

    2011-02-01

    Full Text Available The structure of four synthesized thiosemicarbazones, substituted or not, of benzophenone has been confirmed by spectrometrical analysis IR, NMR 1H and 13C. Their anti-trypanosomal activities were evaluated on Trypanosoma brucei brucei. Among these compounds, benzophenone 4 phenyl-3-thiosemicarbazone 4 has the highest activity with the half-inhibitory concentration (IC50 = 8.48 micromolar (µM. Benzophenone 4-methyl-3-thiosemicarbazone 3 and benzophenone thiosemicarbazone 1 showed moderate anti-trypanosomal activity with IC50 values equal to 23.27 µM and 67.17 µM respectively. Benzophenone 2 methyl-3-thiosemicarbazone 2 showed no activity up to IC50 = 371.74 µM.

  2. Protective effect of magnesium and potassium ions on the permeability of the external mitochondrial membrane.

    Science.gov (United States)

    Gorgoglione, Vincenza; Laraspata, Daniela; La Piana, Gianluigi; Marzulli, Domenico; Lofrumento, Nicola Elio

    2007-05-01

    The data reported are fully consistent with the well-known observation that exogenous cytochrome c (cyto-c) molecules do not permeate through the outer membrane of mitochondria (MOM) incubated in isotonic medium (250 mM sucrose). Cyto-c is unable to accept electrons from the sulfite/cyto-c oxido-reductase (Sox) present in the intermembrane space, unless mitochondria are solubilized. Mitochondria incubated in a very high hypotonic medium (25 mM sucrose), in contrast to any expectation, continue to be not permeable to added cyto-c even if Sox and adenylate kinase are released into the medium. The succinate/exogenous cyto-c reductase activity, very low in isotonic medium, is greatly increased decreasing the osmolarity of the medium but in both cases remains insensitive to proteolysis by added trypsin. In hypotonic medium, magnesium and potassium ions have a protective effect on the release of enzymes and on the reactivity of cyto-c as electron acceptor from both sulfite and succinate; results which are consistent with the view that MOM preserves its identity and remains not permeable to exogenous cyto-c. This report strengthens the proposal, supported by previously published data that in isotonic medium the exogenous NADH/cyto-c electron transport system is catalyzed by intact mitochondria, not permeable to added cyto-c.

  3. Ca2+ and Mg2+-enhanced reduction of arsenazo III to its anion free radical metabolite and generation of superoxide anion by an outer mitochondrial membrane azoreductase.

    Science.gov (United States)

    Moreno, S N; Mason, R P; Docampo, R

    1984-12-10

    At the concentrations usually employed as a Ca2+ indicator, arsenazo III underwent a one-electron reduction by rat liver mitochondria to produce an azo anion radical as demonstrated by electron-spin resonance spectroscopy. Either NADH or NADPH could serve as a source of reducing equivalents for the production of this free radical by intact rat liver mitochondria. Under aerobic conditions, addition of arsenazo III to rat liver mitochondria produced an increase in electron flow from NAD(P)H to molecular oxygen, generating superoxide anion. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H azoreductase reaction unless the mitochondria were solubilized by detergent or anaerobiosis. In addition, NAD(P)H azoreductase activity was higher in the crude outer mitochondrial membrane fraction than in mitoplasts and intact mitochondria. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated cyanide-insensitive oxygen consumption were enhanced by calcium and magnesium, suggesting that, in addition to an enhanced azo anion radical-stabilization by complexation with the metal ions, enhanced reduction of arsenazo III also occurred. Accordingly, addition of cations to crude outer mitochondrial membrane preparations increased arsenazo III-stimulated cyanide-insensitive O2 consumption, H2O2 formation, and NAD(P)H oxidation. Antipyrylazo III was much less effective than arsenazo III in increasing superoxide anion formation by rat liver mitochondria and gave a much weaker electron spin resonance spectrum of an azo anion radical. These results provide direct evidence of an azoreductase activity associated with the outer mitochondrial membrane and of a stimulation of arsenazo III reduction by cations.

  4. Mechanisms Mediating Antigenic Variation in Trypanosoma brucei

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    Rudenko Gloria

    1999-01-01

    Full Text Available Antigenic variation in Trypanosoma brucei is a highly sophisticated survival strategy involving switching between the transcription of one of an estimated thousand variant surface glycoprotein (VSG genes. Switching involves either transcriptional control, resulting in switching between different VSG expression sites; or DNA rearrangement events slotting previously inactive VSG genes into an active VSG expression site. In recent years, considerable progress has been made in techniques allowing us to genetically modify infective bloodstream form trypanosomes. This is allowing us to reengineer VSG expression sites, and look at the effect on the mechanisms subsequently used for antigenic variation. We can now begin a dissection of a highly complicated survival strategy mediated by many different mechanisms operating simultaneously.

  5. KREX2 is not essential for either procyclic or bloodstream form Trypanosoma brucei.

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    Jason Carnes

    Full Text Available BACKGROUND: Most mitochondrial mRNAs in Trypanosoma brucei require RNA editing for maturation and translation. The edited RNAs primarily encode proteins of the oxidative phosphorylation system. These parasites undergo extensive changes in energy metabolism between the insect and bloodstream stages which are mirrored by alterations in RNA editing. Two U-specific exonucleases, KREX1 and KREX2, are both present in protein complexes (editosomes that catalyze RNA editing but the relative roles of each protein are not known. METHODOLOGY/PRINCIPAL FINDINGS: The requirement for KREX2 for RNA editing in vivo was assessed in both procyclic (insect and bloodstream form parasites by methods that use homologous recombination for gene elimination. These studies resulted in null mutant cells in which both alleles were eliminated. The viability of these cells demonstrates that KREX2 is not essential in either life cycle stage, despite certain defects in RNA editing in vivo. Furthermore, editosomes isolated from KREX2 null cells require KREX1 for in vitro U-specific exonuclease activity. CONCLUSIONS: KREX2 is a U-specific exonuclease that is dispensable for RNA editing in vivo in T. brucei BFs and PFs. This result suggests that the U deletion activity, which is required for RNA editing, is primarily mediated in vivo by KREX1 which is normally found associated with only one type of editosome. The retention of the KREX2 gene implies a non-essential role or a role that is essential in other life cycle stages or conditions.

  6. The Mitochondrial Amidoxime-reducing Component (mARC1) Is a Novel Signal-anchored Protein of the Outer Mitochondrial Membrane.

    NARCIS (Netherlands)

    Klein, J.M.; Busch, J.D.; Potting, C.M.; Baker, M.J.; Langer, T.; Schwarz, G.

    2012-01-01

    The mitochondrial amidoxime-reducing component (mARC) was recently discovered as the fifth eukaryotic molybdenum cofactor-containing enzyme. The human genome encodes two mARC proteins, mARC1 and mARC2, sharing significant homologies with respect to sequence and function. Whereas mARC2 was identified

  7. Tauro-β-muricholic acid restricts bile acid-induced hepatocellular apoptosis by preserving the mitochondrial membrane potential.

    Science.gov (United States)

    Denk, Gerald Ulrich; Kleiss, Carl Philipp; Wimmer, Ralf; Vennegeerts, Timo; Reiter, Florian Paul; Schulz, Sabine; Zischka, Hans; Rust, Christian

    2012-08-10

    β-Muricholic acid (βMCA) is a trihydroxylated bile acid that constitutes the major bile acid in rat and mouse. βMCA is more hydrophilic than ursodeoxycholic acid and has been evaluated for dissolution of cholesterol gallstones. Since it is unknown if βMCA has beneficial effects on hepatocyte cell death we determined the effect of tauro-βMCA (TβMCA) on apoptosis in vitro. Human Ntcp-transfected HepG2 cells and primary hepatocytes from rat and mouse were incubated with the proapoptotic glycochenodeoxycholic acid (GCDCA) as well as the free fatty acid palmitate in the absence and presence of TβMCA. Apoptosis was quantified using caspase 3/7-assays and after Hoechst 33342 staining. The mitochondrial membrane potential (MMP) was measured fluorometrically using JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazol-carbocyaniniodide). Immunoblotting was performed against the proapoptotic Bcl-2-protein Bax. In Ntcp-HepG2 cells, GCDCA markedly increased apoptosis after 4h. Co-incubation with TβMCA reduced apoptosis to 49% (p<0.01 vs. GCDCA, each; n=6). While GCDCA (100μmol/L) reduced the MMP to 34% after 6h, combination treatment with TβMCA restored the MMP to control levels at all time points (n=4). TβMCA also restored breakdown of the MMP induced by palmitate. GCDCA induced a translocation of Bax from the cytosol to mitochondria that was inhibited by simultaneous treatment with TβMCA in eqimolar concentrations. TβMCA restricts hepatocellular apoptosis induced by low micromolar concentrations of GCDCA or palmitate via inhibition of Bax translocation to mitochondria and preservation of the MMP. Thus, further studies are warranted to evaluate a potential use of TβMCA in ameliorating liver injury in cholestasis. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM.

    Directory of Open Access Journals (Sweden)

    Daniel O Frank

    Full Text Available The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM. In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20 by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

  9. Extracellular Vesicles from Trypanosoma brucei Mediate Virulence Factor Transfer and Cause Host Anemia.

    Science.gov (United States)

    Szempruch, Anthony J; Sykes, Steven E; Kieft, Rudo; Dennison, Lauren; Becker, Allison C; Gartrell, Anzio; Martin, William J; Nakayasu, Ernesto S; Almeida, Igor C; Hajduk, Stephen L; Harrington, John M

    2016-01-14

    Intercellular communication between parasites and with host cells provides mechanisms for parasite development, immune evasion, and disease pathology. Bloodstream African trypanosomes produce membranous nanotubes that originate from the flagellar membrane and disassociate into free extracellular vesicles (EVs). Trypanosome EVs contain several flagellar proteins that contribute to virulence, and Trypanosoma brucei rhodesiense EVs contain the serum resistance-associated protein (SRA) necessary for human infectivity. T. b. rhodesiense EVs transfer SRA to non-human infectious trypanosomes, allowing evasion of human innate immunity. Trypanosome EVs can also fuse with mammalian erythrocytes, resulting in rapid erythrocyte clearance and anemia. These data indicate that trypanosome EVs are organelles mediating non-hereditary virulence factor transfer and causing host erythrocyte remodeling, inducing anemia. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Levetiracetam differentially alters CD95 expression of neuronal cells and the mitochondrial membrane potential of immune and neuronal cells in vitro

    Directory of Open Access Journals (Sweden)

    Susannah K Rogers

    2014-02-01

    Full Text Available Epilepsy is a neurological seizure disorder that affects over 100 million people worldwide. Levetiracetam, either alone, as monotherapy, or as adjunctive treatment, is widely used to control certain types of seizures. Despite its increasing popularity as a relatively safe and effective anti-convulsive treatment option, its mechanism(s of action are poorly understood. Studies have suggested neuronal, glial, and immune mechanisms of action. Understanding the precise mechanisms of action of Levetiracetam would be extremely beneficial in helping to understand the processes involved in seizure generation and epilepsy. Moreover, a full understanding of these mechanisms would help to create more efficacious treatments while minimizing side effects. The current study examined the effects of Levetiracetam on the mitochondrial membrane potential of neuronal and non-neuronal cells, in vitro, in order to determine if Levetiracetam influences metabolic processes in these cell types. In addition, this study sought to address possible immune-mediated mechanisms by determining if Levetiracetam alters the expression of immune receptor-ligand pairs. The results show that Levetiracetam induces expression of CD95 and CD178 on NGF-treated C17.2 neuronal cells. The results also show that Levetiracetam increases mitochondrial membrane potential on C17.2 neuronal cells in the presence of nerve growth factor. In contrast, Levetiracetam decreases the mitochondrial membrane potential of splenocytes and this effect was dependent on intact invariant chain, thus implicating immune cell interactions. These results suggest that both neuronal and non-neuronal anti-epileptic activities of Levetiracetam involve control over energy metabolism, more specifically, mΔΨ. Future studies are needed to further investigate this potential mechanism of action.

  11. HBCDD-induced sustained reduction in mitochondrial membrane potential, ATP and steroidogenesis in peripubertal rat Leydig cells

    Energy Technology Data Exchange (ETDEWEB)

    Fa, Svetlana; Pogrmic-Majkic, Kristina; Samardzija, Dragana; Hrubik, Jelena; Glisic, Branka; Kovacevic, Radmila; Andric, Nebojsa, E-mail: nebojsa.andric@dbe.uns.ac.rs

    2015-01-01

    Hexabromocyclododecane (HBCDD), a brominated flame retardant added to various consumer products, is a ubiquitous environmental contaminant. We have previously shown that 6-hour exposure to HBCDD disturbs basal and human chorionic gonadotropin (hCG)-induced steroidogenesis in rat Leydig cells. Reduction in mitochondrial membrane potential (ΔΨm) and cAMP production was also observed. Here, we further expanded research on the effect of HBCDD on Leydig cells by using a prolonged exposure scenario. Cells were incubated in the presence of HBCDD during 24 h and then treated with HBCDD + hCG for additional 2 h. Results showed that HBCDD caused a sustained reduction in ATP level after 24 h of exposure, which persisted after additional 2-hour treatment with HBCDD + hCG. cAMP and androgen accumulations measured after 2 h of HBCDD + hCG treatment were also inhibited. Real-time PCR analysis showed significant inhibition in the expression of genes for steroidogenic enzymes, luteinizing hormone receptor, regulatory and transport proteins, and several transcription factors under both treatment conditions. Western blot analysis revealed a decreased level of 30 kDa steroidogenic acute regulatory protein (StAR) after HBCDD + hCG treatment. In addition, HBCDD decreased the conversion of 22-OH cholesterol to pregnenolone and androstenedione to testosterone, indicating loss of the activity of cytochrome P450C11A1 (CYP11A1) and 17β-hydroxysteroid dehydrogenase (HSD17β). Cell survival was not affected, as confirmed by cytotoxicity and trypan blue tests or DNA fragmentation analysis. In summary, our data showed that HBCDD inhibits ATP supply, most likely through a decrease in ΔΨm, and targets multiple sites in the steroidogenic pathway in Leydig cells. - Highlights: • HBCDD causes a sustained reduction in ΔΨm and ATP level in Leydig cells. • Prolonged HBCDD exposure decreases hCG-supported steroidogenesis in Leydig cells. • HBCDD targets StAR, HSD17β and CYP11A1 in Leydig

  12. Phenolic Constituents of Medicinal Plants with Activity against Trypanosoma brucei

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    Ya Nan Sun

    2016-04-01

    Full Text Available Neglected tropical diseases (NTDs affect over one billion people all over the world. These diseases are classified as neglected because they impact populations in areas with poor financial conditions and hence do not attract sufficient research investment. Human African Trypanosomiasis (HAT or sleeping sickness, caused by the parasite Trypanosoma brucei, is one of the NTDs. The current therapeutic interventions for T. brucei infections often have toxic side effects or require hospitalization so that they are not available in the rural environments where HAT occurs. Furthermore, parasite resistance is increasing, so that there is an urgent need to identify novel lead compounds against this infection. Recognizing the wide structural diversity of natural products, we desired to explore and identify novel antitrypanosomal chemotypes from a collection of natural products obtained from plants. In this study, 440 pure compounds from various medicinal plants were tested against T. brucei by in a screening using whole cell in vitro assays. As the result, twenty-two phenolic compounds exhibited potent activity against cultures of T. brucei. Among them, eight compounds—4, 7, 11, 14, 15, 18, 20, and 21—showed inhibitory activity against T. brucei, with IC50 values below 5 µM, ranging from 0.52 to 4.70 μM. Based on these results, we attempt to establish some general trends with respect to structure-activity relationships, which indicate that further investigation and optimization of these derivatives might enable the preparation of potentially useful compounds for treating HAT.

  13. Regulation and spatial organization of PCNA in Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Kaufmann, Doris; Gassen, Alwine [University of Munich (LMU), Department Biology I, Genetics, Grosshaderner Str. 2-4, 82152 Martinsried (Germany); Maiser, Andreas; Leonhardt, Heinrich [University of Munich (LMU), Department Biology II, Grosshaderner Str. 2-4, 82152 Martinsried (Germany); Janzen, Christian J., E-mail: christian.janzen@uni-wuerzburg.de [University of Munich (LMU), Department Biology I, Genetics, Grosshaderner Str. 2-4, 82152 Martinsried (Germany)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Characterization of the proliferating cell nuclear antigen in Trypanosoma brucei (TbPCNA). Black-Right-Pointing-Pointer TbPCNA is a suitable marker to detect replication in T. brucei. Black-Right-Pointing-Pointer TbPCNA distribution and regulation is different compared to closely related parasites T. cruzi and Leishmania donovani. -- Abstract: As in most eukaryotic cells, replication is regulated by a conserved group of proteins in the early-diverged parasite Trypanosoma brucei. Only a few components of the replication machinery have been described in this parasite and regulation, sub-nuclear localization and timing of replication are not well understood. We characterized the proliferating cell nuclear antigen in T. brucei (TbPCNA) to establish a spatial and temporal marker for replication. Interestingly, PCNA distribution and regulation is different compared to the closely related parasites Trypanosoma cruzi and Leishmania donovani. TbPCNA foci are clearly detectable during S phase of the cell cycle but in contrast to T. cruzi they are not preferentially located at the nuclear periphery. Furthermore, PCNA seems to be degraded when cells enter G2 phase in T. brucei suggesting different modes of replication regulation or functions of PCNA in these closely related eukaryotes.

  14. Detection of Trypanosoma brucei in field-captured tsetse flies and identification of host species fed on by the infected flies.

    Science.gov (United States)

    Konnai, Satoru; Mekata, Hirohisa; Odbileg, Raadan; Simuunza, Martin; Chembensof, Mwelwa; Witola, William Harold; Tembo, Mwase Enala; Chitambo, Harrison; Inoue, Noboru; Onuma, Misao; Ohashi, Kazuhiko

    2008-08-01

    The prevalence of trypanosome infections in tsetse flies in the Chiawa area of Lower Zambezi in Zambia, with endemic trypanosomosis, was determined by a polymerase chain reaction (PCR) method that allowed the detection of trypanosome DNA and determination of the type of animal host fed on by the tsetse fly Glossina pallidipes, using tsetse-derived DNA extracts as templates. Ninety G. pallidipes (82 females and 8 males; 18.3%) of the 492 flies captured by baited biconical traps tested positive for the presence of Trypanosoma brucei species genomic DNA. Of the 90 T. brucei-positive flies, 47 (52.2%) also tested positive for vertebrate mitochondrial DNA. Sequence analysis of the vertebrate mitochondrial DNA amplicons established that they originated from 8 different vertebrate species, namely, human (Homo sapiens), African elephant (Loxodonta cyclotis), African buffalo (Syncerus caffer), waterbuck (Kobus ellipsiprymnus), roan antelope (Hippotragus equinus), greater kudu (Tragelaphus strepsiceros), warthog (Phacochoerus africanus), and goat (Capra hircus). Furthermore, to investigate the prevalence of trypanosome infections in domestic goats in the same area where trypanosomes had been detected in tsetse files, a total of 86 goats were randomly selected from 6 different herds. Among the selected goats, 36 (41.9%) were found to be positive for T. brucei species. This combined detection method would be an ideal approach not only for mass screening for infection prevalence in tsetse populations, but also for the prediction of natural reservoirs in areas endemic for trypanosomosis.

  15. Mitochondrial cholesterol import.

    Science.gov (United States)

    Elustondo, Pia; Martin, Laura A; Karten, Barbara

    2017-01-01

    All animal subcellular membranes require cholesterol, which influences membrane fluidity and permeability, fission and fusion processes, and membrane protein function. The distribution of cholesterol among subcellular membranes is highly heterogeneous and the cholesterol content of each membrane must be carefully regulated. Compared to other subcellular membranes, mitochondrial membranes are cholesterol-poor, particularly the inner mitochondrial membrane (IMM). As a result, steroidogenesis can be controlled through the delivery of cholesterol to the IMM, where it is converted to pregnenolone. The low basal levels of cholesterol also make mitochondria sensitive to changes in cholesterol content, which can have a relatively large impact on the biophysical and functional characteristics of mitochondrial membranes. Increased mitochondrial cholesterol levels have been observed in diverse pathological conditions including cancer, steatohepatitis, Alzheimer disease and Niemann-Pick Type C1-deficiency, and are associated with increased oxidative stress, impaired oxidative phosphorylation, and changes in the susceptibility to apoptosis, among other alterations in mitochondrial function. Mitochondria are not included in the vesicular trafficking network; therefore, cholesterol transport to mitochondria is mostly achieved through the activity of lipid transfer proteins at membrane contact sites or by cytosolic, diffusible lipid transfer proteins. Here we will give an overview of the main mechanisms involved in mitochondrial cholesterol import, focusing on the steroidogenic acute regulatory protein StAR/STARD1 and other members of the StAR-related lipid transfer (START) domain protein family, and we will discuss how changes in mitochondrial cholesterol levels can arise and affect mitochondrial function. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Membraner

    DEFF Research Database (Denmark)

    Bach, Finn

    2009-01-01

    Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...

  17. Evaluation of In Vitro Activity of Essential Oils against Trypanosoma brucei brucei and Trypanosoma evansi

    Directory of Open Access Journals (Sweden)

    Nathan Habila

    2010-01-01

    Full Text Available Essential oils (EOs from Cymbopogon citratus (CC, Eucalyptus citriodora (EC, Eucalyptus camaldulensis (ED, and Citrus sinensis (CS were obtained by hydrodistillation process. The EOs were evaluated in vitro for activity against Trypanosoma brucei brucei (Tbb and Trypanosoma evansi (T. evansi. The EOs were found to possess antitrypanosomal activity in vitro in a dose-dependent pattern in a short period of time. The drop in number of parasite over time was achieved doses of 0.4 g/ml, 0.2 g/mL, and 0.1 g/mL for all the EOs. The concentration of 0.4 g/mL CC was more potent at 3 minutes and 2 minutes for Tbb and T. evansi, respectively. The GC-MS analysis of the EOs revealed presence of Cyclobutane (96.09% in CS, 6-octenal (77.11% in EC, Eucalyptol (75% in ED, and Citral (38.32% in CC among several other organic compounds. The results are discussed in relation to trypanosome chemotherapy.

  18. Analysis of the effects of polyphenols on human spermatozoa reveals unexpected impacts on mitochondrial membrane potential, oxidative stress and DNA integrity; implications for assisted reproductive technology.

    Science.gov (United States)

    Aitken, R J; Muscio, L; Whiting, S; Connaughton, H S; Fraser, B A; Nixon, B; Smith, N D; De Iuliis, G N

    2016-12-01

    The need to protect human spermatozoa from oxidative stress during assisted reproductive technology, has prompted a detailed analysis of the impacts of phenolic compounds on the functional integrity of these cells. Investigation of 16 individual compounds revealed a surprising variety of negative effects including: (i) a loss of mitochondrial membrane potential (Δψm) via mechanisms that were not related to opening of the permeability transition pore but associated with a reduction in thiol expression, (ii) a decline in intracellular reduced glutathione, (iii) the stimulation of pro-oxidant activity including the induction of ROS generation from mitochondrial and non-mitochondrial sources, (iv) stimulation of lipid peroxidation, (v) the generation of oxidative DNA damage, and (vi) impaired sperm motility. For most of the polyphenolic compounds examined, the loss of motility was gradual and highly correlated with the induction of lipid peroxidation (r=0.889). The exception was gossypol, which induced a rapid loss of motility due to its inherent alkylating activity; one consequence of which was a marked reduction in carboxymethyl lysine expression on the sperm tail; a post-translational modification that is known to play a key role in the regulation of sperm movement. The only polyphenols that did not appear to have adverse effects on spermatozoa were resveratrol, genistein and THP at doses below 100μM. These compounds could, therefore, have some therapeutic potential in a clinical setting. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Salvianolic acid B inhibits intermittent high glucose-induced INS-1 cell apoptosis through regulation of Bcl-2 proteins and mitochondrial membrane potential.

    Science.gov (United States)

    Tao, Shanjun; Ren, Younan; Zheng, Haowen; Zhao, Mengqiu; Zhang, Xu; Zhu, Yuanmei; Yang, Jieren; Zheng, Shuguo

    2017-11-05

    Blood glucose fluctuations, also referred to as intermittent high glucose, have been validated to be more harmful than sustained high glucose in exacerbating pancreatic dysfunction by inducing β cell apoptosis. Salvianolic acid B (Sal B), an aqueous component of Salvia miltiorrhiza, has been proved beneficial to pancreatic islet function in diabetes, but the underlying mechanisms remain to be elucidated. The present study investigated the protective effect of Sal B on INS-1 cells exposed to intermittent high glucose and the possible mechanisms implicated. The results indicated that Sal B was able to restore cell viability and suppress INS-1 cell apoptosis induced by intermittent high glucose. Preincubation with Sal B led to a significant decrease of caspase-9 and caspase-3 activity and poly ADP-ribose polymerase (PARP) cleavage. Exposure to intermittent high glucose induced significant up-regulation of proapoptotic proteins, down-regulation of antiapoptotic protein and depolarization of mitochondrial membrane potential (MMP) in INS-1 cells, while these changes were reversed effectively in Sal B treated groups. In addition, Sal B markedly attenuated intermittent high glucose-induced oxidative stress as manifested by notably decreased levels of intracellular reactive oxygen species and malondialdehyde (MDA). Taken together, these results indicate that Sal B is able to suppress intermittent high glucose-induced INS-1 cell apoptosis, which might be ascribed to regulation of Bcl-2 family protein expression and preservation of mitochondrial membrane potential. Copyright © 2017. Published by Elsevier B.V.

  20. Brucea javanica Leaf Extract Induced Apoptosis in Human Oral Squamous Cell Carcinoma (HSC2 Cells by Attenuation of Mitochondrial Membrane Permeability

    Directory of Open Access Journals (Sweden)

    Britanto Dani Wicaksono

    2015-08-01

    Full Text Available BACKGROUND: Brucea javanica extract has been reported to have anti-proliferative and cell death induction activities. B. javanica extract was reported to induce apoptosis through caspase cascade. Most of investigated B. javanica extracts were derived from seeds and fruits, or commercially available oil emulsion. Therefore we conducted a study on B. javanica leaf extract (BJLE in oral cancer cells. METHODS: B. javanica leaves were collected, identified, minced, dried, extracted with distilled ethanol at room temperature for 24 hours, filtered and evaporated. Resulted BJLE was stored at 4°C. Human oral squamous cell carcinoma (HSC-2 cells were fasted for 12 hours and treated with BJLE in various concentrations for 24 hours. Cells were then quantified with 3-(4,5-dimethylthiazol-2-yl-2,5-Diphenyltetrazolium bromide (MTT assay, demonstrated with 4',6'-diamidino-2-phenylindole (DAPI staining. To find out mitochondrial membrane permeability (MMP, mitochondrial membrane potential (ΔΨM was analyzed. RESULTS: BJLE reduced percentage of viable HSC-2 cells in a concentration dependent manner. BJLE induced apoptosis in HSC-2 cells. With treatment of 50 μg/ml BJLE, fragmented nuclei were seen. ΔΨM of HSC-2 cells treated with 50 μg/ml BJLE were shifted to the left, meaning that BJLE induced reduction of ΔΨM and attenuation of MMP. CONCLUSIONS: Our results suggested that BJLE could induce apoptosis by attenuating MMP. KEYWORDS: Brucea javanica, leaf, apoptosis, HSC-2, MTT, DAPI, mitochondria, permeability.

  1. A flavivirus protein M-derived peptide directly permeabilizes mitochondrial membranes, triggers cell death and reduces human tumor growth in nude mice.

    Science.gov (United States)

    Brabant, Magali; Baux, Ludwig; Casimir, Richard; Briand, Jean Paul; Chaloin, Olivier; Porceddu, Mathieu; Buron, Nelly; Chauvier, David; Lassalle, Myriam; Lecoeur, Hervé; Langonné, Alain; Dupont, Sylvie; Déas, Olivier; Brenner, Catherine; Rebouillat, Dominique; Muller, Sylviane; Borgne-Sanchez, Annie; Jacotot, Etienne

    2009-10-01

    Dengue viruses belong to the Flavivirus family and are responsible for hemorrhagic fever in Human. Dengue virus infection triggers apoptosis especially through the expression of the small membrane (M) protein. Using isolated mitochondria, we found that synthetic peptides containing the C-terminus part of the M ectodomain caused apoptosis-related mitochondrial membrane permeabilization (MMP) events. These events include matrix swelling and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). Protein M Flavivirus sequence alignments and helical wheel projections reveal a conserved distribution of charged residues. Moreover, when combined to the cell penetrating HIV-1 Tat peptide transduction domain (Tat-PTD), this sequence triggers a caspase-dependent cell death associated with DeltaPsi(m) loss and cytochrome c release. Mutational approaches coupled to functional screening on isolated mitochondria resulted in the selection of a protein M derived sequence containing nine residues with potent MMP-inducing properties on isolated mitochondria. A chimeric peptide composed of a Tat-PTD linked to the 9-mer entity triggers MMP and cell death. Finally, local administration of this chimeric peptide induces growth inhibition of xenograft prostate PC3 tumors in immuno-compromised mice, and significantly enhances animal survival. Together, these findings support the notion of using viral genomes as valuable sources to discover mitochondria-targeted sequences that may lead to the development of new anticancer compounds.

  2. Two Trichothecene Mycotoxins from Myrothecium roridum Induce Apoptosis of HepG-2 Cells via Caspase Activation and Disruption of Mitochondrial Membrane Potential.

    Science.gov (United States)

    Ye, Wei; Chen, Yuchan; Li, Haohua; Zhang, Weimin; Liu, Hongxin; Sun, Zhanghua; Liu, Taomei; Li, Saini

    2016-06-17

    Trichothecene mycotoxins are a type of sesquiterpenoid produced by various kinds of plantpathogenic fungi. In this study, two trichothecene toxins, namely, a novel cytotoxic epiroridin acid and a known trichothecene, mytoxin B, were isolated from the endophytic fungus Myrothecium roridum derived from the medicinal plant Pogostemon cablin. The two trichothecene mytoxins were confirmed to induce the apoptosis of HepG-2 cells by cytomorphology inspection, DNA fragmentation detection, and flow cytometry assay. The cytotoxic mechanisms of the two mycotoxins were investigated by quantitative real time polymerase chain reaction, western blot, and detection of mitochondrial membrane potential. The results showed that the two trichothecene mycotoxins induced the apoptosis of cancer cell HepG-2 via activation of caspase-9 and caspase-3, up-regulation of bax gene expression, down-regulation of bcl-2 gene expression, and disruption of the mitochondrial membrane potential of the HepG-2 cell. This study is the first to report on the cytotoxic mechanism of trichothecene mycotoxins from M. roridum. This study provides new clues for the development of attenuated trichothecene toxins in future treatment of liver cancer.

  3. Nucleoside analysis of DNA from Trypanosoma brucei and Trypanosoma equiperdum.

    Science.gov (United States)

    Crozatier, M; De Brij, R J; Den Engelse, L; Johnson, P J; Borst, P

    1988-11-01

    We have digested trypanosome DNA with a combination of pancreatic DNase I, nuclease P1 and bovine alkaline phosphatase and fractionated the resulting nucleosides on a Supelcosil LC-18-S column by high pressure liquid chromatography. We find less than 0.1% unusual nucleosides, both in Trypanosoma brucei and in a Trypanosoma equiperdum stock, in contrast to a previous report of an unusual nucleoside replacing dC at 1.3% of total nucleosides in T. equiperdum. Our results agree with previous suggestions that the modification of inactive telomeric expression sites for variant-specific surface glycoprotein genes in T. brucei only affects a very small fraction of the total DNA.

  4. Short term exercise induces PGC-1α, ameliorates inflammation and increases mitochondrial membrane proteins but fails to increase respiratory enzymes in aging diabetic hearts.

    Directory of Open Access Journals (Sweden)

    Amy Botta

    Full Text Available PGC-1α, a transcriptional coactivator, controls inflammation and mitochondrial gene expression in insulin-sensitive tissues following exercise intervention. However, attributing such effects to PGC-1α is counfounded by exercise-induced fluctuations in blood glucose, insulin or bodyweight in diabetic patients. The goal of this study was to investigate the role of PGC-1α on inflammation and mitochondrial protein expressions in aging db/db mice hearts, independent of changes in glycemic parameters. In 8-month-old db/db mice hearts with diabetes lasting over 22 weeks, short-term, moderate-intensity exercise upregulated PGC-1α without altering body weight or glycemic parameters. Nonetheless, such a regimen lowered both cardiac (macrophage infiltration, iNOS and TNFα and systemic (circulating chemokines and cytokines inflammation. Curiously, such an anti-inflammatory effect was also linked to attenuated expression of downstream transcription factors of PGC-1α such as NRF-1 and several respiratory genes. Such mismatch between PGC-1α and its downstream targets was associated with elevated mitochondrial membrane proteins like Tom70 but a concurrent reduction in oxidative phosphorylation protein expressions in exercised db/db hearts. As mitochondrial oxidative stress was predominant in these hearts, in support of our in vivo data, increasing concentrations of H2O2 dose-dependently increased PGC-1α expression while inhibiting expression of inflammatory genes and downstream transcription factors in H9c2 cardiomyocytes in vitro. We conclude that short-term exercise-induced oxidative stress may be key in attenuating cardiac inflammatory genes and impairing PGC-1α mediated gene transcription of downstream transcription factors in type 2 diabetic hearts at an advanced age.

  5. Nitroheterocyclic drug resistance mechanisms in Trypanosoma brucei.

    Science.gov (United States)

    Wyllie, Susan; Foth, Bernardo J; Kelner, Anna; Sokolova, Antoaneta Y; Berriman, Matthew; Fairlamb, Alan H

    2016-03-01

    The objective of this study was to identify the mechanisms of resistance to nifurtimox and fexinidazole in African trypanosomes. Bloodstream-form Trypanosoma brucei were selected for resistance to nifurtimox and fexinidazole by stepwise exposure to increasing drug concentrations. Clones were subjected to WGS to identify putative resistance genes. Transgenic parasites modulating expression of genes of interest were generated and drug susceptibility phenotypes determined. Nifurtimox-resistant (NfxR) and fexinidazole-resistant (FxR) parasites shared reciprocal cross-resistance suggestive of a common mechanism of action. Previously, a type I nitroreductase (NTR) has been implicated in nitro drug activation. WGS of resistant clones revealed that NfxR parasites had lost >100 kb from one copy of chromosome 7, rendering them hemizygous for NTR as well as over 30 other genes. FxR parasites retained both copies of NTR, but lost >70 kb downstream of one NTR allele, decreasing NTR transcription by half. A single knockout line of NTR displayed 1.6- and 1.9-fold resistance to nifurtimox and fexinidazole, respectively. Since NfxR and FxR parasites are ∼6- and 20-fold resistant to nifurtimox and fexinidazole, respectively, additional factors must be involved. Overexpression and knockout studies ruled out a role for a putative oxidoreductase (Tb927.7.7410) and a hypothetical gene (Tb927.1.1050), previously identified in a genome-scale RNAi screen. NTR was confirmed as a key resistance determinant, either by loss of one gene copy or loss of gene expression. Further work is required to identify which of the many dozens of SNPs identified in the drug-resistant cell lines contribute to the overall resistance phenotype. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

  6. Differential hydrolysis of erythrocyte and mitochondrial membrane phospholipids by two phospholipase A2 isoenzymes (NK-PLA2-I and NK-PLA2-II) from the venom of the Indian monocled cobra Naja kaouthia.

    Science.gov (United States)

    Doley, Robin; King, Glenn F; Mukherjee, Ashis K

    2004-05-01

    We previously demonstrated that venom from the Indian monocled cobra Naja kaouthia is a rich source of phospholipase A2 enzymes, and we purified and characterized a major PLA2 isoenzyme (NK-PLA2-I) from N. kaouthia venom. In the present study, we report the purification and biochemical characterization of a second PLA2 isoenzyme (NK-PLA2-II) from the same venom. A comparison of the membrane phospholipid hydrolysis patterns by these two PLA2s has revealed that they cause significantly more damage to mitochondrial membranes (NK-PLA2-I > NK-PLA2-II) as compared to erythrocyte membranes due to more efficient binding of the enzymes to mitochondrial membranes. Fatty acid release patterns by these PLA2s from the membrane phospholipid PC-pools indicate that NK-PLA2-I does not discriminate between saturated and unsaturated fatty acids whereas NK-PLA2-II shows a preference for unsaturated fatty acids during the initial phase of attack. The current investigation provides new insight into the molecular arrangement of NK-PLA2-sensitive domains in erythrocyte and mitochondrial membranes and highlights the contribution of polar, but uncharged, amino acids such as serine and cysteine in NK-PLA2 induced membrane damage.

  7. Effects of Morinda lucida leaf extract on Trypanosoma brucei brucei infection in mice.

    Science.gov (United States)

    Asuzu, I U; Chineme, C N

    1990-10-01

    The dried leaves of Morinda lucida were extracted with 50% methanol and the extract was recovered in a 9.7% w/w yield. Acute toxicity tests were performed in mice and the intraperitoneal LD50 of the extract was 2000 mg/kg. The extract induced purgation in mice from the first hour after oral administration and reached its peak between the third and fourth hour. The purgation was not dose-dependent. M. lucida leaf extract i.p. significantly suppressed the level of parasitemia after Trypanosoma brucei infection in mice. Suppression of existing parasitemia appeared dose-dependent with 1000 mg/kg i.p. producing the maximum effect. The best trypanocidal activity was obtained when treatment with M. lucida extract commenced simultaneously with trypanosome inoculation.

  8. Pharmacologic Effects on Mitochondrial Function

    Science.gov (United States)

    Cohen, Bruce H.

    2010-01-01

    The vast majority of energy necessary for cellular function is produced in mitochondria. Free-radical production and apoptosis are other critical mitochondrial functions. The complex structure, electrochemical properties of the inner mitochondrial membrane (IMM), and genetic control from both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) are…

  9. Immunomodulatory activity of Buchholzia coriacea seed methanol extract on Trypanosoma brucei brucei infected mice.

    Science.gov (United States)

    Eze, James I; Ekelozie, Chioma F; Nweze, Nwakego E

    2017-12-01

    The seeds of Buchholzia coriacea Engler (Capparaceae) are used in Eastern Nigeria to treat feverish conditions, and to treat malaria and sleeping sickness that cause fever. The current study assesses the immunomodulatory activity of Buchholzia coriacea seed extract on Trypanosoma brucei brucei infected mice. Delayed hypersensitivity reaction, humoral antibody response and in-vivo leucocyte mobilization tests were assessed in three different experiments to determine the effect of the extract on immune response. Seventy-five (75) mice (25 mice per experiment) were used for the study and were each infected with 1.00 × 106 trypanosomes intra-peritoneally. Groups A, B and C were given 250, 500 and 1000 mg/kg of the extract, respectively, group D received 7.5 mg/kg body weight of levamisole and group E was the control. Sheep RBCs were used as antigen. The acute toxicity tests did not cause clinical signs or death within 24 h post treatment at all the doses tested. The extract inhibited delayed hypersensitivity reaction by 20.9 and 20.8% at 250 and 500 mg/kg, respectively, while at 1000 mg/kg, the paw size increased (-101.9%) when compared with the control. The extract elevated the antibody titre from 1.60 ± 0.40 for control to 8.00 ± 3.58 for 500 mg/kg group. The extract increased in total leucocytes counts. The extract has a very wide safety margin and was able to improve immune response. The results of the present study showed that Buchholzia coriacea seed methanol extract possesses immunostimulatory activity on trypanosome-infected mice.

  10. The antioxidant Trolox restores mitochondrial membrane potential and Ca2+ -stimulated ATP production in human complex I deficiency.

    NARCIS (Netherlands)

    Distelmaier, F.; Visch, H.J.; Smeitink, J.A.M.; Mayatepek, E.; Koopman, W.J.H.; Willems, P.H.G.M.

    2009-01-01

    Malfunction of mitochondrial complex I caused by nuclear gene mutations causes early-onset neurodegenerative diseases. Previous work using cultured fibroblasts of complex-I-deficient patients revealed elevated levels of reactive oxygen species (ROS) and reductions in both total Ca(2+) content of the

  11. ( Nigella sativa ) oil on Trypanosoma brucei -infected rats

    African Journals Online (AJOL)

    The effect of black seed oil (Nigella sativa oil) on parasitaemia, some serum and liver enzymes as well as some haematological parameters in Trypanosoma brucei-infected rats were investigated. The results show there was low parasitaemia and extension of life span of rats from 12 days of the infected untreated (control) ...

  12. The genome of the African trypanosome Trypanosoma brucei.

    Science.gov (United States)

    Berriman, Matthew; Ghedin, Elodie; Hertz-Fowler, Christiane; Blandin, Gaëlle; Renauld, Hubert; Bartholomeu, Daniella C; Lennard, Nicola J; Caler, Elisabet; Hamlin, Nancy E; Haas, Brian; Böhme, Ulrike; Hannick, Linda; Aslett, Martin A; Shallom, Joshua; Marcello, Lucio; Hou, Lihua; Wickstead, Bill; Alsmark, U Cecilia M; Arrowsmith, Claire; Atkin, Rebecca J; Barron, Andrew J; Bringaud, Frederic; Brooks, Karen; Carrington, Mark; Cherevach, Inna; Chillingworth, Tracey-Jane; Churcher, Carol; Clark, Louise N; Corton, Craig H; Cronin, Ann; Davies, Rob M; Doggett, Jonathon; Djikeng, Appolinaire; Feldblyum, Tamara; Field, Mark C; Fraser, Audrey; Goodhead, Ian; Hance, Zahra; Harper, David; Harris, Barbara R; Hauser, Heidi; Hostetler, Jessica; Ivens, Al; Jagels, Kay; Johnson, David; Johnson, Justin; Jones, Kristine; Kerhornou, Arnaud X; Koo, Hean; Larke, Natasha; Landfear, Scott; Larkin, Christopher; Leech, Vanessa; Line, Alexandra; Lord, Angela; Macleod, Annette; Mooney, Paul J; Moule, Sharon; Martin, David M A; Morgan, Gareth W; Mungall, Karen; Norbertczak, Halina; Ormond, Doug; Pai, Grace; Peacock, Chris S; Peterson, Jeremy; Quail, Michael A; Rabbinowitsch, Ester; Rajandream, Marie-Adele; Reitter, Chris; Salzberg, Steven L; Sanders, Mandy; Schobel, Seth; Sharp, Sarah; Simmonds, Mark; Simpson, Anjana J; Tallon, Luke; Turner, C Michael R; Tait, Andrew; Tivey, Adrian R; Van Aken, Susan; Walker, Danielle; Wanless, David; Wang, Shiliang; White, Brian; White, Owen; Whitehead, Sally; Woodward, John; Wortman, Jennifer; Adams, Mark D; Embley, T Martin; Gull, Keith; Ullu, Elisabetta; Barry, J David; Fairlamb, Alan H; Opperdoes, Fred; Barrell, Barclay G; Donelson, John E; Hall, Neil; Fraser, Claire M; Melville, Sara E; El-Sayed, Najib M

    2005-07-15

    African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei. The 26-megabase genome contains 9068 predicted genes, including approximately 900 pseudogenes and approximately 1700 T. brucei-specific genes. Large subtelomeric arrays contain an archive of 806 variant surface glycoprotein (VSG) genes used by the parasite to evade the mammalian immune system. Most VSG genes are pseudogenes, which may be used to generate expressed mosaic genes by ectopic recombination. Comparisons of the cytoskeleton and endocytic trafficking systems with those of humans and other eukaryotic organisms reveal major differences. A comparison of metabolic pathways encoded by the genomes of T. brucei, T. cruzi, and Leishmania major reveals the least overall metabolic capability in T. brucei and the greatest in L. major. Horizontal transfer of genes of bacterial origin has contributed to some of the metabolic differences in these parasites, and a number of novel potential drug targets have been identified.

  13. Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei.

    NARCIS (Netherlands)

    Helfert, S.; Estevez, A.M.; Bakker, B.M.; Michels, P.A.M.; Clayton, C.

    2001-01-01

    Kinetoplastid protozoa compartmentalize the first seven enzymes of glycolysis and two enzymes of glycerol metabolism in a microbody, the glycosome. While in its mammalian host, Trypanosoma brucei depends entirely on glucose for ATP generation. Under aerobic conditions, most of the glucose is

  14. Population genetic structure and cladistic analysis of Trypanosoma brucei isolates

    NARCIS (Netherlands)

    Agbo, E.C.; Clausen, P.H.; Buscher, P.; Majiwa, P.A.O.; Pas, te M.F.W.; Claassen, E.

    2003-01-01

    Using a novel multilocus DNA marker analysis method, we studied the population genetic structure of Trypansoma brucei stocks and derived clones isolated from animal and rhodesiense sleeping sickness patients during a national sleeping sickness control program in Mukono district, Uganda. We then

  15. Role of cytokines in Trypanosoma brucei-induced anaemia: A ...

    African Journals Online (AJOL)

    the blood-brain barrier. The subspecies Trypanosoma brucei rhodesiense in eastern and southern Africa generally causes a more acute form of ... The persistence of anaemia beyond the initial wave of parasitaemia (after which low numbers of parasites are in circulation) indicates that anaemia may be directly induced.

  16. New functions for parts of the Krebs cycle in procyclic Trypanosoma brucei, a cycle not operating as a cycle.

    Science.gov (United States)

    van Weelden, Susanne W H; van Hellemond, Jaap J; Opperdoes, Fred R; Tielens, Aloysius G M

    2005-04-01

    We investigated whether substrate availability influences the type of energy metabolism in procyclic Trypanosoma brucei. We show that absence of glycolytic substrates (glucose and glycerol) does not induce a shift from a fermentative metabolism to complete oxidation of substrates. We also show that glucose (and even glycolysis) is not essential for normal functioning and proliferation of pleomorphic procyclic T. brucei cells. Furthermore, absence of glucose did not result in increased degradation of amino acids. Variations in availability of glucose and glycerol did result, however, in adaptations in metabolism in such a way that the glycosome was always in redox balance. We argue that it is likely that, in procyclic cells, phosphoglycerate kinase is located not only in the cytosol, but also inside glycosomes, as otherwise an ATP deficit would occur in this organelle. We demonstrate that procyclic T. brucei uses parts of the Krebs cycle for purposes other than complete degradation of mitochondrial substrates. We suggest that citrate synthase plus pyruvate dehydrogenase and malate dehydrogenase are used to transport acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, a process we show to occur in proliferating procyclic cells. The part of the Krebs cycle consisting of alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase was used for the degradation of proline and glutamate to succinate. We also demonstrate that the subsequent enzymes of the Krebs cycle, succinate dehydrogenase and fumarase, are most likely used for conversion of succinate into malate, which can then be used in gluconeogenesis.

  17. Channel-forming activities in the glycosomal fraction from the bloodstream form of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Melisa Gualdron-López

    Full Text Available BACKGROUND: Glycosomes are a specialized form of peroxisomes (microbodies present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear. METHODS/PRINCIPAL FINDINGS: We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T. brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70-80 pA, 20-25 pA, and 8-11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte. All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20-25 pA is anion-selective (P(K+/P(Cl-∼0.31, while the other two types of channels are slightly selective for cations (P(K+/P(Cl- ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively. The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel's pore. CONCLUSIONS/SIGNIFICANCE: These results indicate that the membrane of glycosomes

  18. Hepatitis B Virus HBx Protein Localizes to Mitochondria in Primary Rat Hepatocytes and Modulates Mitochondrial Membrane Potential ▿

    OpenAIRE

    Clippinger, Amy J.; Bouchard, Michael J.

    2008-01-01

    Over 350 million people are chronically infected with hepatitis B virus (HBV), and a significant number of chronically infected individuals develop primary liver cancer. HBV encodes seven viral proteins, including the nonstructural X (HBx) protein. The results of studies with immortalized or transformed cells and with HBx-transgenic mice demonstrated that HBx can interact with mitochondria. However, no studies with normal hepatocytes have characterized the precise mitochondrial localization o...

  19. Thiamine deficiency results in release of soluble factors that disrupt mitochondrial membrane potential and downregulate the glutamate transporter splice-variant GLT-1b in cultured astrocytes.

    Science.gov (United States)

    Jhala, Shivraj S; Wang, Dongmei; Hazell, Alan S

    2014-06-06

    Loss of astrocytic glutamate transporters is a major feature of both thiamine deficiency (TD) and Wernicke's encephalopathy. However, the underlying basis of this process is not well understood. In the present study we have investigated the possibility of release of astrocytic soluble factors that might be involved in the regulation of the glutamate transporter GLT-1b in these cells. Treatment of naïve astrocytes with conditioned media from astrocytes exposed to TD conditions resulted in a progressive decrease in glutamate uptake over 24 h. Immunoblotting and flow cytometry measurements indicated this was accompanied by a 20-40% loss of GLT-1b. Astrocytes exposed to either TD or TD conditioned media showed increased disruption of mitochondrial membrane potential compared to control cells, and treatment of astrocytes with TD resulted in an increase in the pro-inflammatory cytokine TNF-α and elevated levels of phospho-IκB fragment, indicative of increased activation of NF-κB. Inhibition of TNF-α activity with the use of a neutralizing antibody blocked the increased NF-κB activation, while inhibition of NF-κB ameliorated the decrease in GLT-1b and reversed the decrease in glutamate uptake occurring with TD treatment. Together, these findings indicate that astrocytes exposed to TD conditions show responses suggesting that soluble factors released by these cells under conditions of TD play a regulatory role in terms of glutamate transport function and mitochondrial integrity. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Ruthenium(II) complexes: DNA-binding, cytotoxicity, apoptosis, cellular localization, cell cycle arrest, reactive oxygen species, mitochondrial membrane potential and western blot analysis.

    Science.gov (United States)

    Li, Wei; Jiang, Guang-Bin; Yao, Jun-Hua; Wang, Xiu-Zhen; Wang, Ji; Han, Bing-Jie; Xie, Yang-Yin; Lin, Gan-Jian; Huang, Hong-Liang; Liu, Yun-Jun

    2014-11-01

    The aim of our study was to investigate DNA-binding and cytotoxic activity of the four new Ru(II) polypyridyl complexes [Ru(dmb)₂(HMHPIP)](ClO₄)₂ (1), [Ru(bpy)₂(HMHPIP)](ClO₄)₂ (2), [Ru(phen)₂(HMHPIP)](ClO₄)₂ (3) and [Ru(dmp)₂(HMHPIP)](ClO₄)₂ (4). The complexes interact with DNA through intercalative mode and show relatively high cytotoxic activity against A549 cells, no cytotoxicity toward MG-63 cells. Complexes 1-4 can enhance the levels of ROS in A549 cells and induce the decrease of the mitochondrial membrane potential. These complexes inhibit the cell growth in A549 cells at G0/G1 or S phase. Complex 3 activated caspase 7, and down-regulated the expression of the anti-apoptotic protein Bcl-2. Complexes 1-4 induce apoptosis in A549 cells through ROS-mediated mitochondrial dysfunction pathway. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

  1. Radiosensitization by fullerene-C60 dissolved in squalene on human malignant melanoma through lipid peroxidation and enhanced mitochondrial membrane potential

    Science.gov (United States)

    Kato, Shinya; Kimura, Masatsugu; Miwa, Nobuhiko

    2014-04-01

    We examined fullerene-C60 dissolved in squalene (C60/Sqe) for the ability to potentiate the radiosensitization under X-ray irradiation on human malignant melanoma HMV-II cells, which were treated with C60/Sqe and thereafter irradiated with X-ray. The cell proliferation for C60/Sqe was inhibited more markedly than for Sqe alone. Meanwhile, cell proliferation was almost unaltered for C60/squalane (Sqa) or Sqa, a hydrogenated form of Sqe, as compared to no-additive control. Thus radiosensitization of C60/Sqe is attributed to peroxidation of unsaturated bonds of squalene by X-ray-excited C60 in contrast to squalane. The fluorescence images of HMV-II cells stained with Rhodamine123, an indicator for mitochondrial membrane potential, were monitored for 6 h after X-ray irradiation. C60/Sqe obviously exhibited more augmented fluorescence intensity on perinuclear region of HMV-II cells than Sqe alone. TBARS assay showed that the lipid peroxidation level as malondialdehyde-equivalent increased by combination of C60/Sqe and X-ray dose-dependently on X-ray doses. C60/Sqe exhibited lipid peroxidation more markedly by 1.2-fold than Sqe alone. Thus the level of lipid peroxidation of squalene was sufficiently higher in C60/Sqe than in Sqe in the absence of C60 under X-ray irradiation, suggesting the combination of C60/Sqe and X-ray irradiation induced radiosensitization on HMV-II cells by peroxidation of absorbed Sqe in mitochondrial membrane via oxidative stress mediated by fullerene-C60.

  2. Mucuna pruriens and its major constituent L-DOPA recover spermatogenic loss by combating ROS, loss of mitochondrial membrane potential and apoptosis.

    Directory of Open Access Journals (Sweden)

    Akhand Pratap Singh

    Full Text Available BACKGROUND: The Ayurvedic medicinal system claims Mucuna pruriens (MP to possess pro-male fertility, aphrodisiac and adaptogenic properties. Some scientific evidence also supports its pro-male fertility properties; however, the mechanism of its action is not yet clear. The present study aimed at demonstrating spermatogenic restorative efficacy of MP and its major constituent L-DOPA (LD, and finding the possible mechanism of action thereof in a rat model. METHODOLOGY/FINDINGS: Ethinyl estradiol (EE was administered at a rate of 3 mg/kg body weight (BW/day for a period of 14 days to generate a rat model with compromised spermatogenesis. MP and LD were administered in two separate groups of these animals starting 15(th day for a period of 56 days, and the results were compared with an auto-recovery (AR group. Sperm count and motility, testis histo-architecture, level of reactive oxygen species (ROS, mitochondrial membrane potential (MMP, apoptosis, peripheral hormone levels and testicular germ cell populations were analysed, in all experimental groups. We observed efficient and quick recovery of spermatogenesis in MP and LD groups in comparison to the auto-recovery group. The treatment regulated ROS level, apoptosis, and mitochondrial membrane potential (MMP, recovered the hypothalamic-pituitary-gonadal axis and the number of testicular germ cells, ultimately leading to increased sperm count and motility. CONCLUSION/SIGNIFICANCE: M. pruriens efficiently recovers the spermatogenic loss induced due to EE administration. The recovery is mediated by reduction in ROS level, restoration of MMP, regulation of apoptosis and eventual increase in the number of germ cells and regulation of apoptosis. The present study simplified the complexity of mechanism involved and provided meaningful insights into MP/LD mediated correction of spermatogenic impairment caused by estrogens exposure. This is the first study demonstrating that L-DOPA largely accounts for pro

  3. Diverse patterns of expression of the cytochrome c oxidase subunit I gene and unassigned reading frames 4 and 5 during the life cycle of Trypanosoma brucei.

    Science.gov (United States)

    Jasmer, D P; Feagin, J E; Stuart, K

    1985-01-01

    Transcription of a maxicircle segment from Trypanosoma brucei 164 that contains nucleotide (nt) sequences corresponding to cytochrome c oxidase subunit I (COI) and unassigned reading frames (URFs) 4 and 5 of other mitochondrial systems was investigated. Two major transcripts that differ in size by ca. 200 nt map to each of the COI and URF4 genes, while a single major transcript maps to URF5. In total RNA, the larger COI transcript is more abundant in procyclic forms (PFs) than in bloodstream forms (BFs), the smaller COI and both URF4 transcripts have similar abundances in both forms, and the single URF5 transcript is more abundant in BF than PF. These patterns of expression differ in poly(A)+ RNA as a result of a higher proportion of poly(A)+ mitochondrial transcripts in PFs than in BFs. In addition, small (300- to 500-nt) RNAs that are transcribed from C-rich sequences located between putative protein-coding genes also exhibit diverse patterns of expression between life cycle stages and differences in polyadenylation in PFs compared with BFs. These observations suggest that multiple processes regulate the differential expression of mitochondrial genes in T. brucei. Images PMID:2427925

  4. Photothermal Treatment of Human Pancreatic Cancer Using PEGylated Multi-Walled Carbon Nanotubes Induces Apoptosis by Triggering Mitochondrial Membrane Depolarization Mechanism.

    Science.gov (United States)

    Mocan, Teodora; Matea, Cristian T; Cojocaru, Iulia; Ilie, Ioana; Tabaran, Flaviu A; Zaharie, Florin; Iancu, Cornel; Bartos, Dana; Mocan, Lucian

    2014-01-01

    Pancreatic cancer (PC) is one of the most lethal solid tumor in humans, with an overall 5-year survival rate of less than 5%. Thermally active carbon nanotubes have already brought to light promising results in PC research and treatment. We report here the construct of a nano-biosystem based on multi-walled carbon nanotubes and polyethylene glycol (PEG) molecules validated through AFM, UV-Vis and DLS. We next studied the photothermal effect of these PEG-ylated multi-walled carbon nanotubes (5, 10 and 50 μg/mL, respectively) on pancreatic cancer cells (PANC-1) and further analyzed the molecular and cellular events involved in cell death occurrence. Using cell proliferation, apoptosis, membrane polarization and oxidative stress assays for ELISA, fluorescence microscopy and flow cytometry we show here that hyperthermia following MWCNTs-PEG laser mediated treatment (808 nm, 2W) leads to mitochondrial membrane depolarization that activates the flux of free radicals within the cell and the oxidative state mediate cellular damage in PC cells via apoptotic pathway. Our results are of decisive importance especially in regard with the development of novel nano-biosystems capable to target mitochondria and to synergically act both as cytotoxic drug as well as thermally active agents in order to overcome one of the most common problem met in oncology, that of intrinsic resistance to chemotherapeutics.

  5. Effects of alkyl alcohols and related chemicals on rat liver structure and function. III. Physiochemical properties of ethanol-, propanol- and butanol- treated rat liver mitochondrial membranes.

    Science.gov (United States)

    Adachi, K; Momota, M; Teranishi, Y; Ueki, R; Hagiwara, M; Wakabayashi, T; Popinigis, J

    1992-08-01

    The physicochemical properties of mitochondria in liver tissue obtained from rats given 32% ethanol, 32% propanol or 6.9% butanol in drinking water for up to 3 months were investigated using differential scanning calorimetry and fluorescence polarization measurements. The results obtained were as follows: 1) Phospholipids extracted from mitochondria showed increases in the relative amounts of phosphatidylcholine, phosphatidylinositol and phosphatidylserine, and a decrease in the relative amount of phosphatidylethanolamine. An increase in the unsaturated/saturated fatty acid ratio of phospholipids was also observed. 2) Elevation of the thermotropic lipid phase transition temperature with a decrease in the enthalpy value (delta H) was revealed by differential scanning calorimetry. 3) The elevation of the lipid phase transition temperature was detected also by fluorescence polarization measurements using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. Elevation of mitochondrial membrane fluidity was found in some of the experimental animals, but most showed no changes in comparison with the control. A possible role of membrane fusion in the mechanism of formation of ethanol-, propanol- and butanol-induced hepatic megamitochondria is discussed on the basis of these results.

  6. Effect of Seminal Plasma Removal on Cell Membrane, Acrosomal Integrity and Mitochondrial Activity of Cooled Stallion Semen

    Directory of Open Access Journals (Sweden)

    Dhafer M. Aziz

    2012-07-01

    Full Text Available Fresh semen samples were collected from 11 warm blood stallions, each ejaculate was distributed into three equal parts. The first part was diluted in a skim milk-glucose diluent (SMG, the second part was diluted in a skim milk-glucose supplemented with Tyrode's medium (SMG-T, the third part was centrifuged to remove the seminal plasma, then the sperm was resuspended in the second diluent (SMG-T-C. The diluted semen were evaluated immediately after dilution (0 hour and at 24, 48, 72, and 96 hours of storage at 5°C. Flow cytometry was performed to determine sperm viability, mitochondrial activity and acrosomal integrity. Immediately after dilution the tested parameters of sperms that diluted in SMG-T was significantly (P<0.001 higher than those diluted with SMG and SMG-T-C, and with SMG-T-C were higher significantly (P<0.05 than those diluted with SMG. The decreasing rate in tested sperm parameter was greater significantly (P<0.001 in semen samples which were diluted with SMG than those diluted with SMG-T and SMG-T-C. In conclusion, the present study indicated that viability, acrosomal integrity, and mitochondrial activity of stallion sperms were better preserved in SMG-T in comparison with SMG, also centrifugation and removal of the seminal plasma have an adverse effect on these three sperm parameters.

  7. Crystal structure of arginine methyltransferase 6 from Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Chongyuan Wang

    Full Text Available Arginine methylation plays vital roles in the cellular functions of the protozoan Trypanosoma brucei. The T. brucei arginine methyltransferase 6 (TbPRMT6 is a type I arginine methyltransferase homologous to human PRMT6. In this study, we report the crystal structures of apo-TbPRMT6 and its complex with the reaction product S-adenosyl-homocysteine (SAH. The structure of apo-TbPRMT6 displays several features that are different from those of type I PRMTs that were structurally characterized previously, including four stretches of insertion, the absence of strand β15, and a distinct dimerization arm. The comparison of the apo-TbPRMT6 and SAH-TbPRMT6 structures revealed the fine rearrangements in the active site upon SAH binding. The isothermal titration calorimetry results demonstrated that SAH binding greatly increases the affinity of TbPRMT6 to a substrate peptide derived from bovine histone H4. The western blotting and mass spectrometry results revealed that TbPRMT6 methylates bovine histone H4 tail at arginine 3 but cannot methylate several T. brucei histone tails. In summary, our results highlight the structural differences between TbPRMT6 and other type I PRMTs and reveal that the active site rearrangement upon SAH binding is important for the substrate binding of TbPRMT6.

  8. Membranes

    OpenAIRE

    Junbo Hou; Min Yang

    2012-01-01

    Lithium ion batteries have proven themselves the main choice of power sources for portable electronics. Besides consumer electronics, lithium ion batteries are also growing in popularity for military, electric vehicle, and aerospace applications. The present review attempts to summarize the knowledge about some selected membranes in lithium ion batteries. Based on the type of electrolyte used, literature concerning ceramic-glass and polymer solid ion conductors, microporous filter type separa...

  9. Effect of diminazene aceturate, levamisole and vitamin C combination therapy in rats experimentally infected with Trypanosoma brucei brucei.

    Science.gov (United States)

    Chekwube, Adieme Ijeoma; Onyema, Ezeh Ikenna; Ikenna, Ugochukwu Emmanuel; Ezeokonkwo, Romanus Chukwuduruo

    2014-06-01

    To investigate the effect of diminazene aceturate (DA) alone or in combination with either levamisole and/or Vitamin C in albino rats experimentally infected with Trypanosoma brucei brucei. Thirty adult male albino rats, randomly assigned into 6 groups (A-F) of 5 rats each were used. They were either infected with 1×10(6) trypanosomes intraperitoneally (groups A-E) or uninfected (group F). The different groups were treated respectively as follows: group A-with 3.5 mg/kg DA; group B-3.5 mg/kg DA and 7.5 mg/kg levamisole; group C-3.5 mg/kg DA and 100 mg/kg vitamin C; and group D-3.5 mg/kg DA and 7.5 mg/kg levamisole and 100 mg/kg vitamin C. Group E was left untreated. Parameters assessed include: rectal temperature, body weight changes, packed cell volume (PCV), Haemoglobin concentration (Hb), total leucocyte count (TLC) differential leucocyte count (DLC), parasitaemia, clinical signs and survivability. Average pre-patent period of 5 days was recorded. Parasites in the blood were cleared in all treated groups (A-D) within 48 hours post treatment (PT). Untreated rats in group E died between 25 and 32 days post infection (PI). Relapse was not recorded in all the treated groups (A-D). The initial reduction in PCV, Hb, TLC and increases in rectal temperature following infection were reversed by the treatments. The rats that received drug combinations (groups B, C and D) showed faster and higher recovery rates than the uninfected control and group A. Levamisole and/or Vitamin C combination with DA were more effective in the treatment of rats infected with Trypanosoma brucei brucei. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  10. A model of mitochondrial creatine kinase binding to membranes: adsorption constants, essential amino acids and the effect of ionic strength

    DEFF Research Database (Denmark)

    Fedosov, Sergey; Belousova, Lubov; Plesner, Igor

    1993-01-01

    is is suggested as the main candidate to form the adsorption site of mitCK. Deprotonated octameric mitCK easily dissociated from the membrane (View the MathML source at ionic strength View the MathML source and 5°C); after protonation its affinity increased many times (View the MathML source). Determination...... on the enzyme adsorption. An analysis of our own data as well as of the data from the literature is consistent with the adsorption site of the octameric mitCK being composed of 4 amino acid residues with pK = 8.8 in the free enzyme. The pK value changes to 9.8 upon binding of the protein to the membrane. Lysine...

  11. Activation of c-Jun N-terminal kinase is essential for mitochondrial membrane potential change and apoptosis induced by doxycycline in melanoma cells

    Science.gov (United States)

    Shieh, Jiunn-Min; Huang, Tur-Fu; Hung, Chi-Feng; Chou, Kuan-Hsien; Tsai, Yih-Jeng; Wu, Wen-Bin

    2010-01-01

    Background and purpose: Tetracyclines were recently found to induce tumour cell death, but the early processes involved in this cytotoxic effect remain unclear. Experimental approach: Viability of human and mouse melanoma cells was determined by MTT assay and flow cytometry. Kinase/protein/caspase activation was measured by Western blotting and mitochondrial membrane potential (ΔΨm) was analyzed by fluorescence microscopy and flow cytometry. Key results: Human and mouse melanoma cells were treated with doxycycline or minocycline but only doxycycline was cytotoxic. This cell death (apoptosis) in A2058 cells involved activation of caspase-3, -7 and -9 and contributed to inhibition, by doxycycline, of matrix metalloproteinase (MMP) activity and migration of these cells. Doxycycline induced intra-cellular reactive oxygen species (ROS) production, apoptosis signal-regulated kinase 1 (ASK1), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activation at an early stage of treatment and induced mitochondrial cytochrome c release into cytosol and ΔΨm change during apoptosis. The JNK inhibitor/small interference RNA inhibited doxycycline-induced JNK activation, ΔΨm change and apoptosis, but did not affect ASK1 activation, suggesting a role of ASK1 for JNK activation in melanoma cell apoptosis. Two ROS scavengers reduced doxycycline-induced JNK and caspase activation, and apoptosis. Taken together, the results suggest the involvement of a ROS-ASK1-JNK pathway in doxycycline-induced melanoma cell apoptosis. Conclusions and implications: We have shown a promising cytotoxic effect of doxycycline on melanoma cells, have identified ROS and ASK1 as the possible initiators and have demonstrated that JNK activation is necessary for doxycycline-induced melanoma cell apoptosis. PMID:20590610

  12. trans-10,cis-12 Conjugated linoleic acid induces depolarization of mitochondrial membranes in HT-29 human colon cancer cells: a possible mechanism for induction of apoptosis.

    Science.gov (United States)

    Cho, Han Jin; Kwon, Gyoo Taik; Park, Jung Han Yoon

    2009-10-01

    Conjugated linoleic acid (CLA), which is naturally present in a variety of foods such as milk fat and the meat of ruminant animals, has been demonstrated to exert chemoprotective effects in several tissues in experimental animals. CLA is a collective term, which denotes one or more positional and geometric isomers of octadecadienoic acid, with cis-9,trans-11 (c9t11) and trans-10,cis-12 CLA (t10c12) being the principal isomers in commercial preparations. We observed previously that physiological levels of CLA inhibited HT-29 cell growth, and the growth inhibitory effects of CLA were attributed to the effect of t10c12, but not c9t11. In the present study, we assessed the mechanisms by which physiological levels of CLA and t10c12 induce apoptosis in HT-29 cells. HT-29 cells were cultured for 3 days in serum-free medium in the presence of various concentrations of CLA (0-20 micromol/L) or t10c12 (0-4 micromol/L). Addition of CLA or t10c12 to culture medium resulted in a dose-dependent increase in the numbers of apoptotic cells. The results of western blot analysis of total cell lysates showed that CLA and t10c12 increased the levels of cleaved caspase-9, caspase-3, and poly(ADP-ribose) polymerase but did not alter the levels of Bcl-2 family member proteins. However, these fatty acids were shown to increase the translocation of Bad and Bax to the mitochondria, increase mitochondrial membrane permeability, and induce the release of cytochrome c and Smac/Diablo from the mitochondria. In addition, CLA and t10c12 diminished Akt content and Akt phosphorylation. These findings indicate that physiological levels of t10c12 induce apoptosis in HT-29 colon cancer cells, which is mediated via mitochondrion-mediated events associated with a decline in Akt activity, an increase in the translocation of the pro-apototic Bad and Bax to the mitochondria, and the subsequent disruption of normal mitochondrial membrane potential.

  13. A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes.

    Directory of Open Access Journals (Sweden)

    Pavel Montes de Oca Balderas

    Full Text Available Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA receptor (NMDAR is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF labeling in rat cultured cortical astrocytes (rCCA of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa2+ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(--2-amino-5-phosphonopentanoic acid (APV and Kynurenic acid (KYNA and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase.

  14. Molecular basis for mitochondrial signaling

    CERN Document Server

    2017-01-01

    This book covers recent advances in the study of structure, function, and regulation of metabolite, protein and ion translocating channels, and transporters in mitochondria. A wide array of cutting-edge methods are covered, ranging from electrophysiology and cell biology to bioinformatics, as well as structural, systems, and computational biology. At last, the molecular identity of two important channels in the mitochondrial inner membrane, the mitochondrial calcium uniporter and the mitochondrial permeability transition pore have been established. After years of work on the physiology and structure of VDAC channels in the mitochondrial outer membrane, there have been multiple discoveries on VDAC permeation and regulation by cytosolic proteins. Recent breakthroughs in structural studies of the mitochondrial cholesterol translocator reveal a set of novel unexpected features and provide essential clues for defining therapeutic strategies. Molecular Basis for Mitochondrial Signaling covers these and many more re...

  15. Relevance of the inner mitochondrial membrane enzyme F1F0-ATPase as an autoantigen in autoimmune liver disorders.

    Science.gov (United States)

    Preuss, Beate; Berg, Christoph; Dengjel, Jörn; Stevanovic, Stefan; Klein, Reinhild

    2012-02-01

    Recently, a non-M2-related mitochondrial 60 kDa protein found to be recognized by antimitochondrial antibody (AMA) negative sera from patients with primary biliary cirrhosis (PBC) has been shown to contain parts of the five F(1)-ATPase subunits α, β, γ, δ and ε. In this study, we examined whether this enzyme is, indeed, a target antigen in PBC. Analysed were 60 AMA-positive/anti-M2-negative and 103 anti-M2-positive PBC patients, 46 patients with autoimmune hepatitis (AIH), 35 patients with primary sclerosing cholangitis (PSC), 110 patients with viral hepatitis, 40 patients with inflammatory bowel diseases (IBD), 33 patients with connective tissue diseases (systemic lupus erythematosus, mixed connective tissue disease, Sjögren disease, systemic sclerosis) and 25 blood donors. The F(1)-ATPase-subunits α-δ were recombinantly expressed in Escherichia coli, purified and applied to ELISA and Western blotting. In all, 40 of the 60 AMA-positive/anti-M2-negative (67%) and 44 (43%) of the 103 anti-M2-positive PBC-sera reacted with at least one of the F(1)-subunits α-δ. The β- and γ-subunits were preferentially recognized. However, also up to 57% of patients with AIH and 34% of patients with PSC had anti-β- or γ-antibodies, while patients with viral hepatitis had these antibodies in up to 13%. Patients with IBD had anti-β and anti-γ-antibodies in up to 20 and 5% respectively. None of the patients with connective tissue diseases had antibodies to the β- and only 6% to the γ-subunit. Sera from healthy blood donors were negative. Antibodies to the β- and γ-subunits of F(1)-ATPase are further AMAs in PBC but occur also in other autoimmune liver disorders; they may be, therefore, indicators for a general autoimmune process of the liver. © 2011 John Wiley & Sons A/S.

  16. The production of reactive oxygen species and the mitochondrial membrane potential are modulated during onion oil-induced cell cycle arrest and apoptosis in A549 cells.

    Science.gov (United States)

    Wu, Xin-jiang; Stahl, Thorsten; Hu, Ying; Kassie, Fekadu; Mersch-Sundermann, Volker

    2006-03-01

    Protective effects of Allium vegetables against cancers have been shown extensively in experimental animals and epidemiologic studies. We investigated cell proliferation and the induction of apoptosis by onion oil extracted from Allium cepa, a widely consumed Allium vegetable, in human lung cancer A549 cells. GC/MS analysis suggested that propyl sulfides but not allyl sulfides are major sulfur-containing constituents of onion oil. Onion oil at 12.5 mg/L significantly induced apoptosis (13% increase of apoptotic cells) as indicated by sub-G1 DNA content. It also caused cell cycle arrest at the G2/M phase; 25 mg/L onion oil increased the percentage of G2/M cells almost 6-fold compared with the dimethyl sulfoxide control. The action of onion oil may occur via a reactive oxygen species-dependent pathway because cell cycle arrest and apoptosis were blocked by the antioxidants N-acetylcysteine and exogenous glutathione. Marked collapse of the mitochondrial membrane potential suggested that dysfunction of the mitochondria may be involved in the oxidative burst and apoptosis induced by onion oil. Expression of phospho-cdc2 and phospho-cyclin B1 were downregulated by onion oil, perhaps accounting for the G2/M arrest. Overall, these results suggest that onion oil may exert chemopreventive action by inducing cell cycle arrest and apoptosis in tumor cells.

  17. Cleavage of Bid by executioner caspases mediates feed forward amplification of mitochondrial outer membrane permeabilization during genotoxic stress-induced apoptosis in Jurkat cells.

    Science.gov (United States)

    Shelton, Shary N; Shawgo, Mary E; Robertson, John D

    2009-04-24

    The extent to which the BH3-only protein Bid is important for intrinsic (mitochondria-mediated) apoptotic cell death induced by genotoxic stress remains controversial. In the present study, we examine this issue using a panel of gene-manipulated Bax-deficient Jurkat T-lymphocytes. Cells stably depleted of Bid were far less sensitive than control-transfected cells to etoposide-induced apoptosis. In particular, drug-induced Bak activation, cytochrome c release, loss of mitochondrial membrane potential, and caspase activation were all decreased in cells lacking Bid. Reconstitution experiments using recombinant proteins and permeabilized Bid-deficient cells demonstrated that truncated Bid (tBid), but not full-length Bid, potently induced Bak activation and the release of cytochrome c. Further, caspase-8-deficient Jurkat cells efficiently cleaved Bid and were sensitive to drug-induced apoptosis. By comparison, Apaf-1-deficient cells, as well as cells overexpressing full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP, failed to cleave Bid in response to genotoxic stress. These data suggest that tBid plays an important regulatory role in the execution of DNA damage-induced cytochrome c release and apoptosis. However, the fact that cleavage of Bid to tBid is mediated by executioner caspases suggests that a self-amplifying feed forward loop involving caspases, Bid, and mitochondria may help determine irreversible commitment to apoptosis.

  18. Determination of high mitochondrial membrane potential in spermatozoa loaded with the mitochondrial probe 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) using flow cytometry.

    Science.gov (United States)

    A flow cytometric method was developed to identify viable, energized sperm cells with high mitochondrial inner transmembrane potential (''m), > 80-100 mV using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and the impermeant nuclear ...

  19. Caenorhabditis elegans ATPase inhibitor factor 1 (IF1 MAI-2 preserves the mitochondrial membrane potential (Δψm and is important to induce germ cell apoptosis.

    Directory of Open Access Journals (Sweden)

    L P Fernández-Cárdenas

    Full Text Available When the electrochemical proton gradient is disrupted in the mitochondria, IF1 (Inhibitor Factor-1 inhibits the reverse hydrolytic activity of the F1Fo-ATP synthase, thereby allowing cells to conserve ATP at the expense of losing the mitochondrial membrane potential (Δψm. The function of IF1 has been studied mainly in different cell lines, but these studies have generated contrasting results, which have not been helpful to understand the real role of this protein in a whole organism. In this work, we studied IF1 function in Caenorhabditis elegans to understand IF1´s role in vivo. C. elegans has two inhibitor proteins of the F1Fo-ATPase, MAI-1 and MAI-2. To determine their protein localization in C. elegans, we generated translational reporters and found that MAI-2 is expressed ubiquitously in the mitochondria; conversely, MAI-1 was found in the cytoplasm and nuclei of certain tissues. By CRISPR/Cas9 genome editing, we generated mai-2 mutant alleles. Here, we showed that mai-2 mutant animals have normal progeny, embryonic development and lifespan. Contrasting with the results previously obtained in cell lines, we found no evident defects in the mitochondrial network, dimer/monomer ATP synthase ratio, ATP concentration or respiration. Our results suggest that some of the roles previously attributed to IF1 in cell lines could not reflect the function of this protein in a whole organism and could be attributed to specific cell lines or methods used to silence, knockout or overexpress this protein. However, we did observe that animals lacking IF1 had an enhanced Δψm and lower physiological germ cell apoptosis. Importantly, we found that mai-2 mutant animals must be under stress to observe the role of IF1. Accordingly, we observed that mai-2 mutant animals were more sensitive to heat shock, oxidative stress and electron transport chain blockade. Furthermore, we observed that IF1 is important to induce germ cell apoptosis under certain types of

  20. Minimum Information Loss Based Multi-kernel Learning for Flagellar Protein Recognition in Trypanosoma Brucei

    KAUST Repository

    Wang, Jim Jing-Yan

    2014-12-01

    Trypanosma brucei (T. Brucei) is an important pathogen agent of African trypanosomiasis. The flagellum is an essential and multifunctional organelle of T. Brucei, thus it is very important to recognize the flagellar proteins from T. Brucei proteins for the purposes of both biological research and drug design. In this paper, we investigate computationally recognizing flagellar proteins in T. Brucei by pattern recognition methods. It is argued that an optimal decision function can be obtained as the difference of probability functions of flagella protein and the non-flagellar protein for the purpose of flagella protein recognition. We propose to learn a multi-kernel classification function to approximate this optimal decision function, by minimizing the information loss of such approximation which is measured by the Kull back-Leibler (KL) divergence. An iterative multi-kernel classifier learning algorithm is developed to minimize the KL divergence for the problem of T. Brucei flagella protein recognition, experiments show its advantage over other T. Brucei flagellar protein recognition and multi-kernel learning methods. © 2014 IEEE.

  1. Telomeric expression sites are highly conserved in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Christiane Hertz-Fowler

    Full Text Available Subtelomeric regions are often under-represented in genome sequences of eukaryotes. One of the best known examples of the use of telomere proximity for adaptive purposes are the bloodstream expression sites (BESs of the African trypanosome Trypanosoma brucei. To enhance our understanding of BES structure and function in host adaptation and immune evasion, the BES repertoire from the Lister 427 strain of T. brucei were independently tagged and sequenced. BESs are polymorphic in size and structure but reveal a surprisingly conserved architecture in the context of extensive recombination. Very small BESs do exist and many functioning BESs do not contain the full complement of expression site associated genes (ESAGs. The consequences of duplicated or missing ESAGs, including ESAG9, a newly named ESAG12, and additional variant surface glycoprotein genes (VSGs were evaluated by functional assays after BESs were tagged with a drug-resistance gene. Phylogenetic analysis of constituent ESAG families suggests that BESs are sequence mosaics and that extensive recombination has shaped the evolution of the BES repertoire. This work opens important perspectives in understanding the molecular mechanisms of antigenic variation, a widely used strategy for immune evasion in pathogens, and telomere biology.

  2. Mitochondrial small conductance SK2 channels prevent glutamate-induced oxytosis and mitochondrial dysfunction.

    Science.gov (United States)

    Dolga, Amalia M; Netter, Michael F; Perocchi, Fabiana; Doti, Nunzianna; Meissner, Lilja; Tobaben, Svenja; Grohm, Julia; Zischka, Hans; Plesnila, Nikolaus; Decher, Niels; Culmsee, Carsten

    2013-04-12

    Small conductance calcium-activated potassium (SK2/K(Ca)2.2) channels are known to be located in the neuronal plasma membrane where they provide feedback control of NMDA receptor activity. Here, we provide evidence that SK2 channels are also located in the inner mitochondrial membrane of neuronal mitochondria. Patch clamp recordings in isolated mitoplasts suggest insertion into the inner mitochondrial membrane with the C and N termini facing the intermembrane space. Activation of SK channels increased mitochondrial K(+) currents, whereas channel inhibition attenuated these currents. In a model of glutamate toxicity, activation of SK2 channels attenuated the loss of the mitochondrial transmembrane potential, blocked mitochondrial fission, prevented the release of proapoptotic mitochondrial proteins, and reduced cell death. Neuroprotection was blocked by specific SK2 inhibitory peptides and siRNA targeting SK2 channels. Activation of mitochondrial SK2 channels may therefore represent promising targets for neuroprotective strategies in conditions of mitochondrial dysfunction.

  3. Mitochondrial Small Conductance SK2 Channels Prevent Glutamate-induced Oxytosis and Mitochondrial Dysfunction*

    Science.gov (United States)

    Dolga, Amalia M.; Netter, Michael F.; Perocchi, Fabiana; Doti, Nunzianna; Meissner, Lilja; Tobaben, Svenja; Grohm, Julia; Zischka, Hans; Plesnila, Nikolaus; Decher, Niels; Culmsee, Carsten

    2013-01-01

    Small conductance calcium-activated potassium (SK2/KCa2.2) channels are known to be located in the neuronal plasma membrane where they provide feedback control of NMDA receptor activity. Here, we provide evidence that SK2 channels are also located in the inner mitochondrial membrane of neuronal mitochondria. Patch clamp recordings in isolated mitoplasts suggest insertion into the inner mitochondrial membrane with the C and N termini facing the intermembrane space. Activation of SK channels increased mitochondrial K+ currents, whereas channel inhibition attenuated these currents. In a model of glutamate toxicity, activation of SK2 channels attenuated the loss of the mitochondrial transmembrane potential, blocked mitochondrial fission, prevented the release of proapoptotic mitochondrial proteins, and reduced cell death. Neuroprotection was blocked by specific SK2 inhibitory peptides and siRNA targeting SK2 channels. Activation of mitochondrial SK2 channels may therefore represent promising targets for neuroprotective strategies in conditions of mitochondrial dysfunction. PMID:23430260

  4. Antitrypanosomal effect of methanolic extract of Zingiber officinale (ginger on Trypanosoma brucei brucei-infected Wistar mice

    Directory of Open Access Journals (Sweden)

    P. I. Kobo

    2014-10-01

    Full Text Available Aim: The study was carried out to determine the in vivo antitrypanosomal effect of methanolic extract of Zingiber officinale (ginger in Trypanosoma brucei brucei-infected mice. Materials and Methods: Twenty-five mice were randomly allocated into five groups of five animals each. Group I and II were given Tween 80 (1 ml/kg and diminazene aceturate (3.5 mg/kg to serve as untreated and treated controls, respectively. Groups III-V received the extract at 200, 400 and 800 mg/kg body weight, respectively. All treatments were given for 6 consecutive days and through the oral route. The mean body weight, mean survival period and daily level of parasitaemia were evaluated. Results: Acute toxicity showed the extract to be relatively safe. There was an insignificant increase in body weight and survival rate of mice treated with the extract. The level of parasitaemia in the extract treated groups was decreased. Conclusion: This study shows the in vivo potential of methanolic extract of Z. officinale in the treatment of trypanosomiasis.

  5. Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins.

    Directory of Open Access Journals (Sweden)

    Cornelia Klein

    Full Text Available We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites.

  6. Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation

    NARCIS (Netherlands)

    Davila, M Plaza; Muñoz, P Martin; Bolaños, J M Gallardo; Stout, T A E|info:eu-repo/dai/nl/304828831; Gadella, B M|info:eu-repo/dai/nl/115389873; Tapia, J A; da Silva, C Balao; Ferrusola, C Ortega; Peña, F J

    2016-01-01

    To investigate the hypothesis that oxidative phosphorylation is a major source of ATP to fuel stallion sperm motility, oxidative phosphorylation was suppressed using the mitochondrial uncouplers CCCP and 2,4,-dinitrophenol (DNP) and by inhibiting mitochondrial respiration at complex IV using sodium

  7. In or out? On the tightness of glycosomal compartmentalizatin of metabolites and enzymes in Trypanosoma brucei.

    NARCIS (Netherlands)

    Haanstra, J.R.; Bakker, B.M.; Michels, P.A.M.

    2014-01-01

    Trypanosomatids sequester large parts of glucose metabolism inside specialised peroxisomes, called glycosomes. Many studies have shown that correct glycosomal compartmentalization of glycolytic enzymes is essential for bloodstream-form Trypanosoma brucei. The recent finding of pore-forming

  8. 1H, 13C and 15N resonance assignment of Urm1 from Trypanosoma brucei.

    Science.gov (United States)

    Zhang, Wen; Ding, Husheng; Zhang, Jiahai; Tu, Xiaoming

    2008-06-01

    Urm1 (ubiquitin-related modifier), involved in diverse biological processes in yeast, is proved to be a "molecular fossil" in ubiquitin superfamily. Here we report the resonance assignment of Urm1 from Trypanosoma brucei.

  9. The Phosphoproteome of Bloodstream Form Trypanosoma brucei, Causative Agent of African Sleeping Sickness

    OpenAIRE

    Nett, Isabelle R. E.; Martin, David M. A.; Miranda-Saavedra, Diego; Lamont, Douglas; Barber, Jonathan D.; Mehlert, Angela; Ferguson, Michael A. J.

    2009-01-01

    The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of severa...

  10. Steroid Alkaloids from Holarrhena africana with Strong Activity against Trypanosoma brucei rhodesiense

    OpenAIRE

    Charles Okeke Nnadi; Ngozi Justina Nwodo; Marcel Kaiser; Reto Brun; Thomas J. Schmidt

    2017-01-01

    In our continued search for natural compounds with activity against Trypanosoma brucei, causative agent of human African trypanosomiasis (HAT, “sleeping sickness”), we have investigated extracts from the leaves and bark of the West African Holarrhena africana (syn. Holarrhena floribunda; Apocynaceae). The extracts and their alkaloid-enriched fractions displayed promising in vitro activity against bloodstream forms of T. brucei rhodesiense (Tbr; East African HAT). Bioactivity-guided chromatogr...

  11. β-elemene reverses the drug resistance of A549/DDP lung cancer cells by activating intracellular redox system, decreasing mitochondrial membrane potential and P-glycoprotein expression, and inducing apoptosis.

    Science.gov (United States)

    Yao, Chengcai; Jiang, Jie; Tu, Yuanrong; Ye, Shefang; Du, Haoxin; Zhang, Yi

    2014-07-01

    β-elemene (β-ELE) injection is a new anticancer drug extracted from Curcuma zedoaria Roscoe that has been widely used to treat malignant tumors. Recent studies show that β-ELE reverses the drug resistance of tumor cells. To explore the possible mechanisms of β-ELE, we investigated its effects on cisplatin (DDP)-resistant human lung adenocarcinoma A549/DDP cells. The effects of β-ELE on the growth of A549/DDP cells in vitro were determined by MTT assay. Apoptosis was assessed by fluorescence microscopy with Hoechst 33258 staining, flow cytometry with Annexin V-FITC/propium iodide double staining; mitochondrial membrane potential using JC-1 fluorescence probe and laser confocal scanning microscopy, and intracellular reactive oxygen species levels were measured by 2',7'-dichlorfluorescein-diacetate staining and flow cytometry; and contents of cytosolic glutathione were determined by glutathione assay kits. Intracellular Rhodamine-123 fluorescence intensity was detected by flow cytometry, and the expression of P-glycoprotein (P-gp) was detected by Western blotting. β-ELE inhibited the proliferation of A549/DDP cells in a time- and dose-dependent manner. Furthermore, β-ELE enhanced the sensitivity of A549/DDP cells to cisplatin and reversed the drug resistance of A549/DDP cells. Consistent with a role in activating apoptosis, β-ELE decreased mitochondrial membrane potential, increased intracellular reactive oxygen species concentration and intracellular accumulation of Rhodamine-123, decreased the cytoplasmic glutathione levels and the expression of P-gp in a time- and dose-dependent manner. These results define a pathway of β-ELE function that involves decreased mitochondrial membrane potential and P-gp expression activated intracellular redox system, and induced apoptosis leading to reverse drug resistance.

  12. Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies.

    Science.gov (United States)

    Arakaki, Tracy L; Buckner, Frederick S; Gillespie, J Robert; Malmquist, Nicholas A; Phillips, Margaret A; Kalyuzhniy, Oleksandr; Luft, Joseph R; Detitta, George T; Verlinde, Christophe L M J; Van Voorhis, Wesley C; Hol, Wim G J; Merritt, Ethan A

    2008-04-01

    Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluorouracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection. Specifically, a combination therapy including a T. brucei-specific DHODH inhibitor plus 5-FU may prove to be an effective therapeutic strategy. We also show that this trypanosomal enzyme is inhibited by known inhibitors of bacterial Class 1A DHODH, in distinction to the sensitivity of DHODH from human and other higher eukaryotes. This selectivity is supported by the crystal structure of the T. brucei enzyme, which is reported here at a resolution of 1.95 A. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH.

  13. Mitochondrial haplogroups

    DEFF Research Database (Denmark)

    Benn, Marianne; Schwartz, Marianne; Nordestgaard, Børge G

    2008-01-01

    Rare mutations in the mitochondrial genome may cause disease. Mitochondrial haplogroups defined by common polymorphisms have been associated with risk of disease and longevity. We tested the hypothesis that common haplogroups predict risk of ischemic cardiovascular disease, morbidity from other...

  14. Kinetic activity, membrane mitochondrial potential, lipid peroxidation, intracellular pH and calcium of frozen/thawed bovine spermatozoa treated with metabolic enhancers.

    Science.gov (United States)

    Boni, R; Gallo, A; Cecchini, S

    2017-01-01

    Owing to the progressive decline of sperm motility during storage there is a need to find substances capable of enhancing sperm energy metabolism and motility and/or preserving it from oxidative damage. The aim of this study was to evaluate in frozen/thawed bovine spermatozoa the effect of several compounds, such as myo-inositol, pentoxifylline, penicillamine + hypotaurine + epinephrine mixture (PHE), caffeine and coenzyme Q10+ zinc + d-aspartate mixture (CZA), on either kinetic or metabolic parameters. Sperm kinetics was evaluated by Sperm Class Analyser whereas specific fluorochromes were used to evaluated mitochondrial membrane potential (MMP), intracellular pH, intracellular calcium concentration and lipid peroxidation. Lipid peroxidation was also evaluated by TBARS analysis. Treatments significantly affected total and progressive motility with different dynamics in relation to the incubation time. After the first hour of incubation, CZA treatment produced the best performance in total and progressive sperm motility as well as in curvilinear velocity, average path velocity and amplitude of head displacement, whereas pentoxifylline stimulated the highest straight-line velocity. MMP showed higher values (p lipid peroxidation were significantly (p < 0.05) affected by the incubation time rather than the treatments. Intracellular pH varied significantly (p < 0.01) in relation to either the incubation time or treatments. In particular, it showed a progressive increase throughout incubation with values in control group significantly higher than in myo-inositol, PHE, caffeine, pentoxifylline and CZA groups (7.37 ± 0.03 vs. 7.29 ± 0.03, 7.28 ± 0.03, 7.26 ± 0.03, 7.22 ± 0.03 and 7.00 ± 0.03, respectively; p < 0.01).; however, among treatments, CZA displayed the lowest values. Significant correlations were found between sperm kinetic and metabolic parameters. These findings provide new comparative information on the effects of putative metabolic

  15. Grape proanthocyanidins induce apoptosis by loss of mitochondrial membrane potential of human non-small cell lung cancer cells in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Tripti Singh

    Full Text Available Lung cancer remains the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC represents approximately 80% of total lung cancer cases. The use of non-toxic dietary phytochemicals can be considered as a chemotherapeutic strategy for the management of the NSCLC. Here, we report that grape seed proanthocyanidins (GSPs induce apoptosis of NSCLC cells, A549 and H1299, in vitro which is mediated through increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose polymerase (PARP. Pre-treatment of A549 and H1299 cells with the caspase-3 inhibitor (z-DEVD-fmk significantly blocked the GSPs-induced apoptosis of these cells confirmed that GSPs-induced apoptosis is mediated through activation of caspases-3. Treatments of A549 and H1299 cells with GSPs resulted in an increase in G1 arrest. G0/G1 phase of the cell cycle is known to be controlled by cyclin dependent kinases (Cdk, cyclin-dependent kinase inhibitors (Cdki and cyclins. Our western blot analyses showed that GSPs-induced G1 cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27, and a simultaneous decrease in the levels of Cdk2, Cdk4, Cdk6 and cyclins. Further, administration of 50, 100 or 200 mg GSPs/kg body weight of mice by oral gavage (5 d/week markedly inhibited the growth of s.c. A549 and H1299 lung tumor xenografts in athymic nude mice, which was associated with the induction of apoptotic cell death, increased expression of Bax, reduced expression of anti-apoptotic proteins and activation of caspase-3 in tumor xenograft cells. Based on the data obtained in animal study, human equivalent dose of GSPs was calculated, which seems affordable and attainable. Together, these results suggest that GSPs may represent a potential therapeutic agent for the non

  16. Role of oxidative stress, mitochondrial membrane potential, and calcium homeostasis in human lymphocyte death induced by nickel carbonate hydroxide in vitro

    Energy Technology Data Exchange (ETDEWEB)

    M' Bemba-Meka, Prosper [Faculty of Medicine, Universite de Montreal, QC (Canada); University of Louisville, Department of Pharmacology and Toxicology, Center for Genetics and Molecular Medicine, Louisville, KY (United States); Lemieux, Nicole [Universite de Montreal, Department of Pathology and Cellular Biology, Main Station, P.O. Box 6128, Montreal, QC (Canada); Chakrabarti, Saroj K. [Faculty of Medicine, Universite de Montreal, QC (Canada)

    2006-07-15

    When isolated human lymphocytes were treated in vitro with various concentrations of soluble form of nickel carbonate hydroxide (NiCH) (0-1 mM), at 37 C for 4 h, both concentration- and time-dependent effects of NiCH on lymphocyte death were observed. Increased generation of hydrogen peroxide (H{sub 2}O{sub 2}), superoxide anion (O{sub 2} {sup -}), depletion of both no protein (NP-) and protein (P-) sulfhydryl (SH) contents and lipid peroxidation (LPO) were induced by NiCH. Pretreatment of lymphocytes with either catalase (H{sub 2}O{sub 2} scavenger), or deferoxamine (DFO) (iron chelator), or excess glutathione (GSH) (an antioxidant) not only significantly reduced the NiCH-induced generation of H{sub 2}O{sub 2} and LPO, but also increased the NP-SH and P-SH contents initially reduced by NiCH. NiCH-induced generation of excess O{sub 2} {sup -} but not excess LPO was significantly reduced by pretreatment with superoxide dismutase (SOD). NiCH-induced lymphocyte death was significantly prevented by pre-treatment with either catalase, or dimethylthiourea/mannitol (hydroxyl radical scavengers), or DFO, or excess GSH/N-acetylcysteine. NiCH-induced lymphocyte death was also significantly prevented by pretreatment with excess SOD. Thus, various types of oxidative stresses play an important role in NiCH-induced lymphocyte death. Cotreatment with cyclosporin A, a specific inhibitor of alteration in mitochondrial membrane potential ({delta}{psi}{sub m}), not only inhibited NiCH-induced alteration in {delta}{psi}{sub m}, but also significantly prevented Ni-compound-induced lymphocyte death. Furthermore, NiCH-induced destabilization of cellular calcium homeostasis. As such, NiCH-induced lymphocyte death was significantly prevented by modulating intracellular calcium fluxes such as Ca{sup 2+} channel blockers and intracellular Ca{sup 2+} antagonist. Thus, the mechanism of NiCH (soluble form)-induced activation of lymphocyte death signalling pathways involves not only the excess

  17. Effect of short-term exposure to diesel exhaust particles and carboxylic acids on mitochondrial membrane disruption in airway epithelial cells

    Science.gov (United States)

    Rationale: Diesel exhaust has been shown to induce adverse pulmonary health effects; however, the underlying mechanisms for these effects are still unclear. Previous studies have imlplicated mitochondrial dysfunction in the toxicity of diesel exhaust particles (DEP). DEP contain...

  18. Regulators of Trypanosoma brucei cell cycle progression and differentiation identified using a kinome-wide RNAi screen.

    Directory of Open Access Journals (Sweden)

    Nathaniel G Jones

    2014-01-01

    Full Text Available The African trypanosome, Trypanosoma brucei, maintains an integral link between cell cycle regulation and differentiation during its intricate life cycle. Whilst extensive changes in phosphorylation have been documented between the mammalian bloodstream form and the insect procyclic form, relatively little is known about the parasite's protein kinases (PKs involved in the control of cellular proliferation and differentiation. To address this, a T. brucei kinome-wide RNAi cell line library was generated, allowing independent inducible knockdown of each of the parasite's 190 predicted protein kinases. Screening of this library using a cell viability assay identified ≥42 PKs that are required for normal bloodstream form proliferation in culture. A secondary screen identified 24 PKs whose RNAi-mediated depletion resulted in a variety of cell cycle defects including in G1/S, kinetoplast replication/segregation, mitosis and cytokinesis, 15 of which are novel cell cycle regulators. A further screen identified for the first time two PKs, named repressor of differentiation kinase (RDK1 and RDK2, depletion of which promoted bloodstream to procyclic form differentiation. RDK1 is a membrane-associated STE11-like PK, whilst RDK2 is a NEK PK that is essential for parasite proliferation. RDK1 acts in conjunction with the PTP1/PIP39 phosphatase cascade to block uncontrolled bloodstream to procyclic form differentiation, whilst RDK2 is a PK whose depletion efficiently induces differentiation in the absence of known triggers. Thus, the RNAi kinome library provides a valuable asset for functional analysis of cell signalling pathways in African trypanosomes as well as drug target identification and validation.

  19. Characterization of Two Mitochondrial Flavin Adenine Dinucleotide-Dependent Glycerol-3-Phosphate Dehydrogenases in Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Škodová, Ingrid; Verner, Zdeněk; Bringaud, F.; Fabian, P.; Lukeš, Julius; Horváth, A.

    2013-01-01

    Roč. 12, č. 12 (2013), s. 1664-1673 ISSN 1535-9778 R&D Projects: GA ČR(CZ) GAP305/11/2179; GA ČR GD206/09/H026; GA MŠk LH12104 Institutional support: RVO:60077344 Keywords : alternative NADH dehydrogenase * inducible expression system * blood-stream forms * complex-I * procyclic trypanosomes * sleeping sickness * oxidase * localization * metabolism * cycle Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.179, year: 2013

  20. Paclitaxel resistance development is associated with biphasic changes in reactive oxygen species, mitochondrial membrane potential and autophagy with elevated energy production capacity in lung cancer cells: A chronological study.

    Science.gov (United States)

    Datta, Satabdi; Choudhury, Diptiman; Das, Amlan; Das Mukherjee, Dipanwita; Das, Nabanita; Roy, Sib Sankar; Chakrabarti, Gopal

    2017-02-01

    Paclitaxel (Tx) is one of the first-line chemotherapeutic drugs used against lung cancer, but acquired resistance to this drug is a major challenge against successful chemotherapy. In this work, we have focused on the chronological changes of various cellular parameters and associated effect on Tx (10 nM) resistance development in A549 cell line. It was observed, at initial stage, the cell death percentage due to drug treatment had increased up to 20 days, and thereafter, it started declining and became completely resistant by 40 days. Expressions of βIII tubulin and drug efflux pumps also increased over the period of resistance development. Changes in cellular autophagy and reactive oxygen species generation showed a biphasic pattern and increased gradually over the course of upto 20 days, thereafter declined gradually; however, their levels remained higher than untreated cells when resistance was acquired. Increase in extracellular acidification rates and oxygen consumption rates was found to be directly correlated with acquisition of resistance. The depolarisation of mitochondrial membrane potential was also biphasic; first, it increased with increase of cell death up to 20 days, thereafter, it gradually decreased to normal level along with resistance development. Increase in activity of catalase, glutathione peroxidase and glutathione content over these periods may attribute in bringing down the reactive oxygen species levels and normalisation of mitochondrial membrane potential in spite of comparatively higher reactive oxygen species production by the Tx-resistant cells.

  1. Mitochondrial vasculopathy.

    Science.gov (United States)

    Finsterer, Josef; Zarrouk-Mahjoub, Sinda

    2016-05-26

    Mitochondrial disorders (MIDs) are usually multisystem disorders (mitochondrial multiorgan disorder syndrome) either on from onset or starting at a point during the disease course. Most frequently affected tissues are those with a high oxygen demand such as the central nervous system, the muscle, endocrine glands, or the myocardium. Recently, it has been shown that rarely also the arteries may be affected (mitochondrial arteriopathy). This review focuses on the type, diagnosis, and treatment of mitochondrial vasculopathy in MID patients. A literature search using appropriate search terms was carried out. Mitochondrial vasculopathy manifests as either microangiopathy or macroangiopathy. Clinical manifestations of mitochondrial microangiopathy include leukoencephalopathy, migraine-like headache, stroke-like episodes, or peripheral retinopathy. Mitochondrial macroangiopathy manifests as atherosclerosis, ectasia of arteries, aneurysm formation, dissection, or spontaneous rupture of arteries. The diagnosis relies on the documentation and confirmation of the mitochondrial metabolic defect or the genetic cause after exclusion of non-MID causes. Treatment is not at variance compared to treatment of vasculopathy due to non-MID causes. Mitochondrial vasculopathy exists and manifests as micro- or macroangiopathy. Diagnosing mitochondrial vasculopathy is crucial since appropriate treatment may prevent from severe complications.

  2. Glucose uptake occurs by facilitated diffusion in procyclic forms of Trypanosoma brucei.

    Science.gov (United States)

    Wille, U; Seyfang, A; Duszenko, M

    1996-02-15

    The glucose transporter of Trypanosoma brucei procyclic forms was characterized and compared with its bloodstream form counterpart. Measuring the glucose consumption enzymatically, we determined a saturable uptake process of relatively high affinity (Km = 80 microM, Vmax = 4 nmol min-1 10(-8) cells), which showed substrate inhibition at glucose concentrations above 1.5 mM (Ki = 21 mM). Control experiments measuring deoxy-D-[3H]Glc uptake under zero-trans conditions indicated that substrate inhibition occurred on the level of glycolysis. Temperature-dependent kinetics revealed a temperature quotient of Q10 = 2.33 and an activation energy of Ea = 64 kJ mol-1. As shown by trans-stimulation experiments, glucose uptake was stereospecific for the D isomer, whereas L-glucose was not recognized. Inhibitor studies using either the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (5 microM), the H+/ATPase inhibitor N,N'-dicyclohexylcarbodiimide (20 microM), the ionophor monensin (1 microM), or the Na+/K+-ATPase inhibitor ouabain (1 mM) showed insignificant effects on transport efficiency. The procyclic glucose transporter was subsequently enriched in a plasma-membrane fraction and functionally reconstituted into proteoliposomes. Using Na+-free conditions in the absence of a proton gradient, the specific activity of D-[14C]glucose transport was determined as 2.9 nmol min-1 (mg protein)-1 at 0.2 mM glucose. From these cumulative results, we conclude that glucose uptake by the procyclic insect form of the parasite occurs by facilitated diffusion, similar to the hexose-transport system expressed in bloodstream forms. However, the markedly higher substrate affinity indicates a differential expression of different transporter isoforms throughout the lifecycle.

  3. Role of centrins 2 and 3 in organelle segregation and cytokinesis in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Angamuthu Selvapandiyan

    Full Text Available Centrins are calcium binding proteins involved in cell division in eukaryotes. Previously, we have shown that depletion of centrin1 in Trypanosoma brucei (T. brucei displayed arrested organelle segregation resulting in loss of cytokinesis. In this study we analyzed the role of T. brucei centrin2 (TbCen2 and T. brucei 3 (TbCen3 in the early events of T. brucei procyclic cell cycle. Both the immunofluorescence assay and electron microscopy showed that TbCen2 and 3-deficient cells were enlarged in size with duplicated basal bodies, multinuclei and new flagella that are detached along the length of the cell body. In both TbCen2 and TbCen3 depleted cells segregation of the organelles i.e. basal bodies, kinetoplast and nucleus was disrupted. Further analysis of the cells with defective organelle segregation identified three different sub configurations of organelle mis-segregations (Type 1-3. In addition, in majority of the TbCen2 depleted cells and in nearly half of the TbCen3 depleted cells, the kinetoplasts were enlarged and undivided. The abnormal segregations ultimately led to aborted cytokinesis and hence affected growth in these cells. Therefore, both centrin2 and 3 are involved in organelle segregation similar to centrin1 as was previously observed. In addition, we identified their role in kinetoplast division which may be also linked to overall mis-segregation.

  4. Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model.

    Science.gov (United States)

    Dauchy, Frédéric-Antoine; Bonhivers, Mélanie; Landrein, Nicolas; Dacheux, Denis; Courtois, Pierrette; Lauruol, Florian; Daulouède, Sylvie; Vincendeau, Philippe; Robinson, Derrick R

    2016-11-01

    Trypanosoma brucei gambiense is the main causative agent of Human African Trypanosomiasis (HAT), also known as sleeping sickness. Because of limited alternatives and treatment toxicities, new therapeutic options are urgently needed for patients with HAT. Sterol 14alpha-demethylase (CYP51) is a potential drug target but its essentiality has not been determined in T. brucei. We used a tetracycline-inducible RNAi system to assess the essentiality of CYP51 in T. brucei bloodstream form (BSF) cells and we evaluated the effect of posaconazole, a well-tolerated triazole drug, within a panel of virulent strains in vitro and in a murine model. Expression of CYP51 in several T. brucei cell lines was demonstrated by western blot and its essentiality was demonstrated by RNA interference (CYP51RNAi) in vitro. Following reduction of TbCYP51 expression by RNAi, cell growth was reduced and eventually stopped compared to WT or non-induced cells, showing the requirement of CYP51 in T. brucei. These phenotypes were rescued by addition of ergosterol. Additionally, CYP51RNAi induction caused morphological defects with multiflagellated cells (pnifurtimox-eflornithine combinations showed similar improvement in mice survival (p≤0.001). Our results provide support for a CYP51 targeting based treatment in HAT. Thus posaconazole used in combination may represent a therapeutic alternative for trypanosomiasis.

  5. Mitochondrial Myopathy

    Science.gov (United States)

    ... symptoms of mitochondrial myopathies include muscle weakness or exercise intolerance, heart failure or rhythm disturbances, dementia, movement disorders, stroke-like episodes, deafness, blindness, droopy ...

  6. High-throughput Gene Tagging in Trypanosoma brucei.

    Science.gov (United States)

    Dyer, Philip; Dean, Samuel; Sunter, Jack

    2016-08-12

    Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

  7. Protective effect of humus extract against Trypanosoma brucei infection in mice.

    Science.gov (United States)

    Kodama, Hiroshi; Denso; Okazaki, Fumi; Ishida, Saeko

    2008-11-01

    Humic substances are formed during the decomposition of organic matter in humus, and are found in many natural environments in which organic materials and microorganisms are present. Oral administration of humus extract to mice successfully induced effective protection against experimental challenge by the two subspecies, Trypanosoma brucei brucei and T. brucei gambiense. Mortality was most reduced among mice who received a 3% humus extract for 21 days in drinking water ad libitum. Spleen cells from humus-administered mice exhibited significant non-specific cytotoxic activity against L1210 mouse leukemia target cells. Also, spleen cells produced significantly higher amounts of Interferon-gamma when stimulated in vitro with Concanavalin A than cells from normal controls. These results clearly show that administration to mice of humus extract induced effective resistance against Trypanosoma infection. Enhancement of the innate immune system may be involved in host defense against trypanosomiasis.

  8. Isolation and analysis of the genetic diversity of repertoires of VSG expression site containing telomeres from Trypanosoma brucei gambiense, T. b. brucei and T. equiperdum

    Directory of Open Access Journals (Sweden)

    Becker Marion

    2008-08-01

    Full Text Available Abstract Background African trypanosomes (including Trypanosoma brucei are unicellular parasites which multiply in the mammalian bloodstream. T. brucei has about twenty telomeric bloodstream form Variant Surface Glycoprotein (VSG expression sites (BESs, of which one is expressed at a time in a mutually exclusive fashion. BESs are polycistronic transcription units, containing a variety of families of expression site associated genes (ESAGs in addition to the telomeric VSG. These polymorphic ESAG families are thought to play a role in parasite-host adaptation, and it has been proposed that ESAG diversity might be related to host range. Analysis of the genetic diversity of these telomeric gene families has been confounded by the underrepresentation of telomeric sequences in standard libraries. We have previously developed a method to selectively isolate sets of trypanosome BES containing telomeres using Transformation associated recombination (TAR cloning in yeast. Results Here we describe the isolation of repertoires of BES containing telomeres from three trypanosome subspecies: Trypanosoma brucei gambiense DAL 972 (causative agent of West-African trypanosomiasis, T. b. brucei EATRO 2340 (a nonhuman infective strain and T. equiperdum STIB 818 (which causes a sexually transmitted disease in equines. We have sequenced and analysed the genetic diversity at four BES loci (BES promoter region, ESAG6, ESAG5 and ESAG2 from these three trypanosome BES repertoires. Conclusion With the exception of ESAG2, the BES sequence repertoires derived from T. b. gambiense are both less diverse than and nearly reciprocally monophyletic relative to those from T. b. brucei and T. equiperdum. Furthermore, although we find evidence for adaptive evolution in all three ESAG repertoires in T. b. brucei and T. equiperdum, only ESAG2 appears to be under diversifying selection in T. b. gambiense. This low level of variation in the T. b. gambiense BES sequence repertoires is

  9. Isolation and analysis of the genetic diversity of repertoires of VSG expression site containing telomeres from Trypanosoma brucei gambiense, T. b. brucei and T. equiperdum.

    Science.gov (United States)

    Young, Rosanna; Taylor, Jesse E; Kurioka, Ayako; Becker, Marion; Louis, Edward J; Rudenko, Gloria

    2008-08-12

    African trypanosomes (including Trypanosoma brucei) are unicellular parasites which multiply in the mammalian bloodstream. T. brucei has about twenty telomeric bloodstream form Variant Surface Glycoprotein (VSG) expression sites (BESs), of which one is expressed at a time in a mutually exclusive fashion. BESs are polycistronic transcription units, containing a variety of families of expression site associated genes (ESAGs) in addition to the telomeric VSG. These polymorphic ESAG families are thought to play a role in parasite-host adaptation, and it has been proposed that ESAG diversity might be related to host range. Analysis of the genetic diversity of these telomeric gene families has been confounded by the underrepresentation of telomeric sequences in standard libraries. We have previously developed a method to selectively isolate sets of trypanosome BES containing telomeres using Transformation associated recombination (TAR) cloning in yeast. Here we describe the isolation of repertoires of BES containing telomeres from three trypanosome subspecies: Trypanosoma brucei gambiense DAL 972 (causative agent of West-African trypanosomiasis), T. b. brucei EATRO 2340 (a nonhuman infective strain) and T. equiperdum STIB 818 (which causes a sexually transmitted disease in equines). We have sequenced and analysed the genetic diversity at four BES loci (BES promoter region, ESAG6, ESAG5 and ESAG2) from these three trypanosome BES repertoires. With the exception of ESAG2, the BES sequence repertoires derived from T. b. gambiense are both less diverse than and nearly reciprocally monophyletic relative to those from T. b. brucei and T. equiperdum. Furthermore, although we find evidence for adaptive evolution in all three ESAG repertoires in T. b. brucei and T. equiperdum, only ESAG2 appears to be under diversifying selection in T. b. gambiense. This low level of variation in the T. b. gambiense BES sequence repertoires is consistent both with the relatively narrow host range

  10. The use of yellow fluorescent hybrids to indicate mating in Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Ferris Vanessa

    2008-02-01

    Full Text Available Abstract Background Trypanosoma brucei undergoes genetic exchange in its insect vector, the tsetse fly, by an unknown mechanism. The difficulties of working with this experimental system of genetic exchange have hampered investigation, particularly because the trypanosome life cycle stages involved cannot be cultured in vitro and therefore must be examined in the insect. Searching for small numbers of hybrid trypanosomes directly in the fly has become possible through the incorporation of fluorescent reporter genes, and we have previously carried out a successful cross using a reporter-repressor strategy. However, we could not be certain that all fluorescent trypanosomes observed in that cross were hybrids, due to mutations of the repressor leading to spontaneous fluorescence, and we have therefore developed an alternative strategy. Results To visualize the production of hybrids in the fly, parental trypanosome clones were transfected with a gene encoding Green Fluorescent Protein (GFP or Red Fluorescent Protein (RFP. Co-infection of flies with red and green fluorescent parental trypanosomes produced yellow fluorescent hybrids, which were easily visualized in the fly salivary glands. Yellow trypanosomes were not seen in midgut or proventricular samples and first appeared in the glands as epimastigotes as early as 13 days after fly infection. Cloned progeny originating from individual salivary glands had yellow, red, green or no fluorescence and were confirmed as hybrids by microsatellite, molecular karyotype and kinetoplast (mitochondrial DNA analyses. Hybrid clones showed biparental inheritance of both nuclear and kinetoplast genomes. While segregation and reassortment of the reporter genes and microsatellite alleles were consistent with Mendelian inheritance, flow cytometry measurement of DNA content revealed both diploid and polyploid trypanosomes among the hybrid progeny clones. Conclusion The strategy of using production of yellow hybrids

  11. The role of the PI(3,5)P2 kinase TbFab1 in endo/lysosomal trafficking in Trypanosoma brucei.

    Science.gov (United States)

    Gilden, Julia K; Umaer, Khan; Kruzel, Emilia K; Hecht, Oliver; Correa, Renan O; Mansfield, John M; Bangs, James D

    2017-06-01

    Protein trafficking through endo/lysosomal compartments is critically important to the biology of the protozoan parasite Trypanosoma brucei, but the routes material may take to the lysosome, as well as the molecular factors regulating those routes, remain incompletely understood. Phosphoinositides are signaling phospholipids that regulate many trafficking events by recruiting specific effector proteins to discrete membrane subdomains. In this study, we investigate the role of one phosphoinositide, PI(3,5)P2 in T. brucei. We find a low steady state level of PI(3,5)P2 in bloodstream form parasites comparable to that of other organisms. RNAi knockdown of the putative PI(3)P-5 kinase TbFab1 decreases the PI(3,5)P2 pool leading to rapid cell death. TbFab1 and PI(3,5)P2 both localize strongly to late endo/lysosomes. While most trafficking functions were intact in TbFab1 deficient cells, including both endocytic and biosynthetic trafficking to the lysosome, lysosomal turnover of an endogenous ubiquitinylated membrane protein, ISG65, was completely blocked suggesting that TbFab1 plays a role in the ESCRT-mediated late endosomal/multivesicular body degradative pathways. Knockdown of a second component of PI(3,5)P2 metabolism, the PI(3,5)P2 phosphatase TbFig4, also resulted in delayed turnover of ISG65. Together, these results demonstrate an essential role for PI(3,5)P2 in the turnover of ubiquitinylated membrane proteins and in trypanosome endomembrane biology. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Mitochondrial respiration is sensitive to cytoarchitectural breakdown.

    Science.gov (United States)

    Kandel, Judith; Angelin, Alessia A; Wallace, Douglas C; Eckmann, David M

    2016-11-07

    An abundance of research suggests that cellular mitochondrial and cytoskeletal disruption are related, but few studies have directly investigated causative connections between the two. We previously demonstrated that inhibiting microtubule and microfilament polymerization affects mitochondrial motility on the whole-cell level in fibroblasts. Since mitochondrial motility can be indicative of mitochondrial function, we now further characterize the effects of these cytoskeletal inhibitors on mitochondrial potential, morphology and respiration. We found that although they did not reduce mitochondrial inner membrane potential, cytoskeletal toxins induced significant decreases in basal mitochondrial respiration. In some cases, basal respiration was only affected after cells were pretreated with the calcium ionophore A23187 in order to stress mitochondrial function. In most cases, mitochondrial morphology remained unaffected, but extreme microfilament depolymerization or combined intermediate doses of microtubule and microfilament toxins resulted in decreased mitochondrial lengths. Interestingly, these two particular exposures did not affect mitochondrial respiration in cells not sensitized with A23187, indicating an interplay between mitochondrial morphology and respiration. In all cases, inducing maximal respiration diminished differences between control and experimental groups, suggesting that reduced basal respiration originates as a largely elective rather than pathological symptom of cytoskeletal impairment. However, viability experiments suggest that even this type of respiration decrease may be associated with cell death.

  13. Use of Human Cancer Cell Lines Mitochondria to Explore the Mechanisms of BH3 Peptides and ABT-737-Induced Mitochondrial Membrane Permeabilization

    OpenAIRE

    Nelly Buron; Mathieu Porceddu; Magali Brabant; Diana Desgué; Cindy Racoeur; Myriam Lassalle; Christine Péchoux; Pierre Rustin; Etienne Jacotot; Annie Borgne-Sanchez

    2010-01-01

    Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria....

  14. Nanoparticle-Mediated Delivery of Mitochondrial Division Inhibitor 1 to the Myocardium Protects the Heart From Ischemia-Reperfusion Injury Through Inhibition of Mitochondria Outer Membrane Permeabilization: A New Therapeutic Modality for Acute Myocardial Infarction.

    Science.gov (United States)

    Ishikita, Ayako; Matoba, Tetsuya; Ikeda, Gentaro; Koga, Jun-Ichiro; Mao, Yajing; Nakano, Kaku; Takeuchi, Osamu; Sadoshima, Junichi; Egashira, Kensuke

    2016-07-22

    Mitochondria-mediated cell death plays a critical role in myocardial ischemia-reperfusion (IR) injury. We hypothesized that nanoparticle-mediated drug delivery of mitochondrial division inhibitor 1 (Mdivi1) protects hearts from IR injury through inhibition of mitochondria outer membrane permeabilization (MOMP), which causes mitochondrial-mediated cell death. We formulated poly (lactic-co-glycolic acid) nanoparticles containing Mdivi1 (Mdivi1-NP). We recently demonstrated that these nanoparticles could be successfully delivered to the cytosol and mitochondria of cardiomyocytes under H2O2-induced oxidative stress that mimicked IR injury. Pretreatment with Mdivi1-NP ameliorated H2O2-induced cell death in rat neonatal cardiomyocytes more potently than Mdivi1 alone, as indicated by a lower estimated half-maximal effective concentration and greater maximal effect on cell survival. Mdivi1-NP treatment of Langendorff-perfused mouse hearts through the coronary arteries at the time of reperfusion reduced infarct size after IR injury more effectively than Mdivi1 alone. Mdivi1-NP treatment also inhibited Drp1-mediated Bax translocation to the mitochondria and subsequent cytochrome c leakage into the cytosol, namely, MOMP, in mouse IR hearts. MOMP inhibition was also observed in cyclophilin D knockout (CypD-KO) mice, which lack the mitochondrial permeability transition pore (MPTP) opening. Intravenous Mdivi1-NP treatment in vivo at the time of reperfusion reduced IR injury in wild-type and CypD-KO mice, but not Bax-KO mice. Mdivi1-NP treatment reduced IR injury through inhibition of MOMP, even in the absence of a CypD/MPTP opening. Thus, nanoparticle-mediated drug delivery of Mdivi1 may be a novel treatment strategy for IR injury. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

  15. MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content

    Directory of Open Access Journals (Sweden)

    Elisa Balboa

    2017-08-01

    Full Text Available MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria.

  16. MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content.

    Science.gov (United States)

    Balboa, Elisa; Castro, Juan; Pinochet, María-José; Cancino, Gonzalo I; Matías, Nuria; Sáez, P J; Martínez, Alexis; Álvarez, Alejandra R; Garcia-Ruiz, Carmen; Fernandez-Checa, José C; Zanlungo, Silvana

    2017-08-01

    MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH) levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Melatonin and human mitochondrial diseases

    Directory of Open Access Journals (Sweden)

    Reza Sharafati-Chaleshtori

    2017-01-01

    Full Text Available Mitochondrial dysfunction is one of the main causative factors in a wide variety of complications such as neurodegenerative disorders, ischemia/reperfusion, aging process, and septic shock. Decrease in respiratory complex activity, increase in free radical production, increase in mitochondrial synthase activity, increase in nitric oxide production, and impair in electron transport system and/or mitochondrial permeability are considered as the main factors responsible for mitochondrial dysfunction. Melatonin, the pineal gland hormone, is selectively taken up by mitochondria and acts as a powerful antioxidant, regulating the mitochondrial bioenergetic function. Melatonin increases the permeability of membranes and is the stimulator of antioxidant enzymes including superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase. It also acts as an inhibitor of lipoxygenase. Melatonin can cause resistance to oxidation damage by fixing the microsomal membranes. Melatonin has been shown to retard aging and inhibit neurodegenerative disorders, ischemia/reperfusion, septic shock, diabetes, cancer, and other complications related to oxidative stress. The purpose of the current study, other than introducing melatonin, was to present the recent findings on clinical effects in diseases related to mitochondrial dysfunction including diabetes, cancer, gastrointestinal diseases, and diseases related to brain function.

  18. Aqueous cinnamon extract (ACE-c) from the bark of Cinnamomum cassia causes apoptosis in human cervical cancer cell line (SiHa) through loss of mitochondrial membrane potential.

    Science.gov (United States)

    Koppikar, Soumya J; Choudhari, Amit S; Suryavanshi, Snehal A; Kumari, Shweta; Chattopadhyay, Samit; Kaul-Ghanekar, Ruchika

    2010-05-18

    Chemoprevention, which includes the use of synthetic or natural agents (alone or in combination) to block the development of cancer in human beings, is an extremely promising strategy for cancer prevention. Cinnamon is one of the most widely used herbal medicines with diverse biological activities including anti-tumor activity. In the present study, we have reported the anti-neoplastic activity of cinnamon in cervical cancer cell line, SiHa. The aqueous cinnamon extract (ACE-c) was analyzed for its cinnamaldehyde content by HPTLC analysis. The polyphenol content of ACE-c was measured by Folin-Ciocalteau method. Cytotoxicity analysis was performed by MTT assay. We studied the effect of cinnamon on growth kinetics by performing growth curve, colony formation and soft agar assays. The cells treated with ACE-c were analyzed for wound healing assay as well as for matrix metalloproteinase-2 (MMP-2) expression at mRNA and protein level by RT-PCR and zymography, respectively. Her-2 protein expression was analyzed in the control and ACE-c treated samples by immunoblotting as well as confocal microscopy. Apoptosis studies and calcium signaling assays were analyzed by FACS. Loss of mitochondrial membrane potential (Deltapsim) in cinnamon treated cells was studied by JC-1 staining and analyzed by confocal microscopy as well as FACS. Cinnamon alters the growth kinetics of SiHa cells in a dose-dependent manner. Cells treated with ACE-c exhibited reduced number of colonies compared to the control cells. The treated cells exhibited reduced migration potential that could be explained due to downregulation of MMP-2 expression. Interestingly, the expression of Her-2 oncoprotein was significantly reduced in the presence of ACE-c. Cinnamon extract induced apoptosis in the cervical cancer cells through increase in intracellular calcium signaling as well as loss of mitochondrial membrane potential. Cinnamon could be used as a potent chemopreventive drug in cervical cancer.

  19. Melatonin: A Mitochondrial Targeting Molecule Involving Mitochondrial Protection and Dynamics

    Directory of Open Access Journals (Sweden)

    Dun-Xian Tan

    2016-12-01

    Full Text Available Melatonin has been speculated to be mainly synthesized by mitochondria. This speculation is supported by the recent discovery that aralkylamine N-acetyltransferase/serotonin N-acetyltransferase (AANAT/SNAT is localized in mitochondria of oocytes and the isolated mitochondria generate melatonin. We have also speculated that melatonin is a mitochondria-targeted antioxidant. It accumulates in mitochondria with high concentration against a concentration gradient. This is probably achieved by an active transportation via mitochondrial melatonin transporter(s. Melatonin protects mitochondria by scavenging reactive oxygen species (ROS, inhibiting the mitochondrial permeability transition pore (MPTP, and activating uncoupling proteins (UCPs. Thus, melatonin maintains the optimal mitochondrial membrane potential and preserves mitochondrial functions. In addition, mitochondrial biogenesis and dynamics is also regulated by melatonin. In most cases, melatonin reduces mitochondrial fission and elevates their fusion. Mitochondrial dynamics exhibit an oscillatory pattern which matches the melatonin circadian secretory rhythm in pinealeocytes and probably in other cells. Recently, melatonin has been found to promote mitophagy and improve homeostasis of mitochondria.

  20. Melatonin: A Mitochondrial Targeting Molecule Involving Mitochondrial Protection and Dynamics.

    Science.gov (United States)

    Tan, Dun-Xian; Manchester, Lucien C; Qin, Lilan; Reiter, Russel J

    2016-12-16

    Melatonin has been speculated to be mainly synthesized by mitochondria. This speculation is supported by the recent discovery that aralkylamine N-acetyltransferase/serotonin N-acetyltransferase (AANAT/SNAT) is localized in mitochondria of oocytes and the isolated mitochondria generate melatonin. We have also speculated that melatonin is a mitochondria-targeted antioxidant. It accumulates in mitochondria with high concentration against a concentration gradient. This is probably achieved by an active transportation via mitochondrial melatonin transporter(s). Melatonin protects mitochondria by scavenging reactive oxygen species (ROS), inhibiting the mitochondrial permeability transition pore (MPTP), and activating uncoupling proteins (UCPs). Thus, melatonin maintains the optimal mitochondrial membrane potential and preserves mitochondrial functions. In addition, mitochondrial biogenesis and dynamics is also regulated by melatonin. In most cases, melatonin reduces mitochondrial fission and elevates their fusion. Mitochondrial dynamics exhibit an oscillatory pattern which matches the melatonin circadian secretory rhythm in pinealeocytes and probably in other cells. Recently, melatonin has been found to promote mitophagy and improve homeostasis of mitochondria.

  1. Protons Trigger Mitochondrial Flashes.

    Science.gov (United States)

    Wang, Xianhua; Zhang, Xing; Huang, Zhanglong; Wu, Di; Liu, Beibei; Zhang, Rufeng; Yin, Rongkang; Hou, Tingting; Jian, Chongshu; Xu, Jiejia; Zhao, Yan; Wang, Yanru; Gao, Feng; Cheng, Heping

    2016-07-26

    Emerging evidence indicates that mitochondrial flashes (mitoflashes) are highly conserved elemental mitochondrial signaling events. However, which signal controls their ignition and how they are integrated with other mitochondrial signals and functions remain elusive. In this study, we aimed to further delineate the signal components of the mitoflash and determine the mitoflash trigger mechanism. Using multiple biosensors and chemical probes as well as label-free autofluorescence, we found that the mitoflash reflects chemical and electrical excitation at the single-organelle level, comprising bursting superoxide production, oxidative redox shift, and matrix alkalinization as well as transient membrane depolarization. Both electroneutral H(+)/K(+) or H(+)/Na(+) antiport and matrix proton uncaging elicited immediate and robust mitoflash responses over a broad dynamic range in cardiomyocytes and HeLa cells. However, charge-uncompensated proton transport, which depolarizes mitochondria, caused the opposite effect, and steady matrix acidification mildly inhibited mitoflashes. Based on a numerical simulation, we estimated a mean proton lifetime of 1.42 ns and diffusion distance of 2.06 nm in the matrix. We conclude that nanodomain protons act as a novel, to our knowledge, trigger of mitoflashes in energized mitochondria. This finding suggests that mitoflash genesis is functionally and mechanistically integrated with mitochondrial energy metabolism. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  2. Mitochondrial DNA.

    Science.gov (United States)

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  3. Arterial blood pressure changes in acute T. brucei infection of dogs ...

    African Journals Online (AJOL)

    The aim of this study is to find out the usefulness of serial arterial blood pressure measurements in predicting severity and outcome of acute Trypanosoma brucei infection in dogs. Twenty adult dogs of mixed sexes and aged between 2 and 5 years were used for this study. The dogs were of good cardiac health and were ...

  4. Biosynthesis of SUMOylated Proteins in Bacteria Using the Trypanosoma brucei Enzymatic System

    Science.gov (United States)

    Iribarren, Paula Ana; Berazategui, María Agustina; Cazzulo, Juan José; Alvarez, Vanina Eder

    2015-01-01

    Post-translational modification with the Small Ubiquitin-like Modifier (SUMO) is conserved in eukaryotic organisms and plays important regulatory roles in proteins affecting diverse cellular processes. In Trypanosoma brucei, member of one of the earliest branches in eukaryotic evolution, SUMO is essential for normal cell cycle progression and is likely to be involved in the epigenetic control of genes crucial for parasite survival, such as those encoding the variant surface glycoproteins. Molecular pathways modulated by SUMO have started to be discovered by proteomic studies; however, characterization of functional consequences is limited to a reduced number of targets. Here we present a bacterial strain engineered to produce SUMOylated proteins, by transferring SUMO from T. brucei together with the enzymes essential for its activation and conjugation. Due to the lack of background in E. coli, this system is useful to express and identify SUMOylated proteins directly in cell lysates by immunoblotting, and SUMOylated targets can be eventually purified for biochemical or structural studies. We applied this strategy to describe the ability of TbSUMO to form chains in vitro and to detect SUMOylation of a model substrate, PCNA both from Saccharomyces cerevisiae and from T. brucei. To further validate targets, we applied an in vitro deconjugation assay using the T. brucei SUMO-specific protease capable to revert the pattern of modification. This system represents a valuable tool for target validation, mutant generation and functional studies of SUMOylated proteins in trypanosomatids. PMID:26258470

  5. Phosphatidylinositol 4-kinase III-beta is required for Golgi maintenance and cytokinesis in Trypanosoma brucei.

    Science.gov (United States)

    Rodgers, Melissa J; Albanesi, Joseph P; Phillips, Margaret A

    2007-07-01

    The parasitic protozoan Trypanosoma brucei contains two type III phosphatidylinositol 4-kinases (alpha and beta). We have cloned the gene encoding the T. brucei type III phosphatidylinositol 4-kinase beta (TbPI4KIII-beta), expressed the protein in COS-7 cells, and confirmed that the protein catalyzes the phosphorylation of phosphatidylinositol. Depletion of TbPI4KIII-beta in procyclic T. brucei by RNA interference (RNAi) resulted in inhibition of cell growth and a distorted cellular morphology. RNAi cells had a distorted Golgi apparatus, and lysosomal and flagellar pocket proteins were mislocalized. Ultrastructural analysis revealed the internal accumulation of a heterogeneous population of vesicles, abnormal positioning of organelles, and a loss of cell polarity. Scanning electron microcopy revealed a twisted phenotype, and dividing cells often exhibited a detached daughter flagellum and lacked a cleavage furrow. Cell cycle analysis confirmed that cells depleted of TbPI4KIII-beta have a postmitotic cytokinesis block that occurs after a single round of mitosis, suggestive of a specific cell cycle block. In summary, TbPI4KIII-beta is an essential protein in procyclic T. brucei, required for maintenance of Golgi structure, protein trafficking, normal cellular shape, and cytokinesis.

  6. The Flagellar Arginine Kinase in Trypanosoma brucei Is Important for Infection in Tsetse Flies.

    Directory of Open Access Journals (Sweden)

    Cher-Pheng Ooi

    Full Text Available African trypanosomes are flagellated parasites that cause sleeping sickness. Parasites are transmitted from one mammalian host to another by the bite of a tsetse fly. Trypanosoma brucei possesses three different genes for arginine kinase (AK including one (AK3 that encodes a protein localised to the flagellum. AK3 is characterised by the presence of a unique amino-terminal insertion that specifies flagellar targeting. We show here a phylogenetic analysis revealing that flagellar AK arose in two independent duplication events in T. brucei and T. congolense, the two species of African trypanosomes that infect the tsetse midgut. In T. brucei, AK3 is detected in all stages of parasite development in the fly (in the midgut and in the salivary glands as well as in bloodstream cells, but with predominance at insect stages. Genetic knockout leads to a slight reduction in motility and impairs parasite infectivity towards tsetse flies in single and competition experiments, both phenotypes being reverted upon expression of an epitope-tagged version of AK3. We speculate that this flagellar arginine kinase is important for T. brucei infection of tsetse, especially in the context of mixed infections and that its flagellar targeting relies on a system equivalent to that discovered for calflagins, a family of trypanosome flagellum calcium binding proteins.

  7. The Flagellar Arginine Kinase in Trypanosoma brucei Is Important for Infection in Tsetse Flies.

    Science.gov (United States)

    Ooi, Cher-Pheng; Rotureau, Brice; Gribaldo, Simonetta; Georgikou, Christina; Julkowska, Daria; Blisnick, Thierry; Perrot, Sylvie; Subota, Ines; Bastin, Philippe

    2015-01-01

    African trypanosomes are flagellated parasites that cause sleeping sickness. Parasites are transmitted from one mammalian host to another by the bite of a tsetse fly. Trypanosoma brucei possesses three different genes for arginine kinase (AK) including one (AK3) that encodes a protein localised to the flagellum. AK3 is characterised by the presence of a unique amino-terminal insertion that specifies flagellar targeting. We show here a phylogenetic analysis revealing that flagellar AK arose in two independent duplication events in T. brucei and T. congolense, the two species of African trypanosomes that infect the tsetse midgut. In T. brucei, AK3 is detected in all stages of parasite development in the fly (in the midgut and in the salivary glands) as well as in bloodstream cells, but with predominance at insect stages. Genetic knockout leads to a slight reduction in motility and impairs parasite infectivity towards tsetse flies in single and competition experiments, both phenotypes being reverted upon expression of an epitope-tagged version of AK3. We speculate that this flagellar arginine kinase is important for T. brucei infection of tsetse, especially in the context of mixed infections and that its flagellar targeting relies on a system equivalent to that discovered for calflagins, a family of trypanosome flagellum calcium binding proteins.

  8. Particle-bound enzymes in the bloodstream form of Trypanosoma brucei

    NARCIS (Netherlands)

    Opperdoes, F. R.; Borst, P.; Spits, H.

    1977-01-01

    We have screened the bloodstream form of Trypanosoma brucei for the presence of enzymes that could serve as markers for the microbodies and the highly repressed mitochondrion of this organism. None of seven known microbody enzymes were detected at all, but glycerol-3-phosphate oxidase, ATPase,

  9. Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation

    NARCIS (Netherlands)

    Weelden, van S.W.H.; Fast, B.; Vogt, A.; Meer, van der P.; Saas, J.; Hellemond, van J.J.; Tielens, A.G.M.; Boshart, M.

    2003-01-01

    The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene

  10. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei

    NARCIS (Netherlands)

    Zomerdijk, J. C.; Ouellette, M.; ten Asbroek, A. L.; Kieft, R.; Bommer, A. M.; Clayton, C. E.; Borst, P.

    1990-01-01

    The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site

  11. Maf1 is a negative regulator of transcription in Trypanosoma brucei.

    Science.gov (United States)

    Romero-Meza, Gabriela; Vélez-Ramírez, Daniel E; Florencio-Martínez, Luis E; Román-Carraro, Fiordaliso C; Manning-Cela, Rebeca; Hernández-Rivas, Rosaura; Martínez-Calvillo, Santiago

    2017-02-01

    RNA polymerase III (Pol III) produces small RNA molecules that play essential roles in mRNA processing and translation. Maf1, originally described as a negative regulator of Pol III transcription, has been studied from yeast to human. Here we characterized Maf1 in the parasitic protozoa Trypanosoma brucei (TbMaf1), representing the first report to analyse Maf1 in an early-diverged eukaryote. While Maf1 is generally encoded by a single-copy gene, the T. brucei genome contains two almost identical TbMaf1 genes. The TbMaf1 protein has the three conserved sequences and is predicted to fold into a globular structure. Unlike in yeast, TbMaf1 localizes to the nucleus in procyclic forms of T. brucei under normal growth conditions. Cell lines that either downregulate or overexpress TbMaf1 were generated, and growth curve analysis with them suggested that TbMaf1 participates in the regulation of cell growth of T. brucei. Nuclear run-on and chromatin immunoprecipitation analyses demonstrated that TbMaf1 represses Pol III transcription of tRNA and U2 snRNA genes by associating with their promoters. Interestingly, 5S rRNA levels do not change after TbMaf1 ablation or overexpression. Notably, our data also revealed that TbMaf1 regulates Pol I transcription of procyclin gene and Pol II transcription of SL RNA genes. © 2016 John Wiley & Sons Ltd.

  12. Isolation and characterization of kinetoplast DNA from the bloodstream form of Trypanosoma brucei.

    NARCIS (Netherlands)

    A.H. Fairlamb; P.O. Weislogel; J.H.J. Hoeijmakers (Jan); P. Borst (Piet)

    1978-01-01

    textabstractWe have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in kinetoplast DNA (kDNA) networks of Trypanosoma brucei (East African

  13. Deletion of the Trypanosoma brucei Superoxide Dismutase Gene sodb1 Increases Sensitivity to Nifurtimox and Benznidazole▿

    Science.gov (United States)

    Prathalingham, S. Radhika; Wilkinson, Shane R.; Horn, David; Kelly, John M.

    2007-01-01

    It has been more than 25 years since it was first reported that nifurtimox and benznidazole promote superoxide production in trypanosomes. However, there has been no direct evidence of an association between the drug-induced free radicals and trypanocidal activity. Here, we identify a superoxide dismutase required to protect Trypanosoma brucei from drug-generated superoxide. PMID:17145786

  14. Deletion of the Trypanosoma brucei superoxide dismutase gene sodb1 increases sensitivity to nifurtimox and benznidazole.

    Science.gov (United States)

    Prathalingham, S Radhika; Wilkinson, Shane R; Horn, David; Kelly, John M

    2007-02-01

    It has been more than 25 years since it was first reported that nifurtimox and benznidazole promote superoxide production in trypanosomes. However, there has been no direct evidence of an association between the drug-induced free radicals and trypanocidal activity. Here, we identify a superoxide dismutase required to protect Trypanosoma brucei from drug-generated superoxide.

  15. Trypanosoma brucei Infection in a herd of sedentary cattle in Danja ...

    African Journals Online (AJOL)

    Trypanosoma brucei Infection in a herd of sedentary cattle in Danja Local Government Area, Katsina State, Northern Nigeria– A possible resurgence of Tsetse flies ... resulted in advocacy for pastoralists to settle down into productive cattle production compared to the nomadic behavior which does not enhance productivity.

  16. Novel molecular mechanism for targeting the parasite Trypanosoma brucei with snake venom toxins

    DEFF Research Database (Denmark)

    Martos Esteban, Andrea; Laustsen, Andreas Hougaard; Carrington, Mark

    Trypanosoma brucei is a parasitic protozoan species capable to infecting insect vectors whose bite further produces African sleeping sickness inhuman beings. During parasites’extracellular lives in the mammalian host, its outer coat, mainly composedof Variable surface glycoproteins (VSGs)[2], und...

  17. Aquaglyceroporins Are the Entry Pathway of Boric Acid in Trypanosoma brucei.

    Science.gov (United States)

    Marsiccobetre, Sabrina; Rodríguez-Acosta, Alexis; Lang, Florian; Figarella, Katherine; Uzcátegui, Néstor L

    2017-05-01

    The boron element possesses a range of different effects on living beings. It is essential to beneficial at low concentrations, but toxic at excessive concentrations. Recently, some boron-based compounds have been identified as promising molecules against Trypanosoma brucei, the causative agent of sleeping sickness. However, until now, the boron metabolism and its access route into the parasite remained elusive. The present study addressed the permeability of T. brucei aquaglyceroporins (TbAQPs) for boric acid, the main natural boron species. To this end, the three TbAQPs were expressed in Saccharomyces cerevisiae and Xenopus laevis oocytes. Our findings in both expression systems showed that all three TbAQPs are permeable for boric acid. Especially TbAQP2 is highly permeable for this compound, displaying one of the highest conductances reported for a solute in these channels. Additionally, T. brucei aquaglyceroporin activities were sensitive to pH. Taken together, these results establish that TbAQPs are channels for boric acid and are highly efficient entry pathways for boron into the parasite. Our findings stress the importance of studying the physiological functions of boron and their derivatives in T. brucei, as well as the pharmacological implications of their uptake by trypanosome aquaglyceroporins. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Biosynthesis of SUMOylated Proteins in Bacteria Using the Trypanosoma brucei Enzymatic System.

    Directory of Open Access Journals (Sweden)

    Paula Ana Iribarren

    Full Text Available Post-translational modification with the Small Ubiquitin-like Modifier (SUMO is conserved in eukaryotic organisms and plays important regulatory roles in proteins affecting diverse cellular processes. In Trypanosoma brucei, member of one of the earliest branches in eukaryotic evolution, SUMO is essential for normal cell cycle progression and is likely to be involved in the epigenetic control of genes crucial for parasite survival, such as those encoding the variant surface glycoproteins. Molecular pathways modulated by SUMO have started to be discovered by proteomic studies; however, characterization of functional consequences is limited to a reduced number of targets. Here we present a bacterial strain engineered to produce SUMOylated proteins, by transferring SUMO from T. brucei together with the enzymes essential for its activation and conjugation. Due to the lack of background in E. coli, this system is useful to express and identify SUMOylated proteins directly in cell lysates by immunoblotting, and SUMOylated targets can be eventually purified for biochemical or structural studies. We applied this strategy to describe the ability of TbSUMO to form chains in vitro and to detect SUMOylation of a model substrate, PCNA both from Saccharomyces cerevisiae and from T. brucei. To further validate targets, we applied an in vitro deconjugation assay using the T. brucei SUMO-specific protease capable to revert the pattern of modification. This system represents a valuable tool for target validation, mutant generation and functional studies of SUMOylated proteins in trypanosomatids.

  19. BAPTA-AM decreases cellular pH, inhibits acidocalcisome acidification and autophagy in amino acid-starved T. brucei.

    Science.gov (United States)

    Li, Feng-Jun; Tan, Kevin S W; He, Cynthia Y

    2017-04-01

    To investigate the role of Ca2+ signaling in starvation-induced autophagy in Trypanosoma brucei, the causative agent of human African trypanosomiasis, we used cell-permeant Ca2+ chelator BAPTA-AM and cell impermeant chelator EGTA, and examined the potential involvement of several intracellular Ca2+ signaling pathways in T. brucei autophagy. The results showed an unexpected effect of BAPTA-AM in decreasing cellular pH and inhibiting acidocalcisome acidification in starved cells. The implication of these results in the role of Ca2+ signaling and cellular/organellar pH in T. brucei autophagy is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model.

    Directory of Open Access Journals (Sweden)

    Frédéric-Antoine Dauchy

    2016-11-01

    Full Text Available Trypanosoma brucei gambiense is the main causative agent of Human African Trypanosomiasis (HAT, also known as sleeping sickness. Because of limited alternatives and treatment toxicities, new therapeutic options are urgently needed for patients with HAT. Sterol 14alpha-demethylase (CYP51 is a potential drug target but its essentiality has not been determined in T. brucei. We used a tetracycline-inducible RNAi system to assess the essentiality of CYP51 in T. brucei bloodstream form (BSF cells and we evaluated the effect of posaconazole, a well-tolerated triazole drug, within a panel of virulent strains in vitro and in a murine model. Expression of CYP51 in several T. brucei cell lines was demonstrated by western blot and its essentiality was demonstrated by RNA interference (CYP51RNAi in vitro. Following reduction of TbCYP51 expression by RNAi, cell growth was reduced and eventually stopped compared to WT or non-induced cells, showing the requirement of CYP51 in T. brucei. These phenotypes were rescued by addition of ergosterol. Additionally, CYP51RNAi induction caused morphological defects with multiflagellated cells (p<0.05, suggesting cytokinesis dysfunction. The survival of CYP51RNAi Doxycycline-treated mice (p = 0.053 and of CYP51RNAi 5-day pre-induced Doxycycline-treated mice (p = 0.008 were improved compared to WT showing a CYP51 RNAi effect on trypanosomal virulence in mice. The posaconazole concentrations that inhibited parasite growth by 50% (IC50 were 8.5, 2.7, 1.6 and 0.12 μM for T. b. brucei 427 90-13, T. b. brucei Antat 1.1, T. b. gambiense Feo (Feo/ITMAP/1893 and T. b. gambiense Biyamina (MHOM/SD/82, respectively. During infection with these last three virulent strains, posaconazole-eflornithine and nifurtimox-eflornithine combinations showed similar improvement in mice survival (p≤0.001. Our results provide support for a CYP51 targeting based treatment in HAT. Thus posaconazole used in combination may represent a therapeutic

  1. Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells

    NARCIS (Netherlands)

    Y.S. Ju (Young Seok); J.M.C. Tubio (Jose M.); W. Mifsud (William); B. Fu (Beiyuan); H. Davies (Helen); M. Ramakrishna (Manasa); Y. Li (Yilong); L.R. Yates (Lucy); G. Gundem (Gunes); P.S. Tarpey (Patrick); S. Behjati (Sam); E. Papaemmanuil (Elli); S. Martin (Sandra); A. Fullam (Anthony); M. Gerstung (Moritz); J. Nangalia (Jyoti); A.R. Green (Anthony R.); C. Caldas (Carlos); Å. Borg (Åke); A. Tutt (Andrew); M.T. Michael Lee (Ming Ta); L.J. van 't Veer (Laura); B.K.T. Tan (Benita K.T.); S.A.J.R. Aparicio (Samuel A. J.); P.N. Span (Paul); J.W.M. Martens (John W. M.); S. Knappskog (Stian); A. Vincent-Salomon (Anne); A.-L. Borresen-Dale (Anne-Lise); J. Eyfjord; A.M. Flanagan (Adrienne); C.S. Foster; D. Neal (David); C. Cooper (Colin); R. Eeles (Rosalind); S. Lakhani (Sunil); C. Desmedt (Christine); G. Thomas (Gilles); A.L. Richardson (Andrea); C.A. Purdie (Colin A.); A.M. Thompson (Alastair M.); U. McDermott (Ultan); F. Yang (Fengtang); S. Nik-Zainal (Serena); P.J. Campbell (Peter); M.R. Stratton (Michael)

    2015-01-01

    textabstractMitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion

  2. The phosphoproteome of bloodstream form Trypanosoma brucei, causative agent of African sleeping sickness.

    Science.gov (United States)

    Nett, Isabelle R E; Martin, David M A; Miranda-Saavedra, Diego; Lamont, Douglas; Barber, Jonathan D; Mehlert, Angela; Ferguson, Michael A J

    2009-07-01

    The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of several T. brucei kinases, very few specific phosphorylation sites have been determined in this organism. Using a gel-free, phosphopeptide enrichment-based proteomics approach we performed the first large scale phosphorylation site analyses for T.brucei. Serine, threonine, and tyrosine phosphorylation sites were determined for a cytosolic protein fraction of the bloodstream form of the parasite, resulting in the identification of 491 phosphoproteins based on the identification of 852 unique phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in this study are predicted from their genome annotations to participate in a wide variety of biological processes, including signal transduction, processing of DNA and RNA, protein synthesis, and degradation and to a minor extent in metabolic pathways. The analysis of phosphopeptides and phosphorylation sites was facilitated by in-house developed software, and this automated approach was validated by manual annotation of spectra of the kinase subset of proteins. Analysis of the cytosolic bloodstream form T. brucei kinome revealed the presence of 44 phosphorylated protein kinases in our data set that could be classified into the major eukaryotic protein kinase groups by applying a multilevel hidden Markov model library of the kinase catalytic domain. Identification of the kinase phosphorylation sites showed conserved phosphorylation sequence motifs in several kinase activation segments, supporting the view that

  3. Probing for primary functions of prohibitin in Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Týč, Jiří; Faktorová, Drahomíra; Kriegová, Eva; Jirků, Milan; Vávrová, Zuzana; Maslov, D. A.; Lukeš, Julius

    2010-01-01

    Roč. 40, č. 1 (2010), s. 73-83 ISSN 0020-7519 R&D Projects: GA ČR GA204/09/1667; GA AV ČR IAA500960705; GA ČR(CZ) GP204/06/P423 Institutional research plan: CEZ:AV0Z60220518 Keywords : prohibitin * mitochondrion * morphology * mitochondrial translation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.822, year: 2010

  4. Structure of the Human Mitochondrial Ribosome Studied In Situ by Cryoelectron Tomography

    NARCIS (Netherlands)

    Englmeier, Robert; Pfeffer, Stefan; Förster, Friedrich

    2017-01-01

    Mitochondria maintain their own genome and its corresponding protein synthesis machine, the mitochondrial ribosome (mitoribosome). Mitoribosomes primarily synthesize highly hydrophobic proteins of the inner mitochondrial membrane. Recent studies revealed the complete structure of the isolated

  5. Aqueous Cinnamon Extract (ACE-c from the bark of Cinnamomum cassia causes apoptosis in human cervical cancer cell line (SiHa through loss of mitochondrial membrane potential

    Directory of Open Access Journals (Sweden)

    Chattopadhyay Samit

    2010-05-01

    Full Text Available Abstract Background Chemoprevention, which includes the use of synthetic or natural agents (alone or in combination to block the development of cancer in human beings, is an extremely promising strategy for cancer prevention. Cinnamon is one of the most widely used herbal medicines with diverse biological activities including anti-tumor activity. In the present study, we have reported the anti-neoplastic activity of cinnamon in cervical cancer cell line, SiHa. Methods The aqueous cinnamon extract (ACE-c was analyzed for its cinnamaldehyde content by HPTLC analysis. The polyphenol content of ACE-c was measured by Folin-Ciocalteau method. Cytotoxicity analysis was performed by MTT assay. We studied the effect of cinnamon on growth kinetics by performing growth curve, colony formation and soft agar assays. The cells treated with ACE-c were analyzed for wound healing assay as well as for matrix metalloproteinase-2 (MMP-2 expression at mRNA and protein level by RT-PCR and zymography, respectively. Her-2 protein expression was analyzed in the control and ACE-c treated samples by immunoblotting as well as confocal microscopy. Apoptosis studies and calcium signaling assays were analyzed by FACS. Loss of mitochondrial membrane potential (Δψm in cinnamon treated cells was studied by JC-1 staining and analyzed by confocal microscopy as well as FACS. Results Cinnamon alters the growth kinetics of SiHa cells in a dose-dependent manner. Cells treated with ACE-c exhibited reduced number of colonies compared to the control cells. The treated cells exhibited reduced migration potential that could be explained due to downregulation of MMP-2 expression. Interestingly, the expression of Her-2 oncoprotein was significantly reduced in the presence of ACE-c. Cinnamon extract induced apoptosis in the cervical cancer cells through increase in intracellular calcium signaling as well as loss of mitochondrial membrane potential. Conclusion Cinnamon could be used as a

  6. Mitochondrial Fusion Proteins and Human Diseases

    Directory of Open Access Journals (Sweden)

    Michela Ranieri

    2013-01-01

    Full Text Available Mitochondria are highly dynamic, complex organelles that continuously alter their shape, ranging between two opposite processes, fission and fusion, in response to several stimuli and the metabolic demands of the cell. Alterations in mitochondrial dynamics due to mutations in proteins involved in the fusion-fission machinery represent an important pathogenic mechanism of human diseases. The most relevant proteins involved in the mitochondrial fusion process are three GTPase dynamin-like proteins: mitofusin 1 (MFN1 and 2 (MFN2, located in the outer mitochondrial membrane, and optic atrophy protein 1 (OPA1, in the inner membrane. An expanding number of degenerative disorders are associated with mutations in the genes encoding MFN2 and OPA1, including Charcot-Marie-Tooth disease type 2A and autosomal dominant optic atrophy. While these disorders can still be considered rare, defective mitochondrial dynamics seem to play a significant role in the molecular and cellular pathogenesis of more common neurodegenerative diseases, for example, Alzheimer’s and Parkinson’s diseases. This review provides an overview of the basic molecular mechanisms involved in mitochondrial fusion and focuses on the alteration in mitochondrial DNA amount resulting from impairment of mitochondrial dynamics. We also review the literature describing the main disorders associated with the disruption of mitochondrial fusion.

  7. Mitochondrial dysfunction and organophosphorus compounds

    Energy Technology Data Exchange (ETDEWEB)

    Karami-Mohajeri, Somayyeh [Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of); Abdollahi, Mohammad, E-mail: Mohammad.Abdollahi@UToronto.Ca [Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2013-07-01

    Organophosphorous (OPs) pesticides are the most widely used pesticides in the agriculture and home. However, many acute or chronic poisoning reports about OPs have been published in the recent years. Mitochondria as a site of cellular oxygen consumption and energy production can be a target for OPs poisoning as a non-cholinergic mechanism of toxicity of OPs. In the present review, we have reviewed and criticized all the evidences about the mitochondrial dysfunctions as a mechanism of toxicity of OPs. For this purpose, all biochemical, molecular, and morphological data were retrieved from various studies. Some toxicities of OPs are arisen from dysfunction of mitochondrial oxidative phosphorylation through alteration of complexes I, II, III, IV and V activities and disruption of mitochondrial membrane. Reductions of adenosine triphosphate (ATP) synthesis or induction of its hydrolysis can impair the cellular energy. The OPs disrupt cellular and mitochondrial antioxidant defense, reactive oxygen species generation, and calcium uptake and promote oxidative and genotoxic damage triggering cell death via cytochrome C released from mitochondria and consequent activation of caspases. The mitochondrial dysfunction induced by OPs can be restored by use of antioxidants such as vitamin E and C, alpha-tocopherol, electron donors, and through increasing the cytosolic ATP level. However, to elucidate many aspect of mitochondrial toxicity of Ops, further studies should be performed. - Highlights: • As a non-cholinergic mechanism of toxicity, mitochondria is a target for OPs. • OPs affect action of complexes I, II, III, IV and V in the mitochondria. • OPs reduce mitochondrial ATP. • OPs promote oxidative and genotoxic damage via release of cytochrome C from mitochondria. • OP-induced mitochondrial dysfunction can be restored by increasing the cytosolic ATP.

  8. Overexpression of mitochondrial sirtuins alters glycolysis and mitochondrial function in HEK293 cells.

    Directory of Open Access Journals (Sweden)

    Michelle Barbi de Moura

    Full Text Available SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.

  9. Essential Assembly Factor Rpf2 Forms Novel Interactions within the 5S RNP in Trypanosoma brucei.

    Science.gov (United States)

    Kamina, Anyango D; Jaremko, Daniel; Christen, Linda; Williams, Noreen

    2017-01-01

    Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. During this process, there are several assembly steps that function as regulators to ensure proper ribosome formation. One of these steps is the assembly of the 5S ribonucleoprotein particle (5S RNP) in the central protuberance of the 60S ribosomal subunit. In eukaryotes, the 5S RNP is composed of 5S rRNA, ribosomal proteins L5 and L11, and assembly factors Rpf2 and Rrs1. Our laboratory previously showed that in Trypanosoma brucei, the 5S RNP is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37. In this study, we characterize an additional component of the 5S RNP, the T. brucei homolog of Rpf2. This is the first study to functionally characterize interactions mediated by Rpf2 in an organism other than fungi. T. brucei Rpf2 (TbRpf2) was identified from tandem affinity purification using extracts prepared from protein A-tobacco etch virus (TEV)-protein C (PTP)-tagged L5, P34, and P37 cell lines, followed by mass spectrometry analysis. We characterized the binding interactions between TbRpf2 and the previously characterized members of the T. brucei 5S RNP. Our studies show that TbRpf2 mediates conserved binding interactions with 5S rRNA and L5 and that TbRpf2 also interacts with trypanosome-specific proteins P34 and P37. We performed RNA interference (RNAi) knockdown of TbRpf2 and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation. These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in T. brucei. IMPORTANCETrypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of the

  10. Kinetic and mutational analysis of the Trypanosoma brucei NBT1 nucleobase transporter expressed in Saccharomyces cerevisiae reveals structural similarities between ENT and MFS transporters.

    Science.gov (United States)

    Papageorgiou, I; De Koning, H P; Soteriadou, K; Diallinas, G

    2008-05-01

    Parasitic protozoa are unable to synthesise purines de novo and thus depend on the uptake of nucleosides and nucleobases across their plasma membrane through specific transporters. A number of nucleoside and nucleobase transporters from Trypanosoma brucei brucei and Leishmania major have recently been characterised and shown to belong to the equilibrative nucleoside transporter (ENT) family. A number of studies have demonstrated the functional importance of particular transmembrane segments (TMS) in nucleoside-specific ENT proteins. TbNBT1, one of only three bona fide nucleobase-selective members of the ENT family, has previously been shown to be a high-affinity transporter for purine nucleobases and guanosine. In this study, we use the Saccharomyces cerevisiae expression system to build a biochemical model of how TbNBT1 recognises nucleobases. We next performed random in vitro and site-directed mutagenesis to identify residues critical for TbNBT1 function. The identification of residues likely to contribute to permeant binding, when combined with a structural model of TbNBT1 obtained by homology threading, yield a tentative three-dimensional model of the transporter binding site that is consistent with the binding model emerging from the biochemical data. The model strongly suggests the involvement of TMS5, TMS7 and TMS8 in TbNBT1 function. This situation is very similar to that concerning transporters of the major facilitator superfamily (MFS), one of which was used as a template for the threading. This point raises the possibility that ENT and MFS carriers, despite being considered evolutionarily distinct, might in fact share similar topologies and substrate translocations pathways.

  11. Aparasitemic serological suspects in Trypanosoma brucei gambiense human African trypanosomiasis : a potential human reservoir of parasites ?

    OpenAIRE

    Koffi, Mathurin; Solano, Philippe; Denizot, M.; Courtin, D.; Garcia, André; Lejon, V.; Buscher, P.; Cuny, Gérard; Jamonneau, Vincent

    2006-01-01

    The serological and parasitological tests used for Trypanosoma brucei gambiense human African trypanosomiasis (HAT) diagnosis have low specificity and sensitivity, respectively, and in the field, control program teams are faced with subjects with positive serology but negative parasitology who remain untreated. The aim of this work was to explore, using PCR tool, the significance of these aparasitemic serological suspects. Since discordant PCR results have been observed earlier with different...

  12. Differential virulence and tsetse fly transmissibility of Trypanosoma congolense and Trypanosoma brucei strains

    Directory of Open Access Journals (Sweden)

    Purity K. Gitonga

    2017-01-01

    Full Text Available African animal trypanosomiasis causes significant economic losses in sub-Saharan African countries because of livestock mortalities and reduced productivity. Trypanosomes, the causative agents, are transmitted by tsetse flies (Glossina spp.. In the current study, we compared and contrasted the virulence characteristics of five Trypanosoma congolense and Trypanosoma brucei isolates using groups of Swiss white mice (n = 6. We further determined the vectorial capacity of Glossina pallidipes, for each of the trypanosome isolates. Results showed that the overall pre-patent (PP periods were 8.4 ± 0.9 (range, 4–11 and 4.5 ± 0.2 (range, 4–6 for T. congolense and T. brucei isolates, respectively (p < 0.01. Despite the longer mean PP, T. congolense–infected mice exhibited a significantly (p < 0.05 shorter survival time than T. brucei–infected mice, indicating greater virulence. Differences were also noted among the individual isolates with T. congolense KETRI 2909 causing the most acute infection of the entire group with a mean ± standard error survival time of 9 ± 2.1 days. Survival time of infected tsetse flies and the proportion with mature infections at 30 days post-exposure to the infective blood meals varied among isolates, with subacute infection–causing T. congolense EATRO 1829 and chronic infection–causing T. brucei EATRO 2267 isolates showing the highest mature infection rates of 38.5% and 23.1%, respectively. Therefore, our study provides further evidence of occurrence of differences in virulence and transmissibility of eastern African trypanosome strains and has identified two, T. congolense EATRO 1829 and T. brucei EATRO 2267, as suitable for tsetse infectivity and transmissibility experiments.

  13. Mapping of branch sites in trans-spliced pre-mRNAs of Trypanosoma brucei.

    Science.gov (United States)

    Patzelt, E; Perry, K L; Agabian, N

    1989-01-01

    The process of trans splicing is essential to the maturation of all mRNAs in the Trypanosomatidae, a family of protozoan parasites, and to specific mRNAs in several species of nematode. In Trypanosoma brucei, a 39-nucleotide (nt) leader sequence originating from a small, 139-nt donor RNA (the spliced leader [SL] RNA) is spliced to the 5' end of mRNAs. An intermediate in this trans-splicing process is a Y structure which contains the 3' 100 nt of the SL RNA covalently linked to the pre-mRNA via a 2'-5' phosphodiester bond at the branch point residue. We mapped the branch points in T. brucei alpha- and beta-tubulin pre-mRNAs. The primary branch acceptors for the alpha- and beta-tubulins are 44 and 56 nt upstream of the 3' splice sites, respectively, and are A residues. Minor branch acceptors were detected 42 and 49 nt upstream of the alpha-tubulin splice site and 58 nt upstream of the splice site in beta-tubulin. The regions surrounding these branch points lack homology to the consensus sequences determined for mammalian cells and yeasts; there is also no conservation among the sequences themselves. Thus, the identified sequences suggest that the mechanism of branch point recognition in T. brucei differs from the mechanism of recognition by U2 RNA that has been proposed for other eucaryotes. Images PMID:2479824

  14. The inositol pyrophosphate synthesis pathway in Trypanosoma brucei is linked to polyphosphate synthesis in acidocalcisomes.

    Science.gov (United States)

    Cordeiro, Ciro D; Saiardi, Adolfo; Docampo, Roberto

    2017-10-01

    Inositol pyrophosphates are novel signaling molecules possessing high-energy pyrophosphate bonds and involved in a number of biological functions. Here, we report the correct identification and characterization of the kinases involved in the inositol pyrophosphate biosynthetic pathway in Trypanosoma brucei: inositol polyphosphate multikinase (TbIPMK), inositol pentakisphosphate 2-kinase (TbIP5K) and inositol hexakisphosphate kinase (TbIP6K). TbIP5K and TbIP6K were not identifiable by sequence alone and their activities were validated by enzymatic assays with the recombinant proteins or by their complementation of yeast mutants. We also analyzed T. brucei extracts for the presence of inositol phosphates using polyacrylamide gel electrophoresis and high-performance liquid chromatography. Interestingly, we could detect inositol phosphate (IP), inositol 4,5-bisphosphate (IP2 ), inositol 1,4,5-trisphosphate (IP3 ), and inositol hexakisphosphate (IP6 ) in T. brucei different stages. Bloodstream forms unable to produce inositol pyrophosphates, due to downregulation of TbIPMK expression by conditional knockout, have reduced levels of polyphosphate and altered acidocalcisomes. Our study links the inositol pyrophosphate pathway to the synthesis of polyphosphate in acidocalcisomes, and may lead to better understanding of these organisms and provide new targets for drug discovery. © 2017 John Wiley & Sons Ltd.

  15. Molecular variation of Trypanosoma brucei subspecies as revealed by AFLP fingerprinting.

    Science.gov (United States)

    Agbo, E E C; Majiwa, P A O; Claassen, H J H M; te Pas, M F W

    2002-04-01

    Genetic analysis of Trypanosoma spp. depends on the detection of variation between strains. We have used the amplified fragment length polymorphism (AFLP) technique to develop a convenient and reliable method for genetic characterization of Trypanosome (sub)species. AFLP accesses multiple independent sites within the genome and would allow a better definition of the relatedness of different Trypanosome (sub)species. Nine isolates (3 from each T. brucei subspecies) were tested with 40 AFLP primer combinations to identify the most appropriate pairs of restriction endonucleases and selective primers. Primers based on the recognition sequences of EcoRI and BglII were chosen and used to analyse 31 T. brucei isolates. Similarity levels calculated with the Pearson correlation coefficient ranged from 15 to 98%, and clusters were determined using the unweighted pair-group method using arithmetic averages (UPGMA). At the intraspecific level, AFLP fingerprints were grouped by numerical analysis in 2 main clusters, allowing a clear separation of T. b. gambiense (cluster I) from T. b. brucei and T. b. rhodesiense isolates (cluster II). Interspecies evaluation of this customized approach produced heterogeneous AFLP patterns, with unique genetic markers, except for T. evansi and T. equiperdum, which showed identical patterns and clustered together.

  16. NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei.

    Science.gov (United States)

    Narayanan, Mani Shankar; Kushwaha, Manish; Ersfeld, Klaus; Fullbrook, Alexander; Stanne, Tara M; Rudenko, Gloria

    2011-03-01

    Trypanosoma brucei mono-allelically expresses one of approximately 1500 variant surface glycoprotein (VSG) genes while multiplying in the mammalian bloodstream. The active VSG is transcribed by RNA polymerase I in one of approximately 15 telomeric VSG expression sites (ESs). T. brucei is unusual in controlling gene expression predominantly post-transcriptionally, and how ESs are mono-allelically controlled remains a mystery. Here we identify a novel transcription regulator, which resembles a nucleoplasmin-like protein (NLP) with an AT-hook motif. NLP is key for ES control in bloodstream form T. brucei, as NLP knockdown results in 45- to 65-fold derepression of the silent VSG221 ES. NLP is also involved in repression of transcription in the inactive VSG Basic Copy arrays, minichromosomes and procyclin loci. NLP is shown to be enriched on the 177- and 50-bp simple sequence repeats, the non-transcribed regions around rDNA and procyclin, and both active and silent ESs. Blocking NLP synthesis leads to downregulation of the active ES, indicating that NLP plays a role in regulating appropriate levels of transcription of ESs in both their active and silent state. Discovery of the unusual transcription regulator NLP provides new insight into the factors that are critical for ES control.

  17. NIMA-related kinase TbNRKC is involved in basal body separation in Trypanosoma brucei.

    Science.gov (United States)

    Pradel, Lydie C; Bonhivers, Mélanie; Landrein, Nicolas; Robinson, Derrick R

    2006-05-01

    The NIMA-related kinase 2 (NEK 2) has important cell cycle functions related to centriole integrity and splitting. Trypanosoma brucei does not possess centrioles, however, cytokinesis is coupled to basal body separation events. Here we report the first functional characterisation of a T. brucei basal body-cytoskeletal NIMA-related kinase (NRK) protein, TbNRKC. The TbNRKC kinase domain has high amino acid identity with the human NEK1 kinase domain (50%) but also shares 42% identity with human NEK2. TbNRKC is expressed in bloodstream and procyclic cells and functions as a bona fide kinase in vitro. Remarkably, RNAi knockdown of TbNRKC and overexpression of kinase-dead TbNRKC in procyclic forms induces the accumulation of cells with four basal bodies, whereas overexpression of active protein produces supernumary basal bodies and blocks cytokinesis. TbNRKC is located on mature and immature basal bodies and is the first T. brucei NRK to be found associated with the basal body cytokinesis pathway.

  18. ATPaseTb2, a unique membrane-bound FoF1-ATPase component, is essential in bloodstream and dyskinetoplastic trypanosomes.

    Directory of Open Access Journals (Sweden)

    Karolína Šubrtová

    2015-02-01

    Full Text Available In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt membrane potential (Δψm is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential FoF1-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψm that manifests as a decreased growth phenotype, indicating that the FoF1-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk and thus subunit a, an essential component of the proton pore in the membrane Fo-moiety. These Dk cells generate the Δψm by combining the hydrolytic activity of the matrix-facing F1-ATPase and the electrogenic exchange of ATP4- for ADP3- by the ATP/ADP carrier (AAC. Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric FoF1-ATPase complexes were identified. In fact, the immunoprecipitation of a F1-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψm of Dk cells. Thus, even in the absence of subunit a, a portion of the FoF1-ATPase is assembled in Dk cells.

  19. Variation in yeast mitochondrial activity associated with asci.

    Science.gov (United States)

    Swart, Chantel W; van Wyk, Pieter W J; Pohl, Carolina H; Kock, Johan L F

    2008-07-01

    An increase in mitochondrial membrane potential (DeltaPsim) and mitochondrially produced 3-hydroxy (3-OH) oxylipins was experienced in asci of the nonfermentative yeasts Galactomyces reessii and Lipomyces starkeyi and the fermentative yeasts Pichia farinosa and Schizosaccharomyces octosporus. Strikingly, asci of Zygosaccharomyces bailii showed no increase in mitochondrial activity (DeltaPsim and oxylipin production). As expected, oxygen deprivation only inhibited ascus formation in those yeasts with increased ascus mitochondrial activity. We conclude that ascus formation in yeasts is not always dependent on mitochondrial activity. In this case, fermentation may provide enough energy for ascus formation in Z. bailii.

  20. Mitochondrial cristae revealed with focused light.

    OpenAIRE

    Schmidt, R.; Wurm, C.; Punge, A.; Egner, A.; Jakobs, S.; Hell, S.

    2009-01-01

    Because of the diffraction resolution barrier, optical microscopes have so far failed in visualizing the mitochondrial cristae, that is, the folds of the inner membrane of this 200 to 400 nm diameter sized tubular organelle. Realizing a ∼30 nm isotropic subdiffraction resolution in isoSTED fluorescence nanoscopy, we have visualized these essential structures in the mitochondria of intact cells. We find a pronounced heterogeneity in the cristae arrangements even within individual mitochondrial...

  1. Mitochondrial cristae revealed with focused light.

    Science.gov (United States)

    Schmidt, Roman; Wurm, Christian A; Punge, Annedore; Egner, Alexander; Jakobs, Stefan; Hell, Stefan W

    2009-06-01

    Because of the diffraction resolution barrier, optical microscopes have so far failed in visualizing the mitochondrial cristae, that is, the folds of the inner membrane of this 200 to 400 nm diameter sized tubular organelle. Realizing a approximately 30 nm isotropic subdiffraction resolution in isoSTED fluorescence nanoscopy, we have visualized these essential structures in the mitochondria of intact cells. We find a pronounced heterogeneity in the cristae arrangements even within individual mitochondrial tubules.

  2. Mentha aquatica L. extract affects mitochondrial bioenergetics

    OpenAIRE

    Ferreira, Fernanda M.; Pereira, Olívia R.; Cardoso, Susana M.; Oliveira, Paulo J; Moreno, António J. M.

    2013-01-01

    Mentha aquatica extracts are commonly used in food flavoring and pharmacology. In the present work, we evaluated the possible effects of Mentha aquatica L. (water mint) ethanolic extract on rat liver mitochondria bioenergetics. Rat liver mitochondria were isolated using conventional protocols. M. aquatica extracts were evaluated on mitochondrial membrane electric potential by using a tetraphenylphosphonium cation (TPP+)-selective electrode, while mitochondrial respiratory activity was eval...

  3. [Topology of the mitochondrial potassium ion channels].

    Science.gov (United States)

    Laskowski, Michał; Kulawiak, Bogusz

    In the inner mitochondrial membrane several potassium channels have been identified whose activation lead to cytoprotection during ischemic event. It was found that activation of mitochondrial large conductance calcium activated potassium channel (mitoBKCa) and ATP regulated potassium channel (mitoKATP) preserves brain and heart muscle cells against ischemia/reperfusion induced damage. However the detailed cytoprotection mechanism remains unclear. Similarly, the molecular structures and protein interactions of the mitochondrial potassium channels are still unknown. In this article, we summarize the current knowledge of the mitoKATP and mitoBKCa channels topology. Different aspects of this topic are discussed like import and assembly of the channel subunits and biophysical properties of mitochondrial compartments. Additionally, the consequences of different topology models on the cytoprotective function of the mitochondrial potassium channels were analyzed.

  4. DNA import competence and mitochondrial genetics

    Directory of Open Access Journals (Sweden)

    Weber-Lotfi F.

    2014-01-01

    Full Text Available Aim. To understand the mechanism(s underlying mitochondrial competence for DNA uptake and to exploit these pathways for the development of in vivo models of gene therapy. Methods. DNA uptake into isolated mitochondria from plant or from mutant Saccharomyces cerevisiae defective for mitochondrial proteins and carriers, biochemical approaches and transfection of mammalian cells with DNA bound to mitochondriotropic liposomes. Results. Special focus on the inner membrane showed the involvement of isoforms of the adenine nucleotide translocator and the contribution of proteins controlling mitochondrial morphology in DNA uptake into yeast organelles. Transfection assays led to significant incorporation of a mitochondrial construct into mammalian cells and expression of a marker gene. Conclusions. The data imply that there are multiple mitochondrial DNA import pathways. On the other hand, preliminary results suggest that mitochondriotropic liposomes can deliver DNA to mitochondria in live mammalian cells.

  5. Exercise training increases sarcolemmal and mitochondrial fatty acid transport proteins in human skeletal muscle

    National Research Council Canada - National Science Library

    Talanian, Jason L; Holloway, Graham P; Snook, Laelie A; Heigenhauser, George J F; Bonen, Arend; Spriet, Lawrence L

    2010-01-01

    ... examined. Therefore, we determined whether high-intensity interval training (HIIT) increased total skeletal muscle, sarcolemmal, and mitochondrial membrane fatty acid transport protein contents...

  6. A Pre-clinical Animal Model of Trypanosoma brucei Infection Demonstrating Cardiac Dysfunction.

    Directory of Open Access Journals (Sweden)

    Charlotte S McCarroll

    2015-05-01

    Full Text Available African trypanosomiasis (AT, caused by Trypanosoma brucei species, results in both neurological and cardiac dysfunction and can be fatal if untreated. Research on the pathogenesis and treatment of the disease has centred to date on the characteristic neurological symptoms, whereas cardiac dysfunction (e.g. ventricular arrhythmias in AT remains largely unstudied. Animal models of AT demonstrating cardiac dysfunction similar to that described in field cases of AT are critically required to transform our understanding of AT-induced cardiac pathophysiology and identify future treatment strategies. We have previously shown that T. brucei can interact with heart muscle cells (cardiomyocytes to induce ventricular arrhythmias in ex vivo adult rat hearts. However, it is unknown whether the arrhythmias observed ex vivo are also present during in vivo infection in experimental animal models. Here we show for the first time the characterisation of ventricular arrhythmias in vivo in two animal models of AT infection using electrocardiographic (ECG monitoring. The first model utilised a commonly used monomorphic laboratory strain, Trypanosoma brucei brucei Lister 427, whilst the second model used a pleomorphic laboratory strain, T. b. brucei TREU 927, which demonstrates a similar chronic infection profile to clinical cases. The frequency of ventricular arrhythmias and heart rate (HR was significantly increased at the endpoint of infection in the TREU 927 infection model, but not in the Lister 427 infection model. At the end of infection, hearts from both models were isolated and Langendorff perfused ex vivo with increasing concentrations of the β-adrenergic agonist isoproterenol (ISO. Interestingly, the increased frequency of arrhythmias observed in vivo in the TREU 927 infection model was lost upon isolation of the heart ex vivo, but re-emerged with the addition of ISO. Our results demonstrate that TREU 927 infection modifies the substrate of the myocardium

  7. The mitochondrial-targeted antioxidant, MitoQ, increases liver mitochondrial cardiolipin content in obesogenic diet-fed rats.

    Science.gov (United States)

    Fouret, Gilles; Tolika, Evanthia; Lecomte, Jérôme; Bonafos, Béatrice; Aoun, Manar; Murphy, Michael P; Ferreri, Carla; Chatgilialoglu, Chryssostomos; Dubreucq, Eric; Coudray, Charles; Feillet-Coudray, Christine

    2015-10-01

    Cardiolipin (CL), a unique mitochondrial phospholipid, plays a key role in several processes of mitochondrial bioenergetics as well as in mitochondrial membrane stability and dynamics. The present study was designed to determine the effect of MitoQ, a mitochondrial-targeted antioxidant, on the content of liver mitochondrial membrane phospholipids, in particular CL, and its fatty acid composition in obesogenic diet-fed rats. To do this, twenty-four 6week old male Sprague Dawley rats were randomized into three groups of 8 animals and fed for 8weeks with either a control diet, a high fat diet (HF), or a HF diet with MitoQ (HF+MitoQ). Phospholipid classes and fatty acid composition were assayed by chromatographic methods in liver and liver mitochondria. Mitochondrial bioenergetic function was also evaluated. While MitoQ had no or slight effects on total liver fatty acid composition and phospholipid classes and their fatty acid composition, it had major effects on liver mitochondrial phospholipids and mitochondrial function. Indeed, MitoQ both increased CL synthase gene expression and CL content of liver mitochondria and increased 18:2n-6 (linoleic acid) content of mitochondrial phospholipids by comparison to the HF diet. Moreover, mitochondrial CL content was positively correlated to mitochondrial membrane fluidity, membrane potential and respiration, as well as to ATP synthase activity, while it was negatively correlated to mitochondrial ROS production. These findings suggest that MitoQ may decrease pathogenic alterations to CL content and profiles, thereby preserving mitochondrial function and attenuating the development of some of the features of metabolic syndrome in obesogenic diet-fed rats. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Mitochondrial bioenergetics decay in aging: beneficial effect of melatonin.

    Science.gov (United States)

    Paradies, Giuseppe; Paradies, Valeria; Ruggiero, Francesca M; Petrosillo, Giuseppe

    2017-11-01

    Aging is a biological process characterized by progressive decline in physiological functions, increased oxidative stress, reduced capacity to respond to stresses, and increased risk of contracting age-associated disorders. Mitochondria are referred to as the powerhouse of the cell through their role in the oxidative phosphorylation to generate ATP. These organelles contribute to the aging process, mainly through impairment of electron transport chain activity, opening of the mitochondrial permeability transition pore and increased oxidative stress. These events lead to damage to proteins, lipids and mitochondrial DNA. Cardiolipin, a phospholipid of the inner mitochondrial membrane, plays a pivotal role in several mitochondrial bioenergetic processes as well as in mitochondrial-dependent steps of apoptosis and in mitochondrial membrane stability and dynamics. Cardiolipin alterations are associated with mitochondrial bienergetics decline in multiple tissues in a variety of physiopathological conditions, as well as in the aging process. Melatonin, the major product of the pineal gland, is considered an effective protector of mitochondrial bioenergetic function. Melatonin preserves mitochondrial function by preventing cardiolipin oxidation and this may explain, at least in part, the protective role of this compound in mitochondrial physiopathology and aging. Here, mechanisms through which melatonin exerts its protective role against mitochondrial dysfunction associated with aging and age-associated disorders are discussed.

  9. Interaction between the flagellar pocket collar and the hook complex via a novel microtubule-binding protein in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Anna Albisetti

    2017-11-01

    Full Text Available Trypanosoma brucei belongs to a group of unicellular, flagellated parasites that are responsible for human African trypanosomiasis. An essential aspect of parasite pathogenicity is cytoskeleton remodelling, which occurs during the life cycle of the parasite and is accompanied by major changes in morphology and organelle positioning. The flagellum originates from the basal bodies and exits the cell body through the flagellar pocket (FP but remains attached to the cell body via the flagellum attachment zone (FAZ. The FP is an invagination of the pellicular membrane and is the sole site for endo- and exocytosis. The FAZ is a large complex of cytoskeletal proteins, plus an intracellular set of four specialised microtubules (MtQ that elongate from the basal bodies to the anterior end of the cell. At the distal end of the FP, an essential, intracellular, cytoskeletal structure called the flagellar pocket collar (FPC circumvents the flagellum. Overlapping the FPC is the hook complex (HC (a sub-structure of the previously named bilobe that is also essential and is thought to be involved in protein FP entry. BILBO1 is the only functionally characterised FPC protein and is necessary for FPC and FP biogenesis. Here, we used a combination of in vitro and in vivo approaches to identify and characterize a new BILBO1 partner protein-FPC4. We demonstrate that FPC4 localises to the FPC, the HC, and possibly to a proximal portion of the MtQ. We found that the C-terminal domain of FPC4 interacts with the BILBO1 N-terminal domain, and we identified the key amino acids required for this interaction. Interestingly, the FPC4 N-terminal domain was found to bind microtubules. Over-expression studies highlight the role of FPC4 in its association with the FPC, HC and FPC segregation. Our data suggest a tripartite association between the FPC, the HC and the MtQ.

  10. Immunization with recombinant actin from Trypanosoma evansi induces protective immunity against T. evansi, T. equiperdum and T. b. brucei infection.

    Science.gov (United States)

    Li, San-Qiang; Yang, Wu-Biao; Lun, Zhao-Rong; Ma, Ling-Jun; Xi, Shou-Min; Chen, Qun-Li; Song, Xiao-Wei; Kang, Jian; Yang, Lan-Ze

    2009-01-01

    Actin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi actin shows 100%, 98.7%, and 93.1%, homology with Trypanosoma equiperdum, Trypanosoma brucei brucei, and Trypanosoma cruzi. Recombinant actin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant actin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818, and T. b. brucei STIB 940, showing 63.3%, 56.7%, and 53.3% protection, respectively. Serum collected from the rabbit immunized with recombinant actin inhibited the growth of T. evansi, T. equiperdum, and T. b. brucei in vitro cultivation. Serum from mice and rabbits immunized with recombinant actin only recognized T. evansi actin but not mouse actin. The results of this study suggest that the recombinant T. evansi actin induces protective immunity against T. evansi, T. equiperdum, and T. b. brucei infection and may be useful in the development of a vaccine with other cytoskeletal proteins to prevent animal trypanosomiasis caused by these three trypanosome species.

  11. Chemopreventive effect of methanolic extract of Azadirachta indica on experimental Trypanosoma brucei induced oxidative stress in dogs.

    Science.gov (United States)

    Omobowale, Temidayo O; Oyagbemi, Ademola A; Oyewunmi, Oyefunbi A; Adejumobi, Olumuyiwa A

    2015-01-01

    The medicinal properties of Azadirachta indica have been harnessed for many years in the treatment of many diseases in both humans and animals. Twenty-five apparently healthy dogs weighing between 3 and 8 kg were randomly divided into five groups with five dogs in each group. Ameliorative effect of A. indica on erythrocyte antioxidant status and markers of oxidative stress were assessed. Liver and kidney function tests were also performed. Pre-treatment with methanolic extract of Azadirachta indica (MEAI) at different doses did not significantly alter the values of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activity in Trypanosoma brucei infection. Although, serum creatinine significantly (P indica, after 2 weeks of T. brucei infection. However, the reduced glutathione (GSH) content of the erythrocyte increased significantly in animals pre-treated with 50 mg/kg and 200 mg/kg of A. indica respectively. Markers of oxidative stress such as malondialdehyde and hydrogen peroxide generated were higher in animals infected with T. brucei with no significant (P >0.05) difference compared to the values obtained in pre-treated animals. Pre-treatment with 100 mg/kg and 200 mg/kg of A. indica significantly (P < 0.05) decreased serum myeloperoxidase activity at 2 weeks post-infection with T. brucei. From this study, MEAI showed significant ability to attenuate oxidative stress and inflammation during experimental T. brucei infection.

  12. PCR approach for the detection of Trypanosoma brucei and T. equiperdum and their differentiation from T. evansi based on maxicircle kinetoplast DNA.

    Science.gov (United States)

    Li, Feng-Jun; Gasser, Robin B; Lai, De-Hua; Claes, Filip; Zhu, Xing-Quan; Lun, Zhao-Rong

    2007-02-01

    The goal of this study was to develop a PCR approach based on the sequence of maxicircle kinetoplast DNA (kDNA) of Trypanosoma brucei to distinguish T. brucei/T. equiperdum from T. evansi and to evaluate its diagnostic use for their detection in blood samples. Primers derived from the sequence of the maxicircle kDNA of T. brucei, encoding the NADH dehydrogenase subunit 5 (nad5) gene, were used to test the PCR-amplification from T. brucei (including T. b. brucei and T. b. rhodesiense), T. equiperdum, T. evansi, T. vivax and T. congolense. A primer pair to a nuclear DNA region incorporated into a separate PCR was employed to control for the presence of amplifiable genomic DNA (representing the subgenus Trypanozoon) in each sample subjected to the PCR. Products of approximately 395bp were amplified from all T. brucei and T. equiperdum samples tested using the nad5-PCR, but not from T. evansi DNA samples or any of the control samples representing T. vivax, T. congolense, or host. The current PCR approach allows the rapid differentiation of T. brucei/T.equiperdum from T. evansi and can detect the equivalent of 20-25 cells of T. brucei or T. equiperdum in purified genomic DNA or infected blood samples.

  13. What Is Mitochondrial DNA?

    Science.gov (United States)

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  14. Piracetam improves mitochondrial dysfunction following oxidative stress

    Science.gov (United States)

    Keil, Uta; Scherping, Isabel; Hauptmann, Susanne; Schuessel, Katin; Eckert, Anne; Müller, Walter E

    2005-01-01

    Mitochondrial dysfunction including decrease of mitochondrial membrane potential and reduced ATP production represents a common final pathway of many conditions associated with oxidative stress, for example, hypoxia, hypoglycemia, and aging. Since the cognition-improving effects of the standard nootropic piracetam are usually more pronounced under such pathological conditions and young healthy animals usually benefit little by piracetam, the effect of piracetam on mitochondrial dysfunction following oxidative stress was investigated using PC12 cells and dissociated brain cells of animals treated with piracetam. Piracetam treatment at concentrations between 100 and 1000 μM improved mitochondrial membrane potential and ATP production of PC12 cells following oxidative stress induced by sodium nitroprusside (SNP) and serum deprivation. Under conditions of mild serum deprivation, piracetam (500 μM) induced a nearly complete recovery of mitochondrial membrane potential and ATP levels. Piracetam also reduced caspase 9 activity after SNP treatment. Piracetam treatment (100–500 mg kg−1 daily) of mice was also associated with improved mitochondrial function in dissociated brain cells. Significant improvement was mainly seen in aged animals and only less in young animals. Moreover, the same treatment reduced antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase, and glutathione reductase) in aged mouse brain only, which are elevated as an adaptive response to the increased oxidative stress with aging. In conclusion, therapeutically relevant in vitro and in vivo concentrations of piracetam are able to improve mitochondrial dysfunction associated with oxidative stress and/or aging. Mitochondrial stabilization and protection might be an important mechanism to explain many of piracetam's beneficial effects in elderly patients. PMID:16284628

  15. Mitochondrial CB1 receptor is involved in ACEA-induced protective effects on neurons and mitochondrial functions

    OpenAIRE

    Lei Ma; Ji Jia; Wen Niu; Tao Jiang; Qian Zhai; Lei Yang; Fuhai Bai; Qiang Wang; Lize Xiong

    2015-01-01

    Mitochondrial dysfunction contributes to cell death after cerebral ischemia/reperfusion (I/R) injury. Cannabinoid CB1 receptor is expressed in neuronal mitochondrial membranes (mtCB1R) and involved in regulating mitochondrial functions under physiological conditions. However, whether mtCB1R affords neuroprotection against I/R injury remains unknown. We used mouse models of cerebral I/R, primary cultured hippocampal neurons exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) and Ca2+-i...

  16. The phosphoarginine energy-buffering system of trypanosoma brucei involves multiple arginine kinase isoforms with different subcellular locations.

    Directory of Open Access Journals (Sweden)

    Frank Voncken

    Full Text Available Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3. Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide 'SNL'. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90% of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed.

  17. The Phosphoarginine Energy-Buffering System of Trypanosoma brucei Involves Multiple Arginine Kinase Isoforms with Different Subcellular Locations

    Science.gov (United States)

    Wadforth, Cath; Harley, Maggie; Colasante, Claudia

    2013-01-01

    Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3). Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide ‘SNL’. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90%) of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed. PMID:23776565

  18. Mitochondrial fusion, fission, and mitochondrial toxicity.

    Science.gov (United States)

    Meyer, Joel N; Leuthner, Tess C; Luz, Anthony L

    2017-08-05

    Mitochondrial dynamics are regulated by two sets of opposed processes: mitochondrial fusion and fission, and mitochondrial biogenesis and degradation (including mitophagy), as well as processes such as intracellular transport. These processes maintain mitochondrial homeostasis, regulate mitochondrial form, volume and function, and are increasingly understood to be critical components of the cellular stress response. Mitochondrial dynamics vary based on developmental stage and age, cell type, environmental factors, and genetic background. Indeed, many mitochondrial homeostasis genes are human disease genes. Emerging evidence indicates that deficiencies in these genes often sensitize to environmental exposures, yet can also be protective under certain circumstances. Inhibition of mitochondrial dynamics also affects elimination of irreparable mitochondrial DNA (mtDNA) damage and transmission of mtDNA mutations. We briefly review the basic biology of mitodynamic processes with a focus on mitochondrial fusion and fission, discuss what is known and unknown regarding how these processes respond to chemical and other stressors, and review the literature on interactions between mitochondrial toxicity and genetic variation in mitochondrial fusion and fission genes. Finally, we suggest areas for future research, including elucidating the full range of mitodynamic responses from low to high-level exposures, and from acute to chronic exposures; detailed examination of the physiological consequences of mitodynamic alterations in different cell types; mechanism-based testing of mitotoxicant interactions with interindividual variability in mitodynamics processes; and incorporating other environmental variables that affect mitochondria, such as diet and exercise. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. BID links ferroptosis to mitochondrial cell death pathways

    Directory of Open Access Journals (Sweden)

    Sandra Neitemeier

    2017-08-01

    In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by Xc- inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death.

  20. Mitochondrial redox signaling enables repair of injured skeletal muscle cells.

    Science.gov (United States)

    Horn, Adam; Van der Meulen, Jack H; Defour, Aurelia; Hogarth, Marshall; Sreetama, Sen Chandra; Reed, Aaron; Scheffer, Luana; Chandel, Navdeep S; Jaiswal, Jyoti K

    2017-09-05

    Strain and physical trauma to mechanically active cells, such as skeletal muscle myofibers, injures their plasma membranes, and mitochondrial function is required for their repair. We found that mitochondrial function was also needed for plasma membrane repair in myoblasts as well as nonmuscle cells, which depended on mitochondrial uptake of calcium through the mitochondrial calcium uniporter (MCU). Calcium uptake transiently increased the mitochondrial production of reactive oxygen species (ROS), which locally activated the guanosine triphosphatase (GTPase) RhoA, triggering F-actin accumulation at the site of injury and facilitating membrane repair. Blocking mitochondrial calcium uptake or ROS production prevented injury-triggered RhoA activation, actin polymerization, and plasma membrane repair. This repair mechanism was shared between myoblasts, nonmuscle cells, and mature skeletal myofibers. Quenching mitochondrial ROS in myofibers during eccentric exercise ex vivo caused increased damage to myofibers, resulting in a greater loss of muscle force. These results suggest a physiological role for mitochondria in plasma membrane repair in injured cells, a role that highlights a beneficial effect of ROS. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  1. Higher mitochondrial potential and elevated mitochondrial respiration are associated with excessive activation of blood platelets in diabetic rats.

    Science.gov (United States)

    Siewiera, Karolina; Kassassir, Hassan; Talar, Marcin; Wieteska, Lukasz; Watala, Cezary

    2016-03-01

    The high glucose concentration observed in diabetic patients is a recognized factor of mitochondrial damage in various cell types. Its impact on mitochondrial bioenergetics in blood platelets remains largely vague. The aim of the study was to determine how the metabolism of carbohydrates, which has been impaired by streptozotocin-induced diabetes may affect the functioning of platelet mitochondria. Diabetes was induced in Sprague Dawley rats by intraperitoneal injection of streptozotocin. Platelet mitochondrial respiratory capacity was monitored as oxygen consumption (high-resolution respirometry). Mitochondrial membrane potential was assessed using a fluorescent probe, JC-1. Activation of circulating platelets was monitored by flow cytometry measuring of the expressions of CD61 and CD62P on a blood platelet surface. To determine mitochondrial protein density in platelets, Western Blot technique was used. The results indicate significantly elevated mitochondria mass, increased mitochondrial membrane potential (ΔΨm) and enhanced respiration in STZ-diabetic animals, although the respiration control ratios appear to remain unchanged. Higher ΔΨm and elevated mitochondrial respiration were closely related to the excessive activation of circulating platelets in diabetic animals. Long-term diabetes can result in increased mitochondrial mass and may lead to hyperpolarization of blood platelet mitochondrial membrane. These alterations may be a potential underlying cause of abnormal platelet functioning in diabetes mellitus and hence, a potential target for antiplatelet therapies in diabetes. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Genome-wide analysis of chromatin structures in Trypanosoma brucei using high-resolution MNase-ChIP-seq.

    Science.gov (United States)

    Wedel, Carolin; Siegel, T Nicolai

    2017-09-01

    Specific DNA-protein interactions are the basis for many important cellular mechanisms like the regulation of gene expression or replication. Knowledge about the precise genomic locations of DNA-protein interactions is important because it provides insight into the regulation of these processes. Recently, we have adapted an approach that combines micrococcal nuclease (MNase) digestion of chromatin with chromatin immunoprecipitation in Trypanosoma brucei. Here, we describe in detail how this method can be used to map the genome-wide distribution of nucleosomes or other DNA-binding proteins at high resolution in T. brucei. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Pyrimidine Salvage Enzymes Are Essential for De Novo Biosynthesis of Deoxypyrimidine Nucleotides in Trypanosoma brucei.

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    Christopher Leija

    2016-11-01

    Full Text Available The human pathogenic parasite Trypanosoma brucei possess both de novo and salvage routes for the biosynthesis of pyrimidine nucleotides. Consequently, they do not require salvageable pyrimidines for growth. Thymidine kinase (TK catalyzes the formation of dTMP and dUMP and is one of several salvage enzymes that appear redundant to the de novo pathway. Surprisingly, we show through analysis of TK conditional null and RNAi cells that TK is essential for growth and for infectivity in a mouse model, and that a catalytically active enzyme is required for its function. Unlike humans, T. brucei and all other kinetoplastids lack dCMP deaminase (DCTD, which provides an alternative route to dUMP formation. Ectopic expression of human DCTD resulted in full rescue of the RNAi growth phenotype and allowed for selection of viable TK null cells. Metabolite profiling by LC-MS/MS revealed a buildup of deoxypyrimidine nucleosides in TK depleted cells. Knockout of cytidine deaminase (CDA, which converts deoxycytidine to deoxyuridine led to thymidine/deoxyuridine auxotrophy. These unexpected results suggested that T. brucei encodes an unidentified 5'-nucleotidase that converts deoxypyrimidine nucleotides to their corresponding nucleosides, leading to their dead-end buildup in TK depleted cells at the expense of dTTP pools. Bioinformatics analysis identified several potential candidate genes that could encode 5'-nucleotidase activity including an HD-domain protein that we show catalyzes dephosphorylation of deoxyribonucleotide 5'-monophosphates. We conclude that TK is essential for synthesis of thymine nucleotides regardless of whether the nucleoside precursors originate from the de novo pathway or through salvage. Reliance on TK in the absence of DCTD may be a shared vulnerability among trypanosomatids and may provide a unique opportunity to selectively target a diverse group of pathogenic single-celled eukaryotes with a single drug.

  4. Knockdown of asparagine synthetase A renders Trypanosoma brucei auxotrophic to asparagine.

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    Inês Loureiro

    Full Text Available Asparagine synthetase (AS catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A, known as strictly ammonia-dependent, and asparagine synthetase B (AS-B, which can use either ammonia or glutamine. The absence of AS-A in humans, and its presence in trypanosomes, suggested AS-A as a potential drug target that deserved further investigation. We report the presence of functional AS-A in Trypanosoma cruzi (TcAS-A and Trypanosoma brucei (TbAS-A: the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP, ammonia and Mg(2+. TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor, but surprisingly, can also use glutamine, a characteristic so far never described for any AS-A. TbAS-A knockdown by RNAi didn't affect in vitro growth of bloodstream forms of the parasite. However, growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine. As expected, mice infections with induced and non-induced T. brucei RNAi clones were similar to those from wild-type parasites. However, when induced T. brucei RNAi clones were injected in mice undergoing asparaginase treatment, which depletes blood asparagine, the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites. Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting, but is unlikely to be suitable as a drug target.

  5. Chemical characterisation of Nigerian red propolis and its biological activity against Trypanosoma Brucei.

    Science.gov (United States)

    Omar, Ruwida M K; Igoli, John; Gray, Alexander I; Ebiloma, Godwin Unekwuojo; Clements, Carol; Fearnley, James; Ebel, Ru Angeli Edrada; Zhang, Tong; De Koning, Harry P; Watson, David G

    2016-01-01

    A previous study showed the unique character of Nigerian red propolis from Rivers State, Nigeria (RSN), with regards to chemical composition and activity against Trypanosoma brucei in comparison with other African propolis. To carry out fractionation and biological testing of Nigerian propolis in order to isolate compounds with anti-trypanosomal activity. To compare the composition of the RSN propolis with the composition of Brazilian red propolis. Profiling was carried out using HPLC-UV-ELSD and HPLC-Orbitrap-FTMS on extracts of two samples collected from RSN with data extraction using MZmine software. Isolation was carried out by normal phase and reversed phase MPLC. Elucidation of the compounds with a purity > 95% was performed by 1D/2D NMR HRMS and HRLC-MS(n) . Ten phenolic compounds were isolated or in the case of liquiritigenin partially purified. Data for nine of these correlated with literature reports of known compounds i.e. one isoflavanone, calycosin (1); two flavanones, liquiritigenin (2) and pinocembrin (5); an isoflavan, vestitol (3); a pterocarpan, medicarpin (4); two prenylflavanones, 8-prenylnaringenin (7) and 6-prenylnaringenin (8); and two geranyl flavonoids, propolin D (9) and macarangin (10). The tenth was elucidated as a previously undescribed dihydrobenzofuran (6). The isolated compounds were tested against Trypanosoma brucei and displayed moderate to high activity. Some of the compounds tested had similar activity against wild type T. brucei and two strains displaying pentamidine resistance. Nigerian propolis from RSN has some similarities with Brazilian red propolis. The propolis displayed anti-trypanosomal activity at a potentially useful level. Copyright © 2015 John Wiley & Sons, Ltd.

  6. The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.

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    Nikolay G Kolev

    2010-09-01

    Full Text Available The genome of Trypanosoma brucei, the causative agent of African trypanosomiasis, was published five years ago, yet identification of all genes and their transcripts remains to be accomplished. Annotation is challenged by the organization of genes transcribed by RNA polymerase II (Pol II into long unidirectional gene clusters with no knowledge of how transcription is initiated. Here we report a single-nucleotide resolution genomic map of the T. brucei transcriptome, adding 1,114 new transcripts, including 103 non-coding RNAs, confirming and correcting many of the annotated features and revealing an extensive heterogeneity of 5' and 3' ends. Some of the new transcripts encode polypeptides that are either conserved in T. cruzi and Leishmania major or were previously detected in mass spectrometry analyses. High-throughput RNA sequencing (RNA-Seq was sensitive enough to detect transcripts at putative Pol II transcription initiation sites. Our results, as well as recent data from the literature, indicate that transcription initiation is not solely restricted to regions at the beginning of gene clusters, but may occur at internal sites. We also provide evidence that transcription at all putative initiation sites in T. brucei is bidirectional, a recently recognized fundamental property of eukaryotic promoters. Our results have implications for gene expression patterns in other important human pathogens with similar genome organization (Trypanosoma cruzi, Leishmania sp. and revealed heterogeneity in pre-mRNA processing that could potentially contribute to the survival and success of the parasite population in the insect vector and the mammalian host.

  7. Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells.

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    Deborah Frenkel

    2016-07-01

    Full Text Available After infection with T. brucei AnTat 1.1, C57BL/6 mice lost splenic B2 B cells and lymphoid follicles, developed poor parasite-specific antibody responses, lost weight, became anemic and died with fulminating parasitemia within 35 days. In contrast, infected C57BL/6 mice lacking the cytotoxic granule pore-forming protein perforin (Prf1-/- retained splenic B2 B cells and lymphoid follicles, developed high-titer antibody responses against many trypanosome polypeptides, rapidly suppressed parasitemia and did not develop anemia or lose weight for at least 60 days. Several lines of evidence show that T. brucei infection-induced splenic B cell depletion results from natural killer (NK cell-mediated cytotoxicity: i B2 B cells were depleted from the spleens of infected intact, T cell deficient (TCR-/- and FcγRIIIa deficient (CD16-/- C57BL/6 mice excluding a requirement for T cells, NKT cell, or antibody-dependent cell-mediated cytotoxicity; ii administration of NK1.1 specific IgG2a (mAb PK136 but not irrelevant IgG2a (myeloma M9144 prevented infection-induced B cell depletion consistent with a requirement for NK cells; iii splenic NK cells but not T cells or NKT cells degranulated in infected C57BL/6 mice co-incident with B cell depletion evidenced by increased surface expression of CD107a; iv purified NK cells from naïve C57BL/6 mice killed purified splenic B cells from T. brucei infected but not uninfected mice in vitro indicating acquisition of an NK cell activating phenotype by the post-infection B cells; v adoptively transferred C57BL/6 NK cells prevented infection-induced B cell population growth in infected Prf1-/- mice consistent with in vivo B cell killing; vi degranulated NK cells in infected mice had altered gene and differentiation antigen expression and lost cytotoxic activity consistent with functional exhaustion, but increased in number as infection progressed indicating continued generation. We conclude that NK cells in T. brucei

  8. Role of Cardiolipin in Mitochondrial Signaling Pathways

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    Jan Dudek

    2017-09-01

    Full Text Available The phospholipid cardiolipin (CL is an essential constituent of mitochondrial membranes and plays a role in many mitochondrial processes, including respiration and energy conversion. Pathological changes in CL amount or species composition can have deleterious consequences for mitochondrial function and trigger the production of reactive oxygen species. Signaling networks monitor mitochondrial function and trigger an adequate cellular response. Here, we summarize the role of CL in cellular signaling pathways and focus on tissues with high-energy demand, like the heart. CL itself was recently identified as a precursor for the formation of lipid mediators. We highlight the concept of CL as a signaling platform. CL is exposed to the outer mitochondrial membrane upon mitochondrial stress and CL domains serve as a binding site in many cellular signaling events. During mitophagy, CL interacts with essential players of mitophagy like Beclin 1 and recruits the autophagic machinery by its interaction with LC3. Apoptotic signaling pathways require CL as a binding platform to recruit apoptotic factors such as tBid, Bax, caspase-8. CL required for the activation of the inflammasome and plays a role in inflammatory signaling. As changes in CL species composition has been observed in many diseases, the signaling pathways described here may play a general role in pathology.

  9. Mitochondrial DNA Alterations and Reduced Mitochondrial Function in Aging

    OpenAIRE

    Hebert, Sadie L.; Lanza, Ian R.; Nair, K. Sreekumaran

    2010-01-01

    Oxidative damage to mitochondrial DNA increases with aging. This damage has the potential to affect mitochondrial DNA replication and transcription which could alter the abundance or functionality of mitochondrial proteins. This review describes mitochondrial DNA alterations and changes in mitochondrial function that occur with aging. Age-related alterations in mitochondrial DNA as a possible contributor to the reduction in mitochondrial function are discussed.

  10. Mitochondrial Aging: Is There a Mitochondrial Clock?

    Science.gov (United States)

    Zorov, Dmitry B; Popkov, Vasily A; Zorova, Ljubava D; Vorobjev, Ivan A; Pevzner, Irina B; Silachev, Denis N; Zorov, Savva D; Jankauskas, Stanislovas S; Babenko, Valentina A; Plotnikov, Egor Y

    2017-09-01

    Fragmentation (fission) of mitochondria, occurring in response to oxidative challenge, leads to heterogeneity in the mitochondrial population. It is assumed that fission provides a way to segregate mitochondrial content between the "young" and "old" phenotype, with the formation of mitochondrial "garbage," which later will be disposed. Fidelity of this process is the basis of mitochondrial homeostasis, which is disrupted in pathological conditions and aging. The asymmetry of the mitochondrial fission is similar to that of their evolutionary ancestors, bacteria, which also undergo an aging process. It is assumed that mitochondrial markers of aging are recognized by the mitochondrial quality control system, preventing the accumulation of dysfunctional mitochondria, which normally are subjected to disposal. Possibly, oncocytoma, with its abnormal proliferation of mitochondria occupying the entire cytoplasm, represents the case when segregation of damaged mitochondria is impaired during mitochondrial division. It is plausible that mitochondria contain a "clock" which counts the degree of mitochondrial senescence as the extent of flagging (by ubiquitination) of damaged mitochondria. Mitochondrial aging captures the essence of the systemic aging which must be analyzed. We assume that the mitochondrial aging mechanism is similar to the mechanism of aging of the immune system which we discuss in detail. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Mitochondrial translation initiation machinery: conservation and diversification.

    Science.gov (United States)

    Kuzmenko, Anton; Atkinson, Gemma C; Levitskii, Sergey; Zenkin, Nikolay; Tenson, Tanel; Hauryliuk, Vasili; Kamenski, Piotr

    2014-05-01

    The highly streamlined mitochondrial genome encodes almost exclusively a handful of transmembrane components of the respiratory chain complex. In order to ensure the correct assembly of the respiratory chain, the products of these genes must be produced in the correct stoichiometry and inserted into the membrane, posing a unique challenge to the mitochondrial translational system. In this review we describe the proteins orchestrating mitochondrial translation initiation: bacterial-like general initiation factors mIF2 and mIF3, as well as mitochondria-specific components - mRNA-specific translational activators and mRNA-nonspecific accessory initiation factors. We consider how the fast rate of evolution in these organelles has not only created a system that is divergent from that of its bacterial ancestors, but has led to a huge diversity in lineage specific mechanistic features of mitochondrial translation initiation among eukaryotes. Copyright © 2013 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

  12. Melatonin in Mitochondrial Dysfunction and Related Disorders

    Science.gov (United States)

    Srinivasan, Venkatramanujam; Spence, D. Warren; Pandi-Perumal, Seithikurippu R.; Brown, Gregory M.; Cardinali, Daniel P.

    2011-01-01

    Mitochondrial dysfunction is considered one of the major causative factors in the aging process, ischemia/reperfusion (I/R), septic shock, and neurodegenerative disorders like Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's disease (HD). Increased free radical generation, enhanced mitochondrial inducible nitric oxide (NO) synthase activity, enhanced NO production, decreased respiratory complex activity, impaired electron transport system, and opening of mitochondrial permeability transition pore all have been suggested as factors responsible for impaired mitochondrial function. Melatonin, the major hormone of the pineal gland, also acts as an antioxidant and as a regulator of mitochondrial bioenergetic function. Both in vitro and in vivo, melatonin was effective for preventing oxidative stress/nitrosative stress-induced mitochondrial dysfunction seen in experimental models of PD, AD, and HD. In addition, melatonin is known to retard aging and to inhibit the lethal effects of septic shock or I/R lesions by maintaining respiratory complex activities, electron transport chain, and ATP production in mitochondria. Melatonin is selectively taken up by mitochondrial membranes, a function not shared by other antioxidants. Melatonin has thus emerged as a major potential therapeutic tool for treating neurodegenerative disorders such as PD or AD, and for preventing the lethal effects of septic shock or I/R. PMID:21629741

  13. Melatonin in Mitochondrial Dysfunction and Related Disorders

    Directory of Open Access Journals (Sweden)

    Venkatramanujam Srinivasan

    2011-01-01

    Full Text Available Mitochondrial dysfunction is considered one of the major causative factors in the aging process, ischemia/reperfusion (I/R, septic shock, and neurodegenerative disorders like Parkinson's disease (PD, Alzheimer's disease (AD, and Huntington's disease (HD. Increased free radical generation, enhanced mitochondrial inducible nitric oxide (NO synthase activity, enhanced NO production, decreased respiratory complex activity, impaired electron transport system, and opening of mitochondrial permeability transition pore all have been suggested as factors responsible for impaired mitochondrial function. Melatonin, the major hormone of the pineal gland, also acts as an antioxidant and as a regulator of mitochondrial bioenergetic function. Both in vitro and in vivo, melatonin was effective for preventing oxidative stress/nitrosative stress-induced mitochondrial dysfunction seen in experimental models of PD, AD, and HD. In addition, melatonin is known to retard aging and to inhibit the lethal effects of septic shock or I/R lesions by maintaining respiratory complex activities, electron transport chain, and ATP production in mitochondria. Melatonin is selectively taken up by mitochondrial membranes, a function not shared by other antioxidants. Melatonin has thus emerged as a major potential therapeutic tool for treating neurodegenerative disorders such as PD or AD, and for preventing the lethal effects of septic shock or I/R.

  14. Structural and functional insight into ADF/cofilin from Trypanosoma brucei.

    Science.gov (United States)

    Dai, Kun; Liao, Shanhui; Zhang, Jiahai; Zhang, Xuecheng; Tu, Xiaoming

    2013-01-01

    The ADF/cofilin family has been characterized as a group of actin-binding proteins critical for controlling the assembly of actin within the cells. In this study, the solution structure of the ADF/cofilin from Trypanosoma brucei (TbCof) was determined by NMR spectroscopy. TbCof adopts the conserved ADF/cofilin fold with a central β-sheet composed of six β-strands surrounded by five α-helices. Isothermal titration calorimetry experiments denoted a submicromolar affinity between TbCof and G-actin, and the affinity between TbCof and ADP-G-actin was five times higher than that between TbCof and ATP-G-actin at low ionic strength. The results obtained from electron microscopy and actin filament sedimentation assays showed that TbCof depolymerized but did not co-sediment with actin filaments and its ability of F-actin depolymerization was pH independent. Similar to actin, TbCof was distributed throughout the cytoplasm. All our data indicate a structurally and functionally conserved ADF/cofilin from Trypanosoma brucei.

  15. Base J and H3.V Regulate Transcriptional Termination in Trypanosoma brucei.

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    Danae Schulz

    2016-01-01

    Full Text Available Trypanosoma brucei is a protozoan parasite that lacks many transcription factors found in other eukaryotes, such as those whose binding demarcates enhancers. T. brucei retains histone variants and modifications, however, and it is hypothesized that it relies on epigenetic marks to define transcription-related boundaries. The histone H3 variant (H3.V and an alternate nucleotide, base J (ß-D-glucosyl-hydroxymethyluracil, are two chromatin marks found at both transcription termination sites (TTSs and telomeres. Here, we report that the absence of both base J and H3.V result in transcription readthrough and the appearance of antisense transcripts near TTSs. Additionally, we find that maintaining the transcriptional silencing of pol I-transcribed telomeric Variant Surface Glycoprotein (VSG genes appears to be dependent on deposition of H3.V alone. Our study reveals that gene expression depends on different epigenetic cues depending on chromosomal location and on the transcribing polymerase. This work provides insight into how these signals may have evolved into the more nuanced and fine-tuned gene regulatory mechanisms observed in other model systems.

  16. Investigating the Chaperone Properties of a Novel Heat Shock Protein, Hsp70.c, from Trypanosoma brucei

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    Adélle Burger

    2014-01-01

    Full Text Available The neglected tropical disease, African Trypanosomiasis, is fatal and has a crippling impact on economic development. Heat shock protein 70 (Hsp70 is an important molecular chaperone that is expressed in response to stress and Hsp40 acts as its co-chaperone. These proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. A novel cytosolic Hsp70, from Trypanosoma brucei, TbHsp70.c, contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. The ability of a cytosolic Hsp40 from Trypanosoma brucei J protein 2, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective was to functionally characterize TbHsp70.c to further expand our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile MDH and chemically denatured rhodanese. ATPase assays revealed a 2.8-fold stimulation of the ATPase activity of TbHsp70.c by Tbj2. TbHsp70.c and Tbj2 both demonstrated chaperone activity and Tbj2 functions as a co-chaperone of TbHsp70.c. In vivo heat stress experiments indicated upregulation of the expression levels of TbHsp70.c.

  17. Secondary metabolites from Vietnamese marine invertebrates with activity against Trypanosoma brucei and T. cruzi.

    Science.gov (United States)

    Thao, Nguyen Phuong; No, Joo Hwan; Luyen, Bui Thi Thuy; Yang, Gyongseon; Byun, Soo Young; Goo, Junghyun; Kim, Kyung Tae; Cuong, Nguyen Xuan; Nam, Nguyen Hoai; Van Minh, Chau; Schmidt, Thomas J; Kang, Jong Seong; Kim, Young Ho

    2014-06-11

    Marine-derived natural products from invertebrates comprise an extremely diverse and promising source of the compounds from a wide variety of structural classes. This study describes the discovery of five marine natural products with activity against Trypanosoma species by natural product library screening using whole cell in vitro assays. We investigated the anti-trypanosomal activity of the extracts from the soft corals and echinoderms living in Vietnamese seas. Of the samples screened, the methanolic extracts of several marine organisms exhibited potent activities against cultures of Trypanosoma brucei and T. cruzi (EC50 activity against T. brucei with EC50 values ranging from 1.57 ± 0.14 to 14.6 ± 1.36 μM, relative to the positive control, pentamidine (EC50 = 0.015 ± 0.003 μM). Laevigatol B (1) and 5α-cholest-8(14)-ene-3β,7α-diol (5) exhibited also significant inhibitory effects on T. cruzi. The cytotoxic activity of the pure compounds on mammalian cells was also assessed and found to be insignificant in all cases. This is the first report on the inhibitory effects of marine organisms collected in Vietnamese seas against Trypanosoma species responsible for neglected tropical diseases.

  18. An Overview of Trypanosoma brucei Infections: An Intense Host-Parasite Interaction.

    Science.gov (United States)

    Ponte-Sucre, Alicia

    2016-01-01

    Trypanosoma brucei rhodesiense and T. brucei gambiense, the causative agents of Human African Trypanosomiasis, are transmitted by tsetse flies. Within the vector, the parasite undergoes through transformations that prepares it to infect the human host. Sequentially these developmental stages are the replicative procyclic (in which the parasite surface is covered by procyclins) and trypo-epimastigote forms, as well as the non-replicative, infective, metacyclic form that develops in the vector salivary glands. As a pre-adaptation to their life in humans, metacyclic parasites begin to express and be densely covered by the Variant Surface Glycoprotein (VSG). Once the metacyclic form invades the human host the parasite develops into the bloodstream form. Herein the VSG triggers a humoral immune response. To avoid this humoral response, and essential for survival while in the bloodstream, the parasite changes its cover periodically and sheds into the surroundings the expressed VSG, thus evading the consequences of the immune system activation. Additionally, tools comparable to quorum sensing are used by the parasite for the successful parasite transmission from human to insect. On the other hand, the human host promotes clearance of the parasite triggering innate and adaptive immune responses and stimulating cytokine and chemokine secretion. All in all, the host-parasite interaction is extremely active and leads to responses that need multiple control sites to develop appropriately.

  19. Betaine is a positive regulator of mitochondrial respiration

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Icksoo, E-mail: icksoolee@dankook.ac.kr

    2015-01-09

    Highlights: • Betaine enhances cytochrome c oxidase activity and mitochondrial respiration. • Betaine increases mitochondrial membrane potential and cellular energy levels. • Betaine’s anti-tumorigenic effect might be due to a reversal of the Warburg effect. - Abstract: Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro. Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect.

  20. 1H, 13C and 15N resonance assignments for a putative ADF/Cofilin from Trypanosoma brucei.

    Science.gov (United States)

    Dai, Kun; Yuan, Guangfa; Liao, Shanhui; Zhang, Jiahai; Tu, Xiaoming

    2011-10-01

    Actin-depolymerizing factor (ADF)/cofilin proteins are a family of actin-binding proteins expressed in almost all eukaryotic cells, and play a significant role in regulating actin-filament dynamics. Here we report the resonance assignments of a putative ADF/cofilin from Trypanosoma brucei for further understanding of the relationship between its structure and function.

  1. Variations in maxi-circle and mini-circle sequences in kinetoplast DNAs from different Trypanosoma brucei strains.

    NARCIS (Netherlands)

    P. Borst (Piet); F. Fase-Fowler; J.H.J. Hoeijmakers (Jan); A.C.C. Frasch

    1980-01-01

    textabstractWe have compared a total of 30 recognition sites for eight restriction endonucleases on the 20-kilobase-pair maxi-circle of kinetoplast DNAs from five different Trypanosoma brucei strains. In addition to three polymorphic sites were have found a 5 kilobase-pair region that is not cleaved

  2. THE CYTOSOLIC AND GLYCOSOMAL GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM TRYPANOSOMA-BRUCEI - KINETIC-PROPERTIES AND COMPARISON WITH HOMOLOGOUS ENZYMES

    NARCIS (Netherlands)

    LAMBEIR, AM; LOISEAU, AM; KUNTZ, DA; VELLIEUX, FM; MICHELS, PAM; OPPERDOES, FR

    1991-01-01

    The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like

  3. A tropical tale: how Naja nigricollis venom beats Trypanosoma brucei

    DEFF Research Database (Denmark)

    Martos Esteban, Andrea; Laustsen, Andreas Hougaard; Carrington, Mark

    Trypanosoma brucei is a parasitic protozoan species capable to infecting insect vectors whose bite further produces African sleeping sickness inhuman beings [1]. During the parasite’s extracellular life in the mammalian host,its outer coat, mainly composed of Variable Surface Glycoproteins (VSGs...

  4. Haematological indices in Trypanosoma brucei brucei (Federe isolate infected Nigerian donkeys (Equus asinus treated with homidium and isometamidium chloride of ciprofloxacin in broiler chickens after single intravenous and intraingluvial administration

    Directory of Open Access Journals (Sweden)

    Queen Nneka Oparah

    2017-03-01

    Full Text Available The efficacy of intramuscular administration of Homidium chloride (Novidium® and Isometamidium chloride (Sécuridium® in Nigerian donkeys (Equus asinus experimentally infected with T. b. brucei (Federe isolate was investigated. Changes in haematological and serum biochemical indices were evaluated using clinical haematology and biochemistry methods. Red blood cell (RBC count for the negative control group was significantly higher than for the positive control, Novidium® and Sécuridium®-treatment groups. Haemoglobin (Hb concentration significantly reduced in the infected untreated group compared with other groups. Packed cell volume (PCV was significantly different between negative and positive controls, and also between the infected untreated and treatment groups. There was significant reduction in platelet counts post-infection and post-treatment. Mean corpuscular volume (MCV increased significantly in the treatment groups while mean corpuscular haemoglobin concentration (MCHC significantly reduced only in the Sécuridium®-treatment group. Lymphocyte count for infected untreated was non-significantly higher than for the uninfected controls, but treatment with both trypanocides recorded further increases, which were higher compared with that of the uninfected group. Post infection and treatment, aspartate aminotransferase (AST levels increased significantly. There were non-significant differences in electrolyte ion concentrations across the groups except for chloride ion which recorded a significant reduction in the Novidium®-treatment group. This experiment revealed that Nigerian donkeys infected with T. brucei brucei (Federe isolate developed symptoms of trypanosomosis; anaemia, lymphocytosis and thrombocytopenia. Treatment with the trypanocides ameliorated effects of the infection, and results suggest that immunosuppression may not be a substantial clinical manifestation of T. brucei brucei (Federe isolate trypanosomosis in Nigerian

  5. Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS-mediated cardiomyocyte hypertrophy.

    Science.gov (United States)

    Tigchelaar, Wardit; Yu, Hongjuan; de Jong, Anne Margreet; van Gilst, Wiek H; van der Harst, Pim; Westenbrink, B Daan; de Boer, Rudolf A; Silljé, Herman H W

    2015-01-15

    Recently, a locus at the mitochondrial exo/endonuclease EXOG gene, which has been implicated in mitochondrial DNA repair, was associated with cardiac function. The function of EXOG in cardiomyocytes is still elusive. Here we investigated the role of EXOG in mitochondrial function and hypertrophy in cardiomyocytes. Depletion of EXOG in primary neonatal rat ventricular cardiomyocytes (NRVCs) induced a marked increase in cardiomyocyte hypertrophy. Depletion of EXOG, however, did not result in loss of mitochondrial DNA integrity. Although EXOG depletion did not induce fetal gene expression and common hypertrophy pathways were not activated, a clear increase in ribosomal S6 phosphorylation was observed, which readily explains increased protein synthesis. With the use of a Seahorse flux analyzer, it was shown that the mitochondrial oxidative consumption rate (OCR) was increased 2.4-fold in EXOG-depleted NRVCs. Moreover, ATP-linked OCR was 5.2-fold higher. This increase was not explained by mitochondrial biogenesis or alterations in mitochondrial membrane potential. Western blotting confirmed normal levels of the oxidative phosphorylation (OXPHOS) complexes. The increased OCR was accompanied by a 5.4-fold increase in mitochondrial ROS levels. These increased ROS levels could be normalized with specific mitochondrial ROS scavengers (MitoTEMPO, mnSOD). Remarkably, scavenging of excess ROS strongly attenuated the hypertrophic response. In conclusion, loss of EXOG affects normal mitochondrial function resulting in increased mitochondrial respiration, excess ROS production, and cardiomyocyte hypertrophy. Copyright © 2015 the American Physiological Society.

  6. Roles of mitochondrial fragmentation and reactive oxygen species in mitochondrial dysfunction and myocardial insulin resistance

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Tomoyuki [Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192 (Japan); Saotome, Masao, E-mail: msaotome@hama-med.ac.jp [Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192 (Japan); Nobuhara, Mamoru; Sakamoto, Atsushi; Urushida, Tsuyoshi; Katoh, Hideki; Satoh, Hiroshi [Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192 (Japan); Funaki, Makoto [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan); Hayashi, Hideharu [Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192 (Japan)

    2014-05-01

    Purpose: Evidence suggests an association between aberrant mitochondrial dynamics and cardiac diseases. Because myocardial metabolic deficiency caused by insulin resistance plays a crucial role in heart disease, we investigated the role of dynamin-related protein-1 (DRP1; a mitochondrial fission protein) in the pathogenesis of myocardial insulin resistance. Methods and Results: DRP1-expressing H9c2 myocytes, which had fragmented mitochondria with mitochondrial membrane potential (ΔΨ{sub m}) depolarization, exhibited attenuated insulin signaling and 2-deoxy-D-glucose (2-DG) uptake, indicating insulin resistance. Treatment of the DRP1-expressing myocytes with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (TMPyP) significantly improved insulin resistance and mitochondrial dysfunction. When myocytes were exposed to hydrogen peroxide (H{sub 2}O{sub 2}), they increased DRP1 expression and mitochondrial fragmentation, resulting in ΔΨ{sub m} depolarization and insulin resistance. When DRP1 was suppressed by siRNA, H{sub 2}O{sub 2}-induced mitochondrial dysfunction and insulin resistance were restored. Our results suggest that a mutual enhancement between DRP1 and reactive oxygen species could induce mitochondrial dysfunction and myocardial insulin resistance. In palmitate-induced insulin-resistant myocytes, neither DRP1-suppression nor TMPyP restored the ΔΨ{sub m} depolarization and impaired 2-DG uptake, however they improved insulin signaling. Conclusions: A mutual enhancement between DRP1 and ROS could promote mitochondrial dysfunction and inhibition of insulin signal transduction. However, other mechanisms, including lipid metabolite-induced mitochondrial dysfunction, may be involved in palmitate-induced insulin resistance. - Highlights: • DRP1 promotes mitochondrial fragmentation and insulin-resistance. • A mutual enhancement between DRP1 and ROS ipromotes insulin-resistance. • Palmitate increases DRP1 expression and induces insulin

  7. Sulfhydryl reagent susceptibility in proteins with high sequence similarity--triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi and Leishmania mexicana.

    Science.gov (United States)

    Garza-Ramos, G; Cabrera, N; Saavedra-Lira, E; Tuena de Gómez-Puyou, M; Ostoa-Saloma, P; Pérez-Montfort, R; Gómez-Puyou, A

    1998-05-01

    The amino acid sequence of triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68%. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei triosephosphate isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi triosephosphate isomerase was more than 100-fold more sensitive. The sensitivity of wild type triosephosphate isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8-anilino-1-napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half-maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana triosephosphate isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accessibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi triosephosphate isomerase which makes its dimer interface more susceptible for perturbation.

  8. Molecular profiles of Trypanosoma brucei, T. evansi and T. equiperdum stocks revealed by the random amplified polymorphic DNA method.

    Science.gov (United States)

    Lun, Zhao-Rong; Li, An-Xing; Chen, Xiao-Guang; Lu, Li-Xin; Zhu, Xing-Quan

    2004-03-01

    A total of 20 random primers (10-mers) were used to amplify RAPD markers from the genomic DNA of four Trypanosoma brucei stocks from East and West Africa, four T. evansi stocks from Africa, Asia and South America and one T. equiperdum stock from Asia. Between 65 and 88 reproducible fragments ranging from 0.25 to 2.15 kb were generated from these stocks depending on the stock/primer combination. The similarity coefficient (SC) among the stocks of T. brucei from Kenya, Nigeria, Tanzania and Zambia ranged from 62.9% to 74.0% (average: 67.6%). The SC among the stocks of T. evansi from Kenya, China and Brazil was 76.4%-95.5% (average: 86.4%), while the SC between T. evansi stock from China and Brazil was 95.5%. For T. evansi and T. equiperdum, the SC among the stocks ranged from 81.2% to 94.4% (average: 87.6%). As for the SC among the stocks of T. brucei and T. evansi, it was found to be from 54.7% to 80.3% (average: 68.0%) and the SC among stocks of T. brucei and T. equiperdum was from 59.4% to 76.9% (average: 68.1%). Our results indicate that the stocks of T. evansi from China and from Brazil are more closely related to the stock of T. equiperdum from China than to the stocks of T. evansi isolated from Kenya and to the stocks of T. brucei. In addition, our results further support the hypothesis that T. evansi stocks from China and Brazil could have arisen from a single lineage. The possible evolution of T. evansi and T. equiperdum is also discussed.

  9. Parkin suppresses Drp1-independent mitochondrial division

    Energy Technology Data Exchange (ETDEWEB)

    Roy, Madhuparna, E-mail: mroy17@jhmi.edu; Itoh, Kie, E-mail: kito5@jhmi.edu; Iijima, Miho, E-mail: miijima@jhmi.edu; Sesaki, Hiromi, E-mail: hsesaki@jhmi.edu

    2016-07-01

    The cycle of mitochondrial division and fusion disconnect and reconnect individual mitochondria in cells to remodel this energy-producing organelle. Although dynamin-related protein 1 (Drp1) plays a major role in mitochondrial division in cells, a reduced level of mitochondrial division still persists even in the absence of Drp1. It is unknown how much Drp1-mediated mitochondrial division accounts for the connectivity of mitochondria. The role of a Parkinson’s disease-associated protein—parkin, which biochemically and genetically interacts with Drp1—in mitochondrial connectivity also remains poorly understood. Here, we quantified the number and connectivity of mitochondria using mitochondria-targeted photoactivatable GFP in cells. We show that the loss of Drp1 increases the connectivity of mitochondria by 15-fold in mouse embryonic fibroblasts (MEFs). While a single loss of parkin does not affect the connectivity of mitochondria, the connectivity of mitochondria significantly decreased compared with a single loss of Drp1 when parkin was lost in the absence of Drp1. Furthermore, the loss of parkin decreased the frequency of depolarization of the mitochondrial inner membrane that is caused by increased mitochondrial connectivity in Drp1-knockout MEFs. Therefore, our data suggest that parkin negatively regulates Drp1-indendent mitochondrial division. -- Highlights: •A Drp1-mediated mechanism accounts for ∼95% of mitochondrial division. •Parkin controls the connectivity of mitochondria via a mechanism that is independent of Drp1. •In the absence of Drp1, connected mitochondria transiently depolarize. •The transient depolarization is independent of calcium signaling and uncoupling protein 2.

  10. Altered Mitochondrial Dynamics and TBI Pathophysiology

    Science.gov (United States)

    Fischer, Tara D.; Hylin, Michael J.; Zhao, Jing; Moore, Anthony N.; Waxham, M. Neal; Dash, Pramod K.

    2016-01-01

    Mitochondrial function is intimately linked to cellular survival, growth, and death. Mitochondria not only generate ATP from oxidative phosphorylation, but also mediate intracellular calcium buffering, generation of reactive oxygen species (ROS), and apoptosis. Electron leakage from the electron transport chain, especially from damaged or depolarized mitochondria, can generate excess free radicals that damage cellular proteins, DNA, and lipids. Furthermore, mitochondrial damage releases pro-apoptotic factors to initiate cell death. Previous studies have reported that traumatic brain injury (TBI) reduces mitochondrial respiration, enhances production of ROS, and triggers apoptotic cell death, suggesting a prominent role of mitochondria in TBI pathophysiology. Mitochondria maintain cellular energy homeostasis and health via balanced processes of fusion and fission, continuously dividing and fusing to form an interconnected network throughout the cell. An imbalance of these processes, particularly an excess of fission, can be detrimental to mitochondrial function, causing decreased respiration, ROS production, and apoptosis. Mitochondrial fission is regulated by the cytosolic GTPase, dynamin-related protein 1 (Drp1), which translocates to the mitochondrial outer membrane (MOM) to initiate fission. Aberrant Drp1 activity has been linked to excessive mitochondrial fission and neurodegeneration. Measurement of Drp1 levels in purified hippocampal mitochondria showed an increase in TBI animals as compared to sham controls. Analysis of cryo-electron micrographs of these mitochondria also showed that TBI caused an initial increase in the length of hippocampal mitochondria at 24 h post-injury, followed by a significant decrease in length at 72 h. Post-TBI administration of Mitochondrial division inhibitor-1 (Mdivi-1), a pharmacological inhibitor of Drp1, prevented this decrease in mitochondria length. Mdivi-1 treatment also reduced the loss of newborn neurons in the

  11. The Function of the Mitochondrial Calcium Uniporter in Neurodegenerative Disorders

    Directory of Open Access Journals (Sweden)

    Yajin Liao

    2017-02-01

    Full Text Available The mitochondrial calcium uniporter (MCU—a calcium uniporter on the inner membrane of mitochondria—controls the mitochondrial calcium uptake in normal and abnormal situations. Mitochondrial calcium is essential for the production of adenosine triphosphate (ATP; however, excessive calcium will induce mitochondrial dysfunction. Calcium homeostasis disruption and mitochondrial dysfunction is observed in many neurodegenerative disorders. However, the role and regulatory mechanism of the MCU in the development of these diseases are obscure. In this review, we summarize the role of the MCU in controlling oxidative stress-elevated mitochondrial calcium and its function in neurodegenerative disorders. Inhibition of the MCU signaling pathway might be a new target for the treatment of neurodegenerative disorders.

  12. Mitochondrial disease and epilepsy.

    Science.gov (United States)

    Rahman, Shamima

    2012-05-01

    Mitochondrial respiratory chain disorders are relatively common inborn errors of energy metabolism, with a combined prevalence of one in 5000. These disorders typically affect tissues with high energy requirements, and cerebral involvement occurs frequently in childhood, often manifesting in seizures. Mitochondrial diseases are genetically heterogeneous; to date, mutations have been reported in all 37 mitochondrially encoded genes and more than 80 nuclear genes. The major genetic causes of mitochondrial epilepsy are mitochondrial DNA mutations (including those typically associated with the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes [MELAS] and myoclonic epilepsy with ragged red fibres [MERRF] syndromes); mutations in POLG (classically associated with Alpers syndrome but also presenting as the mitochondrial recessive ataxia syndrome [MIRAS], spinocerebellar ataxia with epilepsy [SCAE], and myoclonus, epilepsy, myopathy, sensory ataxia [MEMSA] syndromes in older individuals) and other disorders of mitochondrial DNA maintenance; complex I deficiency; disorders of coenzyme Q(10) biosynthesis; and disorders of mitochondrial translation such as RARS2 mutations. It is not clear why some genetic defects, but not others, are particularly associated with seizures. Epilepsy may be the presenting feature of mitochondrial disease but is often part of a multisystem clinical presentation. Mitochondrial epilepsy may be very difficult to manage, and is often a poor prognostic feature. At present there are no curative treatments for mitochondrial disease. Individuals with mitochondrial epilepsy are frequently prescribed multiple anticonvulsants, and the role of vitamins and other nutritional supplements and the ketogenic diet remain unproven. © The Author. Developmental Medicine & Child Neurology © 2012 Mac Keith Press.

  13. Laminar Shear Stress Promotes Mitochondrial Homeostasis in Endothelial Cells.

    Science.gov (United States)

    Wu, Li-Hong; Chang, Hao-Chun; Ting, Pei-Ching; Wang, Danny Ling

    2017-12-08

    Vascular endothelial cells (ECs) are constantly subjected to flow-induced shear stress that is crucial for endothelial functions. Laminar shear stress (LSS) exerts atheroprotection to ECs. Mitochondrial homeostasis is essential for cellular survival. However, the effects of LSS on mitochondrial homeostasis in ECs remain unclear. Mitochondrial homeostasis in ECs exposed to LSS was examined. Cultured human umbilical vein ECs were subjected to LSS (12 dynes/cm2 ) generated by a parallel-plate flow chamber system. ECs subjected to LSS demonstrated an increment of mitochondria in tubular form coupled with the increase of fusion proteins (Mfn2, OPA1) and the decrease of fission protein (Fis1). An increase of both long- and short- OPA1 along with a higher protease YME1L level were observed. LSS triggered a rapid phosphorylation on S637 but a decrease on S616 of fission-controlled protein Drp1. Consistently, Drp1 translocation to mitochondria was decreased in sheared ECs, suggesting that LSS promotes mitochondrial fusion. Enhanced mitochondrial biogenesis in sheared ECs was shown by the increase of mitochondrial mass and its regulatory proeins (PGC1α, TFAM, Nrf1). LSS enhances the expression of mitochondrial antioxidant enzymes and improves mitochondrial functions indicated by the increase of mitochondrial membrane potential (ΔΨm) and ATP generation. TNF α treatment decreased mitochondrial tubular network and its functions in ECs. LSS mitigated TNFα-induced mitochondrial impairments in ECs. Our results clearly indicate that LSS promotes mitochondrial homeostasis and attenuates inflammation-induced mitochondrial impairments in ECs. Our results provide novel insights into the manner of mitochondrial dynamics and functions modulated by LSS that contribute to endothelial integrity. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  14. Chimerization at the AQP2–AQP3 locus is the genetic basis of melarsoprol–pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates

    Directory of Open Access Journals (Sweden)

    Fabrice E. Graf

    2015-08-01

    Full Text Available Aquaglyceroporin-2 is a known determinant of melarsoprol–pentamidine cross-resistance in Trypanosoma brucei brucei laboratory strains. Recently, chimerization at the AQP2–AQP3 tandem locus was described from melarsoprol–pentamidine cross-resistant Trypanosoma brucei gambiense isolates from sleeping sickness patients in the Democratic Republic of the Congo. Here, we demonstrate that reintroduction of wild-type AQP2 into one of these isolates fully restores drug susceptibility while expression of the chimeric AQP2/3 gene in aqp2–aqp3 null T. b. brucei does not. This proves that AQP2–AQP3 chimerization is the cause of melarsoprol–pentamidine cross-resistance in the T. b. gambiense isolates.

  15. A systematic assessment of mitochondrial function identified novel signatures for drug-induced mitochondrial disruption in cells.

    Science.gov (United States)

    Li, Nianyu; Oquendo, Elisa; Capaldi, Roderick A; Robinson, J Paul; He, Yudong D; Hamadeh, Hisham K; Afshari, Cynthia A; Lightfoot-Dunn, Ruth; Narayanan, Padma Kumar

    2014-11-01

    Mitochondrial perturbation has been recognized as a contributing factor to various drug-induced organ toxicities. To address this issue, we developed a high-throughput flow cytometry-based mitochondrial signaling assay to systematically investigate mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction: mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS), intracellular reduced glutathione (GSH) level, and cell viability. Modulation of these parameters by a training set of compounds, comprised of established mitochondrial poisons and 60 marketed drugs (30 nM to 1mM), was tested in HL-60 cells (a human pro-myelocytic leukemia cell line) cultured in either glucose-supplemented (GSM) or glucose-free (containing galactose/glutamine; GFM) RPMI-1640 media. Post-hoc bio-informatic analyses of IC50 or EC50 values for all parameters tested revealed that MMP depolarization in HL-60 cells cultured in GSM was the most reliable parameter for determining mitochondrial dysfunction in these cells. Disruptors of mitochondrial function depolarized MMP at concentrations lower than those that caused loss of cell viability, especially in cells cultured in GSM; cellular GSH levels correlated more closely to loss of viability in vitro. Some mitochondrial respiratory chain inhibitors increased mitochondrial ROS generation; however, measuring an increase in ROS alone was not sufficient to identify mitochondrial disruptors. Furthermore, hierarchical cluster analysis of all measured parameters provided confirmation that MMP depletion, without loss of cell viability, was the key signature for identifying mitochondrial disruptors. Subsequent classification of compounds based on ratios of IC50s of cell viability:MMP determined that this parameter is the most critical indicator of mitochondrial health in cells and provides a powerful tool to predict whether novel small molecule entities possess this liability. © The Author

  16. Further evidence from SSCP and ITS DNA sequencing support Trypanosoma evansi and Trypanosoma equiperdum as subspecies or even strains of Trypanosoma brucei

    OpenAIRE

    Wen, Y; Lun, Z; Zhu, X; Hide, G; Lai, D

    2016-01-01

    The subgenus Trypanozoon includes three species Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum, which are morphologically identical and indistinguishable even using some molecular methods. In this study, PCR-based single strand conformation polymorphism (PCR-SSCP) was used to analyze the ribosomal DNA of the Trypanozoon species. Data indicate different patterns of ITS2 fragments between T. brucei, T. evansi and T. equiperdum by SSCP. Furthermore, analysis of total ITS seque...

  17. Efavirenz induces neuronal autophagy and mitochondrial alterations.

    Science.gov (United States)

    Purnell, Phillip R; Fox, Howard S

    2014-11-01

    Efavirenz (EFV) is a non-nucleoside reverse-transcriptase inhibitor in wide use for the treatment of human immunodeficiency virus infection. Although EFV is generally well tolerated, neuropsychiatric toxicity has been well documented. The toxic effects of EFV in hepatocytes and keratinocytes have been linked to mitochondrial perturbations and changes in autophagy. Here, we studied the effect of EFV on mitochondria and autophagy in neuronal cell lines and primary neurons. In SH-SY5Y cells, EFV induced a drop in ATP production, which coincided with increased autophagy, mitochondrial fragmentation, and mitochondrial depolarization. EFV-induced mitophagy was also detected by colocalization of mitochondria and autophagosomes and use of an outer mitochondrial membrane tandem fluorescent vector. Pharmacologic inhibition of autophagy with 3-methyladenine increased the cytotoxic effect of EFV, suggesting that autophagy promotes cell survival. EFV also reduces ATP production in primary neurons, induces autophagy, and changes mitochondrial morphology. Overall, EFV is able to acutely induce autophagy and mitochondrial changes in neurons. These changes may be involved in the mechanism leading to central nervous system toxicity observed in clinical EFV use. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  18. Mitochondrial Dysfunction in Lysosomal Storage Disorders.

    Science.gov (United States)

    de la Mata, Mario; Cotán, David; Villanueva-Paz, Marina; de Lavera, Isabel; Álvarez-Córdoba, Mónica; Luzón-Hidalgo, Raquel; Suárez-Rivero, Juan M; Tiscornia, Gustavo; Oropesa-Ávila, Manuel

    2016-10-11

    Lysosomal storage diseases (LSDs) describe a heterogeneous group of rare inherited metabolic disorders that result from the absence or loss of function of lysosomal hydrolases or transporters, resulting in the progressive accumulation of undigested material in lysosomes. The accumulation of substances affects the function of lysosomes and other organelles, resulting in secondary alterations such as impairment of autophagy, mitochondrial dysfunction, inflammation and apoptosis. LSDs frequently involve the central nervous system (CNS), where neuronal dysfunction or loss results in progressive neurodegeneration and premature death. Many LSDs exhibit signs of mitochondrial dysfunction, which include mitochondrial morphological changes, decreased mitochondrial membrane potential (ΔΨm), diminished ATP production and increased generation of reactive oxygen species (ROS). Furthermore, reduced autophagic flux may lead to the persistence of dysfunctional mitochondria. Gaucher disease (GD), the LSD with the highest prevalence, is caused by mutations in the GBA1 gene that results in defective and insufficient activity of the enzyme β-glucocerebrosidase (GCase). Decreased catalytic activity and/or instability of GCase leads to accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph) in the lysosomes of macrophage cells and visceral organs. Mitochondrial dysfunction has been reported to occur in numerous cellular and mouse models of GD. The aim of this manuscript is to review the current knowledge and implications of mitochondrial dysfunction in LSDs.

  19. Mitochondrial Dysfunction in Lysosomal Storage Disorders

    Directory of Open Access Journals (Sweden)

    Mario de la Mata

    2016-10-01

    Full Text Available Lysosomal storage diseases (LSDs describe a heterogeneous group of rare inherited metabolic disorders that result from the absence or loss of function of lysosomal hydrolases or transporters, resulting in the progressive accumulation of undigested material in lysosomes. The accumulation of substances affects the function of lysosomes and other organelles, resulting in secondary alterations such as impairment of autophagy, mitochondrial dysfunction, inflammation and apoptosis. LSDs frequently involve the central nervous system (CNS, where neuronal dysfunction or loss results in progressive neurodegeneration and premature death. Many LSDs exhibit signs of mitochondrial dysfunction, which include mitochondrial morphological changes, decreased mitochondrial membrane potential (ΔΨm, diminished ATP production and increased generation of reactive oxygen species (ROS. Furthermore, reduced autophagic flux may lead to the persistence of dysfunctional mitochondria. Gaucher disease (GD, the LSD with the highest prevalence, is caused by mutations in the GBA1 gene that results in defective and insufficient activity of the enzyme β-glucocerebrosidase (GCase. Decreased catalytic activity and/or instability of GCase leads to accumulation of glucosylceramide (GlcCer and glucosylsphingosine (GlcSph in the lysosomes of macrophage cells and visceral organs. Mitochondrial dysfunction has been reported to occur in numerous cellular and mouse models of GD. The aim of this manuscript is to review the current knowledge and implications of mitochondrial dysfunction in LSDs.

  20. Nitric Oxide Regulation of Mitochondrial Processes: Commonality in Medical Disorders.

    Science.gov (United States)

    Stefano, George B; Kream, Richard M

    2015-07-16

    The vital status of diverse classes of eukaryotic mitochondria is reflected by the high degree of evolutionary modification functionally linked to ongoing multifaceted organelle development. From this teleological perspective, a logistical enhancement of eukaryotic cellular energy requirements indicates a convergence of metabolic processes within the mitochondrial matrix for optimal synthesis of ATP from ADP and inorganic phosphate and necessitates an evolutionarily driven retrofit of the primordial endosymbiont bacterial plasma membrane into the inner mitochondrial membrane. The biochemical complexity of eukaryotic inner membrane electron transport complexes linked to temporally-defined, state-dependent, fluctuations in mitochondrial oxygen utilization is capable of generating deleterious reactive oxygen species. Within this functional context, an extensive neurochemical literature supports the role of the free radical gas nitric oxide (NO) as a key signaling molecule involved in the regulation of multiple aspects of mitochondrial respiration/oxidative phosphorylation. Importantly, the unique chemical properties of NO underlie its rapid metabolism in vivo within a mechanistic spectrum of small oxidative molecules, free and protein-bound thiol adducts, and reversible binding to ferrous heme iron centers. Recent compelling work has identified a medically relevant dual regulation pathway for mitochondrial NO expression mediated by traditionally characterized NO synthases (NOS) and by enzymatic reduction of available cellular nitrite pools by a diverse class of cytosolic and mitochondrial nitrite reductases. Accordingly, our short review presents selected medically-based discussion topics relating to multi-faceted NO regulation of mitochondrial functions in human health and disease states.

  1. Trypanosoma brucei FKBP12 differentially controls motility and cytokinesis in procyclic and bloodstream forms.

    Science.gov (United States)

    Brasseur, Anaïs; Rotureau, Brice; Vermeersch, Marjorie; Blisnick, Thierry; Salmon, Didier; Bastin, Philippe; Pays, Etienne; Vanhamme, Luc; Pérez-Morga, David

    2013-02-01

    FKBP12 proteins are able to inhibit TOR kinases or calcineurin phosphatases upon binding of rapamycin or FK506 drugs, respectively. The Trypanosoma brucei FKBP12 homologue (TbFKBP12) was found to be a cytoskeleton-associated protein with specific localization in the flagellar pocket area of the bloodstream form. In the insect procyclic form, RNA interference-mediated knockdown of TbFKBP12 affected motility. In bloodstream cells, depletion of TbFKBP12 affected cytokinesis and cytoskeleton architecture. These last effects were associated with the presence of internal translucent cavities limited by an inside-out configuration of the normal cell surface, with a luminal variant surface glycoprotein coat lined up by microtubules. These cavities, which recreated the streamlined shape of the normal trypanosome cytoskeleton, might represent unsuccessful attempts for cell abscission. We propose that TbFKBP12 differentially affects stage-specific processes through association with the cytoskeleton.

  2. Sec16 determines the size and functioning of the Golgi in the protist parasite, Trypanosoma brucei.

    Science.gov (United States)

    Sealey-Cardona, Marco; Schmidt, Katy; Demmel, Lars; Hirschmugl, Tatjana; Gesell, Tanja; Dong, Gang; Warren, Graham

    2014-06-01

    The Sec16 homologue in Trypanosoma brucei has been identified and characterized. TbSec16 colocalizes with COPII components at the single endoplasmic reticulum exit site (ERES), which is next to the single Golgi stack in the insect (procyclic) form of this organism. Depletion of TbSec16 reduces the size of the ERES and the Golgi, and slows growth and transport of a secretory marker to the cell surface; conversely, overexpression of TbSec16 increases the size of the ERES and Golgi but has no effect on growth or secretion. Together these data suggest that TbSec16 regulates the size of the ERES and Golgi and this size is set for optimal growth of the organism. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Enhanced succinic acid production in Aspergillus saccharolyticus by heterologous expression of fumarate reductase from Trypanosoma brucei

    DEFF Research Database (Denmark)

    Yang, Lei; Lübeck, Mette; Ahring, Birgitte K.

    2015-01-01

    production medium as well as the complete medium, but the measured enzyme activities were different depending on the media. Furthermore, a soluble NADH-dependent fumarate reductase gene (frd) from Trypanosoma brucei was inserted and expressed in A. saccharolyticus. The expression of the frd gene led......Aspergillus saccharolyticus exhibits great potential as a cell factory for industrial production of dicarboxylic acids. In the analysis of the organic acid profile, A. saccharolyticus was cultivated in an acid production medium using two different pH conditions. The specific activities...... on the pattern and the amount of organic acids produced by A. saccharolyticus. The wild-type strain produced higher amount of malic acid and succinic acid in the pH buffered condition (pH 6.5) compared with the pH non-buffered condition. The enzyme assays showed that the rTCA branch was active in the acid...

  4. Compositional compartmentalization of the nuclear genomes of Trypanosoma brucei and Trypanosoma equiperdum.

    Science.gov (United States)

    Isacchi, A; Bernardi, G; Bernardi, G

    1993-12-06

    High molecular weight DNA preparations from Trypanosoma brucei and Trypanosoma equiperdum were fractionated by preparative centrifugation in a Cs2SO4 density gradient in the presence of BAMD, bis(acetatomercurimethyl)dioxane, a sequence-specific DNA ligand. Analytical centrifugation in CsCl of the DNA fractions so obtained showed that both DNAs had a bimodal distribution with two major peaks banding at 1.702-1.703 and 1.708 g/cm3 and representing 1/3 and 2/3 of total DNA, respectively. Several minor components were also detected. These results indicate that a compositional compartmentalization is not only found in the genome of vertebrates and plants, as already described, but also in those of protozoa such as Trypanosomes.

  5. Zinc finger nuclease technology: A stable tool for high efficiency transformation in bloodstream form T. brucei.

    Science.gov (United States)

    Schumann, Gabriela; Kangussu-Marcolino, Monica M; Doiron, Nicholas; Käser, Sandro; de Assis Burle-Caldas, Gabriela; DaRocha, Wanderson D; Teixeira, Santuza M; Roditi, Isabel

    2017-04-01

    In Trypanosoma brucei, the generation of knockout mutants is relatively easy compared to other organisms as transfection methods are well established. These methods have their limitations, however, when it comes to the generation of genome-wide libraries that require a minimum of several hundred thousand transformants. Double-strand breaks with the meganuclease ISce-I dramatically increase transformation efficiency, but are not widely in use as cell lines need to be generated de novo before each transfection. Here we show that zinc finger nucleases are a robust and stable tool that can enhance transformation in bloodstream forms by more than an order of magnitude. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Population genetics of Trypanosoma brucei rhodesiense: clonality and diversity within and between foci.

    Directory of Open Access Journals (Sweden)

    Craig W Duffy

    2013-11-01

    Full Text Available African trypanosomes are unusual among pathogenic protozoa in that they can undergo their complete morphological life cycle in the tsetse fly vector with mating as a non-obligatory part of this development. Trypanosoma brucei rhodesiense, which infects humans and livestock in East and Southern Africa, has classically been described as a host-range variant of the non-human infective Trypanosoma brucei that occurs as stable clonal lineages. We have examined T. b. rhodesiense populations from East (Uganda and Southern (Malawi Africa using a panel of microsatellite markers, incorporating both spatial and temporal analyses. Our data demonstrate that Ugandan T. b. rhodesiense existed as clonal populations, with a small number of highly related genotypes and substantial linkage disequilibrium between pairs of loci. However, these populations were not stable as the dominant genotypes changed and the genetic diversity also reduced over time. Thus these populations do not conform to one of the criteria for strict clonality, namely stability of predominant genotypes over time, and our results show that, in a period in the mid 1990s, the previously predominant genotypes were not detected but were replaced by a novel clonal population with limited genetic relationship to the original population present between 1970 and 1990. In contrast, the Malawi T. b. rhodesiense population demonstrated significantly greater diversity and evidence for frequent genetic exchange. Therefore, the population genetics of T. b. rhodesiense is more complex than previously described. This has important implications for the spread of the single copy T. b. rhodesiense gene that allows human infectivity, and therefore the epidemiology of the human disease, as well as suggesting that these parasites represent an important organism to study the influence of optional recombination upon population genetic dynamics.

  7. Secondary Metabolites from Vietnamese Marine Invertebrates with Activity against Trypanosoma brucei and T. cruzi

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    Nguyen Phuong Thao

    2014-06-01

    Full Text Available Marine-derived natural products from invertebrates comprise an extremely diverse and promising source of the compounds from a wide variety of structural classes. This study describes the discovery of five marine natural products with activity against Trypanosoma species by natural product library screening using whole cell in vitro assays. We investigated the anti-trypanosomal activity of the extracts from the soft corals and echinoderms living in Vietnamese seas. Of the samples screened, the methanolic extracts of several marine organisms exhibited potent activities against cultures of Trypanosoma brucei and T. cruzi (EC50 < 5.0 μg/mL. Among the compounds isolated from these extracts, laevigatol B (1 from Lobophytum crassum and L. laevigatum, (24S-ergost-4-ene-3-one (2 from Sinularia dissecta, astropectenol A (3 from Astropecten polyacanthus, and cholest-8-ene-3β,5α,6β,7α-tetraol (4 from Diadema savignyi showed inhibitory activity against T. brucei with EC50 values ranging from 1.57 ± 0.14 to 14.6 ± 1.36 μM, relative to the positive control, pentamidine (EC50 = 0.015 ± 0.003 μM. Laevigatol B (1 and 5α-cholest-8(14-ene-3β,7α-diol (5 exhibited also significant inhibitory effects on T. cruzi. The cytotoxic activity of the pure compounds on mammalian cells was also assessed and found to be insignificant in all cases. This is the first report on the inhibitory effects of marine organisms collected in Vietnamese seas against Trypanosoma species responsible for neglected tropical diseases.

  8. Protein functional links in Trypanosoma brucei, identified by gene fusion analysis

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    Trimpalis Philip

    2011-07-01

    Full Text Available Abstract Background Domain or gene fusion analysis is a bioinformatics method for detecting gene fusions in one organism by comparing its genome to that of other organisms. The occurrence of gene fusions suggests that the two original genes that participated in the fusion are functionally linked, i.e. their gene products interact either as part of a multi-subunit protein complex, or in a metabolic pathway. Gene fusion analysis has been used to identify protein functional links in prokaryotes as well as in eukaryotic model organisms, such as yeast and Drosophila. Results In this study we have extended this approach to include a number of recently sequenced protists, four of which are pathogenic, to identify fusion linked proteins in Trypanosoma brucei, the causative agent of African sleeping sickness. We have also examined the evolution of the gene fusion events identified, to determine whether they can be attributed to fusion or fission, by looking at the conservation of the fused genes and of the individual component genes across the major eukaryotic and prokaryotic lineages. We find relatively limited occurrence of gene fusions/fissions within the protist lineages examined. Our results point to two trypanosome-specific gene fissions, which have recently been experimentally confirmed, one fusion involving proteins involved in the same metabolic pathway, as well as two novel putative functional links between fusion-linked protein pairs. Conclusions This is the first study of protein functional links in T. brucei identified by gene fusion analysis. We have used strict thresholds and only discuss results which are highly likely to be genuine and which either have already been or can be experimentally verified. We discuss the possible impact of the identification of these novel putative protein-protein interactions, to the development of new trypanosome therapeutic drugs.

  9. Mitochondrial mutagenesis induced by tumor-specific radiation bystander effects.

    LENUS (Irish Health Repository)

    Gorman, Sheeona

    2012-02-01

    The radiation bystander effect is a cellular process whereby cells not directly exposed to radiation display cellular alterations similar to directly irradiated cells. Cellular targets including mitochondria have been postulated to play a significant role in this process. In this study, we utilized the Random Mutation Capture assay to quantify the levels of random mutations and deletions in the mitochondrial genome of bystander cells. A significant increase in the frequency of random mitochondrial mutations was found at 24 h in bystander cells exposed to conditioned media from irradiated tumor explants (p = 0.018). CG:TA mutations were the most abundant lesion induced. A transient increase in the frequency of random mitochondrial deletions was also detected in bystander cells exposed to conditioned media from tumor but not normal tissue at 24 h (p = 0.028). The increase in both point mutations and deletions was transient and not detected at 72 h. To further investigate mitochondrial dysfunction, mitochondrial membrane potential and reactive oxygen species were assessed in these bystander cells. There was a significant reduction in mitochondrial membrane potential and this was positively associated with the frequency of random point mutation and deletions in bystander cells treated with conditioned media from tumor tissue (r = 0.71, p = 0.02). This study has shown that mitochondrial genome alterations are an acute consequence of the radiation bystander effect secondary to mitochondrial dysfunction and suggests that this cannot be solely attributable to changes in ROS levels alone.

  10. Altered Mitochondrial Dynamics and TBI Pathophysiology

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    Tara Diane Fischer

    2016-03-01

    Full Text Available Mitochondrial function is intimately linked to cellular survival, growth, and death. Mitochondria not only generate ATP from oxidative phosphorylation, but also mediate intracellular calcium buffering, generation of reactive oxygen species (ROS, and apoptosis. Electron leakage from the electron transport chain, especially from damaged or depolarized mitochondria, can generate excess free radicals that damage cellular proteins, DNA, and lipids. Furthermore, mitochondrial damage releases pro-apoptotic factors to initiate cell death. Previous studies have reported that traumatic brain injury (TBI reduces mitochondrial respiration, enhances production of ROS, and triggers apoptotic cell death, suggesting a prominent role of mitochondria in TBI pathophysiology. Mitochondria maintain cellular energy homeostasis and health via balanced processes of fusion and fission, continuously dividing and fusing to form an interconnected network throughout the cell. An imbalance of these processes, particularly an excess of fission, can be detrimental to mitochondrial function, causing decreased respiration, ROS production, and apoptosis. Mitochondrial fission is regulated by the cytosolic GTPase, dynamin-related protein 1 (Drp1, which translocates to the mitochondrial outer membrane to initiate fission. Aberrant Drp1 activity has been linked to excessive mitochondrial fission and neurodegeneration. Measurement of Drp1 levels in purified hippocampal mitochondria showed an increase in TBI animals as compared to sham controls. Analysis of cryo-electron micrographs of these mitochondria also showed that TBI caused an initial increase in the length of hippocampal mitochondria at 24 hours post-injury, followed by a significant decrease in length at 72 hours. Post-TBI administration of Mdivi-1, a pharmacological inhibitor of Drp1, prevented this decrease in mitochondria length. Mdivi-1 treatment also reduced the loss of newborn neurons in the hippocampus and improved

  11. A mitochondrial odyssey.

    Science.gov (United States)

    Neupert, Walter

    2012-01-01

    Good fortune let me be an innocent child during World War II, a hopeful adolescent with encouraging parents during the years of German recovery, and a self-determined adult in a period of peace, freedom, and wealth. My luck continued as a scientist who could entirely follow his fancy. My mind was always set on understanding how things are made. At a certain point, I found myself confronted with the question of how mitochondria and organelles, which cannot be formed de novo, are put together. Intracellular transport of proteins, their translocation across the mitochondrial membranes, and their folding and assembly were the processes that fascinated me. Now, after some 30 years, we have wonderful insights, unimagined views of a complex and at the same time simple machinery and its workings. We have glimpses of how orderly processes are established in the cell to assemble from single molecules our beautiful mitochondria that every day make some 50 kg of ATP for each of us. At the same time, we have learned amazing lessons from the tinkering of evolution that developed mitochondria from bacteria.

  12. Disorders of phospholipid metabolism: an emerging class of mitochondrial disease due to defects in nuclear genes

    Directory of Open Access Journals (Sweden)

    Ya-Wen eLu

    2015-02-01

    Full Text Available The human nuclear and mitochondrial genomes co-exist within each cell. While the mitochondrial genome encodes for a limited number of proteins, transfer RNAs, and ribosomal RNAs, the vast majority of mitochondrial proteins are encoded in the nuclear genome. Of the multitude of mitochondrial disorders known to date, only a fifth are maternally inherited. The recent characterization of the mitochondrial proteome therefore serves as an important step towards delineating the nosology of a large spectrum of phenotypically heterogeneous diseases. Following the identification of the first nuclear gene defect to underlie a mitochondrial disorder, a plenitude of genetic variants that provoke mitochondrial pathophysiology have been molecularly elucidated and classified into six categories that impact: 1 oxidative phosphorylation (subunits and assembly factors; 2 mitochondrial DNA maintenance and expression; 3 mitochondrial protein import and assembly; 4 mitochondrial quality control (chaperones and proteases; 5 iron-sulfur cluster homeostasis; and 6 mitochondrial dynamics (fission and fusion. Here, we propose that an additional class of genetic variant be included in the classification schema to acknowledge the role of genetic defects in phospholipid biosynthesis, remodeling, and metabolism in mitochondrial pathophysiology. This seventh class includes a small but notable group of nuclear-encoded proteins whose dysfunction impacts normal mitochondrial phospholipid metabolism. The resulting human disorders present with a diverse array of pathologic consequences that reflect the variety of functions that phospholipids have in mitochondria and highlight the important role of proper membrane homeostasis in mitochondrial biology.

  13. No gold standard estimation of the sensitivity and specificity of two molecular diagnostic protocols for Trypanosoma brucei spp. in Western Kenya.

    Directory of Open Access Journals (Sweden)

    Barend Mark de Clare Bronsvoort

    2010-01-01

    Full Text Available African animal trypanosomiasis is caused by a range of tsetse transmitted protozoan parasites includingTrypanosoma vivax, Trypanosoma congolense and Trypansoma brucei. In Western Kenya and other parts of East Africa two subspecies of T. brucei, T.b. brucei and the zoonoticT.b. rhodesiense, co-circulate in livestock. A range of polymerase chain reactions (PCR have been developed as important molecular diagnostic tools for epidemiological investigations of T. brucei s.l. in the animal reservoir and of its zoonotic potential. Quantification of the relative performance of different diagnostic PCRs is essential to ensure comparability of studies. This paper describes an evaluation of two diagnostic test systems for T. brucei using a T. brucei s.l. specific PCR [1] and a single nested PCR targeting the Internal Transcribed Spacer (ITS regions of trypanosome ribosomal DNA [2]. A Bayesian formulation of the Hui-Walter latent class model was employed to estimate their test performance in the absence of a gold standard test for detecting T.brucei s.l. infections in ear-vein blood samples from cattle, pig, sheep and goat populations in Western Kenya, stored on Whatman FTA cards. The results indicate that the system employing the T. brucei s.l. specific PCR (Se1=0.760 had a higher sensitivity than the ITS-PCR (Se2=0.640; both have high specificity (Sp1=0.998; Sp2=0.997. The true prevalences for livestock populations were estimated (pcattle=0.091, ppigs=0.066, pgoats=0.005, psheep=0.006, taking into account the uncertainties in the specificity and sensitivity of the two test systems. Implications of test performance include the required survey sample size; due to its higher sensitivity and specificity, the T. brucei s.l. specific PCR requires a consistently smaller sample size than the ITS-PCR for the detection of T. brucei s.l. However the ITS-PCR is able to simultaneously screen samples for other pathogenic trypanosomes and may thus be, overall, a better

  14. Diagnosis of human trypanosomiasis, due to Trypanosoma brucei gambiense in central Africa, by the polymerase chain reaction

    OpenAIRE

    Penchenier, Laurent; Simo, G.; Grébaut, Pascal; Nkinin, S.; Laveissière, Claude; Herder, Stéphane

    2000-01-01

    During a mass screening of sleeping sickness conducted in 1998 and 1999, and involving 27,932 persons in Cameroon and the Central African Republic, we tested the polymerase chain reaction (PCR) on whole blood for the diagnosis of human African trypanosomiasis due to #Trypanosoma brucei gambiense$. The 1858 samples obtained were from 4 groups : 155 infected patients, 1432 serological suspects detected by the card agglutination test for trypanosomiasis (CATT), 222 negative controls living in th...

  15. T. brucei infection reduces B lymphopoiesis in bone marrow and truncates compensatory splenic lymphopoiesis through transitional B-cell apoptosis.

    Directory of Open Access Journals (Sweden)

    Viki Bockstal

    2011-06-01

    Full Text Available African trypanosomes of the Trypanosoma brucei species are extracellular protozoan parasites that cause the deadly disease African trypanosomiasis in humans and contribute to the animal counterpart, Nagana. Trypanosome clearance from the bloodstream is mediated by antibodies specific for their Variant Surface Glycoprotein (VSG coat antigens. However, T. brucei infection induces polyclonal B cell activation, B cell clonal exhaustion, sustained depletion of mature splenic Marginal Zone B (MZB and Follicular B (FoB cells, and destruction of the B-cell memory compartment. To determine how trypanosome infection compromises the humoral immune defense system we used a C57BL/6 T. brucei AnTat 1.1 mouse model and multicolor flow cytometry to document B cell development and maturation during infection. Our results show a more than 95% reduction in B cell precursor numbers from the CLP, pre-pro-B, pro-B, pre-B and immature B cell stages in the bone marrow. In the spleen, T. brucei induces extramedullary B lymphopoiesis as evidenced by significant increases in HSC-LMPP, CLP, pre-pro-B, pro-B and pre-B cell populations. However, final B cell maturation is abrogated by infection-induced apoptosis of transitional B cells of both the T1 and T2 populations which is not uniquely dependent on TNF-, Fas-, or prostaglandin-dependent death pathways. Results obtained from ex vivo co-cultures of living bloodstream form trypanosomes and splenocytes demonstrate that trypanosome surface coat-dependent contact with T1/2 B cells triggers their deletion. We conclude that infection-induced and possibly parasite-contact dependent deletion of transitional B cells prevents replenishment of mature B cell compartments during infection thus contributing to a loss of the host's capacity to sustain antibody responses against recurring parasitemic waves.

  16. A target-based high throughput screen yields Trypanosoma brucei hexokinase small molecule inhibitors with antiparasitic activity.

    Directory of Open Access Journals (Sweden)

    Elizabeth R Sharlow

    2010-04-01

    Full Text Available The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK, an enzyme essential to the parasite that transfers the gamma-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay.Exploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were approximately 20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03brucei parasites, Leishmania promastigotes, and mammalian cell lines. Analysis of two structurally related compounds, ebselen and SID 17387000, revealed that both were mixed inhibitors of TbHK1 with respect to ATP. Additionally, both compounds inhibited parasite lysate-derived HK activity. None of the compounds displayed structural similarity to known hexokinase inhibitors or human African trypanosomiasis therapeutics.The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome.

  17. Mitochondrial Reactive Oxygen Species in Lipotoxic Hearts Induces Post-Translational Modifications of AKAP121, DRP1 and OPA1 That Promote Mitochondrial Fission.

    Science.gov (United States)

    Tsushima, Kensuke; Bugger, Heiko; Wende, Adam R; Soto, Jamie; Jenson, Gregory A; Tor, Austin R; McGlauflin, Rose; Kenny, Helena C; Zhang, Yuan; Souvenir, Rhonda; Hu, Xiao X; Black, Crystal L; Pereira, Renata O; Lira, Vitor A; Spitzer, Kenneth; Sharp, Terry L; Shoghi, Kooresh I; Sparagna, Genevieve C; Rog-Zielinska, Eva A; Kohl, Peter; Khalimonchuk, Oleh; Schaffer, Jean E; Abel, E Dale

    2017-11-01

    Rationale: Cardiac lipotoxicity, characterized by increased uptake, oxidation and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood. Objective: To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo. Methods and Results: Using a transgenic mouse model of cardiac lipotoxicity overexpressing long-chain acyl-CoA synthetase 1 in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes (NRVCs). Palmitate exposure to NRVCs initially activates mitochondrial respiration, coupled with increased mitochondrial membrane potential and adenosine triphosphate (ATP) synthesis. However, long-term exposure to palmitate (> 8h) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of A-kinase anchor protein (AKAP121) leading to reduced phosphorylation of DRP1 at Ser637 and altered proteolytic processing of OPA1. Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro. Conclusions: Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a novel

  18. Involvement of the mitochondrial compartment in human NCL fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Pezzini, Francesco; Gismondi, Floriana [Department of Neurological, Psychological, Morphological and Motor Sciences, Divisions of Neurology (Child Neurology) and Neuropathology, University of Verona Medical School, Verona (Italy); Tessa, Alessandra [IRCCS Fondazione Stella Maris-Molecular Medicine Unit, Pisa (Italy); Tonin, Paola [Department of Neurological, Psychological, Morphological and Motor Sciences, Divisions of Neurology (Child Neurology) and Neuropathology, University of Verona Medical School, Verona (Italy); Carrozzo, Rosalba [IRCCS Bambino Gesu Hospital-Molecular Medicine Unit, Roma (Italy); Mole, Sara E. [MRC Laboratory for Molecular Cell Biology, Molecular Medicines Unit, UCL Institute of Child Health and Department of Genetics, Evolution and Environment, University College London (United Kingdom); Santorelli, Filippo M. [IRCCS Fondazione Stella Maris-Molecular Medicine Unit, Pisa (Italy); Simonati, Alessandro, E-mail: alessandro.simonati@univr.it [Department of Neurological, Psychological, Morphological and Motor Sciences, Divisions of Neurology (Child Neurology) and Neuropathology, University of Verona Medical School, Verona (Italy)

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Mitochondrial reticulum fragmentation occurs in human CLN1 and CLN6 fibroblasts. Black-Right-Pointing-Pointer Likewise mitochondrial shift-to periphery and decreased mitochondrial density are seen. Black-Right-Pointing-Pointer Enhanced caspase-mediated apoptosis occurs following STS treatment in CLN1 fibroblasts. -- Abstract: Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.

  19. Modulation of mitochondrial morphology by bioenergetics defects in primary human fibroblasts

    DEFF Research Database (Denmark)

    Guillery, O.; Malka, F.; Frachon, P.

    2008-01-01

    induced partial but significant mitochondrial fragmentation, whereas dissipation of mitochondrial membrane potential (D Psi m) provoked complete fragmentation, and glycolysis inhibition had no effect. Oxidative phosphorylation defective fibroblasts had essentially normal filamentous mitochondria under...... basal conditions, although when challenged some of them presented with mild alteration of fission or fusion efficacy. Severely defective cells disclosed complete mitochondrial fragmentation under glycolysis inhibition. In conclusion, mitochondrial morphology is modulated by D Psi m but loosely linked...... to mitochondrial oxidative phosphorylation. Its alteration by glycolysis, inhibition points to a severe oxidative phosphorylation defect. (C) 2008 Elsevier B.V. All rights reserved Udgivelsesdato: 2008/4...

  20. Tissue-specific modulation of mitochondrial DNA segregation by a defect in mitochondrial division.

    Science.gov (United States)

    Jokinen, Riikka; Marttinen, Paula; Stewart, James B; Neil Dear, T; Battersby, Brendan J

    2016-02-15

    Mitochondria are dynamic organelles that divide and fuse by remodeling an outer and inner membrane in response to developmental, physiological and stress stimuli. These events are coordinated by conserved dynamin-related GTPases. The dynamics of mitochondrial morphology require coordination with mitochondrial DNA (mtDNA) to ensure faithful genome transmission, however, this process remains poorly understood. Mitochondrial division is linked to the segregation of mtDNA but how it affects cases of mtDNA heteroplasmy, where two or more mtDNA variants/mutations co-exist in a cell, is unknown. Segregation of heteroplasmic human pathogenic mtDNA mutations is a critical factor in the onset and severity of human mitochondrial diseases. Here, we investigated the coupling of mitochondrial morphology to the transmission and segregation of mtDNA in mammals by taking advantage of two genetically modified mouse models: one with a dominant-negative mutation in the dynamin-related protein 1 (Drp1 or Dnm1l) that impairs mitochondrial fission and the other, heteroplasmic mice segregating two neutral mtDNA haplotypes (BALB and NZB). We show a tissue-specific response to mtDNA segregation from a defect in mitochondrial fission. Only mtDNA segregation in the hematopoietic compartment is modulated from impaired Dnm1l function. In contrast, no effect was observed in other tissues arising from the three germ layers during development and in mtDNA transmission through the female germline. Our data suggest a robust organization of a heteroplasmic mtDNA segregating unit across mammalian cell types that can overcome impaired mitochondrial division to ensure faithful transmission of the mitochondrial genome. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Connexin 43 impacts on mitochondrial potassium uptake

    Directory of Open Access Journals (Sweden)

    Kerstin eBoengler

    2013-06-01

    Full Text Available In cardiomyocytes, connexin 43 (Cx43 forms gap junctions and unopposed hemichannels at the plasma membrane, but the protein is also present at the inner membrane of subsarcolemmal mitochondria. Both inhibition and genetic ablation of Cx43 reduce ADP-stimulated complex 1 respiration. Since mitochondrial potassium influx impacts on oxygen consumption, we investigated whether or not inhibition or ablation of mitochondrial Cx43 alters mitochondrial potassium uptake.Subsarcolemmal mitochondria were isolated from rat left ventricular (LV myocardium and loaded with the potassium-sensitive dye PBFI. Intramitochondrial potassium was replaced by TEA (tetraethylammonium. Mitochondria were incubated under control conditions or treated with 250 µM Gap19, a peptide that specifically inhibits Cx43-dependent hemichannels at plasma membranes. Subsequently, 140 mM KCl was added and the slope of the increase in PBFI fluorescence over time was calculated. The slope of the PBFI fluorescence of the control mitochondria was set to 100%. In the presence of Gap19, the mitochondrial potassium influx was reduced from 100±11.6 % in control mitochondria to 65.5±10.7 % (n=6, p<0.05. In addition to the pharmacological inhibition of Cx43, potassium influx was studied in mitochondria isolated from conditional Cx43 knockout mice. Here, the ablation of Cx43 was achieved by the injection of 4-hydroxytamoxifen (Cx43Cre-ER(T/fl + 4-OHT. The mitochondria of the Cx43Cre-ER(T/fl + 4-OHT mice contained 3±1% Cx43 (n=6 of that in control mitochondria (100±11%, n=8, p<0.05. The ablation of Cx43 (n=5 reduced the velocity of the potassium influx from 100±11.2 % in control mitochondria (n=9 to 66.6±5.5 % (p<0.05.Taken together, our data indicate that both pharmacological inhibition and genetic ablation of Cx43 reduce mitochondrial potassium influx.

  2. A role of taurine in mitochondrial function

    DEFF Research Database (Denmark)

    Hansen, Svend Høime; Andersen, Mogens Larsen; Cornett, Claus

    2010-01-01

    The mitochondrial pH gradient across the inner-membrane is stabilised by buffering of the matrix. A low-molecular mass buffer compound has to be localised in the matrix to maintain its alkaline pH value. Taurine is found ubiquitously in animal cells with concentrations in the millimolar range and...... enzymes, which are pivotal for beta-oxidation of fatty acids, are demonstrated to have optimal activity in a taurine buffer. By application of the model presented, taurine depletion caused by hyperglycemia could provide a link between mitochondrial dysfunction and diabetes.......The mitochondrial pH gradient across the inner-membrane is stabilised by buffering of the matrix. A low-molecular mass buffer compound has to be localised in the matrix to maintain its alkaline pH value. Taurine is found ubiquitously in animal cells with concentrations in the millimolar range...... and its pKa value is determined to 9.0 (25 degrees C) and 8.6 (37 degrees C), respectively. Localisation of such a low-molecular buffer in the mitochondrial matrix, transforms the matrix into a biochemical reaction chamber for the important matrix-localised enzyme systems. Three acyl-CoA dehydrogenase...

  3. Mitochondrial DNA mutations induce mitochondrial dysfunction, apoptosis and sarcopenia in skeletal muscle of mitochondrial DNA mutator mice.

    Directory of Open Access Journals (Sweden)

    Asimina Hiona

    2010-07-01

    Full Text Available Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established.We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A of the mitochondrial DNA Polymerase gamma, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35-50% in the content of electron transport chain (ETC complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Deltapsim. Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS production or oxidative damage.These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

  4. Cmc1p Is a Conserved Mitochondrial Twin CX9C Protein Involved in Cytochrome c Oxidase Biogenesis▿ †

    OpenAIRE

    Horn, Darryl; Al-Ali, Hassan; Barrientos, Antoni

    2008-01-01

    Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by metallochaperone proteins which probably use copper from a mitochondrial matrix-localized pool. Here we describe a novel conserved mitochondrial metallochaperone-like protein, Cmc1p, whose function affects both COX and Sod1p. In Saccharomyces cerevisiae, Cmc1p localizes to the mitochondrial inner membrane fac...

  5. Extracellular-signal regulated kinase 8 of Trypanosoma brucei uniquely phosphorylates its proliferating cell nuclear antigen homolog and reveals exploitable properties.

    Science.gov (United States)

    Valenciano, Ana L; Knudsen, Giselle M; Mackey, Zachary B

    2016-10-17

    The Trypanosoma brucei subspecies T. brucei gambiense and T. brucei rhodesiense are vector-borne pathogens that cause sleeping sickness also known as Human African Trypanosomiasis (HAT), which is fatal if left untreated. The drugs that treat HAT are ineffective and cause toxic side effects. One strategy for identifying safer and more effective HAT drugs is to therapeutically exploit essential gene targets in T. brucei. Genes that make up a basic mitogen-activated protein kinase (MAPK) network are present in T. brucei. Tb927.10.5140 encodes an essential MAPK that is homologous to the human extracellular-signal regulated kinase 8 (HsERK8) which forms a tight complex with the replication factor proliferating cell nuclear antigen (PCNA) to stabilize intracellular PCNA levels. Here we demonstrate that (TbPCNA) is uniquely phos-phorylated on serine (S) and threonine (T) residues in T. brucei and that TbERK8 phosphorylates TbPCNA at each of these residues. The ability of an ERK8 homolog to phosphorylate a PCNA homolog is a novel biochemical property that is first demonstrated here in T. brucei and may be unique to this pathogen. We demonstrate that the potent HsERK8 inhibitor Ro318220, has an IC50 for TbERK8 that is several hundred times higher than its reported IC50 for HsERK8. This indicated that the active sites of TbERK8 and HsERK8 can be selectively inhibited, which provides a rational basis for discovering inhibitors that specifically target this essential parasite MAPK to kill the parasite.

  6. Membrane dynamics

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications...... for the lateral organization of membranes as wells as for physical properties like bending, permeability and elasticity...

  7. Differential Binding of Mitochondrial Transcripts by MRB8170 and MRB4160 Regulates Distinct Editing Fates of Mitochondrial mRNA in Trypanosomes

    Directory of Open Access Journals (Sweden)

    Sameer Dixit

    2017-01-01

    Full Text Available A dozen mRNAs are edited by multiple insertions and/or deletions of uridine residues in the mitochondrion of Trypanosoma brucei. Several protein complexes have been implicated in performing this type of RNA editing, including the mitochondrial RNA-binding complex 1 (MRB1. Two paralogous novel RNA-binding proteins, MRB8170 and MRB4160, are loosely associated with the core MRB1 complex. Their roles in RNA editing and effects on target mRNAs are so far not well understood. In this study, individual-nucleotide-resolution UV-cross-linking and affinity purification (iCLAP revealed a preferential binding of both proteins to mitochondrial mRNAs, which was positively correlated with their extent of editing. Integrating additional in vivo and in vitro data, we propose that binding of MRB8170 and/or MRB4160 onto pre-mRNA marks it for the initiation of editing and that initial binding of both proteins may facilitate the recruitment of other components of the RNA editing/processing machinery to ensure efficient editing. Surprisingly, MRB8170 also binds never-edited mRNAs, suggesting that at least this paralog has an additional role outside RNA editing to shape the mitochondrial transcriptome.

  8. A novel mitochondrial sphingomyelinase in zebrafish cells.

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    Yabu, Takeshi; Shimuzu, Akio; Yamashita, Michiaki

    2009-07-24

    Sphingolipids are important signaling molecules in many biological processes, but little is known regarding their physiological roles in the mitochondrion. We focused on the biochemical characters of a novel sphingomyelinase (SMase) and its function in mitochondrial ceramide generation in zebrafish embryonic cells. The cloned SMase cDNA encoded a polypeptide of 545 amino acid residues (putative molecular weight, 61,300) containing a mitochondrial localization signal (MLS) and a predicted transmembrane domain. The mature endogenous enzyme was predicted to have a molecular weight of 57,000, and matrix-assisted laser de sorption ionization time-of-flight mass spectrometry analysis indicated that the N-terminal amino acid residue of the mature enzyme was Ala-36. The purified enzyme optimally hydrolyzed [(14)C]sphingomyelin in the presence of 10 mm Mg(2+) at pH 7.5. In HEK293 cells that overexpressed SMase cDNA, the enzyme was localized to the mitochondrial fraction, whereas mutant proteins lacking MLS or both the MLS and the transmembrane domain were absent from the mitochondrial fraction. Endogenous SMase protein co-localized with a mitochondrial cytostaining marker. Using a protease protection assay, we found that SMase was distributed throughout the intermembrane space and/or the inner membrane of the mitochondrion. Furthermore, the overexpression of SMase in HEK293 cells induced ceramide generation and sphingomyelin hydrolysis in the mitochondrial fraction. Antisense phosphorothioate oligonucleotide-induced knockdown repressed ceramide generation and sphingomyelin hydrolysis in the mitochondrial fraction in zebrafish embryonic cells. These observations indicate that SMase catalyzes the hydrolysis of sphingomyelin and generates ceramide in mitochondria in fish cells.

  9. Soy lecithin interferes with mitochondrial function in frozen-thawed ram spermatozoa.

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    Del Valle, I; Gómez-Durán, A; Holt, W V; Muiño-Blanco, T; Cebrián-Pérez, J A

    2012-01-01

    Egg yolk and milk are the 2 major membrane cryoprotectants commonly used in freezing media for the long-term preservation of semen (alone or in combination with others). However, in recent years, there have been increasing arguments against the use of egg yolk or milk because of the risk of introducing diseases through the use of cryopreserved semen. In this study, we analyzed the protective effect of lecithin as an alternative to egg yolk for the cryopreservation of ram semen, using a range of functional markers for sperm viability, motility, apoptosis, and mitochondrial functionality analyses (mitochondrial inner membrane surface [MIMS], mitochondrial inner membrane potential [MIMP], and cell membrane potential) as methods of assessment in samples diluted in 3 different media: Tris-citrate-glucose as control and 2 media supplemented with soy lecithin or egg yolk. The results showed that lecithin was able to effectively protect certain sperm quality characteristics against freezing-induced damage. However, lecithin induced loss of mitochondrial membrane potential or mitochondrial loss that was not reflected by modifications in sperm motility in fresh semen. MIMS and MIMP values decreased in thawed lecithin-treated samples, concomitant with a lower (P lecithin may have affected the inner mitochondrial membrane in frozenthawed spermatozoa and confirmed that sublethal damages that seriously affect sperm functionality, not detected by classic sperm quality analyses, can be evidenced by changes in the inner mitochondrial membrane surface. These findings strengthen the relationship between mitochondrial membrane potential and motility and show that the mitochondrial alterations induced by the cryopreservation process could be specific targets for the improvement of semen cryopreservation protocols.

  10. Steroid Alkaloids from Holarrhena africana with Strong Activity against Trypanosoma brucei rhodesiense.

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    Nnadi, Charles Okeke; Nwodo, Ngozi Justina; Kaiser, Marcel; Brun, Reto; Schmidt, Thomas J

    2017-07-06

    In our continued search for natural compounds with activity against Trypanosoma brucei, causative agent of human African trypanosomiasis (HAT, "sleeping sickness"), we have investigated extracts from the leaves and bark of the West African Holarrhenaafricana (syn. Holarrhena floribunda; Apocynaceae). The extracts and their alkaloid-enriched fractions displayed promising in vitro activity against bloodstream forms of T. brucei rhodesiense (Tbr; East African HAT). Bioactivity-guided chromatographic fractionation of the alkaloid-rich fractions resulted in the isolation of 17 steroid alkaloids, one nitrogen-free steroid and one alkaloid-like non-steroid. Impressive activities (IC50 in µM) against Tbr were recorded for 3β-holaphyllamine (0.40 ± 0.28), 3α-holaphyllamine (0.37 ± 0.16), 3β-dihydroholaphyllamine (0.67 ± 0.03), N-methylholaphyllamine (0.08 ± 0.01), conessimine (0.17 ± 0.08), conessine (0.42 ± 0.09), isoconessimine (0.17 ± 0.11) and holarrhesine (0.12 ± 0.08) with selectivity indices ranging from 13 to 302. Based on comparison of the structures of this congeneric series of steroid alkaloids and their activities, structure-activity relationships (SARs) could be established. It was found that a basic amino group at position C-3 of the pregnane or pregn-5-ene steroid nucleus is required for a significant anti-trypanosomal activity. The mono-methylated amino group at C-3 represents an optimum for activity. ∆(5,6) unsaturation slightly increased the activity while hydrolysis of C-12β ester derivatives led to a loss of activity. An additional amino group at C-20 engaged in a pyrrolidine ring closed towards C-18 significantly increased the selectivity index of the compounds. Our findings provide useful empirical data for further development of steroid alkaloids as a novel class of anti-trypanosomal compounds which represent a promising starting point towards new drugs to combat human African trypanosomiasis.

  11. Steroid Alkaloids from Holarrhena africana with Strong Activity against Trypanosoma brucei rhodesiense

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    Charles Okeke Nnadi

    2017-07-01

    Full Text Available In our continued search for natural compounds with activity against Trypanosoma brucei, causative agent of human African trypanosomiasis (HAT, “sleeping sickness”, we have investigated extracts from the leaves and bark of the West African Holarrhena africana (syn. Holarrhena floribunda; Apocynaceae. The extracts and their alkaloid-enriched fractions displayed promising in vitro activity against bloodstream forms of T. brucei rhodesiense (Tbr; East African HAT. Bioactivity-guided chromatographic fractionation of the alkaloid-rich fractions resulted in the isolation of 17 steroid alkaloids, one nitrogen-free steroid and one alkaloid-like non-steroid. Impressive activities (IC50 in µM against Tbr were recorded for 3β-holaphyllamine (0.40 ± 0.28, 3α-holaphyllamine (0.37 ± 0.16, 3β-dihydroholaphyllamine (0.67 ± 0.03, N-methylholaphyllamine (0.08 ± 0.01, conessimine (0.17 ± 0.08, conessine (0.42 ± 0.09, isoconessimine (0.17 ± 0.11 and holarrhesine (0.12 ± 0.08 with selectivity indices ranging from 13 to 302. Based on comparison of the structures of this congeneric series of steroid alkaloids and their activities, structure-activity relationships (SARs could be established. It was found that a basic amino group at position C-3 of the pregnane or pregn-5-ene steroid nucleus is required for a significant anti-trypanosomal activity. The mono-methylated amino group at C-3 represents an optimum for activity. ∆5,6 unsaturation slightly increased the activity while hydrolysis of C-12β ester derivatives led to a loss of activity. An additional amino group at C-20 engaged in a pyrrolidine ring closed towards C-18 significantly increased the selectivity index of the compounds. Our findings provide useful empirical data for further development of steroid alkaloids as a novel class of anti-trypanosomal compounds which represent a promising starting point towards new drugs to combat human African trypanosomiasis.

  12. Progress in mitochondrial epigenetics.

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    Manev, Hari; Dzitoyeva, Svetlana

    2013-08-01

    Mitochondria, intracellular organelles with their own genome, have been shown capable of interacting with epigenetic mechanisms in at least four different ways. First, epigenetic mechanisms that regulate the expression of nuclear genome influence mitochondria by modulating the expression of nuclear-encoded mitochondrial genes. Second, a cell-specific mitochondrial DNA content (copy number) and mitochondrial activity determine the methylation pattern of nuclear genes. Third, mitochondrial DNA variants influence the nuclear gene expression patterns and the nuclear DNA (ncDNA) methylation levels. Fourth and most recent line of evidence indicates that mitochondrial DNA similar to ncDNA also is subject to epigenetic modifications, particularly by the 5-methylcytosine and 5-hydroxymethylcytosine marks. The latter interaction of mitochondria with epigenetics has been termed 'mitochondrial epigenetics'. Here we summarize recent developments in this particular area of epigenetic research. Furthermore, we propose the term 'mitoepigenetics' to include all four above-noted types of interactions between mitochondria and epigenetics, and we suggest a more restricted usage of the term 'mitochondrial epigenetics' for molecular events dealing solely with the intra-mitochondrial epigenetics and the modifications of mitochondrial genome.

  13. Dynamic regulation of mitochondrial fission through modification of the dynamin-related protein Drp1

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    Chang, Chuang-Rung; Blackstone, Craig

    2017-01-01

    Mitochondria in cells comprise a tubulovesicular network shaped continuously by complementary fission and fusion events. The mammalian Drp1 protein plays a key role in fission, while Mfn1, Mfn2, and OPA1 are required for fusion. Shifts in the balance between these opposing processes can occur rapidly, indicating that modifications to these proteins may regulate mitochondrial membrane dynamics. We highlight posttranslational modifications of the mitochondrial fission protein Drp1, for which these regulatory mechanisms are best characterized. This dynamin-related GTPase undergoes a number of steps to mediate mitochondrial fission, including translocation from cytoplasm to the mitochondrial outer membrane, higher-order assembly into spirals, GTP hydrolysis associated with a conformational change and membrane deformation, and ultimately disassembly. Many of these steps may be influenced by covalent modification of Drp1. We discuss the dynamic nature of Drp1 modifications and how they contribute not only to the normal regulation of mitochondrial division, but also to neuropathologic processes. PMID:20649536

  14. Phosphorylation of mitochondrial polyubiquitin by PINK1 promotes Parkin mitochondrial tethering.

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    Kahori Shiba-Fukushima

    2014-12-01

    Full Text Available The kinase PINK1 and the E3 ubiquitin (Ub ligase Parkin participate in mitochondrial quality control. The phosphorylation of Ser65 in Parkin's ubiquitin-like (UBl domain by PINK1 stimulates Parkin activation and translocation to damaged mitochondria, which induces mitophagy generating polyUb chain. However, Parkin Ser65 phosphorylation is insufficient for Parkin mitochondrial translocation. Here we report that Ser65 in polyUb chain is also phosphorylated by PINK1, and that phosphorylated polyUb chain on mitochondria tethers Parkin at mitochondria. The expression of Tom70MTS-4xUb SE, which mimics phospho-Ser65 polyUb chains on the mitochondria, activated Parkin E3 activity and its mitochondrial translocation. An E3-dead form of Parkin translocated to mitochondria with reduced membrane potential in the presence of Tom70(MTS-4xUb SE, whereas non-phospho-polyUb mutant Tom70(MTS-4xUb SA abrogated Parkin translocation. Parkin binds to the phospho-polyUb chain through its RING1-In-Between-RING (IBR domains, but its RING0-linker is also required for mitochondrial translocation. Moreover, the expression of Tom70(MTS-4xUb SE improved mitochondrial degeneration in PINK1-deficient, but not Parkin-deficient, Drosophila. Our study suggests that the phosphorylation of mitochondrial polyUb by PINK1 is implicated in both Parkin activation and mitochondrial translocation, predicting a chain reaction mechanism of mitochondrial phospho-polyUb production by which rapid translocation of Parkin is achieved.

  15. Neurodegenerative and Fatiguing Illnesses, Infections and Mitochondrial Dysfunction: Use of Natural Supplements to Improve Mitochondrial Function

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    Garth L. Nicolson

    2014-01-01

    Full Text Available Background: Many chronic diseases and illnesses are associated with one or more chronic infections, dysfunction of mitochondria and reduced production of ATP. This results in fatigue and other symptoms that occur in most if not all chronic conditions and diseases. Methods: This is a review of the published literature on chronic infections in neurodegenerative diseases and fatiguing illnesses that are also typified by mitochondrial dysfunction. This contribution also reviews the use of natural supplements to enhance mitochondrial function and reduce the effects of chronic infections to improve overall function in various chronic illnesses. Results: Mitochondrial function can be enhanced by the use of various natural supplements, notably Lipid Replacement Therapy (LRT using glyerolphospholipids and other mitochondrial supplements. In various chronic illnesses that are characterized by the presence of chronic infections, such as intracellular bacteria (Mycoplasma, Borrelia, Chlamydia and other infections and viruses, LRT has proven useful in multiple clinical trials. For example, in clinical studies on chronic fatigue syndrome, fibromyalgia syndrome and other chronic fatiguing illnesses where a large majority of patients have chronic infections, LRT significantly reduced fatigue by 35-43% in different clinical trials and increased mitochondrial function. In clinical trials on patients with multiple intracellular bacterial infections and intractable fatigue LRT plus other mitochondrial supplements significantly decreased fatigue and improved mood and cognition. Conclusions: LRT formulations designed to improve mitochondrial function appear to be useful as non-toxic dietary supplements for reducing fatigue and restoring mitochondrial and other cellular membrane functions in patients with chronic illnesses and multiple chronic infections.

  16. Dynamic modelling under uncertainty: the case of Trypanosoma brucei energy metabolism.

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    Fiona Achcar

    2012-01-01

    Full Text Available Kinetic models of metabolism require detailed knowledge of kinetic parameters. However, due to measurement errors or lack of data this knowledge is often uncertain. The model of glycolysis in the parasitic protozoan Trypanosoma brucei is a particularly well analysed example of a quantitative metabolic model, but so far it has been studied with a fixed set of parameters only. Here we evaluate the effect of parameter uncertainty. In order to define probability distributions for each parameter, information about the experimental sources and confidence intervals for all parameters were collected. We created a wiki-based website dedicated to the detailed documentation of this information: the SilicoTryp wiki (http://silicotryp.ibls.gla.ac.uk/wiki/Glycolysis. Using information collected in the wiki, we then assigned probability distributions to all parameters of the model. This allowed us to sample sets of alternative models, accurately representing our degree of uncertainty. Some properties of the model, such as the repartition of the glycolytic flux between the glycerol and pyruvate producing branches, are robust to these uncertainties. However, our analysis also allowed us to identify fragilities of the model leading to the accumulation of 3-phosphoglycerate and/or pyruvate. The analysis of the control coefficients revealed the importance of taking into account the uncertainties about the parameters, as the ranking of the reactions can be greatly affected. This work will now form the basis for a comprehensive Bayesian analysis and extension of the model considering alternative topologies.

  17. Functional analysis of TOEFAZ1 uncovers protein domains essential for cytokinesis in Trypanosoma brucei.

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    Sinclair-Davis, Amy N; McAllaster, Michael R; de Graffenried, Christopher L

    2017-10-09

    The parasite Trypanosoma brucei is highly polarized, including a flagellum that is attached along the cell surface by the flagellum attachment zone (FAZ). During cell division, the new FAZ positions the cleavage furrow, which ingresses from the anterior tip of the cell towards the posterior. We recently identified Tip Of the Extending FAZ protein 1 (TOEFAZ1) as an essential protein in trypanosome cytokinesis. We analyzed the localization and function of TOEFAZ1 domains using overexpression and RNAi complementation. TOEFAZ1 comprises three domains with separable functions: an N-terminal α-helical domain that may be involved in FAZ recruitment, a central intrinsically disordered domain that retains the morphogenic kinase TbPLK at the new FAZ tip, and a C-terminal zinc finger domain necessary for TOEFAZ1 oligomerization. Both the N-terminal and C-terminal domains are essential for TOEFAZ1 function, but TbPLK retention at the FAZ is not necessary for cytokinesis. The feasibility of alternate cytokinetic pathways that do not employ TOEFAZ1 are also assessed. Our results show that TOEFAZ1 is a multimeric scaffold for recruiting proteins that control the timing and location of cleavage furrow ingression. © 2017. Published by The Company of Biologists Ltd.

  18. Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei.

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    Daniel Nilsson

    2010-08-01

    Full Text Available Trans-splicing of leader sequences onto the 5'ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5'splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT. The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5' splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.

  19. Isothermal microcalorimetry, a new tool to monitor drug action against Trypanosoma brucei and Plasmodium falciparum.

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    Tanja Wenzler

    Full Text Available Isothermal microcalorimetry is an established tool to measure heat flow of physical, chemical or biological processes. The metabolism of viable cells produces heat, and if sufficient cells are present, their heat production can be assessed by this method. In this study, we investigated the heat flow of two medically important protozoans, Trypanosoma brucei rhodesiense and Plasmodium falciparum. Heat flow signals obtained for these pathogens allowed us to monitor parasite growth on a real-time basis as the signals correlated with the number of viable cells. To showcase the potential of microcalorimetry for measuring drug action on pathogenic organisms, we tested the method with three antitrypanosomal drugs, melarsoprol, suramin and pentamidine and three antiplasmodial drugs, chloroquine, artemether and dihydroartemisinin, each at two concentrations on the respective parasite. With the real time measurement, inhibition was observed immediately by a reduced heat flow compared to that in untreated control samples. The onset of drug action, the degree of inhibition and the time to death of the parasite culture could conveniently be monitored over several days. Microcalorimetry is a valuable element to be added to the toolbox for drug discovery for protozoal diseases such as human African trypanosomiasis and malaria. The method could probably be adapted to other protozoan parasites, especially those growing extracellularly.

  20. Flux Analysis of the Trypanosoma brucei Glycolysis Based on a Multiobjective-Criteria Bioinformatic Approach

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    Amine Ghozlane

    2012-01-01

    Full Text Available Trypanosoma brucei is a protozoan parasite of major of interest in discovering new genes for drug targets. This parasite alternates its life cycle between the mammal host(s (bloodstream form and the insect vector (procyclic form, with two divergent glucose metabolism amenable to in vitro culture. While the metabolic network of the bloodstream forms has been well characterized, the flux distribution between the different branches of the glucose metabolic network in the procyclic form has not been addressed so far. We present a computational analysis (called Metaboflux that exploits the metabolic topology of the procyclic form, and allows the incorporation of multipurpose experimental data to increase the biological relevance of the model. The alternatives resulting from the structural complexity of networks are formulated as an optimization problem solved by a metaheuristic where experimental data are modeled in a multiobjective function. Our results show that the current metabolic model is in agreement with experimental data and confirms the observed high metabolic flexibility of glucose metabolism. In addition, Metaboflux offers a rational explanation for the high flexibility in the ratio between final products from glucose metabolism, thsat is, flux redistribution through the malic enzyme steps.

  1. A transcription-independent epigenetic mechanism is associated with antigenic switching in Trypanosoma brucei.

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    Aresta-Branco, Francisco; Pimenta, Silvia; Figueiredo, Luisa M

    2016-04-20

    Antigenic variation in Trypanosoma brucei relies on periodic switching of variant surface glycoproteins (VSGs), which are transcribed monoallelically by RNA polymerase I from one of about 15 bloodstream expression sites (BES). Chromatin of the actively transcribed BES is depleted of nucleosomes, but it is unclear if this open conformation is a mere consequence of a high rate of transcription, or whether it is maintained by a transcription-independent mechanism. Using an inducible BES-silencing reporter strain, we observed that chromatin of the active BES remains open for at least 24 hours after blocking transcription. This conformation is independent of the cell-cycle stage, but dependent upon TDP1, a high mobility group box protein. For two days after BES silencing, we detected a transient and reversible derepression of several silent BESs within the population, suggesting that cells probe other BESs before commitment to one, which is complete by 48 hours. FACS sorting and subsequent subcloning confirmed that probing cells are switching intermediates capable of returning to the original BES, switch to the probed BES or to a different BES. We propose that regulation of BES chromatin structure is an epigenetic mechanism important for successful antigenic switching. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Duplicated paralogous genes subject to positive selection in the genome of Trypanosoma brucei.

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    Richard D Emes

    2008-05-01

    Full Text Available Whole genome studies have highlighted duplicated genes as important substrates for adaptive evolution. We have investigated adaptive evolution in this class of genes in the human parasite Trypanosoma brucei, as indicated by the ratio of non-synonymous (amino-acid changing to synonymous (amino acid retaining nucleotide substitution rates.We have identified duplicated genes that are most rapidly evolving in this important human parasite. This is the first attempt to investigate adaptive evolution in this species at the codon level. We identify 109 genes within 23 clusters of paralogous gene expansions to be subject to positive selection.Genes identified include surface antigens in both the mammalian and insect host life cycle stage suggesting that competitive interaction is not solely with the adaptive immune system of the mammalian host. Also surface transporters related to drug resistance and genes related to developmental progression are detected. We discuss how adaptive evolution of these genes may highlight lineage specific processes essential for parasite survival. We also discuss the implications of adaptive evolution of these targets for parasite biology and control.

  3. Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

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    Marchissio, Maria Julia; Francés, Daniel Eleazar Antonio; Carnovale, Cristina Ester; Marinelli, Raúl Alberto, E-mail: rmarinel@unr.edu.ar

    2012-10-15

    Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H{sub 2}O{sub 2} across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H{sub 2}O{sub 2} release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72 h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p < 0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H{sub 2}O{sub 2} release, assessed by Amplex Red, was reduced by about 45% (p < 0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+ 120%, p < 0.05) and loss of mitochondrial membrane potential (− 80%, p < 0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H{sub 2}O{sub 2} release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death. -- Highlights: ► Aquaporin-8 is expressed in mitochondria of human hepatoma HepG2 cells. ► Aquaporin-8 knockdown impairs mitochondrial H{sub 2}O{sub 2} release and increases ROS. ► Aquaporin

  4. Nutritional rehabilitation of mitochondrial aberrations in aplastic anaemia.

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    He, Ling; Miao, Xiaoyan; Lv, Guangyan; Yang, Peiman; Wu, Wenguo; Jia, Li

    2011-04-01

    Aplastic anaemia (AA) is a disease characterised by bone marrow hypocellularity and peripheral blood pancytopenia. AA is also associated with mitochondrial aberrations. The present study was undertaken primarily to test the hypothesis that a nutrient mixture could affect the nutritional rehabilitation of mitochondrial aberrations in AA mice. BALB/c AA mice were induced by a combination of hypodermic injections of acetylphenylhydrazine (100 mg/kg), X-rays (2·0 Gy) and intraperitoneal injections of cyclophosphamide (80 mg/kg). We treated these mice with nutrient mixture-supplemented diets in a dose-dependent manner (1445·55, 963·7, 674·59 mg/kg per d), and the effects of the nutrient mixture for mitochondrial rehabilitation were analysed in AA mice. Transmission electron microscopy showed that mitochondrial ultrastructural abnormalities in bone marrow cells, splenocytes and hepatocytes of the nutrient mixture groups were restored markedly, compared with the AA group. Mitochondrial membrane potentials of the nutrient mixture groups were increased remarkably. Western blot analysis also revealed that the nutrient mixture significantly inhibited cytochrome c release of mitochondria in the AA group. Furthermore, the mitochondrial DNA content of the nutrient mixture groups was also increased. Our data suggest that the nutrient mixture may promote the rehabilitation of mitochondrial aberrations, and consequently protects against mitochondrial dysfunction in AA mice.

  5. Linear Discriminant Analysis Identifies Mitochondrially Localized Proteins in Neurospora crassa.

    Science.gov (United States)

    Wirsing, Lisette; Klawonn, Frank; Sassen, Wiebke Anna; Lünsdorf, Heinrich; Probst, Corinna; Hust, Michael; Mendel, Ralf R; Kruse, Tobias; Jänsch, Lothar

    2015-09-04

    Besides their role as powerhouses, mitochondria play a pivotal role in the spatial organization of numerous enzymatic functions. They are connected to the ER, and many pathways are organized through the mitochondrial membranes. Thus, the precise definition of mitochondrial proteomes remains a challenging task. Here, we have established a proteomic strategy to accurately determine the mitochondrial localization of proteins from the fungal model organism Neurospora crassa. This strategy relies on both highly pure mitochondria as well as the quantitative monitoring of mitochondrial components along their consecutive enrichment. Pure intact mitochondria were obtained by a multistep approach combining differential and density Percoll (ultra) centrifugations. When compared with three other intermediate enrichment stages, peptide sequencing and quantitative profiling of pure mitochondrial fractions revealed prototypic regulatory profiles of per se mitochondrial components. These regulatory profiles constitute a distinct cluster defining the mitochondrial compartment and support linear discriminant analyses, which rationalized the annotation process. In total, this approach experimentally validated the mitochondrial localization of 512 proteins including 57 proteins that had not been reported for N. crassa before.

  6. Stomatin-like protein 2 deficiency results in impaired mitochondrial translation.

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    Mitsopoulos, Panagiotis; Lapohos, Orsolya; Weraarpachai, Woranontee; Antonicka, Hana; Chang, Yu-Han; Madrenas, Joaquín

    2017-01-01

    Mitochondria translate the RNAs for 13 core polypeptides of respiratory chain and ATP synthase complexes that are essential for the assembly and function of these complexes. This process occurs in close proximity to the mitochondrial inner membrane. However, the mechanisms and molecular machinery involved in mitochondrial translation are not fully understood, and defects in this process can result in severe diseases. Stomatin-like protein (SLP)-2 is a mainly mitochondrial protein that forms cardiolipin- and prohibitin-enriched microdomains in the mitochondrial inner membrane that are important for the formation of respiratory supercomplexes and their function. Given this regulatory role of SLP-2 in processes closely associated with the mitochondrial inner membrane, we hypothesized that the function of SLP-2 would have an impact on mitochondrial translation. 35S-Methionine/cysteine pulse labeling of resting or activated T cells from T cell-specific Slp-2 knockout mice showed a significant impairment in the production of several mitochondrial DNA-encoded polypeptides following T cell activation, including Cytb, COXI, COXII, COXIII, and ATP6. Measurement of mitochondrial DNA stability and mitochondrial transcription revealed that this impairment was at the post-transcriptional level. Examination of mitochondrial ribosome assembly showed that SLP-2 migrated in sucrose-density gradients similarly to the large ribosomal subunit but that its deletion at the genetic level did not affect mitochondrial ribosome assembly. Functionally, the impairment in mitochondrial translation correlated with decreased interleukin-2 production in activated T cells. Altogether, these data show that SLP-2 acts as a general regulator of mitochondrial translation.

  7. Impaired translocation and activation of mitochondrial Akt1 mitigated mitochondrial oxidative phosphorylation Complex V activity in diabetic myocardium.

    Science.gov (United States)

    Yang, Jia-Ying; Deng, Wu; Chen, Yumay; Fan, Weiwei; Baldwin, Kenneth M; Jope, Richard S; Wallace, Douglas C; Wang, Ping H

    2013-06-01

    Insulin can translocate Akt to mitochondria in cardiac muscle. The goals of this study were to define sub-mitochondrial localization of the translocated Akt, to dissect the effects of insulin on Akt isoform translocation, and to determine the direct effect of mitochondrial Akt activation on Complex V activity in normal and diabetic myocardium. The translocated Akt sequentially localized to the mitochondrial intermembrane space, inner membrane, and matrix. To confirm Akt translocation, in vitro import assay showed rapid entry of Akt into mitochondria. Akt isoforms were differentially regulated by insulin stimulation, only Akt1 translocated into mitochondria. In the insulin-resistant Type 2 diabetes model, Akt1 translocation was blunted. Mitochondrial activation of Akt1 increased Complex V activity by 24% in normal myocardium in vivo and restored Complex V activity in diabetic myocardium. Basal mitochondrial Complex V activity was lower by 22% in the Akt1(-/-) myocardium. Insulin-stimulated Complex V activity was not impaired in the Akt1(-/-) myocardium, due to compensatory translocation of Akt2 to mitochondria. Akt1 is the primary isoform that relayed insulin signaling to mitochondria and modulated mitochondrial Complex V activity. Activation of mitochondrial Akt1 enhanced ATP production and increased phosphocreatine in cardiac muscle cells. Dysregulation of this signal pathway might impair mitochondrial bioenergetics in diabetic myocardium. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Wnt Signaling Prevents the Aβ Oligomer-Induced Mitochondrial Permeability Transition Pore Opening Preserving Mitochondrial Structure in Hippocampal Neurons.

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    Arrázola, Macarena S; Ramos-Fernández, Eva; Cisternas, Pedro; Ordenes, Daniela; Inestrosa, Nibaldo C

    2017-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder mainly known for synaptic impairment and neuronal cell loss, affecting memory processes. Beside these damages, mitochondria have been implicated in the pathogenesis of AD through the induction of the mitochondrial permeability transition pore (mPTP). The mPTP is a non-selective pore that is formed under apoptotic conditions, disturbing mitochondrial structure and thus, neuronal viability. In AD, Aβ oligomers (Aβos) favor the opening of the pore, activating mitochondria-dependent neuronal cell death cascades. The Wnt signaling activated through the ligand Wnt3a has been described as a neuroprotective signaling pathway against amyloid-β (Aβ) peptide toxicity in AD. However, the mechanisms by which Wnt signaling prevents Aβos-induced neuronal cell death are unclear. We proposed here to study whether Wnt signaling protects neurons earlier than the late damages in the progression of the disease, through the preservation of the mitochondrial structure by the mPTP inhibition. To study specific events related to mitochondrial permeabilization we performed live-cell imaging from primary rat hippocampal neurons, and electron microscopy to analyze the mitochondrial morphology and structure. We report here that Wnt3a prevents an Aβos-induced cascade of mitochondrial events that leads to neuronal cell death. This cascade involves (a) mPTP opening, (b) mitochondrial swelling, (c) mitochondrial membrane potential loss and (d) cytochrome c release, thus leading to neuronal cell death. Furthermore, our results suggest that the activation of the Wnt signaling prevents mPTP opening by two possible mechanisms, which involve the inhibition of mitochondrial GSK-3β and/or the modulation of mitochondrial hexokinase II levels and activity. This study suggests a possible new approach for the treatment of AD from a mitochondrial perspective, and will also open new lines of study in the field of Wnt signaling in neuroprotection.

  9. Avocado Oil Improves Mitochondrial Function and Decreases Oxidative Stress in Brain of Diabetic Rats

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    Omar Ortiz-Avila

    2015-01-01

    Full Text Available Diabetic encephalopathy is a diabetic complication related to the metabolic alterations featuring diabetes. Diabetes is characterized by increased lipid peroxidation, altered glutathione redox status, exacerbated levels of ROS, and mitochondrial dysfunction. Although the pathophysiology of diabetic encephalopathy remains to be clarified, oxidative stress and mitochondrial dysfunction play a crucial role in the pathogenesis of chronic diabetic complications. Taking this into consideration, the aim of this work was to evaluate the effects of 90-day avocado oil intake in brain mitochondrial function and oxidative status in streptozotocin-induced diabetic rats (STZ rats. Avocado oil improves brain mitochondrial function in diabetic rats preventing impairment of mitochondrial respiration and mitochondrial membrane potential ΔΨm, besides increasing complex III activity. Avocado oil also decreased ROS levels and lipid peroxidation and improved the GSH/GSSG ratio as well. These results demonstrate that avocado oil supplementation prevents brain mitochondrial dysfunction induced by diabetes in association with decreased oxidative stress.

  10. Avocado Oil Improves Mitochondrial Function and Decreases Oxidative Stress in Brain of Diabetic Rats.

    Science.gov (United States)

    Ortiz-Avila, Omar; Esquivel-Martínez, Mauricio; Olmos-Orizaba, Berenice Eridani; Saavedra-Molina, Alfredo; Rodriguez-Orozco, Alain R; Cortés-Rojo, Christian

    2015-01-01

    Diabetic encephalopathy is a diabetic complication related to the metabolic alterations featuring diabetes. Diabetes is characterized by increased lipid peroxidation, altered glutathione redox status, exacerbated levels of ROS, and mitochondrial dysfunction. Although the pathophysiology of diabetic encephalopathy remains to be clarified, oxidative stress and mitochondrial dysfunction play a crucial role in the pathogenesis of chronic diabetic complications. Taking this into consideration, the aim of this work was to evaluate the effects of 90-day avocado oil intake in brain mitochondrial function and oxidative status in streptozotocin-induced diabetic rats (STZ rats). Avocado oil improves brain mitochondrial function in diabetic rats preventing impairment of mitochondrial respiration and mitochondrial membrane potential (ΔΨ m ), besides increasing complex III activity. Avocado oil also decreased ROS levels and lipid peroxidation and improved the GSH/GSSG ratio as well. These results demonstrate that avocado oil supplementation prevents brain mitochondrial dysfunction induced by diabetes in association with decreased oxidative stress.

  11. Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense.

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    Baltz, T; Baltz, D; Giroud, C; Crockett, J

    1985-05-01

    A semi-defined medium for the cultivation of bloodstream forms of the African trypanosome brucei subgroup was developed. Out of 14 different strains tested, 10 could be cultured including Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. The presence of a reducing agent (2-mercaptoethanol or thioglycerol) was found to be essential for growth. The standard medium consisted of Hepes buffered minimum essential medium with Earle's salts supplemented with 0.2 mM 2-mercaptoethanol, 2 mM pyruvate and 10% inactivated serum either from rabbit (T. brucei, T. equiperdum, T. evansi and T. rhodesiense) or human (T. gambiense). Although a general medium could be defined for the long-term maintenance of trypanosome cultures, the initiation to culture nevertheless required particular conditions for the different strains. The cultured trypanosomes had all the characteristics of the in vivo bloodstream forms including: morphology, infectivity, antigenic variation and glucose metabolism.

  12. Further evidence from SSCP and ITS DNA sequencing support Trypanosoma evansi and Trypanosoma equiperdum as subspecies or even strains of Trypanosoma brucei.

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    Wen, Yan-Zi; Lun, Zhao-Rong; Zhu, Xing-Quan; Hide, Geoff; Lai, De-Hua

    2016-07-01

    The subgenus Trypanozoon includes three species Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum, which are morphologically identical and indistinguishable even using some molecular methods. In this study, PCR-based single strand conformation polymorphism (PCR-SSCP) was used to analyze the ribosomal DNA of the Trypanozoon species. Data indicate different patterns of ITS2 fragments between T. brucei, T. evansi and T. equiperdum by SSCP. Furthermore, analysis of total ITS sequences within these three members of the subgenus Trypanozoon showed a high degree of homology using phylogenetic analysis but were polyphyletic in haplotype networks. These data provide novel nuclear evidence to further support the notion that T. evansi and T. equiperdum should be subspecies or even strains of T. brucei. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Mitochondrial fragmentation in human macrophages attenuates palmitate-induced inflammatory responses.

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    Zezina, Ekaterina; Snodgrass, Ryan G; Schreiber, Yannick; Zukunft, Sven; Schürmann, Christoph; Heringdorf, Dagmar Meyer Zu; Geisslinger, Gerd; Fleming, Ingrid; Brandes, Ralf P; Brüne, Bernhard; Namgaladze, Dmitry

    2018-02-02

    Macrophages in adipose tissue contribute to inflammation and the development of insulin resistance in obesity. Exposure of macrophages to saturated fatty acids alters cell metabolism and activates pro-inflammatory signaling. How fatty acids influence macrophage mitochondrial dynamics is unclear. We investigated the mechanism of palmitate-induced mitochondrial fragmentation and its impact on inflammatory responses in primary human macrophages. Fatty acids, such as palmitate, caused mitochondrial fragmentation in human macrophages. Increased mitochondrial fragmentation was also observed in peritoneal macrophages from hyperlipidemic apolipoprotein E knockout mice. Fatty acid-induced mitochondrial fragmentation was independent of the fatty acid chain saturation and required dynamin-related protein 1 (DRP1). Mechanistically, mitochondrial fragmentation was regulated by incorporation of palmitate into mitochondrial phospholipids and their precursors. Palmitate-induced endoplasmic reticulum stress and loss of mitochondrial membrane potential did not contribute to mitochondrial fragmentation. Macrophages treated with palmitate maintained intact mitochondrial respiration and ATP levels. Pharmacological or genetic inhibition of DRP1 enhanced palmitate-induced mitochondrial ROS production, c-Jun phosphorylation, and inflammatory cytokine expression. Our results indicate that mitochondrial fragmentation is a protective mechanism attenuating inflammatory responses induced by palmitate in human macrophages. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Optogenetic control of mitochondrial metabolism and Ca2+ signaling by mitochondria-targeted opsins

    OpenAIRE

    Tkatch, Tatiana; Greotti, Elisa; Baranauskas, Gytis; Pendin, Diana; Roy, Soumitra; Nita, Luliaoana I.; Wettmarshausen, Jennifer; Prigge, Matthias; Yizhar, Ofer; Shirihai, Orian S.; Fishman, Daniel; Hershfinkel, Michal; Fleidervish, Ilya A; Perocchi, Fabiana; Pozzan, Tullio

    2017-01-01

    Mitochondrial functions depend on the steep H+ electrochemical gradient (??H+) across their inner membrane. The available tools for controlling this gradient are essentially limited to inhibitors of the respiratory chain or of the H+ ATPase or to uncouplers, poisons plagued by important side effects and that lack both cell and spatial specificity. We show here that, by transfecting cells with the cDNA encoding channelrhodopsins specifically targeted to the inner mitochondrial membrane, we can...

  15. Diagnostic accuracy of loopamp Trypanosoma brucei detection kit for diagnosis of human African trypanosomiasis in clinical samples.

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    Patrick Mitashi

    Full Text Available BACKGROUND: Molecular methods have great potential for sensitive parasite detection in the diagnosis of human African trypanosomiasis (HAT, but the requirements in terms of laboratory infrastructure limit their use to reference centres. A recently developed assay detects the Trypanozoon repetitive insertion mobile element (RIME DNA under isothermal amplification conditions and has been transformed into a ready-to-use kit format, the Loopamp Trypanosoma brucei. In this study, we have evaluated the diagnostic performance of the Loopamp Trypanosoma brucei assay (hereafter called LAMP in confirmed T.b. gambiense HAT patients, HAT suspects and healthy endemic controls from the Democratic Republic of the Congo (DRC. METHODOLOGY/PRINCIPAL FINDINGS: 142 T.b. gambiense HAT patients, 111 healthy endemic controls and 97 HAT suspects with unconfirmed status were included in this retrospective evaluation. Reference standard tests were parasite detection in blood, lymph or cerebrospinal fluid. Archived DNA from blood of all study participants was analysed in duplicate with LAMP. Sensitivity of LAMP in parasitologically confirmed cases was 87.3% (95% CI 80.9-91.8% in the first run and 93.0% (95% CI 87.5-96.1% in the second run. Specificity in healthy controls was 92.8% (95% CI 86.4-96.3% in the first run and 96.4% (95% CI 91.1-98.6% in the second run. Reproducibility was excellent with a kappa value of 0.81. CONCLUSIONS/SIGNIFICANCE: In this laboratory-based study, the Loopamp Trypanosoma brucei Detection Kit showed good diagnostic accuracy and excellent reproducibility. Further studies are needed to assess the feasibility of its routine use for diagnosis of HAT under field conditions.

  16. Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids.

    Science.gov (United States)

    Nanavaty, Vishal; Sandhu, Ranjodh; Jehi, Sanaa E; Pandya, Unnati M; Li, Bibo

    2017-06-02

    Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, thereby evading the host's immune response. VSGs are monoallelically expressed from subtelomeric expression sites (ESs), and VSG switching exploits subtelomere plasticity. However, subtelomere integrity is essential for T. brucei viability. The telomeric transcript, TERRA, was detected in T. brucei previously. We now show that the active ES-adjacent telomere is transcribed. We find that TbRAP1, a telomere protein essential for VSG silencing, suppresses VSG gene conversion-mediated switching. Importantly, TbRAP1 depletion increases the TERRA level, which appears to result from longer read-through into the telomere downstream of the active ES. Depletion of TbRAP1 also results in more telomeric RNA:DNA hybrids and more double strand breaks (DSBs) at telomeres and subtelomeres. In TbRAP1-depleted cells, expression of excessive TbRNaseH1, which cleaves the RNA strand of the RNA:DNA hybrid, brought telomeric RNA:DNA hybrids, telomeric/subtelomeric DSBs and VSG switching frequency back to WT levels. Therefore, TbRAP1-regulated appropriate levels of TERRA and telomeric RNA:DNA hybrid are fundamental to subtelomere/telomere integrity. Our study revealed for the first time an important role of a long, non-coding RNA in antigenic variation and demonstrated a link between telomeric silencing and subtelomere/telomere integrity through TbRAP1-regulated telomere transcription. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Betaine is a positive regulator of mitochondrial respiration.

    Science.gov (United States)

    Lee, Icksoo

    2015-01-09

    Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro. Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Preliminary X-ray crystallographic studies of yeast mitochondrial protein Tom70p

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    Wu, Yunkun [Department of Cell Biology, Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States); McCombs, Debbie; Nagy, Lisa; DeLucas, Lawrence [Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States); Sha, Bingdong, E-mail: bdsha@uab.edu [Department of Cell Biology, Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States)

    2006-03-01

    Tom70p is an important translocase of the outer membrane complex member and a major surface receptor of the protein-translocation machinery in the outer mitochondrial membrane. To investigate the mechanism by which Tom70p functions to deliver the mitochondrial protein precursors, the cytosolic fragment of yeast Tom70p (cTom70p) has been crystallized. Protein translocations across mitochondrial membranes play critical roles in mitochondrion biogenesis. Protein transport from the cell cytosol to the mitochondrial matrix is carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tom70p is an important TOM-complex member and a major surface receptor of the protein-translocation machinery in the outer mitochondrial membrane. To investigate the mechanism by which Tom70p functions to deliver the mitochondrial protein precursors, the cytosolic fragment of yeast Tom70p (cTom70p) was crystallized. The crystals diffract to 3.2 Å using a synchrotron X-ray source and belong to space group P2{sub 1}, with unit-cell parameters a = 44.89, b = 168.78, c = 83.41 Å, α = 90.00, β = 102.74, γ = 90.00°. There are two Tom70p molecules in one asymmetric unit, which corresponds to a solvent content of approximately 51%. Structure determination by MAD methods is under way.

  19. Inhibition of NAPDH Oxidase 2 (NOX2 Prevents Oxidative Stress and Mitochondrial Abnormalities Caused by Saturated Fat in Cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Leroy C Joseph

    Full Text Available Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The molecular mechanisms are poorly understood. We hypothesized that physiologic levels of saturated fat could increase mitochondrial reactive oxygen species (ROS in cardiomyocytes, leading to abnormalities of calcium homeostasis and mitochondrial function. We investigated the effect of saturated fat on mitochondrial function and calcium homeostasis in isolated ventricular myocytes. The saturated fatty acid palmitate causes a decrease in mitochondrial respiration in cardiomyocytes. Palmitate, but not the monounsaturated fatty acid oleate, causes an increase in both total cellular ROS and mitochondrial ROS. Palmitate depolarizes the mitochondrial inner membrane and causes mitochondrial calcium overload by increasing sarcoplasmic reticulum calcium leak. Inhibitors of PKC or NOX2 prevent mitochondrial dysfunction and the increase in ROS, demonstrating that PKC-NOX2 activation is also required for amplification of palmitate induced-ROS. Cardiomyocytes from mice with genetic deletion of NOX2 do not have palmitate-induced ROS or mitochondrial dysfunction. We conclude that palmitate induces mitochondrial ROS that is amplified by NOX2, causing greater mitochondrial ROS generation and partial depolarization of the mitochondrial inner membrane. The abnormal sarcoplasmic reticulum calcium leak caused by palmitate could promote arrhythmia and heart failure. NOX2 inhibition is a potential therapy for heart disease caused by diabetes or obesity.

  20. Allicin Protects PC12 Cells Against 6-OHDA-Induced Oxidative Stress and Mitochondrial Dysfunction via Regulating Mitochondrial Dynamics.

    Science.gov (United States)

    Liu, Hao; Mao, Ping; Wang, Jia; Wang, Tuo; Xie, Chang-Hou

    2015-01-01

    Parkinson disease (PD) is a common adult-onset neurodegenerative disorder, and PD related neuronal injury is associated with oxidative stress and mitochondrial dysfunction. Allicin, the main biologically active compound derived from garlic, has been shown to exert various anti-oxidative and anti-apoptotic activities in in vitro and in vivo studies. The present study aimed to investigate the potential protective role of allicin in an in vitro PD model induced by 6-hydroxydopamine (6-OHDA) in PC12 cells. The protective effects were measured by cell viability, decreased lactate dehydrogenase (LDH) release and flow cytometry, and the anti-oxidative activity was determined by reactive oxygen species (ROS) generation, lipid peroxidation and the endogenous antioxidant enzyme activities. Mitochondrial function in PC12 cells was detected by mitochondrial membrane potential (MMP) collapse, cytochrome c release, mitochondrial ATP synthesis, and the mitochondrial Ca(2+) buffering capacity. To investigate the potential mechanism, we also measured the expression of mitochondrial biogenesis factors, mitochondrial morphological dynamic changes, as well as detected mitochondrial dynamic proteins by western blot. We found that allicin treatment significant increased cell viability, and decreased LDH release and apoptotic cell death after 6-OHDA exposure. Allicin also inhibited ROS generation, reduced lipid peroxidation and preserved the endogenous antioxidant enzyme activities. These protective effects were associated with suppressed mitochondrial dysfunction, as evidenced by decreased MMP collapse and cytochrome c release, preserved mitochondrial ATP synthesis, and the promotion of mitochondrial Ca(2+) buffering capacity. In addition, allicin significantly enhanced mitochondrial biogenesis and prevented fragmentation of mitochondrial network after 6-OHDA treatment. The results of western blot analysis showed that the 6-OHDA induced decrease in the expression of optic atrophy type 1

  1. Allicin Protects PC12 Cells Against 6-OHDA-Induced Oxidative Stress and Mitochondrial Dysfunction via Regulating Mitochondrial Dynamics

    Directory of Open Access Journals (Sweden)

    Hao Liu

    2015-06-01

    Full Text Available Background: Parkinson disease (PD is a common adult-onset neurodegenerative disorder, and PD related neuronal injury is associated with oxidative stress and mitochondrial dysfunction. Allicin, the main biologically active compound derived from garlic, has been shown to exert various anti-oxidative and anti-apoptotic activities in in vitro and in vivo studies. Methods: The present study aimed to investigate the potential protective role of allicin in an in vitro PD model induced by 6-hydroxydopamine (6-OHDA in PC12 cells. The protective effects were measured by cell viability, decreased lactate dehydrogenase (LDH release and flow cytometry, and the anti-oxidative activity was determined by reactive oxygen species (ROS generation, lipid peroxidation and the endogenous antioxidant enzyme activities. Mitochondrial function in PC12 cells was detected by mitochondrial membrane potential (MMP collapse, cytochrome c release, mitochondrial ATP synthesis, and the mitochondrial Ca2+ buffering capacity. To investigate the potential mechanism, we also measured the expression of mitochondrial biogenesis factors, mitochondrial morphological dynamic changes, as well as detected mitochondrial dynamic proteins by western blot. Results: We found that allicin treatment significant increased cell viability, and decreased LDH release and apoptotic cell death after 6-OHDA exposure. Allicin also inhibited ROS generation, reduced lipid peroxidation and preserved the endogenous antioxidant enzyme activities. These protective effects were associated with suppressed mitochondrial dysfunction, as evidenced by decreased MMP collapse and cytochrome c release, preserved mitochondrial ATP synthesis, and the promotion of mitochondrial Ca2+ buffering capacity. In addition, allicin significantly enhanced mitochondrial biogenesis and prevented fragmentation of mitochondrial network after 6-OHDA treatment. The results of western blot analysis showed that the 6-OHDA induced decrease

  2. Mitochondrial benzodiazepine receptors regulate steroid biosynthesis

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    Mukhin, A.G.; Papadopoulos, V.; Costa, E.; Krueger, K.E. (Georgetown Univ. School of Medicine, Washington, DC (USA))

    1989-12-01

    Recent observations on the steroid synthetic capability within the brain open the possibility that benzodiazepines may influence steroid synthesis in nervous tissue through interactions with peripheral-type benzodiazepine recognition sites, which are highly expressed in steroidogenic cells and associated with the outer mitochondrial membrane. To examine this possibility nine molecules that exhibit a greater than 10,000-fold difference in their affinities for peripheral-type benzodiazepine binding sites were tested for their effects on a well-established steroidogenic model system, the Y-1 mouse adrenal tumor cell line. 4{prime}-Chlorodiazepam, PK 11195, and PK 14067 stimulated steroid production by 2-fold in Y-1 cells, whereas diazepam, flunitrazepam, zolpidem, and PK 14068 displayed a lower (1.2- to 1.5-fold) maximal stimulation. In contrast, clonazepam and flumazenil did not stimulate steroid synthesis. The potencies of these compounds to inhibit {sup 3}H-labeled PK 11195 binding to peripheral-type benzodiazepine recognition sites correlated with their potencies to stimulate steroid production. Similar findings were observed in bovine and rat adrenocortical cell preparations. These results suggest that ligands of the peripheral-type benzodiazepine recognition site acting on this mitochondrial receptor can enhance steroid production. This action may contribute specificity to the pharmacological profile of drugs preferentially acting on the benzodiazepine recognition site associated with the outer membrane of certain mitochondrial populations.

  3. Innate Immune Function of Mitochondrial Metabolism.

    Science.gov (United States)

    Sancho, David; Enamorado, Michel; Garaude, Johan

    2017-01-01

    Sensing of microbe-associated molecular patterns or danger signals by innate immune receptors drives a complex exchange of information. Innate receptor signaling not only triggers transcriptional events but also induces profound changes in metabolic fluxes, redox balance, and metabolite abundance thereby influencing immune cell function. Mitochondria are at the core of metabolic adaptation to the changing environment. The close interaction between mitochondrial metabolism and immune signaling has emerged as a central regulator of innate sensing. Metabolic processes generate a constant flow of electrons that eventually end up in the mitochondrial electron transport chain (ETC). Two electron carriers and four respiratory complexes that can assemble as larger molecular supercomplexes compose the ETC in the mitochondrial inner membrane. While the meaning and biological relevance of such structural organization is a matter of passionate debates, recent data support that innate stimuli remodel the ETC. We will review the function of mitochondrial metabolism and ETC dynamics as innate rheostats that regulate signaling, transcription, and epigenetics to orchestrate innate immune responses.

  4. Pseudomonas aeruginosa pyocyanin induces neutrophil death via mitochondrial reactive oxygen species and mitochondrial acid sphingomyelinase.

    Science.gov (United States)

    Managò, Antonella; Becker, Katrin Anne; Carpinteiro, Alexander; Wilker, Barbara; Soddemann, Matthias; Seitz, Aaron P; Edwards, Michael J; Grassmé, Heike; Szabò, Ildiko; Gulbins, Erich

    2015-05-01

    Pulmonary infections with Pseudomonas aeruginosa are a serious clinical problem and are often lethal. Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited. Neutrophils play an important role in the host's early acute defense against pulmonary P. aeruginosa. Therefore, it is important to define the mechanisms by which P. aeruginosa interacts with host cells, particularly neutrophils. Here, we report that pyocyanin, a membrane-permeable pigment and toxin released by P. aeruginosa, induces the death of wild-type neutrophils; its interaction with the mitochondrial respiratory chain results in the release of reactive oxygen species (ROS), the activation of mitochondrial acid sphingomyelinase, the formation of mitochondrial ceramide, and the release of cytochrome c from mitochondria. A genetic deficiency in acid sphingomyelinase prevents both the activation of this pathway and pyocyanin-induced neutrophil death. This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells. These studies identified the mechanisms by which pyocyanin induces the release of mitochondrial ROS and by which ROS induce neutrophil death via mitochondrial acid sphingomyelinase. These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions.

  5. 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction.

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    Johanna L Höög

    2016-01-01

    Full Text Available Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility.We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum.The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life cycle of this human parasite.

  6. Regulation of Trypanosoma brucei Total and Polysomal mRNA during Development within Its Mammalian Host.

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    Paul Capewell

    Full Text Available The gene expression of Trypanosoma brucei has been examined extensively in the blood of mammalian hosts and in forms found in the midgut of its arthropod vector, the tsetse fly. However, trypanosomes also undergo development within the mammalian bloodstream as they progress from morphologically 'slender forms' to transmissible 'stumpy forms' through morphological intermediates. This transition is temporally progressive within the first wave of parasitaemia such that gene expression can be monitored in relatively pure slender and stumpy populations as well as during the progression between these extremes. The development also represents the progression of cells from translationally active forms adapted for proliferation in the host to translationally quiescent forms, adapted for transmission. We have used metabolic labelling to quantitate translational activity in slender forms, stumpy forms and in forms undergoing early differentiation to procyclic forms in vitro. Thereafter we have examined the cohort of total mRNAs that are enriched throughout development in the mammalian bloodstream (slender, intermediate and stumpy forms, irrespective of strain, revealing those that exhibit consistent developmental regulation rather than sample specific changes. Transcripts that cosediment with polysomes in stumpy forms and slender forms have also been enriched to identify transcripts that escape translational repression prior to transmission. Combined, the expression and polysomal association of transcripts as trypanosomes undergo development in the mammalian bloodstream have been defined, providing a resource for trypanosome researchers. This facilitates the identification of those that undergo developmental regulation in the bloodstream and therefore those likely to have a role in the survival and capacity for transmission of stumpy forms.

  7. 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction

    Science.gov (United States)

    Höög, Johanna L.; Lacomble, Sylvain; Bouchet-Marquis, Cedric; Briggs, Laura; Park, Kristin; Hoenger, Andreas; Gull, Keith

    2016-01-01