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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

    Science.gov (United States)

    Munday, Jane C.; Tagoe, Daniel N. A.; Stelmanis, Valters; Schnaufer, Achim

    2016-01-01

    Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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

  2. 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.

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

    Science.gov (United States)

    de Macêdo, Juan P; Schumann Burkard, Gabriela; Niemann, Moritz; Barrett, Michael P; Vial, Henri; Mäser, Pascal; Roditi, Isabel; Schneider, André; Bütikofer, Peter

    2015-05-01

    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 action or targeting. PMID

  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. Pentatricopeptide repeat proteins in Trypanosoma brucei function in mitochondrial ribosomes

    OpenAIRE

    Pusnik, Mascha; Small, Ian; Read, Laurie K.; Fabbro, Thomas; Schneider, André

    2008-01-01

    The pentatricopeptide repeat (PPR), a degenerate 35-amino-acid motif, defines a novel eukaryotic protein family. Plants have 400 to 500 distinct PPR proteins, whereas other eukaryotes generally have fewer than 5. The few PPR proteins that have been studied have roles in organellar gene expression, probably via direct interaction with RNA. Here we show that the parasitic protozoan Trypanosoma brucei encodes 28 distinct PPR proteins, an extraordinarily high number for a nonplant organism. A com...

  6. Identification of the mitochondrially encoded subunit 6 of F1FO ATPase in Trypanosoma brucei.

    Science.gov (United States)

    Škodová-Sveráková, Ingrid; Horváth, Anton; Maslov, Dmitri A

    2015-06-01

    Kinetoplast maxicircle DNA of trypanosomatids encodes eighteen proteins. RNA editing is required to confer translatability to mRNA for twelve of these. Sequence conservation of the predicted hydrophobic polypeptides indicates that they represent functional components of the respiratory chain. Yet, so far only two of those, cytochrome c oxidase subunit I and apocytochrome b of cytochrome c reductase, have been identified with biochemical methods. Here we report on identification of A6 subunit of F1FO ATPase encoded by a pan-edited mRNA in Trypanosoma brucei. The polypeptide was present among the (35)S-labeled mitochondrial translation products characterized by anomalous migration in denaturing 2D gels. It was identified as an ATPase subunit by co-migration with this complex in Blue Native 2D gels. A partial N-terminal sequence of the corresponding polypeptide present in the gel-purified ATPase complex from Leishmania tarentolae was consistent with the predicted A6 sequence. PMID:26276057

  7. Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei.

    Science.gov (United States)

    Turrens, J F; Bickar, D; Lehninger, A L

    1986-06-01

    The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase.

  8. Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei.

    Science.gov (United States)

    Turrens, J F; Bickar, D; Lehninger, A L

    1986-06-01

    The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase. PMID:3016533

  9. The isolation and partial characterization of the plasma membrane from Trypanosoma brucei.

    Science.gov (United States)

    Voorheis, H P; Gale, J S; Owen, M J; Edwards, W

    1979-04-15

    Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before secrose-density gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na+ + K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-alpha-oxoglutarate aminotransferase, L-asparte-alpha-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase. PMID:486094

  10. 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.

  11. TbPIF5 is a Trypanosoma brucei mitochondrial DNA helicase involved in processing of minicircle Okazaki fragments.

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    Beiyu Liu

    2009-09-01

    Full Text Available Trypanosoma brucei's mitochondrial genome, kinetoplast DNA (kDNA, is a giant network of catenated DNA rings. The network consists of a few thousand 1 kb minicircles and several dozen 23 kb maxicircles. Here we report that TbPIF5, one of T. brucei's six mitochondrial proteins related to Saccharomyces cerevisiae mitochondrial DNA helicase ScPIF1, is involved in minicircle lagging strand synthesis. Like its yeast homolog, TbPIF5 is a 5' to 3' DNA helicase. Together with other enzymes thought to be involved in Okazaki fragment processing, TbPIF5 localizes in vivo to the antipodal sites flanking the kDNA. Minicircles in wild type cells replicate unidirectionally as theta-structures and are unusual in that Okazaki fragments are not joined until after the progeny minicircles have segregated. We now report that overexpression of TbPIF5 causes premature removal of RNA primers and joining of Okazaki fragments on theta structures. Further elongation of the lagging strand is blocked, but the leading strand is completed and the minicircle progeny, one with a truncated H strand (ranging from 0.1 to 1 kb, are segregated. The minicircles with a truncated H strand electrophorese on an agarose gel as a smear. This replication defect is associated with kinetoplast shrinkage and eventual slowing of cell growth. We propose that TbPIF5 unwinds RNA primers after lagging strand synthesis, thus facilitating processing of Okazaki fragments.

  12. Platelet mitochondrial membrane potential in Parkinson's disease

    OpenAIRE

    Antony, P.M.; Boyd, O.; Trefois, C.; Ammerlaan, W; Ostaszewski, M.; Baumuratov, A.S.; Longhino, L.; Antunes, L; Koopman, W.J.H.; Balling, R; Diederich, N.J.

    2014-01-01

    OBJECTIVE: Mitochondrial dysfunction is a hallmark of idiopathic Parkinson's disease (IPD), which has been reported not to be restricted to striatal neurons. However, studies that analyzed mitochondrial function at the level of selected enzymatic activities in peripheral tissues have produced conflicting data. We considered the electron transport chain as a complex system with mitochondrial membrane potential as an integrative indicator for mitochondrial fitness. METHODS: Twenty-five IPD pati...

  13. Differential expression of glycosomal and mitochondrial proteins in the two major life-cycle stages of Trypanosoma brucei.

    Science.gov (United States)

    Vertommen, Didier; Van Roy, Joris; Szikora, Jean-Pierre; Rider, Mark H; Michels, Paul A M; Opperdoes, Fred R

    2008-04-01

    Label-free semi-quantitative differential three-dimensional liquid chromatography coupled to mass spectrometry (3D-LC-MS/MS) was used to compare the glycosomal and mitochondrial proteomes of the bloodstream- and insect-form of Trypanosoma brucei. The abundance of glycosomal marker proteins identified in the two life-cycle stages corresponded well with the relative importance of biochemical pathways present in the glycosomes of the two stages and the peptide spectral count ratios of selected enzymes were in good agreement with published data about their enzymatic specific activities. This approach proved extremely useful for the generation of large scale proteomics data for the comparison of different life-cycle stages. Several proteins involved in oxidative stress protection, sugar-nucleotide synthesis, purine salvage, nucleotide-monophosphate formation and purine-nucleotide cycle were identified as glycosomal proteins. PMID:18242729

  14. Reconstitutions of mitochondrial inner membrane remodeling.

    Science.gov (United States)

    Barbot, Mariam; Meinecke, Michael

    2016-10-01

    Biological membranes exhibit function-related shapes, leading to a plethora of complex and beautiful cell and cell organellar morphologies. Most if not all of these structures have evolved for a particular physiological reason. The shapes of these structures are formed by physical forces that operate on membranes. To create particular shaped cells and cell organelles, membranes must undergo deformations which are determined by the structure and elasticity of the membrane and this process is most probable driven by proteins, lipids and/or interplay of both Zimmerberg and Kozlov (2006). Therefore, an important question of current cell biology in conjunction with physics and mathematics is to elucidate the functional cause for these different membrane morphologies as well as how they are formed. One of the most peculiar membrane shapes is observed in mitochondria. These organelles are surrounded by two membranes and especially the convoluted inner membrane displays a complex ultra-structure. A molecular understanding of how this membrane is shaped is missing to a large extent. Unlike membrane remodeling in classical curvature-dependent processes like clathrin-mediated endocytosis, mitochondria are most likely shaped by integral membrane proteins. Following, we will review the current knowledge of inner mitochondrial membrane architecture and discuss recent findings and advances in understanding the factors that shape this membrane. We will address pending questions especially with regard to the experimentally challenging nature of investigating membrane bending by hydrophobic integral membrane proteins. PMID:27456366

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

    Science.gov (United States)

    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.

  16. A mitochondrially targeted compound delays aging in yeast through a mechanism linking mitochondrial membrane lipid metabolism to mitochondrial redox biology

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    Michelle T. Burstein

    2014-01-01

    Full Text Available A recent study revealed a mechanism of delaying aging in yeast by a natural compound which specifically impacts mitochondrial redox processes. In this mechanism, exogenously added lithocholic bile acid enters yeast cells, accumulates mainly in the inner mitochondrial membrane, and elicits an age-related remodeling of phospholipid synthesis and movement within both mitochondrial membranes. Such remodeling of mitochondrial phospholipid dynamics progresses with the chronological age of a yeast cell and ultimately causes significant changes in mitochondrial membrane lipidome. These changes in the composition of membrane phospholipids alter mitochondrial abundance and morphology, thereby triggering changes in the age-related chronology of such longevity-defining redox processes as mitochondrial respiration, the maintenance of mitochondrial membrane potential, the preservation of cellular homeostasis of mitochondrially produced reactive oxygen species, and the coupling of electron transport to ATP synthesis.

  17. 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.

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

    OpenAIRE

    Michael Wink; Vera Rosenkranz

    2008-01-01

    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, c...

  19. Proteolytic cleavage of Opa1 stimulates mitochondrial inner membrane fusion and couples fusion to oxidative phosphorylation

    OpenAIRE

    Mishra, Prashant; Carelli, Valerio; Manfredi, Giovanni; Chan, David C.

    2014-01-01

    Mitochondrial fusion is essential for maintenance of mitochondrial function. The mitofusin GTPases control mitochondrial outer membrane fusion, whereas the dynamin-related GTPase Opa1 mediates inner membrane fusion. We show that mitochondrial inner membrane fusion is tuned by the level of oxidative phosphorylation (OXPHOS), whereas outer membrane fusion is insensitive. Consequently, cells from patients with pathogenic mtDNA mutations show a selective defect in mitochondrial inner membrane fus...

  20. 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

  1. Dysfunction of Rice Mitochondrial Membrane Induced by Yb3+.

    Science.gov (United States)

    Gao, Jia-Ling; Wu, Man; Liu, Wen; Feng, Zhi-Jiang; Zhang, Ye-Zhong; Jiang, Feng-Lei; Liu, Yi; Dai, Jie

    2015-12-01

    Ytterbium (Yb), a widely used rare earth element, is treated as highly toxic to human being and adverseness to plant. Mitochondria play a significant role in plant growth and development, and are proposed as a potential target for ytterbium toxicity. In this paper, the biological effect of Yb(3+) on isolated rice mitochondria was investigated. We found that Yb(3+) with high concentrations (200 ~ 600 μM) not only induced mitochondrial membrane permeability transition (mtMPT), but also disturbed the mitochondrial ultrastructure. Moreover, Yb(3+) caused the respiratory chain damage, ROS formation, membrane potential decrease, and mitochondrial complex II activity reverse. The results above suggested that Yb(3+) with high concentrations could induce mitochondrial membrane dysfunction. These findings will support some valuable information to the safe application of Yb-based agents. PMID:26305923

  2. Platelet mitochondrial membrane potential in Parkinson's disease

    NARCIS (Netherlands)

    Antony, P.M.; Boyd, O.; Trefois, C.; Ammerlaan, W.; Ostaszewski, M.; Baumuratov, A.S.; Longhino, L.; Antunes, L.; Koopman, W.J.H.; Balling, R.; Diederich, N.J.

    2015-01-01

    OBJECTIVE: Mitochondrial dysfunction is a hallmark of idiopathic Parkinson's disease (IPD), which has been reported not to be restricted to striatal neurons. However, studies that analyzed mitochondrial function at the level of selected enzymatic activities in peripheral tissues have produced confli

  3. 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.

  4. 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.

  5. 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...

  6. Mitochondrial Swelling and Incipient Outer Membrane Rupture in Preapoptotic and Apoptotic Cells

    OpenAIRE

    Sesso, A.; Belizário, JE; Marques, MM; Higuchi, ML; Schumacher, RI; Colquhoun, A; Ito, E.; Kawakami, J.

    2012-01-01

    Outer mitochondrial membrane (OMM) rupture was first noted in isolated mitochondria in which the inner mitochondrial membrane (IMM) had lost its selective permeability. This phenomenon referred to as mitochondrial permeability transition (MPT) refers to a permeabilized inner membrane that originates a large swelling in the mitochondrial matrix, which distends the outer membrane until it ruptures. Here, we have expanded previous electron microscopic observations that in apoptotic cells, OMM ru...

  7. The glycosylphosphatidylinositol-PLC in Trypanosoma brucei forms a linear array on the exterior of the flagellar membrane before and after activation.

    Science.gov (United States)

    Hanrahan, Orla; Webb, Helena; O'Byrne, Robert; Brabazon, Elaine; Treumann, Achim; Sunter, Jack D; Carrington, Mark; Voorheis, H Paul

    2009-06-01

    Bloodstream forms of Trypanosoma brucei contain a glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) that cleaves the GPI-anchor of the variable surface glycoprotein (VSG). Its location in trypanosomes has been controversial. Here, using confocal microscopy and surface labelling techniques, we show that the GPI-PLC is located exclusively in a linear array on the outside of the flagellar membrane, close to the flagellar attachment zone, but does not co-localize with the flagellar attachment zone protein, FAZ1. Consequently, the GPI-PLC and the VSG occupy the same plasma membrane leaflet, which resolves the topological problem associated with the cleavage reaction if the VSG and the GPI-PLC were on opposite sides of the membrane. The exterior location requires the enzyme to be tightly regulated to prevent VSG release under basal conditions. During stimulated VSG release in intact cells, the GPI-PLC did not change location, suggesting that the release mechanism involves lateral diffusion of the VSG in the plane of the membrane to the fixed position of the GPI-PLC.

  8. 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

    The 25 kDa branched polyethylenimine (PEI) is a highly efficient synthetic polycation used in transfection protocols, but also triggers mitochondrial-mediated apoptotic cell death processes where the mechanistic issues are poorly understood. We now demonstrate that PEI in a concentration- and time......-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...... some effects on mitochondrial morphology and respiration, and these may be related to the inherent membrane perturbing properties of this polycation. The PEI-mediated mitochondrial swelling phase is biphasic, with a fast decaying initial period (most prominent from 4 µg/mL PEI) followed by a slower...

  9. Mitochondrial outer-membrane permeabilization and remodelling in apoptosis

    OpenAIRE

    Jourdain, Alexis; Martinou, Jean-Claude

    2009-01-01

    Many human pathologies are associated with defects in mitochondria such as diabetes, neurodegenerative diseases or cancer. This tiny organelle is involved in a plethora of processes in mammalian cells, including energy production, lipid metabolism and cell death. In the so-called intrinsic apoptotic pathway, the outer mitochondrial membrane (MOM) is premeabilized by the pro-apoptotic Bcl-2 members Bax and Bak, allowing the release of apoptogenic factors such as cytochrome c from the inter-mem...

  10. Calcium Flux across Plant Mitochondrial Membranes: Possible Molecular Players

    Science.gov (United States)

    Carraretto, Luca; Checchetto, Vanessa; De Bortoli, Sara; Formentin, Elide; Costa, Alex; Szabó, Ildikó; Teardo, Enrico

    2016-01-01

    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 (MCU), LETM1, a plant glutamate receptor family member, adenine nucleotide/phosphate carriers and the permeability transition pore (PTP), 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. PMID:27065186

  11. 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......-handling stability, and thus is suitable for large-scale screening efforts. In summary, the DASPEI assay is simple and rapid and may be of use in toxicological testing, drug target discovery, and mechanistic models of diseases involving mitochondrial dysfunction....... potential assay using 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI), a rarely used mitochondrial potentiometric probe, in a 96-well format using a fluorescent plate reader. The assay was validated using 2 protonophores (CCCP, DNP), which are known uncouplers, and the neuroleptic thioridazine...

  12. p53's mitochondrial translocation and MOMP action is independent of Puma and Bax and severely disrupts mitochondrial membrane integrity

    Institute of Scientific and Technical Information of China (English)

    Sonja Wolff; Susan Erster; Gustavo Palacios; Ute M Moll

    2008-01-01

    p53's apoptotic program consists of transcription-dependent and transcription-independent pathways. In the latter, physical interactions between mitochondrial p53 and anti-and pro-apoptotic members of the Bcl2 family of mitochondrial permeability regulators are central. Using isogenic cell systems with defined deficiencies, we characterize in detail how mitochondrial p53 contributes to mitochondrial permeabilization, to what extent its action depends on other key Bcl2 family members and define its release activity. We show that mitochondrial p53 is highly efficient in inducing the release of soluble and insoluble apoptogenic factors by severely disrupting outer and inner mitochondrial membrane integrity. This action is associated with wild-type p53-induced oligomerization of Bax, Bak and VDAC and the formation of a stress-induced endogenous complex between p53 and cyclophilin D, normally located at the inner membrane. Tumor-derived p53 mutants are deficient in activating the Bax/Bak lipid pore. These actions are independent of Puma and Bax. Importantly, the latter distinguishes the mitochondrial from the cytosolic p53 death pathway.

  13. Mitochondrial matrix delivery using MITO-Porter, a liposome-based carrier that specifies fusion with mitochondrial membranes

    Energy Technology Data Exchange (ETDEWEB)

    Yasuzaki, Yukari; Yamada, Yuma [Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812 (Japan); Harashima, Hideyoshi, E-mail: harasima@pharm.hokudai.ac.jp [Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812 (Japan)

    2010-06-25

    Mitochondria are the principal producers of energy in cells of higher organisms. It was recently reported that mutations and defects in mitochondrial DNA (mtDNA) are associated with various mitochondrial diseases including a variety of neurodegenerative and neuromuscular diseases. Therefore, an effective mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve this, therapeutic agents need to be delivered into the innermost mitochondrial space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the development of MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. In this study, we provide a demonstration of mitochondrial matrix delivery and the visualization of mitochondrial genes (mtDNA) in living cells using the MITO-Porter. We first prepared MITO-Porter containing encapsulated propidium iodide (PI), a fluorescent dye used to stain nucleic acids to detect mtDNA. We then confirmed the emission of red-fluorescence from PI by conjugation with mtDNA, when the carriers were incubated in the presence of isolated rat liver mitochondria. Finally, intracellular observation by confocal laser scanning microscopy clearly verified that the MITO-Porter delivered PI to the mitochondrial matrix.

  14. Replication factors transiently associate with mtDNA at the mitochondrial inner membrane to facilitate replication

    NARCIS (Netherlands)

    Rajala, N.; Gerhold, J.M.; Martinsson, P.; Klymov, A.; Spelbrink, H.

    2014-01-01

    Mitochondrial DNA (mtDNA) is organized in discrete protein-DNA complexes, nucleoids, that are usually considered to be mitochondrial-inner-membrane associated. Here we addressed the association of replication factors with nucleoids and show that endogenous mtDNA helicase Twinkle and single-stranded

  15. 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.

  16. 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 ...

  17. 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.

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

    Science.gov (United States)

    Korenić, Andrej; Boltze, Johannes; Deten, Alexander; Peters, Myriam; Andjus, Pavle; Radenović, Lidija

    2014-01-01

    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. PMID:24587410

  19. 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)

  20. Lipid unsaturation per se does not explain the physical state of mitochondrial membranes in Mytilus galloprovincialis.

    Science.gov (United States)

    Fiorini, Rosamaria; Pagliarani, Alessandra; Nesci, Salvatore; Trombetti, Fabiana; Pirini, Maurizio; Fabbri, Micaela; Ventrella, Vittoria

    2016-01-01

    Through a multiple approach, the present study on the mitochondrial membranes from mussel gills and swine heart combines some biochemical information on fatty acid composition, sterol pattern, and temperature dependence of the F1FO-ATPase activity (EC 3.6.3.14.) with fluorescence data on mitochondrial membranes and on liposomes obtained from lipid extracts of mitochondria. The physical state of mussel gills and swine heart was investigated by Laurdan steady state fluorescence. Quite surprisingly, the similar temperature dependence of the F1FO complex, illustrated as Arrhenius plot which in both mitochondria exhibits the same discontinuity at approximately 21°C and overlapping activation energies above and below the discontinuity, is apparently compatible with a different composition and physical state of mitochondrial membranes. Accordingly, mussel membranes contain highly unsaturated fatty acids, abundant sterols, including phytosterols, while mammalian membranes only contain cholesterol and in prevalence shorter and less unsaturated fatty acids, leading to a lower membrane unsaturation with respect to mussel mitochondria. As suggested by fluorescence data, the likely formation of peculiar microdomains interacting with the membrane-bound enzyme complex in mussel mitochondria could produce an environment which somehow approaches the physical state of mammalian mitochondrial membranes. Thus, as an adaptive strategy, the interaction between sterols, highly unsaturated phospholipids and proteins in mussel gill mitochondria could allow the F1FO-ATPase activity to maintain the same activation energy as the mammalian enzyme.

  1. 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.

  2. Separate fusion of outer and inner mitochondrial membranes

    OpenAIRE

    Malka, Florence; Guillery, Olwenn; Cifuentes-Diaz, Carmen; Guillou, Emmanuelle; Belenguer, Pascale; Lombès, Anne; Rojo, Manuel

    2005-01-01

    Mitochondria are enveloped by two closely apposed boundary membranes with different properties and functions. It is known that they undergo fusion and fission, but it has remained unclear whether outer and inner membranes fuse simultaneously, coordinately or separately. We set up assays for the study of inner and outer membrane fusion in living human cells. Inner membrane fusion was more sensitive than outer membrane fusion to inhibition of glycolysis. Fusion of the inner membrane, but not of...

  3. Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS.

    Science.gov (United States)

    Gubbens, Jacob; Slijper, Monique; de Kruijff, Ben; de Kroon, Anton I P M

    2008-12-01

    Previously, a 2D gel electrophoresis approach was developed for the Escherichia coli inner membrane, which detects membrane protein complexes that are stable in sodium dodecyl sulfate (SDS) at room temperature, and dissociate under the influence of trifluoroethanol [R. E. Spelbrink et al., J. Biol. Chem. 280 (2005), 28742-8]. Here, the method was applied to the evolutionarily related mitochondrial inner membrane that was isolated from the yeast Saccharomyces cerevisiae. Surprisingly, only very few proteins were found to be dissociated by trifluoroethanol of which Lpd1p, a component of multiple protein complexes localized in the mitochondrial matrix, is the most prominent. Usage of either milder or more stringent conditions did not yield any additional proteins that were released by fluorinated alcohols. This strongly suggests that membrane protein complexes in yeast are less stable in SDS solution than their E. coli counterparts, which might be due to the overall reduced hydrophobicity of mitochondrial transmembrane proteins. PMID:18817900

  4. Stabilization of mitochondrial membrane potential prevents doxorubicin-induced cardiotoxicity in isolated rat heart

    International Nuclear Information System (INIS)

    The present study was undertaken to examine the effects of doxorubicin on left ventricular function and cellular energy state in intact isolated hearts, and, to test whether inhibition of mitochondrial membrane potential dissipation would prevent doxorubicin-induced mitochondrial and myocardial dysfunction. Myocardial contractile performance and mitochondrial respiration were evaluated by left ventricular tension and its first derivatives and cardiac fiber respirometry, respectively. NADH levels, mitochondrial membrane potential and glucose uptake were monitored non-invasively via epicardial imaging of the left ventricular wall of Langendorff-perfused rat hearts. Heart performance was reduced in a time-dependent manner in isolated rat hearts perfused with Krebs-Henseleit solution containing 1 μM doxorubicin. Compared with controls, doxorubicin induced acute myocardial dysfunction (dF/dtmax of 105 ± 8 mN/s in control hearts vs. 49 ± 7 mN/s in doxorubicin-treated hearts; *p < 0.05). In cardiac fibers prepared from perfused hearts, doxorubicin induced depression of mitochondrial respiration (respiratory control ratio of 4.0 ± 0.2 in control hearts vs. 2.2 ± 0.2 in doxorubicin-treated hearts; *p < 0.05) and cytochrome c oxidase kinetic activity (24 ± 1 μM cytochrome c/min/mg in control hearts vs. 14 ± 3 μM cytochrome c/min/mg in doxorubicin-treated hearts; *p < 0.05). Acute cardiotoxicity induced by doxorubicin was accompanied by NADH redox state, mitochondrial membrane potential, and glucose uptake reduction. Inhibition of mitochondrial permeability transition pore opening by cyclosporine A largely prevented mitochondrial membrane potential dissipation, cardiac energy state and dysfunction. These results suggest that in intact hearts an impairment of mitochondrial metabolism is involved in the development of doxorubicin cardiotoxicity.

  5. Biophysical significance of the inner mitochondrial membrane structure on the electrochemical potential of mitochondria

    Science.gov (United States)

    Song, Dong Hoon; Park, Jonghyun; Maurer, Laura L.; Lu, Wei; Philbert, Martin A.; Sastry, Ann Marie

    2013-12-01

    The available literature supports the hypothesis that the morphology of the inner mitochondrial membrane is regulated by different energy states, that the three-dimensional morphology of cristae is dynamic, and that both are related to biochemical function. Examination of the correlation between the inner mitochondrial membrane (IMM) structure and mitochondrial energetic function is critical to an understanding of the links between mesoscale morphology and function in progressive mitochondrial dysfunction such as aging, neurodegeneration, and disease. To investigate this relationship, we develop a model to examine the effects of three-dimensional IMM morphology on the electrochemical potential of mitochondria. The two-dimensional axisymmetric finite element method is used to simulate mitochondrial electric potential and proton concentration distribution. This simulation model demonstrates that the proton motive force (Δp) produced on the membranes of cristae can be higher than that on the inner boundary membrane. The model also shows that high proton concentration in cristae can be induced by the morphology-dependent electric potential gradient along the outer side of the IMM. Furthermore, simulation results show that a high Δp is induced by the large surface-to-volume ratio of an individual crista, whereas a high capacity for ATP synthesis can primarily be achieved by increasing the surface area of an individual crista. The mathematical model presented here provides compelling support for the idea that morphology at the mesoscale is a significant driver of mitochondrial function.

  6. Translocation of chicken heart apocytochrome c and its mutants (C17S, H18D) across mitochondrial membrane

    Institute of Scientific and Technical Information of China (English)

    朱勇; 韩学海; 杨福愉

    1999-01-01

    Cytochrome c is a component of mitochondrial respiratory chain, located at the outer side of mitochondrial inner membrane. Its precursor, apocytochrome c, is encoded by a nuclear gene, synthesized on cytoplasmic ribosomes, and posttranslationally imported into mitochondria, but apocytochrome c is unique in the translocation compared with most mitochondrial proteins. It does not carry a cleavable amino terminal targeting sequence; no proteinous receptor on the mitochondrial outer membrane is identified for its import and its translocation does not compete with other preproteins for translocation machinery in the outer membrane. Besides, neither ATP nor membrane potential is required for its translocation across mitochonctria.

  7. Knockdown of cytosolic glutaredoxin 1 leads to loss of mitochondrial membrane potential: implication in neurodegenerative diseases.

    Directory of Open Access Journals (Sweden)

    Uzma Saeed

    Full Text Available Mitochondrial dysfunction including that caused by oxidative stress has been implicated in the pathogenesis of neurodegenerative diseases. Glutaredoxin 1 (Grx1, a cytosolic thiol disulfide oxido-reductase, reduces glutathionylated proteins to protein thiols and helps maintain redox status of proteins during oxidative stress. Grx1 downregulation aggravates mitochondrial dysfunction in animal models of neurodegenerative diseases, such as Parkinson's and motor neuron disease. We examined the mechanism underlying the regulation of mitochondrial function by Grx1. Downregulation of Grx1 by shRNA results in loss of mitochondrial membrane potential (MMP, which is prevented by the thiol antioxidant, alpha-lipoic acid, or by cyclosporine A, an inhibitor of mitochondrial permeability transition. The thiol groups of voltage dependent anion channel (VDAC, an outer membrane protein in mitochondria but not adenosine nucleotide translocase (ANT, an inner membrane protein, are oxidized when Grx1 is downregulated. We then examined the effect of beta-N-oxalyl amino-L-alanine (L-BOAA, an excitatory amino acid implicated in neurolathyrism (a type of motor neuron disease, that causes mitochondrial dysfunction. Exposure of cells to L-BOAA resulted in loss of MMP, which was prevented by overexpression of Grx1. Grx1 expression is regulated by estrogen in the CNS and treatment of SH-SY5Y cells with estrogen upregulated Grx1 and protected from L-BOAA mediated MMP loss. Our studies demonstrate that Grx1, a cytosolic oxido-reductase, helps maintain mitochondrial integrity and prevents MMP loss caused by oxidative insult. Further, downregulation of Grx1 leads to mitochondrial dysfunction through oxidative modification of the outer membrane protein, VDAC, providing support for the critical role of Grx1 in maintenance of MMP.

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

    OpenAIRE

    L. S. L. S. Reis; A.A. Ramos; A.S. Camargos; E. Oba

    2016-01-01

    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 e...

  9. Protective role of mitochondrial K-ATP channel and mitochondrial membrane transport pore in rat kidney ischemic postconditioning

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wei-liang; ZHAO Yan-li; LIU Xiao-ming; CHEN Jing; ZHANG Dong

    2011-01-01

    Background Previous studies suggested that mechanical intervention during early reperfusion, or ischemia postconditioning (Ipo), could protect kidneys against renal ischemia reperfusion injury (RIRI). However, the mechanisms responsible for this protection remain unclear. This study therefore investigated the protection afforded by Ipo in rat kidneys in vivo, and the roles of mitochondrial KATP channels (mitOKATP) and mitochondrial permeability transition pores (MPTPs), by inhibiting mitOKATP with 5-hydroxydecanoate (5-HD), and by directly detecting open MPTPs using calcein-AM and CoCl2.Methods Thirty-five male Sprague-Dawley rats were randomly assigned to sham-operation (S), ischemia-reperfusion (I/R),Ipo, ischemia reperfusion with 5-HD (I/R+5-HD), or Ipo with 5-HD (Ipo +5-HD) groups. Rats in each group were sacrificed after 6 hours of reperfusion by heart exsanguination or cervical dislocation under anesthesia. RIRI was assessed by determination of creatinine and blood urea nitrogen (BUN), and by examination of histologic sections. The roles of mitoKATP and MPTP were investigated by analyzing fluorescence intensities of mitochondria, mitochondrial membrane potential,intracellular reactive oxygen species (ROS) and intracellular calcium, using appropriate fluorescent markers. The relationship between apoptosis and RIRI was assessed by determining the apoptotic index (Al) of kidney tubular epithelial cells.Results The RIRI model was shown to be successful. Significantly higher levels of creatinine and BUN, and abnormal pathology of histologic sections, were observed in group I/R, compared with group S. 5-HD eliminated the renoprotective effects of Ipo. Mitochondrial and mitochondrial membrane potential fluorescence intensities increased, and intracellular calcium, ROS fluorescence intensities and AI decreased in group Ipo, compared with group I/R. However, mitochondrial and mitochondrial membrane potential fluorescence intensities decreased, and intracellular

  10. 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

  11. 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.

  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. Localization of HPV-18 E2 at Mitochondrial Membranes Induces ROS Release and Modulates Host Cell Metabolism

    OpenAIRE

    Deborah Lai; Chye Ling Tan; Jayantha Gunaratne; Ling Shih Quek; Wenlong Nei; Françoise Thierry; Sophie Bellanger

    2013-01-01

    Papillomavirus E2 proteins are predominantly retained in the nuclei of infected cells, but oncogenic (high-risk) HPV-18 and 16 E2 can shuttle between the host nucleus and cytoplasm. We show here that cytoplasmic HPV-18 E2 localizes to mitochondrial membranes, and independent mass spectrometry analyses of the E2 interactome revealed association to the inner mitochondrial membrane including components of the respiratory chain. Mitochondrial E2 association modifies the cristae morphology when an...

  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. 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

  16. 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...

  17. Outer membrane VDAC1 controls permeability transition of the inner mitochondrial membrane in cellulo during stress-induced apoptosis

    Institute of Scientific and Technical Information of China (English)

    Flora Tomasello; Angela Messina; Lydia Lartigue; Laura Schembri; Chantal Medina; Simona Reina; Didier Thorava; Marc Crouzet; Francois Ichas; Vito De Pinto; Francesca De Giorgi

    2009-01-01

    Voltage-dependent anion channel (VDAC)l is the main channel of the mitochondrial outer membrane (MOM) and it has been proposed to be part of the permeability transition pore (PTP), a putative multiprotein complex candidate agent of the mitochondrial permeability transition (MPT). Working at the single live cell level, we found that over-expression of VDAC1 triggers MPT at the mitochondrial inner membrane (MIM). Conversely, silencing VDAC1 ex-pression results in the inhibition of MPT caused by selenite-induced oxidative stress. This MOM-M1M crosstalk was modulated by Cyclosporin A and mitochondrial Cyclophilin D, but not by Bcl-2 and Bcl-XL, indicative of PTP opera-tion. VDAC1-dependent MPT engages a positive feedback loop involving reactive oxygen species and p38-MAPK, and secondarily triggers a canonical apoptotic response including Bax activation, cytochrome c release and caspase 3 activation. Our data thus support a model of the PTP complex involving VDAC1 at the MOM, and indicate that VDAC1-dependent MPT is an upstream mechanism playing a causal role in oxidative stress-induced apoptosis.

  18. Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis.

    Directory of Open Access Journals (Sweden)

    Vera Kozjak-Pavlovic

    2009-10-01

    Full Text Available The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (DeltaPsi(m. Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of DeltaPsi(m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce DeltaPsi(m loss and apoptosis, demonstrating that dissipation of DeltaPsi(m is a requirement for cell death caused by neisserial infection.

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

    Science.gov (United States)

    Rosales-Corral, Sergio A.; Lopez-Armas, Gabriela; Cruz-Ramos, Jose; Melnikov, Valery G.; Tan, Dun-Xian; Manchester, Lucien C.; Munoz, Ruben; Reiter, Russel J.

    2012-01-01

    Alzheimer pathogenesis involves mitochondrial dysfunction, which is closely related to amyloid-β (Aβ) 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-β (Aβ). The purpose was to determine how Aβ 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 Aβ 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 β was injected, favoring an endogenous anti-inflammatory pathway. PMID:22666620

  20. Simultaneous monitoring of ionophore- and inhibitor-mediated plasma and mitochondrial membrane potential changes in cultured neurons.

    Science.gov (United States)

    Nicholls, David G

    2006-05-26

    Although natural and synthetic ionophores are widely exploited in cell studies, for example, to influence cytoplasmic free calcium concentrations and to depolarize in situ mitochondria, their inherent lack of membrane selectivity means that they affect the ion permeability of both plasma and mitochondrial membranes. A similar ambiguity affects the interpretation of signals from fluorescent membrane-permeant cations (usually termed "mitochondrial membrane potential indicators"), because the accumulation of these probes is influenced by both plasma and mitochondrial membrane potentials. To resolve some of these problems a technique is developed to allow simultaneous monitoring of plasma and mitochondrial membrane potentials at single-cell resolution using a cationic and anionic fluorescent probe. A computer program is described that transforms the fluorescence changes into dynamic estimates of changes in plasma and mitochondrial potentials. Exploiting this technique, primary cultures of rat cerebellar granule neurons display a concentration-dependent response to ionomycin: low concentrations mimic nigericin by hyperpolarizing the mitochondria while slowly depolarizing the plasma membrane and maintaining a stable elevated cytoplasmic calcium. Higher ionomycin concentrations induce a stochastic failure of calcium homeostasis that precedes both mitochondrial depolarization and an enhanced rate of plasma membrane depolarization. In addition, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone only selectively depolarizes mitochondria at submicromolar concentrations. ATP synthase reversal following respiratory chain inhibition depolarizes the mitochondria by 26 mV. PMID:16551630

  1. 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.

  2. 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...... reductions in inner mitochondrial membrane leak and increased efficiency of mitochondria. This change in mitochondrial physiology could contribute to the weight gain seen with antihyperglycaemic treatment....

  3. Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release

    Directory of Open Access Journals (Sweden)

    Seong‑Woon Yu

    2009-11-01

    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.

  4. Evidence for several cysteine transport mechanisms in the mitochondrial membranes of Arabidopsis thaliana.

    Science.gov (United States)

    Lee, Chun Pong; Wirtz, Markus; Hell, Rüdiger

    2014-01-01

    Cysteine is essential for many mitochondrial processes in plants, including translation, iron-sulfur cluster biogenesis and cyanide detoxification. Its biosynthesis is carried out by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which can be found in the cytosol, plastids and mitochondria. Mutants lacking one compartment-specific OAS-TL isoform show viable phenotypes, leading to the hypothesis that the organellar membranes are permeable to substrates and products of the cysteine biosynthetic pathway. In this report, we show that exogenouslly supplied [(35)S]cysteine accumulates in the mitochondrial fraction and is taken up into isolated mitochondria for in organello protein synthesis. Analysis of cysteine uptake by isolated mitochondria and mitoplasts indicates that cysteine is transported by multiple facilitated mechanisms that operate in a concentration gradient-dependent manner. In addition, cysteine uptake is dependent mainly on the ΔpH across the inner membrane. The rates of mitochondrial cysteine transport can be mildly altered by specific metabolites in the cyanide detoxification-linked sulfide oxidation, but not by most substrates and products of the cysteine biosynthetic pathway. Based on these results, we propose that the transport of cysteine plays a pivotal role in regulating cellular cysteine biosynthesis as well as modulating the availability of sulfur for mitochondrial metabolism.

  5. Mitochondrial OXA Translocase Plays a Major Role in Biogenesis of Inner-Membrane Proteins.

    Science.gov (United States)

    Stiller, Sebastian B; Höpker, Jan; Oeljeklaus, Silke; Schütze, Conny; Schrempp, Sandra G; Vent-Schmidt, Jens; Horvath, Susanne E; Frazier, Ann E; Gebert, Natalia; van der Laan, Martin; Bohnert, Maria; Warscheid, Bettina; Pfanner, Nikolaus; Wiedemann, Nils

    2016-05-10

    The mitochondrial inner membrane harbors three protein translocases. Presequence translocase and carrier translocase are essential for importing nuclear-encoded proteins. The oxidase assembly (OXA) translocase is required for exporting mitochondrial-encoded proteins; however, different views exist about its relevance for nuclear-encoded proteins. We report that OXA plays a dual role in the biogenesis of nuclear-encoded mitochondrial proteins. First, a systematic analysis of OXA-deficient mitochondria led to an unexpected expansion of the spectrum of OXA substrates imported via the presequence pathway. Second, biogenesis of numerous metabolite carriers depends on OXA, although they are not imported by the presequence pathway. We show that OXA is crucial for the biogenesis of the Tim18-Sdh3 module of the carrier translocase. The export translocase OXA is thus required for the import of metabolite carriers by promoting assembly of the carrier translocase. We conclude that OXA is of central importance for the biogenesis of the mitochondrial inner membrane.

  6. The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

    International Nuclear Information System (INIS)

    Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 μM) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca2+ efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP+ transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. - Graphical abstract: Guttiferone-A permeabilizes mitochondrial membrane and induces cancer cell death Display Omitted Highlights: → We addressed the involvement of mitochondria in guttiferone (GA) toxicity toward cancer cells. → GA promoted membrane permeabilization, membrane potential dissipation, NAD(P)H depletion, ROS accumulation and ATP depletion. → These actions could be

  7. Feline immunodeficiency virus decreases cell-cell communication and mitochondrial membrane potential.

    OpenAIRE

    Danave, I R; Tiffany-Castiglioni, E; Zenger, E; Barhoumi, R.; Burghardt, R C; Collisson, E W

    1994-01-01

    The in vitro effects of viral replication on mitochondrial membrane potential (MMP) and gap junctional intercellular communication (GJIC) were evaluated as two parameters of potential cellular injury. Two distinct cell types were infected with the Petaluma strain of feline immunodeficiency virus (FIV). Primary astroglia supported acute FIV infection, resulting in syncytia within 3 days of infection, whereas immortalized Crandell feline kidney (CRFK) cells of epithelial origin supported persis...

  8. 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.

  9. A monoclonal antibody marker for the exclusion-zone filaments of Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Decossas Marion

    2008-07-01

    Full Text Available Abstract Background Trypanosoma brucei is a haemoflagellate pathogen of man, wild animals and domesticated livestock in central and southern Africa. In all life cycle stages this parasite has a single mitochondrion that contains a uniquely organised genome that is condensed into a flat disk-like structure called the kinetoplast. The kinetoplast is essential for insect form procyclic cells and therefore is a potential drug target. The kinetoplast is unique in nature because it consists of novel structural proteins and thousands of circular, interlocking, DNA molecules (kDNA. Secondly, kDNA replication is critically timed to coincide with nuclear S phase and new flagellum biogenesis. Thirdly, the kinetoplast is physically attached to the flagellum basal bodies via a structure called the tripartite attachment complex (TAC. The TAC consists of unilateral filaments (within the mitochondrion matrix, differentiated mitochondrial membranes and exclusion-zone filaments that extend from the distal end of the basal bodies. To date only one protein, p166, has been identified to be a component of the TAC. Results In the work presented here we provide data based on a novel EM technique developed to label and characterise cytoskeleton structures in permeabilised cells without extraction of mitochondrion membranes. We use this protocol to provide data on a new monoclonal antibody reagent (Mab 22 and illustrate the precise localisation of basal body-mitochondrial linker proteins. Mab 22 binds to these linker proteins (exclusion-zone filaments and provides a new tool for the characterisation of cytoskeleton mediated kinetoplast segregation. Conclusion The antigen(s recognised by Mab 22 are cytoskeletal, insensitive to extraction by high concentrations of non-ionic detergent, extend from the proximal region of basal bodies and bind to the outer mitochondrial membrane. This protein(s is the first component of the TAC exclusion-zone fibres to be identified. Mab 22

  10. A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria.

    Science.gov (United States)

    Hoppins, Suzanne; Collins, Sean R; Cassidy-Stone, Ann; Hummel, Eric; Devay, Rachel M; Lackner, Laura L; Westermann, Benedikt; Schuldiner, Maya; Weissman, Jonathan S; Nunnari, Jodi

    2011-10-17

    To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane-associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria.

  11. The major transcripts of the kinetoplast Trypanosoma brucei are very small ribosomal RNA's.

    NARCIS (Netherlands)

    I.C. Eperon; J.W.G. Janssen; J.H.J. Hoeijmakers (Jan); P. Borst (Piet)

    1983-01-01

    textabstractThe nucleotide sequence has been determined of a 2.2 kb segment of kinetoplast DNA, which encodes the major mitochondrial transcripts (12S and 9S) of Trypanosoma brucei. The sequence shows that the 12S RNA is a large subunit rRNA, although sufficiently unusual for resistance to chloramph

  12. VDAC electronics: 1. VDAC-hexo(gluco)kinase generator of the mitochondrial outer membrane potential.

    Science.gov (United States)

    Lemeshko, Victor V

    2014-05-01

    The simplest mechanism of the generation of the mitochondrial outer membrane potential (OMP) by the VDAC (voltage-dependent anion channel)-hexokinase complex (VHC), suggested earlier, and by the VDAC-glucokinase complex (VGC), was computationally analyzed. Even at less than 4% of VDACs bound to hexokinase, the calculated OMP is high enough to trigger the electrical closure of VDACs beyond the complexes at threshold concentrations of glucose. These results confirmed our previous hypothesis that the Warburg effect is caused by the electrical closure of VDACs, leading to global restriction of the outer membrane permeability coupled to aerobic glycolysis. The model showed that the inhibition of the conductance and/or an increase in the voltage sensitivity of a relatively small fraction of VDACs by factors like tubulin potentiate the electrical closure of the remaining free VDACs. The extrusion of calcium ions from the mitochondrial intermembrane space by the generated OMP, positive inside, might increase cancer cell resistance to death. Within the VGC model, the known effect of induction of ATP release from mitochondria by accumulated glucose-6-phosphate in pancreatic beta cells might result not only of the known effect of GK dissociation from the VDAC-GK complex, but also of a decrease in the free energy of glucokinase reaction, leading to the OMP decrease and VDAC opening. We suggest that the VDAC-mediated electrical control of the mitochondrial outer membrane permeability, dependent on metabolic conditions, is a fundamental physiological mechanism of global regulation of mitochondrial functions and of cell death. PMID:24412217

  13. Localization of HPV-18 E2 at mitochondrial membranes induces ROS release and modulates host cell metabolism.

    Directory of Open Access Journals (Sweden)

    Deborah Lai

    Full Text Available Papillomavirus E2 proteins are predominantly retained in the nuclei of infected cells, but oncogenic (high-risk HPV-18 and 16 E2 can shuttle between the host nucleus and cytoplasm. We show here that cytoplasmic HPV-18 E2 localizes to mitochondrial membranes, and independent mass spectrometry analyses of the E2 interactome revealed association to the inner mitochondrial membrane including components of the respiratory chain. Mitochondrial E2 association modifies the cristae morphology when analyzed by electron microscopy and increases production of mitochondrial ROS. This ROS release does not induce apoptosis, but instead correlates with stabilization of HIF-1α and increased glycolysis. These mitochondrial functions are not shared by the non-oncogenic (low-risk HPV-6 E2 protein, suggesting that modification of cellular metabolism by high-risk HPV E2 proteins could play a role in carcinogenesis by inducing the Warburg effect.

  14. The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax.

    Science.gov (United States)

    Lidman, Martin; Pokorná, Šárka; Dingeldein, Artur P G; Sparrman, Tobias; Wallgren, Marcus; Šachl, Radek; Hof, Martin; Gröbner, Gerhard

    2016-06-01

    Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface. PMID:26947183

  15. Vimentin is involved in regulation of mitochondrial motility and membrane potential by Rac1

    Directory of Open Access Journals (Sweden)

    Elena A. Matveeva

    2015-10-01

    Full Text Available In this study we show that binding of mitochondria to vimentin intermediate filaments (VIF is regulated by GTPase Rac1. The activation of Rac1 leads to a redoubling of mitochondrial motility in murine fibroblasts. Using double-mutants Rac1(G12V, F37L and Rac1(G12V, Y40H that are capable to activate different effectors of Rac1, we show that mitochondrial movements are regulated through PAK1 kinase. The involvement of PAK1 kinase is also confirmed by the fact that expression of its auto inhibitory domain (PID blocks the effect of activated Rac1 on mitochondrial motility. The observed effect of Rac1 and PAK1 kinase on mitochondria depends on phosphorylation of the Ser-55 of vimentin. Besides the effect on motility Rac1 activation also decreases the mitochondrial membrane potential (MMP which is detected by ∼20% drop of the fluorescence intensity of mitochondria stained with the potential sensitive dye TMRM. One of important consequences of the discovered regulation of MMP by Rac1 and PAK1 is a spatial differentiation of mitochondria in polarized fibroblasts: at the front of the cell they are less energized (by ∼25% than at the rear part.

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

    OpenAIRE

    Sutapa Mukherjee; Luna Samanta; Anita Roy; Shravani Bhanja; 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 ...

  17. Ocean acidification impacts on sperm mitochondrial membrane potential bring sperm swimming behaviour near its tipping point.

    Science.gov (United States)

    Schlegel, Peter; Binet, Monique T; Havenhand, Jonathan N; Doyle, Christopher J; Williamson, Jane E

    2015-04-01

    Broadcast spawning marine invertebrates are susceptible to environmental stressors such as climate change, as their reproduction depends on the successful meeting and fertilization of gametes in the water column. Under near-future scenarios of ocean acidification, the swimming behaviour of marine invertebrate sperm is altered. We tested whether this was due to changes in sperm mitochondrial activity by investigating the effects of ocean acidification on sperm metabolism and swimming behaviour in the sea urchin Centrostephanus rodgersii. We used a fluorescent molecular probe (JC-1) and flow cytometry to visualize mitochondrial activity (measured as change in mitochondrial membrane potential, MMP). Sperm MMP was significantly reduced in ΔpH -0.3 (35% reduction) and ΔpH -0.5 (48% reduction) treatments, whereas sperm swimming behaviour was less sensitive with only slight changes (up to 11% decrease) observed overall. There was significant inter-individual variability in responses of sperm swimming behaviour and MMP to acidified seawater. We suggest it is likely that sperm exposed to these changes in pH are close to their tipping point in terms of physiological tolerance to acidity. Importantly, substantial inter-individual variation in responses of sperm swimming to ocean acidification may increase the scope for selection of resilient phenotypes, which, if heritable, could provide a basis for adaptation to future ocean acidification.

  18. 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.

  19. Calpeptin, not calpain, directly inhibits an ion channel of the inner mitochondrial membrane.

    Science.gov (United States)

    Derksen, Maria; Vorwerk, Christian; Siemen, Detlef

    2016-05-01

    The permeability transition pore (PTP) of inner mitochondrial membranes is a large conductance pathway for ions up to 1500 Da which opening is responsible for ion equilibration and loss of membrane potential in apoptosis and thus in several neurodegenerative diseases. The PTP can be regulated by the Ca(2+)-activated mitochondrial K channel (BK). Calpains are Ca(2+)-activated cystein proteases; calpeptin is an inhibitor of calpains. We wondered whether calpain or calpeptin can modulate activity of PTP or BK. Patch clamp experiments were performed on mitoplasts of rat liver (PTP) and of an astrocytoma cell line (BK). Channel-independent open probability (P o) was determined (PTP) and, taking into account the number of open levels, NPo by single channel analysis (BK). We find that PTP in the presence of Ca(2+) (200 μM) is uninfluenced by calpain (13 nM) and shows insignificant decrease by the calpain inhibitor calpeptin (1 μM). The NPo of the BK is insensitive to calpain (54 nM), too. However, it is significantly and reversibly inhibited by the calpain inhibitor calpeptin (IC50 = 42 μM). The results agree with calpeptin-induced activation of the PTP via inhibition of the BK. Screening experiments with respirometry show calpeptin effects, fitting to inhibition of the BK by calpeptin, and strong inhibition of state 3 respiration. PMID:26108743

  20. 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. PMID:17719569

  1. Changes of proton transportation across the inner mitochondrial membrane and H+-ATPase in endotoxic shock rats

    Institute of Scientific and Technical Information of China (English)

    LU Song-min 陆松敏; SONG Shuang-ming 宋双明; LIU Jian-cang 刘建仓; YANG He-ming 杨鹤鸣; LI Ping 李萍; WANG Zheng-guo 王正国

    2003-01-01

    Objective: To investigate the changes of proton transportation across the inner mitochondrial membrane (IMM) and H+-ATPase of hepatocytes in endotoxic shock rats.Methods: Endotoxin from E.Coil of 5.0 mg/kg or saline of 1 ml/kg was injected into the femoral vein.The rats were sacrificed pre-injection and 1, 3, 5, 8 hours after injection, and plasma and liver tissue samples were collected respectively.The liver tissue samples were used for preparation of mitochondria and submitochondrial particles (SMPs).The proton-translocation of SMPs and H+-ATPase, phospholipase A2 (PLA2) activities and malondialdehyde (MDA) content, membrane fluidities of different level of mitochondria membrane and plasma MDA content were assayed.Results: (1) Five hours after E.Coli.O111B4 injection, the maximum fluorescence quenching ACMA after adding ATP, nicotinamide adenin dinucleoacid hydrogen (NADH), and the succinate were significantly decreased (P<0.05).The time of maximum fluorescent quenching and the half time of fluorescent quenching were significantly prolonged (P<0.01), especially when NADH was used as a substrate.(2) The mitochondrial H+-ATPase activity was significantly increased at early stage of endotoxic shock (P<0.05), and significantly decreased at late stage of endotoxic shock (P<0.01).(3) The mitochondrial membrane bound PLA2 activity, plasmal and mitochondrial MDA content were significantly increased and succinate dehydrogenase (SDH) activity of mitochondria decreased markedly in endotoxic shock rats (P<0.05).(4) The mitochondrial membrane fluidity of different lipid regions was decreased, especially in the head of phospholipid.Conclusions: Proton transportation across IMM and mitochondrial H+-ATPase activity are significantly decreased in endotoxic shock.

  2. Wild chimpanzees are infected by Trypanosoma brucei.

    Science.gov (United States)

    Jirků, Milan; Votýpka, Jan; Petrželková, Klára J; Jirků-Pomajbíková, Kateřina; Kriegová, Eva; Vodička, Roman; Lankester, Felix; Leendertz, Siv Aina J; Wittig, Roman M; Boesch, Christophe; Modrý, David; Ayala, Francisco J; Leendertz, Fabian H; Lukeš, Julius

    2015-12-01

    Although wild chimpanzees and other African great apes live in regions endemic for African sleeping sickness, very little is known about their trypanosome infections, mainly due to major difficulties in obtaining their blood samples. In present work, we established a diagnostic ITS1-based PCR assay that allows detection of the DNA of all four Trypanosoma brucei subspecies (Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense, Trypanosoma brucei gambiense, and Trypanosoma brucei evansi) in feces of experimentally infected mice. Next, using this assay we revealed the presence of trypanosomes in the fecal samples of wild chimpanzees and this finding was further supported by results obtained using a set of primate tissue samples. Phylogenetic analysis of the ITS1 region showed that the majority of obtained sequences fell into the robust T. brucei group, providing strong evidence that these infections were caused by T. b. rhodesiense and/or T. b. gambiense. The optimized technique of trypanosome detection in feces will improve our knowledge about the epidemiology of trypanosomes in primates and possibly also other endangered mammals, from which blood and tissue samples cannot be obtained. 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. PMID:26110113

  3. 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.

  4. Therapeutic Modulation of Apoptosis: Targeting the BCL-2 Family at the Interface of the Mitochondrial Membrane

    Science.gov (United States)

    Nemec, Kathleen N.

    2008-01-01

    A vast portion of human disease results when the process of apoptosis is defective. Disorders resulting from inappropriate cell death range from autoimmune and neurodegenerative conditions to heart disease. Conversely, prevention of apoptosis is the hallmark of cancer and confounds the efficacy of cancer therapeutics. In the search for optimal targets that would enable the control of apoptosis, members of the BCL-2 family of anti- and pro-apoptotic factors have figured prominently. Development of BCL-2 antisense approaches, small molecules, and BH3 peptidomimetics has met with both success and failure. Success-because BCL-2 proteins play essential roles in apoptosis. Failure-because single targets for drug development have limited scope. By examining the activity of the BCL-2 proteins in relation to the mitochondrial landscape and drawing attention to the significant mitochondrial membrane alterations that ensue during apoptosis, we demonstrate the need for a broader based multi-disciplinary approach for the design of novel apoptosis-modulating compounds in the treatment of human disease. PMID:18972587

  5. Steady-state coupling of four membrane systems in mitochondrial oxidative phosphorylation.

    Science.gov (United States)

    Hill, T L

    1979-05-01

    According to Alexandre, Reynafarje, and Lehninger, four different membrane systems are involved, with definite stoichiometry, in the mitochondrial synthesis of ATP by electron transport, via proton transport. We adopt this model and pursue some of its thermodynamic consequences. At steady state, each of the four systems must have the same flux J through the membrane and the overall thermodynamic force X for oxidative phosphorylation is the sum of the four separate forces. From these properties, using an empirical linear flux-force relation for each system, it is easy to obtain J as a function of X. In turn, X depends on the inside [NAD+]/[NADH] and the outside [ATP]/[ADP][Pi] quotients (and on the pH inside). Thus, J is related to these quotients. The relationship we derive is similar to that described by Erecińska and Wilson, as deduced from a quite different model of oxidative phosphorylation. Proton transport is involved explicitly in three of the four systems of the present model. However, because of the steady-state stoichiometric coupling of the four systems, proton transport does not appear in the overall reaction. On the other hand, Erecińska and Wilson use, in their model, a direct connection between electron transport and ATP synthesis. The present paper demonstrates that J can be related to the quotients mentioned above without this direct connection. PMID:287064

  6. Mitochondrial Ca(2+) uniporter (MCU)-dependent and MCU-independent Ca(2+) channels coexist in the inner mitochondrial membrane.

    Science.gov (United States)

    Bondarenko, Alexander I; Jean-Quartier, Claire; Parichatikanond, Warisara; Alam, Muhammad Rizwan; Waldeck-Weiermair, Markus; Malli, Roland; Graier, Wolfgang F

    2014-07-01

    A protein referred to as CCDC109A and then renamed to mitochondrial calcium uniporter (MCU) has recently been shown to accomplish mitochondrial Ca(2+) uptake in different cell types. In this study, we investigated whole-mitoplast inward cation currents and single Ca(2+) channel activities in mitoplasts prepared from stable MCU knockdown HeLa cells using the patch-clamp technique. In whole-mitoplast configuration, diminution of MCU considerably reduced inward Ca(2+) and Na(+) currents. This was accompanied by a decrease in occurrence of single channel activity of the intermediate conductance mitochondrial Ca(2+) current (i-MCC). However, ablation of MCU yielded a compensatory 2.3-fold elevation in the occurrence of the extra large conductance mitochondrial Ca(2+) current (xl-MCC), while the occurrence of bursting currents (b-MCC) remained unaltered. These data reveal i-MCC as MCU-dependent current while xl-MCC and b-MCC seem to be rather MCU-independent, thus, pointing to the engagement of at least two molecularly distinct mitochondrial Ca(2+) channels.

  7. 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...

  8. Mapping of VSG similarities in Trypanosoma brucei

    OpenAIRE

    Weirather, Jason L.; Wilson, Mary E; Donelson, John E.

    2011-01-01

    The protozoan parasite Trypanosoma brucei switches its variant surface glycoprotein (VSG) to subvert its mammalian hosts’ immune responses. The T. brucei genome contains as many as 1600 VSG genes (VSGs), but most are silent noncoding pseudogenes. Only one functional VSG, located in a telomere-linked expression site, is transcribed at a time. Silent VSGs are copied into a VSG expression site through gene conversion. Truncated gene conversion events can generate new mosaic VSGs with segments of...

  9. What happens when Trypanosoma brucei leaves Africa

    OpenAIRE

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

    2008-01-01

    Julius Lukeš 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, w...

  10. The Novel Tail-anchored Membrane Protein Mff Controls Mitochondrial and Peroxisomal Fission in Mammalian Cells

    OpenAIRE

    Gandre-Babbe, Shilpa; van der Bliek, Alexander M.

    2008-01-01

    Few components of the mitochondrial fission machinery are known, even though mitochondrial fission is a complex process of vital importance for cell growth and survival. Here, we describe a novel protein that controls mitochondrial fission. This protein was identified in a small interfering RNA (siRNA) screen using Drosophila cells. The human homologue of this protein was named Mitochondrial fission factor (Mff). Mitochondria of cells transfected with Mff siRNA form a closed network similar t...

  11. Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways

    OpenAIRE

    Rival, Thomas; Macchi, Marc; Arnauné-Pelloquin, Laetitia; Poidevin, Mickael; Maillet, Frédéric; Richard, Fabrice; Fatmi, Ahmed; Belenguer, Pascale; Royet, Julien

    2011-01-01

    This report identifies Drosophila PMI and its human ortholog TMEM11 as novel regulators of mitochondrial morphogenesis. PMI and TMEM11 are inner membrane proteins that control mitochondria dynamics independently of the DRP-1/MFN-1 pathways.

  12. Effects of tea on survival rates and liver pathology of Trypanosoma brucei brucei infected mice

    OpenAIRE

    Mbuthia, S.K; Wachira, N.W; Ngure, R.M; Ouma, J; Kagira, J. M.

    2011-01-01

    The current study investigated the effects of different types of Kenyan tea extracts on the pathogenesis ofTrypanosoma brucei brucei in a Swiss White mice model. Following infection with trypanosomes, the micewere monitored for survival and liver pathology. Tea significantly (P

  13. Anti-trypanosomal effect of Peristrophe bicalyculata extract on Trypanosoma brucei brucei-infected rats

    Institute of Scientific and Technical Information of China (English)

    Abdulazeez Mansurah Abimbola; Ibrahim Abdulrazak Baba; Edibo Zakari Yenusa; Sidali Joseph Omanibe; Idris Habeeb Oladimeji

    2013-01-01

    Objective: To investigate the in vitro and in vivo effect of whole plant extracts of Peristrophe bicalyculata on Trypanosoma brucei brucei-infected rats. Methods: The experiment was divided into two phases: In the first phase, the anti-trypanosomal activity of the hot water, cold water, methanol and butanol extracts of the whole plant were determined by incubating with Trypanosoma brucei brucei. The cold water extract was partially-purified and the anti-trypanosomal activity of the fractions determined. In the second phase, Trypanosoma brucei brucei-infected rats were treated with fraction 2c for nine days. Packed cell volume (PCV), high density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol (TC), triacylglycerol (TAG), aspartate aminotransferase, alanine aminotransferases (ALT), alkaline phosphatase (ALP), total and direct bilirubin levels were determined at the end of the experiment. Results:Cold water extract immobilized 90%of the parasites after 60 min of incubation, and fraction 2c completely immobilized the parasites after 35 min. It significantly increased PCV in Trypanosoma brucei brucei-infected rats. Decreased TC, TAG, HDL and LDL levels of infected rats increased significantly when rats were treated with the fraction, while elevated levels of total bilirubin and ALT also decreased. The difference in urea, direct bilirubin and ALP was not significant when infected rats were compared to rats in other groups. Conclusions:The ability of the plant to ameliorate the infection-induced biochemical changes calls for detailed investigation of the potentials of the plant for antitrypanosomiasis drug delivery.

  14. Evidence for a degradosome-like complex in the mitochondria of Trypanosoma brucei

    OpenAIRE

    Mattiacio, Jonelle L.; Read, Laurie K.

    2009-01-01

    Mitochondrial RNA turnover in yeast involves the degradosome, composed of DSS-1 exoribonuclease and SUV3 RNA helicase. Here, we describe a degradosome-like complex, containing SUV3 and DSS-1 homologues, in the early branching protozoan, Trypanosoma brucei. TbSUV3 is mitochondrially localized and co-sediments with TbDSS-1 on glycerol gradients. Co-immunoprecipitation demonstrates that TbSUV3 and TbDSS-1 associate in a stable complex, which differs from the yeast degradosome in that it is not s...

  15. A method of determining electrical potential gradient across mitochondrial membrane in perfused rat hearts.

    Science.gov (United States)

    Wan, B; Doumen, C; Duszynski, J; Salama, G; LaNoue, K F

    1993-08-01

    The electrical potential gradient across the mitochondrial membrane (delta psi m) in perfused rat hearts was estimated by calculating the equilibrium distribution of the lipophilic cation tetraphenylphosphonium (TPP+), using measured kinetic constants of uptake and release of TPP+. First-order rate constants of TPP+ uptake were measured during 30-min perfusions of intact rat hearts with tracer amounts (5.0 nM) of tritium-labeled TPP+ ([3H]TPP+) in the perfusate. This was followed by a 30-min washout, during which the first-order rate constant of efflux was estimated. Values of [3H]TPP+ outside the heart and total [3H]TPP+ inside the heart at equilibrium were calculated. From this information and separately estimated time-averaged plasma membrane potentials (delta psi c) it was possible to calculate free cytosolic [3H]TPP+ at equilibrium. It was also possible to calculate free intramitochondrial [3H]TPP+ at equilibrium as the difference between total tissue [3H]TPP+ minus free cytosolic TPP+ and the sum of all the bound [3H]TPP+. Bound [3H]TPP+ was determined from [3H]TPP+ binding constants measured in separate experiments, using both isolated mitochondria and isolated cardiac myocytes under conditions where both delta psi m and delta psi c were zero. Delta psi m was calculated from the intramitochondrial and cytosolic free TPP+ concentrations using the Nernst equation. Values of delta psi m were 144.9 +/- 2.0 mV in hearts perfused with 5 mM pyruvate and 118.2 +/- 1.4 mV in hearts perfused with 11 mM glucose, in good agreement with delta psi m obtained from isolated rat heart mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8368347

  16. Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins.

    Science.gov (United States)

    Mikkelsen, R B; Wallach, D F

    1976-05-21

    (1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone. PMID:6061

  17. 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.

  18. Targeting of a Tail-anchored Protein to Endoplasmic Reticulum and Mitochondrial Outer Membrane by Independent but Competing Pathways

    OpenAIRE

    Borgese, Nica; Gazzoni, Ilaria; Barberi, Massimo; Colombo, Sara; Pedrazzini, Emanuela

    2001-01-01

    Many mitochondrial outer membrane (MOM) proteins have a transmembrane domain near the C terminus and an N-terminal cytosolic moiety. It is not clear how these tail-anchored (TA) proteins posttranslationally select their target, but C-terminal charged residues play an important role. To investigate how discrimination between MOM and endoplasmic reticulum (ER) occurs, we used mammalian cytochrome b5, a TA protein existing in two, MOM or ER localized, versions. Substi...

  19. Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway.

    Science.gov (United States)

    Sinzel, Monika; Tan, Tao; Wendling, Philipp; Kalbacher, Hubert; Özbalci, Cagakan; Chelius, Xenia; Westermann, Benedikt; Brügger, Britta; Rapaport, Doron; Dimmer, Kai Stefan

    2016-07-01

    Mitochondria are separated from the remainder of the eukaryotic cell by the mitochondrial outer membrane (MOM). The MOM plays an important role in different transport processes like lipid trafficking and protein import. In yeast, the ER-mitochondria encounter structure (ERMES) has a central, but poorly defined role in both activities. To understand the functions of the ERMES, we searched for suppressors of the deficiency of one of its components, Mdm10, and identified a novel mitochondrial protein that we named Mdm10 complementing protein 3 (Mcp3). Mcp3 partially rescues a variety of ERMES-related phenotypes. We further demonstrate that Mcp3 is an integral protein of the MOM that follows a unique import pathway. It is recognized initially by the import receptor Tom70 and then crosses the MOM via the translocase of the outer membrane. Mcp3 is next relayed to the TIM23 translocase at the inner membrane, gets processed by the inner membrane peptidase (IMP) and finally integrates into the MOM. Hence, Mcp3 follows a novel biogenesis route where a MOM protein is processed by a peptidase of the inner membrane. PMID:27226123

  20. Effect of Qingkailing injection on rat embryonic neuronal apoptosis and mitochondrial membrane potential

    Institute of Scientific and Technical Information of China (English)

    He Pang; Lingqun Zhu; Shuoren Wang; Fuing Niu; Wei Cui

    2006-01-01

    BACKGROUND:The decrease of mitochondrial membrane potential(MMP)is an irreversible marker of neuronal apoptosis during ischemla/reperfusion(I/R)injury of brain tissue.Qingkaiing injection is proved to have protective effect on neuronal ischemic injury.Whether inhibiting the decrease of MMP can inhibit apoptosis when I/R injury of brain tissue occurs is unclear.OBJECTIVE:To observe the effect of Qingkaiing injection on rat embryonic hippocampal neuronal apoptosis,MMP and mitochondroal activity after hypoxia/hypoglycamia and reoxygenation,and make a comparison of therapeutic effect on I/R injury between Oingkaiing injection and nimodipine.DESIGN:Observation and controlled trial.SETTING:Peropheral Vascular Center,Dongzhimen Hospital, Beijing University of Chinese Medicine;the Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing Key Laboratory.Dongzhimen Hospital,Beijing University of Chinese Medicine.MATERIALS:Eight Wistar rats at embryonic 18 days,provided by Breeding Farm of Experimental Animals,Chinese Academy of Medical Sciences(Permission No.SCXK-11-00-0006) were employed in this trial.Qingkaiing injection (Pharmaceutical Factory of Beijing University of Chinese Medicine,Batch No.213710A,10 Ml each,baicalin 50 g and total nitrogen 25 mg included)and nimodipine(ICN company,USA)were also used.METHODS:This experiment was carried out in the Key Laboratory of Chinese Internal Medicine of Ministry of Education,Dongzhimen Hospital,Beijing University of Chinese Medicine and Beijing Key Laboratory from January 2003 to December 2005.①The pregnant rats were anesthetized and fetal rats were isolated for culturong fetal rat hippocampal neurons.The neurons cultured for 10 days were used for expedment.The neurons were divided into 5 groups:model group,control group,nimodipine group.Qingkailing high-dose group and Oingkailing low-dose group.Hypoxia/hypoglycemia and reoxygenation models served as model group,and they were used to simulate reperfusion

  1. Fibrates inhibit the apoptosis of Batten disease lymphoblast cells via autophagy recovery and regulation of mitochondrial membrane potential.

    Science.gov (United States)

    Hong, Minho; Song, Ki Duk; Lee, Hak-Kyo; Yi, SunShin; Lee, Yong Seok; Heo, Tae-Hwe; Jun, Hyun Sik; Kim, Sung-Jo

    2016-03-01

    Batten disease (BD; also known as juvenile neuronal ceroid lipofuscinosis) is a genetic disorder inherited as an autosomal recessive trait and is characterized by blindness, seizures, cognitive decline, and early death resulting from the inherited mutation of the CLN3 gene. Mitochondrial oxidative stress, endoplasmic reticulum (ER) stress, disrupted autophagy, and enhanced apoptosis have been suggested to play a role in BD pathogenesis. Fibrates, a class of lipid-lowering drugs that induce peroxisome proliferator-activated receptor-α (PPAR-α) activation, are the most commonly used PPAR agonists. Assuming that fibrates have a neuroprotective effect, we studied the effects of fibrates, fenofibrate, bezafibrate, and gemfibrozil on apoptosis, depolarization of mitochondrial membrane, and defective autophagy in BD lymphoblast cells. The viability of fibrate-treated BD lymphoblast cells increased to levels of normal lymphoblast cells. In addition, treatment with fibrates inhibited depolarization of mitochondrial membrane potential in BD lymphoblast cells. Defective autophagy in BD lymphoblast cells was normalized when treated with fibrates as indicated by increased acridine orange staining. The recovery of autophagy in BD lymphoblast cells is most likely attributed to the upregulation of autophagy proteins, lysosomal-associated membrane protein 1 (LAMP1), and LC3 I/II, after treatment with fibrates. This study therefore suggests that fibrates may have a therapeutic potential against BD. PMID:26659390

  2. Inhibition of mitochondrial permeability transition by low pH is associated with less extensive membrane protein thiol oxidation.

    Science.gov (United States)

    Teixeira, B M; Kowaltowski, A J; Castilho, R F; Vercesi, A E

    1999-12-01

    Ca2+ and inorganic phosphate-induced mitochondrial swelling and membrane protein thiol oxidation, which are associated with mitochondrial permeability transition, are inhibited by progressively decreasing the incubation medium pH between 7.2 and 6.0. Nevertheless, the detection of mitochondrial H2O2 production under these conditions is increased. Permeability transition induced by phenylarsine oxide, which promotes membrane protein thiol cross-linkage in a process independent of Ca2+ or reactive oxygen species, is also strongly inhibited in acidic incubation media. In addition, we observed that the decreased protein thiol reactivity with phenylarsine oxide or phenylarsine oxide-induced swelling at pH 6.0 is reversed by diethyl pyrocarbonate, in a hydroxylamine-sensitive manner. These results provide evidence that the inhibition of mitrochondrial permeability transition observed at lower incubation medium pH is mediated by a decrease in membrane protein thiol reactivity, related to the protonation of protein histidyl residues. PMID:10841269

  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. Differential effects of insecticides on mitochondrial membrane lfuidity and ATPase activity between the wolf spider and the rice stem borer

    Institute of Scientific and Technical Information of China (English)

    LI Hai-ping; CHANG Jing; FENG Tao; GAO Xi-wu

    2015-01-01

    Differential effects of methamidophos and three pyrethroids on ATPase activity and membrane lfuidity of mitochondria were investigated between the wolf spider (Pirata subpiraticus(Boes. et Str.)) and the rice stem borer (Chilo suppressalis (Walker)). Based on a comparison of LD50values, the toxicities of the tested insecticides were higher to the wolf spider than to the rice stem borer. Cyhalothrin at 1×10–4 mmol L–1 caused inhibition of the mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities, and it’s inhibitions on Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities were signiifcantly higher in the wolf spider (44 and 28%) than in the rice stem borer (19 and 11%). Methamidophos at 1×10–4 mmol L–1 decreased Ca2+-Mg2+-ATPase activity by 16 and 27% in the wolf spider and the rice stem borer, respectively, but no signiifcant effect on the speciifc activity of Na+-K+-ATPase was observed. The DPH (1,6-diphenyl-1,3,5-hexatriene) lfuorescence polarization values of mitochondrial membranes were not signiifcantly affected by methamidophos in either species. However, cyhalothrin and alpha-cyperme-thrin induced the values of DPH polarization of mitochondrial membrane increasing with the concentration of cyhalothrin and alpha-cypermethrin from 20 to 100 µmol L–1 in the rice stem borer and the wolf spider. Effect of ethofenprox on lfuidity of the wolf spider and the rice stem borer was contrary. These results suggest that both inhibition of membrane ATPase and changes of membrane lfuidity could be appended to the action mechanisms of pyrethroid insecticides.

  5. 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.

  6. Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria.

    Science.gov (United States)

    Chowdhury, Subir Roy; Djordjevic, Jelena; Albensi, Benedict C; Fernyhough, Paul

    2016-01-01

    Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30-35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer's disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances. PMID:26647379

  7. Assessing the Mitochondrial Membrane Potential in Cells and In Vivo using Targeted Click Chemistry and Mass Spectrometry

    Science.gov (United States)

    Logan, Angela; Pell, Victoria R.; Shaffer, Karl J.; Evans, Cameron; Stanley, Nathan J.; Robb, Ellen L.; Prime, Tracy A.; Chouchani, Edward T.; Cochemé, Helena M.; Fearnley, Ian M.; Vidoni, Sara; James, Andrew M.; Porteous, Carolyn M.; Partridge, Linda; Krieg, Thomas; Smith, Robin A.J.; Murphy, Michael P.

    2016-01-01

    Summary The mitochondrial membrane potential (Δψm) is a major determinant and indicator of cell fate, but it is not possible to assess small changes in Δψm within cells or in vivo. To overcome this, we developed an approach that utilizes two mitochondria-targeted probes each containing a triphenylphosphonium (TPP) lipophilic cation that drives their accumulation in response to Δψm and the plasma membrane potential (Δψp). One probe contains an azido moiety and the other a cyclooctyne, which react together in a concentration-dependent manner by “click” chemistry to form MitoClick. As the mitochondrial accumulation of both probes depends exponentially on Δψm and Δψp, the rate of MitoClick formation is exquisitely sensitive to small changes in these potentials. MitoClick accumulation can then be quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach enables assessment of subtle changes in membrane potentials within cells and in the mouse heart in vivo. PMID:26712463

  8. Assessing the Mitochondrial Membrane Potential in Cells and In Vivo using Targeted Click Chemistry and Mass Spectrometry.

    Science.gov (United States)

    Logan, Angela; Pell, Victoria R; Shaffer, Karl J; Evans, Cameron; Stanley, Nathan J; Robb, Ellen L; Prime, Tracy A; Chouchani, Edward T; Cochemé, Helena M; Fearnley, Ian M; Vidoni, Sara; James, Andrew M; Porteous, Carolyn M; Partridge, Linda; Krieg, Thomas; Smith, Robin A J; Murphy, Michael P

    2016-02-01

    The mitochondrial membrane potential (Δψm) is a major determinant and indicator of cell fate, but it is not possible to assess small changes in Δψm within cells or in vivo. To overcome this, we developed an approach that utilizes two mitochondria-targeted probes each containing a triphenylphosphonium (TPP) lipophilic cation that drives their accumulation in response to Δψm and the plasma membrane potential (Δψp). One probe contains an azido moiety and the other a cyclooctyne, which react together in a concentration-dependent manner by "click" chemistry to form MitoClick. As the mitochondrial accumulation of both probes depends exponentially on Δψm and Δψp, the rate of MitoClick formation is exquisitely sensitive to small changes in these potentials. MitoClick accumulation can then be quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach enables assessment of subtle changes in membrane potentials within cells and in the mouse heart in vivo.

  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. PMID:24173598

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

    OpenAIRE

    Georges C. Accrombessi; Jacques Poupaert; Raymond H. Fatondji; Salomé D. S. Kpoviessi; Gbaguidi, Fernand A.; Bienvenu Glinma

    2011-01-01

    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 a...

  11. Ionomycin-induced Ca2+ overload is not accompanied by mitochondrial membrane potential dissipation in murine pro-B cells

    OpenAIRE

    Gabriela Ildiko Zonda; Ancuta Goriuc; Marcel Costuleanu

    2010-01-01

    There are extremely few data concerning the involvement of Ca2+ fluxes in the apoptosis of the pro-B cell type Ba/F3. Thus, we aimed the characterization of ionomycin-induced effects on Ba/F3 cells in vitro. Our obtained data show that cytosolic Ca2+ increased in Ba/F3 cells by 1 μM ionomycin in the presence of 1 mM Ca2+ for 24 hours did not induced significant effects on the mitochondrial membrane potential as compared with control cells. The same effects were also associated by ...

  12. Mitochondrial membrane potential is a suitable candidate for assessing pollution toxicity in fish

    International Nuclear Information System (INIS)

    Fish inhabiting polluted estuaries are highly exposed to severe stress characterized by an oxidant-antioxidant imbalance. The aim of the study was to explore the use of stress parameters such as adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio, mitochondrial membrane potential (Δψm) and total protein expression patterns as biomarkers against oxidant exposures in hepatocytes of Mugil cephalus living in either a contaminated (Test; Ennore) or uncontaminated (Control; Kovalam) estuary. Earlier, the pollutant stress impact was determined through light and electron microscopy studies. The ATP/ADP ratio was measured using high performance liquid chromatography; Δψm by fluorescent probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethyl benzimidazolcarbocyanine iodide (JC-1) dye and total protein expression patterns by protein profiling. The preponderance of stress impact was confirmed through microscopy studies that featured cytological alterations, disturbances in the surface morphology and in the cell organelles at the ultrastructural levels. Hepatocytes of test fish demonstrated a decrease in ATP and an increase in ADP and thereby alteration in ATP/ADP ratio (p < 0.05; 20.75%). A significant disturbance (p < 0.05; 26.57%) in Δψm with a ratio of J-aggregates/JC-1 monomer of 1 was observed for test fish hepatocytes compared to control group with a J-aggregates/JC-1 monomer ratio of 1.5. Quantitative assessment of protein expression levels also revealed enhanced induction of both low and high molecular weight proteins in test fish hepatocytes. The findings highlight the use of these parameters as the highly sensitive biomarkers in response to contaminant exposure compared to the routinely used antioxidant and oxidant stress parameters in biomonitoring programs. Among the measured parameters, the determination of Δψm may be suggested as a novel candidate as a biomarker because of its greater specificity and rapid quantitative risk assessment of pollutant

  13. 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.

  14. A transient increase in lipid peroxidation primes preadipocytes for delayed mitochondrial inner membrane permeabilization and ATP depletion during prolonged exposure to fatty acids

    OpenAIRE

    Rogers, Carlyle; Davis, Barbara; Neufer, P. Darrell; Murphy, Michael P.; Anderson, Ethan J.; Robidoux, Jacques

    2013-01-01

    Preadipocytes are periodically subjected to fatty acid (FA) concentrations that are potentially cytotoxic. We tested the hypothesis that prolonged exposure of preadipocytes of human origin to a physiologically relevant mix of FAs leads to mitochondrial inner membrane (MIM) permeabilization and ultimately to mitochondrial crisis. We found that exposure of preadipocytes to FAs led to progressive cyclosporin A-sensitive MIM permeabilization, which in turn caused reduction in MIM potential (ΔΨM),...

  15. 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.

  16. Photoactive mitochondria: in vivo transfer of a light-driven proton pump into the inner mitochondrial membrane of Schizosaccharomyces pombe.

    Science.gov (United States)

    Hoffmann, A; Hildebrandt, V; Heberle, J; Büldt, G

    1994-09-27

    The light-driven proton pump bacteriorhodopsin (bR) from Halobacterium salinarium has been genetically transferred into the inner mitochondrial membrane (IM) of the eukaryotic cell Schizosaccharomyces pombe, where the archaebacterial proton pump replaces or increases the proton gradient usually formed by the respiratory chain. For targeting and integration, as well as for the correct orientation of bR in the IM, the bacterioopsin gene (bop) was fused to signal sequences of IM proteins. Northern and Western blot analysis proved that all hybrid gene constructs containing the bop gene and a mitochondrial signal sequence were expressed and processed to mature bR. Fast transient absorption spectroscopy showed photocycle activity of bR integrated in the IM by formation of the M intermediate. Experiments with the pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein revealed bR-mediated proton pumping from the mitochondrial matrix into the intermembrane space. Glucose uptake measurements under anaerobic conditions showed that yeast cells containing photoactive mitochondria need less sugar under illumination. In summary, our experiments demonstrate the functional genetic transfer of a light energy converter to a naturally nonphotoactive eukaryotic organism. PMID:7937771

  17. 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

    of mitCK adsorption capacity by another method at pH 7.4, when the enzyme is almost protonated, gave View the MathML source. The effect of ionic strength on mitCK adsorption may be described in terms of Debye-Hückel's theory for activity coefficients assuming the charges of the interacting species......The quantitative aspects of mitochondrial creatinekinase (mitCK) binding to mitochondrial membranes were investigated. A simple adsorption and binding model was used for data fitting, taking into account the influence of protein concentration, pH, ionic strength and substrate concentration...... 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...

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

    International Nuclear Information System (INIS)

    The hepatic plasma membrane fatty acid-binding protein (h-FABPPM) 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-FABPPM have GOT enzymatic activity. Monospecific rabbit antiserum against h-FABPPM 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 [3H]oleate but not that of [35S]sulfobromophthalein or [14C]taurocholate. The inhibition of oleate uptake produced by anti-h-FABPPM 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-FABPPM and mGOT are closely related

  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. 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.

  1. A Splice-Isoform of Vesicle-associated Membrane Protein-1 (VAMP-1) Contains a Mitochondrial Targeting Signal

    Science.gov (United States)

    Isenmann, Sandra; Khew-Goodall, Yeesim; Gamble, Jennifer; Vadas, Mathew; Wattenberg, Binks W.

    1998-01-01

    Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell. PMID:9658161

  2. Butachlor induced dissipation of mitochondrial membrane potential, oxidative DNA damage and necrosis in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A; Musarrat, Javed

    2012-12-01

    Butachlor is a systemic herbicide widely applied on rice, tea, wheat, beans and other crops; however, it concurrently exerts toxic effects on beneficial organisms like earthworms, aquatic invertebrates and other non-target animals including humans. Owing to the associated risk to humans, this chloroacetanilide class of herbicide was investigated with the aim to assess its potential for the (i) interaction with DNA, (ii) mitochondria membrane damage and DNA strand breaks and (iii) cell cycle arrest and necrosis in butachlor treated human peripheral blood mononuclear (PBMN) cells. Fluorescence quenching data revealed the binding constant (Ka=1.2×10(4)M(-1)) and binding capacity (n=1.02) of butachlor with ctDNA. The oxidative potential of butachlor was ascertained based on its capacity of inducing reactive oxygen species (ROS) and substantial amounts of promutagenic 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) adducts in DNA. Also, the discernible butachlor dose-dependent reduction in fluorescence intensity of a cationic dye rhodamine (Rh-123) and increased fluorescence intensity of 2',7'-dichlorodihydro fluorescein diacetate (DCFH-DA) in treated cells signifies decreased mitochondrial membrane potential (ΔΨm) due to intracellular ROS generation. The comet data revealed significantly greater Olive tail moment (OTM) values in butachlor treated PBMN cells vs untreated and DMSO controls. Treatment of cultured PBMN cells for 24h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). The flow cytometry analysis of annexin V(-)/7-AAD(+) stained cells demonstrated substantial reduction in live population due to complete loss of cell membrane integrity. Overall the data suggested the formation of butachlor-DNA complex, as an initiating event in butachlor-induced DNA damage. The results elucidated the oxidative role of butachlor in intracellular ROS production, and

  3. 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

  4. 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

    Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We...... reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR)=50 more than 105 FomA proteins could be incorporated...

  5. Cell cycle regulation in Trypanosoma brucei

    OpenAIRE

    Tansy C Hammarton

    2007-01-01

    Cell division is regulated by intricate and interconnected signal transduction pathways that precisely coordinate, in time and space, the complex series of events involved in replicating and segregating the component parts of the cell. In Trypanosoma brucei, considerable progress has been made over recent years in identifying molecular regulators of the cell cycle and elucidating their functions, although many regulators undoubtedly remain to be identified, and there is still a long way to go...

  6. Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis.

    Science.gov (United States)

    Huang, Kai; Zhang, Jingjing; O'Neill, Katelyn L; Gurumurthy, Channabasavaiah B; Quadros, Rolen M; Tu, Yaping; Luo, Xu

    2016-05-27

    The BH3-only protein Bid is known as a critical mediator of the mitochondrial pathway of apoptosis following death receptor activation. However, since full-length Bid possesses potent apoptotic activity, the role of a caspase-mediated Bid cleavage is not established in vivo In addition, due to the fact that multiple caspases cleave Bid at the same site in vitro, the identity of the Bid-cleaving caspase during death receptor signaling remains uncertain. Moreover, as Bid maintains its overall structure following its cleavage by caspase 8, it remains unclear how Bid is activated upon cleavage. Here, Bid-deficient (Bid KO) colon cancer cells were generated by gene editing, and were reconstituted with wild-type or mutants of Bid. While the loss of Bid blocked apoptosis following treatment by TNF-related apoptosis inducing ligand (TRAIL), this blockade was relieved by re-introduction of the wild-type Bid. In contrast, the caspase-resistant mutant Bid(D60E) and a BH3 defective mutant Bid(G94E) failed to restore TRAIL-induced apoptosis. By generating Bid/Bax/Bak-deficient (TKO) cells, we demonstrated that Bid is primarily cleaved by caspase 8, not by effector caspases, to give rise to truncated Bid (tBid) upon TRAIL treatment. Importantly, despite the presence of an intact BH3 domain, a tBid mutant lacking the mitochondrial targeting helices (α6 and α7) showed diminished apoptotic activity. Together, these results for the first time establish that cleavage by caspase 8 and the subsequent association with the outer mitochondrial membrane are two critical events that activate Bid during death receptor-mediated apoptosis. PMID:27053107

  7. Motility modes of the parasite Trypanosoma brucei

    Science.gov (United States)

    Temel, Fatma Zeynep; Qu, Zijie; McAllaster, Michael; de Graffenried, Christopher; Breuer, Kenneth

    2015-11-01

    The parasitic single-celled protozoan Trypanosoma brucei causes African Sleeping Sickness, which is a fatal disease in humans and animals that threatens more than 60 million people in 36 African countries. Cell motility plays a critical role in the developmental phases and dissemination of the parasite. Unlike many other motile cells such as bacteria Escherichia coli or Caulobacter crescentus, the flagellum of T. brucei is attached along the length of its awl-like body, producing a unique mode of motility that is not fully understood or characterized. Here, we report on the motility of T. brucei, which swims using its single flagellum employing both rotating and undulating propulsion modes. We tracked cells in real-time in three dimensions using fluorescent microscopy. Data obtained from experiments using both short-term tracking within the field of view and long-term tracking using a tracking microscope were analyzed. Motility modes and swimming speed were analyzed as functions of cell size, rotation rate and undulation pattern. Research supported by NSF.

  8. Megaconial muscular dystrophy caused by mitochondrial membrane homeostasis defect, new insights from skeletal and heart muscle analyses.

    Science.gov (United States)

    Vanlander, Arnaud V; Muiño Mosquera, Laura; Panzer, Joseph; Deconinck, Tine; Smet, Joél; Seneca, Sara; Van Dorpe, Jo; Ferdinande, Liesbeth; Ceuterick-de Groote, Chantal; De Jonghe, Peter; Van Coster, Rudy; Baets, Jonathan

    2016-03-01

    Megaconial congenital muscular dystrophy is a disease caused by pathogenic mutations in the gene encoding choline kinase beta (CHKB). Microscopically, the disease is hallmarked by the presence of enlarged mitochondria at the periphery of skeletal muscle fibres leaving the centre devoid of mitochondria. Clinical characteristics are delayed motor development, intellectual disability and dilated cardiomyopathy in half of reported cases. This study describes a patient presenting with the cardinal clinical features, in whom a homozygous nonsense mutation (c.248_249insT; p.Arg84Profs*209) was identified in CHKB and who was treated by heart transplantation. Microscopic evaluation of skeletal and heart muscles typically showed enlarged mitochondria. Spectrophotometric evaluation in both tissues revealed a mild decrease of all OXPHOS complexes. Using BN-PAGE analysis followed by activity staining subcomplexes of complex V were detected in both tissues, indicating incomplete complex V assembly. Mitochondrial DNA content was not depleted in analysed tissues. This is the first report describing the microscopic and biochemical abnormalities in the heart from an affected patient. A likely hypothesis is that the biochemical findings are caused by an abnormal lipid profile in the inner mitochondrial membrane resulting from a defective choline kinase B activity.

  9. Mitochondrial membrane potential in human neutrophils is maintained by complex III activity in the absence of supercomplex organisation.

    Directory of Open Access Journals (Sweden)

    Bram J van Raam

    Full Text Available BACKGROUND: Neutrophils depend mainly on glycolysis for their energy provision. Their mitochondria maintain a membrane potential (Deltapsi(m, which is usually generated by the respiratory chain complexes. We investigated the source of Deltapsi(m in neutrophils, as compared to peripheral blood mononuclear leukocytes and HL-60 cells, and whether neutrophils can still utilise this Deltapsi(m for the generation of ATP. METHODS AND PRINCIPAL FINDINGS: Individual activity of the oxidative phosphorylation complexes was significantly reduced in neutrophils, except for complex II and V, but Deltapsi(m was still decreased by inhibition of complex III, confirming the role of the respiratory chain in maintaining Deltapsi(m. Complex V did not maintain Deltapsi(m by consumption of ATP, as has previously been suggested for eosinophils. We show that complex III in neutrophil mitochondria can receive electrons from glycolysis via the glycerol-3-phosphate shuttle. Furthermore, respiratory supercomplexes, which contribute to efficient coupling of the respiratory chain to ATP synthesis, were lacking in neutrophil mitochondria. When HL-60 cells were differentiated to neutrophil-like cells, they lost mitochondrial supercomplex organisation while gaining increased aerobic glycolysis, just like neutrophils. CONCLUSIONS: We show that neutrophils can maintain Deltapsi(m via the glycerol-3-phosphate shuttle, whereby their mitochondria play an important role in the regulation of aerobic glycolysis, rather than producing energy themselves. This peculiar mitochondrial phenotype is acquired during differentiation from myeloid precursors.

  10. Megaconial muscular dystrophy caused by mitochondrial membrane homeostasis defect, new insights from skeletal and heart muscle analyses.

    Science.gov (United States)

    Vanlander, Arnaud V; Muiño Mosquera, Laura; Panzer, Joseph; Deconinck, Tine; Smet, Joél; Seneca, Sara; Van Dorpe, Jo; Ferdinande, Liesbeth; Ceuterick-de Groote, Chantal; De Jonghe, Peter; Van Coster, Rudy; Baets, Jonathan

    2016-03-01

    Megaconial congenital muscular dystrophy is a disease caused by pathogenic mutations in the gene encoding choline kinase beta (CHKB). Microscopically, the disease is hallmarked by the presence of enlarged mitochondria at the periphery of skeletal muscle fibres leaving the centre devoid of mitochondria. Clinical characteristics are delayed motor development, intellectual disability and dilated cardiomyopathy in half of reported cases. This study describes a patient presenting with the cardinal clinical features, in whom a homozygous nonsense mutation (c.248_249insT; p.Arg84Profs*209) was identified in CHKB and who was treated by heart transplantation. Microscopic evaluation of skeletal and heart muscles typically showed enlarged mitochondria. Spectrophotometric evaluation in both tissues revealed a mild decrease of all OXPHOS complexes. Using BN-PAGE analysis followed by activity staining subcomplexes of complex V were detected in both tissues, indicating incomplete complex V assembly. Mitochondrial DNA content was not depleted in analysed tissues. This is the first report describing the microscopic and biochemical abnormalities in the heart from an affected patient. A likely hypothesis is that the biochemical findings are caused by an abnormal lipid profile in the inner mitochondrial membrane resulting from a defective choline kinase B activity. PMID:26855408

  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. Effects of transmembrane potential and pH gradient on the cytochrome c-promoted fusion of mitochondrial mimetic membranes.

    Science.gov (United States)

    Kawai, Cintia; Pessoto, Felipe S; Graves, Catharine V; Carmona-Ribeiro, Ana Maria; Nantes, Iseli L

    2013-08-01

    The present study investigated the effects of ΔΨ and ΔpH (pH gradient) on the interaction of cytochrome c with a mitochondrial mimetic membrane composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL) leading to vesicle fusion. ΔpH generated by lowered bulk pH (pH(out)) of PCPECL liposomes, with an internal pH (pH(in)) of 8.0, favored vesicle fusion with a titration sigmoidal profile (pK(a) ~ 6.9). Conversely, ΔpH generated by enhanced pH(in) of PCPECL at a pH(out) of 6.0 favored the fusion of vesicles with a linear profile. We did not observe a significant amount of liposome fusion when ΔpH was generated by lowered pH(in) at a pH(out) of 8.0. At bulk acidic pH, ΔΨ generated by Na⁺ gradient also favored cyt c-promoted vesicle fusion. At acidic and alkaline pH(out), the presence of ΔpH and ΔΨ did not affect cytochrome c binding affinity measured by pyrene quenching. Therefore, cytochrome c-mediated PC/PE/CL vesicle fusion is dependent of ionization of the protein site L (acidic pH) and the presence of transmembrane potential. The effect of transmembrane potential is probably related to the generation of defects on the lipid bilayer. These results are consistent with previous reports showing that cytochrome c release prior to the dissipation of the ΔΨ(M) blocks inner mitochondrial membrane fusion during apoptosis.

  13. Three-dimensional organization of the endoplasmic reticulum membrane around the mitochondrial constriction site in mammalian cells revealed by using focused-ion beam tomography.

    Science.gov (United States)

    Ohta, Keisuke; Okayama, Satoko; Togo, Akinobu; Nakamura, Kei-Ichiro

    2014-11-01

    The endoplasmic reticulum (ER) and mitochondria associate at multiple contact sites to form specific domains known as mitochondria-ER associated membranes (MAMs) that play a role in the regulation of various cellular processes such as Ca2+ transfer, autophagy, and inflammation. Recently, it has been suggested that MAMs are also involved in mitochondrial dynamics, especially fission events. Cytological analysis showed that ER tubules were frequently located close to each other in mitochondrial fission sites that accumulate fission-related proteins. Three-dimensional (3D) imaging of ER-mitochondrial contacts in yeast mitochondria by using cryo-electron tomography also showed that ER tubules were attached near the constriction site, which is considered to be a fission site1). MAMs have been suggested to play a role in the initiation of mitochondrial fission, although the molecular relationships between MAMs and the mitochondrial fission process have not been established. Although an ER-mitochondrial membrane association has also been observed at the fission site in mammalian mitochondria, the detailed organization of MAMs around mammalian mitochondria remains to be established. To visualize the 3D distribution of the ER-mitochondrial contacts around the mitochondria, especially around the constriction site in mammalian cells, we attempted 3D structural analysis of the mammalian cytoplasm using high-resolution focused ion-beam scanning electron microscopy (FIB-SEM) tomography, and observed the distribution pattern of ER contacts around the mammalian mitochondrial constriction site.Rat hepatocytes and HeLa cells were used. Liver tissue was obtained from male rats (Wistar, 6W) fixed by transcardial perfusion of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) under deep anesthesia. HeLa cells were fixed with the same fixative. The specimens were then stained en bloc to enhance membrane contrast and embedded in epoxy resin2). The surface of

  14. Interventional effect of phycocyanin on mitochondrial membrane potential and activity of PC12 cells after hypoxia/reoxygenation

    Institute of Scientific and Technical Information of China (English)

    Nan Jiang; Yunliang Guo; Hongbing Chen

    2006-01-01

    BACKGROUND: Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation.OBJECTIVE: To observe the effect of phycocyanin on activity of PC12 cells and mitochondrial membrane potential after hypoxia/reoxygenation.DESIGN: Randomized controlled study.SETTING: Cerebrovascular Disease Institute of Affiliated Hospital, Medical College of Qingdao University.MATERIALS: The experiment was carried out at the Key Laboratory of Prevention and Cure for cerebropathia in Shandong Province from October to December 2005. PC12 cells, rat chromaffin tumor cells,were provided by Storage Center of Wuhan University; phycocyanin was provided by Ocean Institute of Academia Sinica; Thiazoyl blue tetrazolium bromide (MTT) and rhodamine 123 were purchased from Sigma Company, USA; RPMI-1640 medium, fetal bovine serum and equine serum were purchased from Gibco Company, USA.METHODS: ① Culture of PC12 cells: PC12 cells were put into RPMI-1640 medium which contained 100 g/L heat inactivation equine serum and 0.05 volume fraction of fetal bovine serum and incubated in CO2 incubator at 37 ℃. Number of cells was regulated to 4 × 105 L-1, and cells were inoculated at 96-well culture plate.The final volume was 100 μL. ② Model establishing and grouping: Cultured PC12 cells were randomly divided into three groups: phycocyanin group, model control group and non-hypoxia group. At 24 hours before hypoxia, culture solution in phycocyanin group was added with phycocyanin so as to make sure the final concentration of 3 g/L, but cells in model control group did not add with phycocyanin. Cells in non-hypoxia group were also randomly divided into adding phycocyanin group (the final concentration of 3 g/L) and non-adding phycocyanin group. Cells in model control group and phycocyanin group were cultured with hypoxia for 1 hour and reoxygenation for 1, 2 and 3 hours; meanwhile, cells in non

  15. Catalytic properties, localization, and in vivo role of Px IV, a novel tryparedoxin peroxidase of Trypanosoma brucei.

    Science.gov (United States)

    Liu, Ilon; Bogacz, Marta; Schaffroth, Corinna; Dirdjaja, Natalie; Krauth-Siegel, R Luise

    2016-06-01

    Px IV is a distant relative of the known glutathione peroxidase-type enzymes of African trypanosomes. Immunofluorescence microscopy of bloodstream cells expressing C-terminally Myc6-tagged Px IV revealed a mitochondrial localization. Recombinant Px IV possesses very low activity as glutathione peroxidase but catalyzes the trypanothione/tryparedoxin-dependent reduction of hydrogen peroxide and, even more efficiently, of arachidonic acid hydroperoxide. Neither overexpression in bloodstream cells nor the deletion of both alleles in bloodstream or procyclic parasites affected the in vitro proliferation. Trypanosoma brucei Px IV shares 58% of all residues with TcGPXII. The orthologous enzymes have in common their substrate preference for fatty acid hydroperoxides. However, the T. cruzi protein has been reported to be localized in the endoplasmic reticulum and to be specific for glutathione as reducing agent. Taken together, our data show that Px IV is a low abundant tryparedoxin peroxidase of T. brucei that is not essential, at least under culture conditions. PMID:27262262

  16. 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.

  17. Effect of inorganic phosphate concentration on the nature of inner mitochondrial membrane alterations mediated by Ca2+ ions. A proposed model for phosphate-stimulated lipid peroxidation.

    Science.gov (United States)

    Kowaltowski, A J; Castilho, R F; Grijalba, M T; Bechara, E J; Vercesi, A E

    1996-02-01

    Addition of high concentrations (>1 mm) of inorganic phosphate (Pi) or arsenate to Ca2+-loaded mitochondria was followed by increased rates of H2O2 production, membrane lipid peroxidation, and swelling. Mitochondrial swelling was only partially prevented either by butylhydroxytoluene, an inhibitor of lipid peroxidation, or cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. This swelling was totally prevented by the simultaneous presence of these compounds. At lower Pi concentrations (1 mm), mitochondrial swelling is reversible and prevented by cyclosporin A, but not by butylhydroxytoluene. In any case (low or high phosphate concentration) exogenous catalase prevented mitochondrial swelling, suggesting that reactive oxygen species (ROS) participate in these mechanisms. Altogether, the data suggest that, at low Pi concentrations, membrane permeabilization is reversible and mediated by opening of the mitochondrial permeability transition pore, whereas at high Pi concentrations, membrane permeabilization is irreversible because lipid peroxidation also takes place. Under these conditions, lipid peroxidation is strongly inhibited by sorbate, a putative quencher of triplet carbonyl species. This suggests that high Pi or arsenate concentrations stimulate propagation of the peroxidative reactions initiated by mitochondrial-generated ROS because these anions are able to catalyze Cn-aldehyde tautomerization producing enols, which can be oxidized by hemeproteins to yield the lower Cn - 1-aldehyde in the triplet state. This proposition was also supported by experiments using a model system consisting of phosphatidylcholine/dicethylphosphate liposomes and the triplet acetone-generating system isobutanal/horseradish peroxidase, where phosphate and Ca2+ cooperate to increase the yield of thiobarbituric acid-reactive substances. PMID:8621682

  18. 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.

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

    DEFF Research Database (Denmark)

    Andersen, Ann Zahle; Poulsen, Allan K; Olsen, Lars Folke;

    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 that the ......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...

  20. 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.

  1. New proteomic approaches to study the organization of yeast mitochondrial membranes

    NARCIS (Netherlands)

    Gubbens, J.

    2008-01-01

    Despite their importance, membrane proteins have traditionally been underrepresented in proteomics studies due to their incompatibility with common methods used in this field. Therefore, new methods have to be developed for studying this class of proteins. In this thesis, new approaches are describe

  2. Triiodothyronine facilitates weaning from extracorporeal membrane oxygenation by improved mitochondrial substrate utilization

    Energy Technology Data Exchange (ETDEWEB)

    Files, Matthew D.; Kajimoto, Masaki; Priddy, Colleen M.; Ledee, Dolena R.; Xu, Chun; Des Rosiers, Christine; Isern, Nancy G.; Portman, Michael A.

    2014-03-20

    Extracorporeal membrane oxygenation (ECMO) provides a bridge to recovery after myocardial injury in infants and children, yet morbidity and mortality remain high. Weaning from the circuit requires adequate cardiac contractile function, which can be impaired by metabolic disturbances induced either by ischemia-reperfusion and / or by ECMO.

  3. 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.

  4. 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.

  5. Mapping of VSG similarities in Trypanosoma brucei.

    Science.gov (United States)

    Weirather, Jason L; Wilson, Mary E; Donelson, John E

    2012-02-01

    The protozoan parasite Trypanosoma brucei switches its variant surface glycoprotein (VSG) to subvert its mammalian hosts' immune responses. The T. brucei genome contains as many as 1600 VSG genes (VSGs), but most are silent noncoding pseudogenes. Only one functional VSG, located in a telomere-linked expression site, is transcribed at a time. Silent VSGs are copied into a VSG expression site through gene conversion. Truncated gene conversion events can generate new mosaic VSGs with segments of sequence identity to other VSGs. To examine the VSG family sub-structure within which these events occur, we combined the available VSG sequences and annotations with scripted BLAST searches to map the relationships among VSGs in the T. brucei genome. Clusters of related VSGs were visualized in 2- and 3-dimensions for different N- and C-terminal regions. Five types of N-termini (N1-N5) were observed, within which gene recombinational events are likely to occur, often with fully-coding 'functional' or 'atypical'VSGs centrally located between more dissimilar VSGs. Members of types N1, N3 and N4 are most closely related in the middle of the N-terminal region, whereas type N2 members are more similar near the N-terminus. Some preference occurs in pairing between specific N- and C-terminal types. Statistical analyses indicated no overall tendency for more related VSGs to be located closer in the genome than less related VSGs, although exceptions were noted. Many potential mosaic gene formation events within each N-terminal type were identified, contrasted by only one possible mosaic gene formation between N-terminal types (N1 and N2). These data suggest that mosaic gene formation is a major contributor to the overall VSG diversity, even though gene recombinational events between members of different N-terminal types occur only rarely. PMID:22079099

  6. Apaf-1-deficient fog mouse cell apoptosis involves hypopolarization of the mitochondrial inner membrane,ATP depletion and citrate accumulation

    Institute of Scientific and Technical Information of China (English)

    Iyoko Katoh; Shingo Sato; Nahoko Fukunishi; Hiroki Yoshida; Takasuke Imai; Shun-ichi Kurata

    2008-01-01

    To explore how the intrinsic apoptosis pathway is controlled in the spontaneous fog (forebrain overgrowth) mutant mice with an Apaf1 splicing deficiency,we examined spleen and bone marrow cells from Apaf1+/+(+/+) and Apaf1fog/fog (fog/fog) mice for initiator caspase-9 activation by cellular stresses.When the mitochondrial inner membrane potential (△Ψm) was disrupted by staurosporine,+/+ cells but not fog/fog cells activated caspase-9 to cause apoptosis,indicating the lack of apoptosomc (apoptosis protease activating factor 1 (Apaf-1)/cytochrome c/(d)ATP/procaspase-9) function in fog/fog cells.However,when a marginal (~20%) decrease in △Ψm was caused by hydrogen peroxide (0.1 mM),peroxynitrite donor 3-morpholinosydnonimine (0.1 mM) and UV-C irradiation (20 J/m2),both +/+ and fog/fog cells triggeredprocaspase-9 auto-processing and its downstream cascade activation.Supporting our previous results,procaspase-9 pre-existing in the mitochondria induced its auto-processing before the cytosolic caspase activation regardless of the geuotypes.Cellular ATP concentration significantly decreased under the hypoactive AΨm condition.Furthermore,we detected accumulation of citrate,a kosmotrope known to facilitate procaspase-9 dimerization,probably due to a feedback control of the Krebs cycle by the electron transfer system.Thus,mitochondrial in situ caspase-9 activation may be caused by the major metabolic reactions in response to physiological stresses,which may represent a mode of Apaf-1-independent apoptosis hypothesized from recent genetic studies.

  7. [Effect of ethylmaleimide on the transport of Ca+ and K+ ions across mitochondrial membranes].

    Science.gov (United States)

    Lofrumento, N E; Zanotti, F; Pavone, A

    1979-04-30

    As already reported, it has been found that the gradient of protons, set up across the inner membrane during the Ca2+ uptake by rat liver mitochondria, can be completely reversed by the addition of NEM. Identical results have been obtained by following the energy dependent K+ uptake. In these last conditions, the rate of H+ efflux supported by succinate oxidation is greatly enhanced only when NEM is added after rotenone. It is proposed that the increased rate other than to the inhibition of Pi uptake, as suggested by Reynafarje and Lehninger, could also be ascribed to a further decrease in the energetic level of the membrane as well as to an increased rate of succinate-Pi exchange diffusion reaction induced by NEM. A possible direct effect of NEM on succinate oxidation has been also considered to account for the inhibition observed when it is added before rotenone. PMID:554640

  8. 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; Merker, Marilyn P

    2011-05-01

    Our goal was to quantify mitochondrial and plasma potential (Δψ(m) and Δψ(p)) 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 ([R(e)]) were measured over time. R123 [R(e)] fell from 10 nM to 6.6 ± 0.1 (SE) nM over 120 min. TMRE [R(e)] 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 [R(e)], and P-glycoprotein (Pgp) inhibition had the opposite effect, demonstrating the qualitative impact of these processes on [R(e)]. A kinetic model incorporating a modified Goldman-Hodgkin-Katz model was fit to [R(e)] vs. time data for R123 and TMRE, respectively, under various conditions to obtain (means ± 95% confidence intervals) Δψ(m) (-130 ± 7 and -133 ± 4 mV), Δψ(p) (-36 ± 4 and -49 ± 4 mV), and a Pgp activity parameter (K(Pgp), 25 ± 5 and 51 ± 11 μl/min). The higher membrane permeability of TMRE also allowed application of steady-state analysis to obtain Δψ(m) (-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 Δψ(m) and Δψ(p.) Finally, steady-state analysis revealed that although room air- and hyperoxia-exposed (95% O(2) for 48 h) cells have equivalent resting Δψ(m), hyperoxic cell Δψ(m) was more sensitive to depolarization with protonophore, consistent with previous observations of pulmonary endothelial hyperoxia-induced mitochondrial dysfunction.

  9. 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

    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...

  10. Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells.

    Science.gov (United States)

    Gerencser, Akos A; Mookerjee, Shona A; Jastroch, Martin; Brand, Martin D

    2016-01-01

    The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic β-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic β-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed β-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with β-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited β-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions.

  11. 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.

  12. Cell membrane penetration and mitochondrial targeting by platinum-decorated ceria nanoparticles.

    Science.gov (United States)

    Torrano, Adriano A; Herrmann, Rudolf; Strobel, Claudia; Rennhak, Markus; Engelke, Hanna; Reller, Armin; Hilger, Ingrid; Wixforth, Achim; Bräuchle, Christoph

    2016-07-01

    In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and focus on their fast uptake and association with mitochondria, the cell's powerhouse. Using live-cell imaging and electron microscopy we clearly show that 46 nm platinum-decorated ceria nanoparticles can rapidly penetrate cell membranes and reach the cytosol. Moreover, if suitably targeted, these particles are able to selectively attach to mitochondria. These results are complemented by cytotoxicity assays, thus providing insights into the biological effects of these particles on cells. Interestingly, no permanent membrane disruption or any other significant adverse effects on cells were observed. The unusual uptake behavior observed for 46 nm nanoparticles was not observed for equivalent but larger 143 nm and 285 nm platinum-decorated particles. Our results demonstrate a remarkable particle size effect in which particles smaller than ∼50-100 nm escape the usual endocytic pathway and translocate directly into the cytosol, while particles larger than ∼150 nm are internalized by conventional endocytosis. Since the small particles are able to bypass endocytosis they could be explored as drug and gene delivery vehicles. Platinum-decorated nanoparticles are therefore highly interesting in the fields of nanotoxicology and nanomedicine. PMID:27341699

  13. MitoNEET Is a Uniquely Folded 2Fe-2S Outer Mitochondrial Membrane Protein Stabilized By Pioglitazone

    Energy Technology Data Exchange (ETDEWEB)

    Paddock, M.L.; Wiley, S.E.; Axelrod, H.L.; Cohen, A.E.; Roy, M.; Abresch, E.C.; Capraro, D.; Murphy, A.N.; Nechushtai, R.; Dixon, J.E.; Jennings, P.A.; /UC, San Diego /SLAC, SSRL /Hebrew U.

    2007-10-19

    Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Angstrom x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the {approx}650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a {beta}-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer.

  14. 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.

  15. Cell membrane penetration and mitochondrial targeting by platinum-decorated ceria nanoparticles

    Science.gov (United States)

    Torrano, Adriano A.; Herrmann, Rudolf; Strobel, Claudia; Rennhak, Markus; Engelke, Hanna; Reller, Armin; Hilger, Ingrid; Wixforth, Achim; Bräuchle, Christoph

    2016-07-01

    In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and focus on their fast uptake and association with mitochondria, the cell's powerhouse. Using live-cell imaging and electron microscopy we clearly show that 46 nm platinum-decorated ceria nanoparticles can rapidly penetrate cell membranes and reach the cytosol. Moreover, if suitably targeted, these particles are able to selectively attach to mitochondria. These results are complemented by cytotoxicity assays, thus providing insights into the biological effects of these particles on cells. Interestingly, no permanent membrane disruption or any other significant adverse effects on cells were observed. The unusual uptake behavior observed for 46 nm nanoparticles was not observed for equivalent but larger 143 nm and 285 nm platinum-decorated particles. Our results demonstrate a remarkable particle size effect in which particles smaller than ~50-100 nm escape the usual endocytic pathway and translocate directly into the cytosol, while particles larger than ~150 nm are internalized by conventional endocytosis. Since the small particles are able to bypass endocytosis they could be explored as drug and gene delivery vehicles. Platinum-decorated nanoparticles are therefore highly interesting in the fields of nanotoxicology and nanomedicine.In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and

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

    DEFF Research Database (Denmark)

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

    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...... in vivo (zebrafish embryo) and in vitro tests. We applied a passive dosing method to expose zebrafish embryos up to 5 days post fertilization to linear dilution series of a set of non-polar narcotics (phenanthrene and three chlorobenzene structure analogues). In addition to increasing mortality, we...... membrane-bound process and is therefore a potential target. We found that in zebrafish embryos ETC activity was increased at low exposure concentrations, suggesting a compensatory response, while it decreased when exposure concentrations reached levels causing reduced motility, heart rate and eventually...

  17. Classical clinical signs in rats experimemtally infected with Trypanosoma brucei

    Institute of Scientific and Technical Information of China (English)

    Nwoha Rosemary Ijeoma Ogechi; Omamegbe Joseph Omolathebu

    2015-01-01

    Objective:To investigate clinical signs in Trypanosoma brucei infection in albino rats. Methods:Fourteen rats grouped into 2 with 7 rats in each group were used to determine classical clinical manifestation of Trypanosoma brucei infection in rats. Group A rats were uninfected control and Group B rats were infected with Trypanosoma brucei. Results:Parasitaemia was recorded in Group B by (3.86±0.34) d and the peak of parasitaemia was observed at Day 5 post infection. Classical signs observed included squint eyes, raised whiskers, lethargy, no weight loss, pyrexia, isolation from the other rats, and starry hair coat. Conclusions:These signs could be diagnostic or aid in diagnosis of Trypanosoma brucei infection in rats.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. A transient increase in lipid peroxidation primes preadipocytes for delayed mitochondrial inner membrane permeabilization and ATP depletion during prolonged exposure to fatty acids.

    Science.gov (United States)

    Rogers, Carlyle; Davis, Barbara; Neufer, P Darrell; Murphy, Michael P; Anderson, Ethan J; Robidoux, Jacques

    2014-02-01

    Preadipocytes are periodically subjected to fatty acid (FA) concentrations that are potentially cytotoxic. We tested the hypothesis that prolonged exposure of preadipocytes of human origin to a physiologically relevant mix of FAs leads to mitochondrial inner membrane (MIM) permeabilization and ultimately to mitochondrial crisis. We found that exposure of preadipocytes to FAs led to progressive cyclosporin A-sensitive MIM permeabilization, which in turn caused a reduction in MIM potential, oxygen consumption, and ATP synthetic capacity and, ultimately, death. Additionally, we showed that FAs induce a transient increase in intramitochondrial reactive oxygen species (ROS) and lipid peroxide production, lasting roughly 30 and 120min for the ROS and lipid peroxides, respectively. MIM permeabilization and its deleterious consequences including mitochondrial crisis and cell death were prevented by treating the cells with the mitochondrial FA uptake inhibitor etomoxir, the mitochondrion-selective superoxide and lipid peroxide antioxidants MitoTempo and MitoQ, or the lipid peroxide and reactive carbonyl scavenger l-carnosine. FAs also promoted a delayed oxidative stress phase. However, the beneficial effects of etomoxir, MitoTempo, and l-carnosine were lost by delaying the treatment by 2h, suggesting that the initial phase was sufficient to prime the cells for the delayed MIM permeabilization and mitochondrial crisis. It also suggested that the second ROS production phase is a consequence of this loss in mitochondrial health. Altogether, our data suggest that approaches designed to diminish intramitochondrial ROS or lipid peroxide accumulation, as well as MIM permeabilization, are valid mechanism-based therapeutic avenues to prevent the loss in preadipocyte metabolic fitness associated with prolonged exposure to elevated FA levels. PMID:24269897

  20. Role of expression site switching in the development of resistance to human Trypanosome Lytic Factor-1 in Trypanosoma brucei brucei.

    Science.gov (United States)

    Kieft, Rudo; Stephens, Natalie A; Capewell, Paul; MacLeod, Annette; Hajduk, Stephen L

    2012-05-01

    Human high-density lipoproteins (HDLs) play an important role in human innate immunity to infection by African trypanosomes with a minor subclass, Trypanosome Lytic Factor-1 (TLF-1), displaying highly selective cytotoxicity to the veterinary pathogen Trypanosoma brucei brucei but not against the human sleeping sickness pathogens Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense. T. b. rhodesiense has evolved the serum resistance associated protein (SRA) that binds and confers resistance to TLF-1 while T. b. gambiense lacks the gene for SRA indicating that these parasites have diverse mechanisms of resistance to TLF-1. Recently, we have shown that T. b. gambiense (group 1) resistance to TLF-1 correlated with the loss of the haptoglobin/hemoglobin receptor (HpHbR) expression, the protein responsible for high affinity binding and uptake of TLF-1. In the course of these studies we also examined TLF-1 resistant T. b. brucei cell lines, generated by long-term in vitro selection. We found that changes in TLF-1 susceptibility in T. b. brucei correlated with changes in variant surface glycoprotein (VSG) expression in addition to reduced TLF-1 binding and uptake. To determine whether the expressed VSG or expression site associated genes (ESAGs) contribute to TLF-1 resistance we prepared a TLF-1 resistant T. b. brucei with a selectable marker in a silent bloodstream expression site (BES). Drug treatment allowed rapid selection of trypanosomes that activated the tagged BES. These studies show that TLF-1 resistance in T. b. brucei is largely independent of the expressed VSG or ESAGs further supporting the central role of HpHbR expression in TLF-1 susceptibility in these cells. PMID:22226682

  1. Gene synthesis, bacterial expression, and 1H NMR spectroscopic studies of the rat outer mitochondrial membrane cytochrome b5.

    Science.gov (United States)

    Rivera, M; Barillas-Mury, C; Christensen, K A; Little, J W; Wells, M A; Walker, F A

    1992-12-01

    The gene coding for the water-soluble domain of the outer mitochondrial membrane cytochrome b5 (OM cytochrome b5) from rat liver has been synthetized and expressed in Escherichia coli. The DNA sequence was obtained by back-translating the known amino acid sequence [Lederer, F., Ghrir, R., Guiard, B., Cortial, S., & Ito, A. (1983) Eur. J. Biochem. 132, 95-102]. The recombinant OM cytochrome b5 was characterized by UV-visible, EPR, and 1H NMR spectroscopy. The UV-visible and EPR spectra of the OM cytochrome b5 are almost identical to the ones obtained from the overexpressed rat microsomal cytochrome b5 [Bodman, S. B. V., Schyler, M. A., Jollie, D. R., & Sligar, S. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9443-9447]. The one-dimensional 1H NMR spectrum of the OM cytochrome b5 indicates that the rhombic perturbation of the ferric center is essentially identical to that in the microsomal beef, rabbit, chicken, and rat cytochromes b5. Two-dimensional 1H NMR spectroscopy (NOESY) and one-dimensional NOE difference spectroscopy were used to assign the contact-shifted resonances that correspond to each of the two isomers that result from the rotation of the heme around its alpha-gamma-meso axis. The assignment of the resonances allowed the determination of the heme orientation ratio in the OM cytochrome b5, which was found to be 1.0 +/- 0.1. It is noteworthy that the two cytochromes b5 that have similar populations of the two heme isomers (large heme disorder) originate from the rat liver. PMID:1333795

  2. 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.

  3. Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells.

    Science.gov (United States)

    Gerencser, Akos A; Mookerjee, Shona A; Jastroch, Martin; Brand, Martin D

    2016-01-01

    The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic β-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic β-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed β-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with β-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited β-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions. PMID:27404273

  4. The assembly of mitochondrial complex I : a product of nuclear-mitochondrial synergy

    NARCIS (Netherlands)

    Vogel, Rutger Oscar

    2007-01-01

    Mitochondria are essential to cellular energy production. Embedded in the mitochondrial inner membrane, the engine of the mitochondrial powerhouse is formed by the five enzymatic complexes of the oxidative phosphorylation (OXPHOS) system. Dysfunction of this system results in mitochondrial disease,

  5. 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. PMID:26771494

  6. 氟康唑对热带念珠菌活性氧和%Effect of fluconazole on reactive oxygen species and mitochondrial membrane potential of Candida tropicalis

    Institute of Scientific and Technical Information of China (English)

    邱莲女; 周永列; 胡庆丰; 朱永泽; 郭伟; 吕火烊

    2011-01-01

    To explore fluconazole's effect mechanism, we investigated the changes of viability rate, reactive oxygen species (ROS), mitochondrial membrane potential (△Ψm) and cell cycle of Candida tropicalis after treatment with fluconazole. The minimum inhibitory concentration (MIC) of the clinical isolates Candida tropicalis to fluconazole were tested by NCCLS M27-A microdilution method. After treatment wth different concentration of fluconazole, viability rate, the intracellular accumulation of ROS, the loss of mitochondrial membrane potential △Ψm and cell cycle of Candida tropicalis were detected with flow cytometry, respectively. After treatment with fluconazole, there were no significant variation among viability rate, ROS, mitochondrial membrane potential △Ψm and cell cycle in fluconazole-resistant strains, but a decrease of mitochondrial membrane potential △Ψm and viability rate,an increase of ROS accumulation were detected in a time-dose-dependent manner in fluconazole-susceptibile strains. A majority of Candida tropicalis were arrested in G2/M phase and apoptosis peak was seen. Free radicals scavenger glutathione inhibited ROS production, prevented G2/M arrest and decreased apoptosis in fluconazole-susceptibile strains. According to it, fluconazole maybe induce intracellular accumulation of ROS and decrease of mitochondrial membrane potential △Ψm, which could result in apoptosis of Candida tropicalis.%为了探讨氟康唑作用机制,观察它对热带念珠菌作用后存活率、活性氧(Reactive oxygenspecies,ROS)、线粒体膜电位(Mitochondrial membrane potential,,△Ψm)和细胞周期的变化.参照NCCLS M27-A 方案的微量稀释法测定氟康唑对热带念珠菌的最低抑菌浓度(MIC); 热带念珠菌与不同浓度氟康唑共同培养后用流式细胞术(Flow cytometry,FCM)分析热带念珠菌存活率、ROS、线粒体膜电位△Ψm 和细胞周期的变化.结果表明,氟康唑作用后,热带念珠菌氟康唑耐药株的

  7. Methyl glycol, methanol and DMSO effects on post-thaw motility, velocities, membrane integrity and mitochondrial function of Brycon orbignyanus and Prochilodus lineatus (Characiformes) sperm.

    Science.gov (United States)

    Viveiros, Ana T M; Nascimento, Ariane F; Leal, Marcelo C; Gonçalves, Antônio C S; Orfão, Laura H; Cosson, Jacky

    2015-02-01

    The aim of this study was to use more accurate techniques to investigate the effects of cryoprotectants (CPAs) and extenders on post-thaw sperm quality of Brycon orbignyanus and Prochilodus lineatus. Six freezing media comprising the combination of three CPAs (DMSO, methanol and methyl glycol) and two extenders (BTS and glucose) were used. Sperm was diluted in each medium, loaded into 0.5-mL straws, frozen in a nitrogen vapor vessel (dry-shipper), and stored in liquid nitrogen at -196 °C. Post-thaw sperm motility rate and velocities (curvilinear = VCL; straight line = VSL; average path = VAP) were evaluated using a computer-assisted sperm analyzer. Membrane integrity and mitochondrial function were determined using fluorochromes. Post-thaw quality was considered high when samples presented the following minimum values: 60 % motile sperm, 140 µm/s of VCL, 50 % intact sperm membrane and 50 % mitochondrial function integrity. High post-thaw quality was observed in B. orbignyanus sperm frozen in BTS-methyl glycol and in P. lineatus sperm frozen in BTS-methyl glycol, glucose-methyl glycol and glucose-methanol. All samples frozen in DMSO yielded low quality. The presence of ions in the BTS extender affected post-thaw sperm quality positively in B. orbignyanus and negatively in P. lineatus. Methyl glycol was the most suitable CPA for both fish species, leading to a good protection of cell membrane, mitochondrial function and motility apparatus during the cryopreservation process. For an improved protection, B. orbignyanus sperm should be frozen in an ionic freezing medium. PMID:25433690

  8. Overexpression of human SOD1 in VDAC1-less yeast restores mitochondrial functionality modulating beta-barrel outer membrane protein genes.

    Science.gov (United States)

    Magrì, Andrea; Di Rosa, Maria Carmela; Tomasello, Marianna Flora; Guarino, Francesca; Reina, Simona; Messina, Angela; De Pinto, Vito

    2016-06-01

    Cu/Zn Superoxide Dismutase (SOD1), the most important antioxidant defense against ROS in eukaryotic cells, localizes in cytosol and intermembrane space of mitochondria (IMS). Several evidences show a SOD1 intersection with both fermentative and respiratory metabolism. The Voltage Dependent Anion Channel (VDAC) is the main pore-forming protein in the mitochondrial outer membrane (MOM), and is considered the gatekeeper of mitochondrial metabolism. Saccharomyces cerevisiae lacking VDAC1 (Δpor1) is a very convenient model system, since it shows an impaired growth rate on non-fermentable carbon source. Transformation of Δpor1 yeast with human SOD1 completely restores the cell growth deficit in non-fermentative conditions and re-establishes the physiological levels of ROS, as well as the mitochondrial membrane potential. No similar result was found upon yeast SOD1 overexpression. A previous report highlighted the action of SOD1 as a transcription factor. Quantitative Real-Time PCR showed that β-barrel outer-membrane encoding-genes por2, tom40, sam50 are induced by hSOD1, but the same effect was not obtained in Δpor1Δpor2 yeast, indicating a crucial function for yVDAC2. Since the lack of VDAC1 in yeast can be considered a stress factor for the cell, hSOD1 could relieve it stimulating the expression of genes bringing to the recovery of the MOM function. Our results suggest a direct influence of SOD1 on VDAC. PMID:26947057

  9. A novel high-throughput activity assay for the Trypanosoma brucei editosome enzyme REL1 and other RNA ligases

    Science.gov (United States)

    Zimmermann, Stephan; Hall, Laurence; Riley, Sean; Sørensen, Jesper; Amaro, Rommie E.; Schnaufer, Achim

    2016-01-01

    The protist parasite Trypanosoma brucei causes Human African trypanosomiasis (HAT), which threatens millions of people in sub-Saharan Africa. Without treatment the infection is almost always lethal. Current drugs for HAT are difficult to administer and have severe side effects. Together with increasing drug resistance this results in urgent need for new treatments. T. brucei and other trypanosomatid pathogens require a distinct form of post-transcriptional mRNA modification for mitochondrial gene expression. A multi-protein complex called the editosome cleaves mitochondrial mRNA, inserts or deletes uridine nucleotides at specific positions and re-ligates the mRNA. RNA editing ligase 1 (REL1) is essential for the re-ligation step and has no close homolog in the mammalian host, making it a promising target for drug discovery. However, traditional assays for RELs use radioactive substrates coupled with gel analysis and are not suitable for high-throughput screening of compound libraries. Here we describe a fluorescence-based REL activity assay. This assay is compatible with a 384-well microplate format and sensitive, satisfies statistical criteria for high-throughput methods and is readily adaptable for other polynucleotide ligases. We validated the assay by determining kinetic properties of REL1 and by identifying REL1 inhibitors in a library of small, pharmacologically active compounds. PMID:26400159

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

    Directory of Open Access Journals (Sweden)

    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.

  11. 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.

  12. 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

  13. A comparative study on the susceptibility of male and female albino mice to Trypanosoma brucei brucei

    Directory of Open Access Journals (Sweden)

    A.A. Turay, G.O. Nwobu, G.R.A. Okogun, C.U. Igwe, K. Adeyeye, K.E. Aghatise, H.O. Okpal & Y.M. Tatfeng

    2005-03-01

    Full Text Available Background & objectives: Trypanosomiasis has remained a major set-back in the development oflivestock farming in tropical Africa. Thus the need for ascertaining the trypanotolerant levels ofdomestic animal breeds and possible improvement on them cannot be over-emphasised.Methods: Level of trypanotolerance in animals was compared between sexes using albino mice infectedwith a Nigerian strain of Trypanosoma brucei brucei at a 50% mouse lethal dose (MLD50.Results: The male mice showed unrestrained parasite growth with a prepatent period (PP of two daysand a mean survival period (MSP of six days corresponding to a gradual decrease in packed cellvolume (PCV, body weight, diet response and white blood cells (WBC count to the time of death.Their female counterparts showed a PP of three days and MSP of ten days with a similar PCV gradientbut a refractory WBC count. There was no significant difference in the differential leucocytes countin both sexes. However, the eosinophils count was significantly higher in the infected animals. It wasfound that female albino mice exercised more parasite restraint than their male counterparts.Interpretation & conclusion: The result suggests that the female animals may be more trypanotoleranthence may be more useful in protein production in trypanosomiasis endemic areas. However, furtherresearch using large domestic breeds like goats and sheep may be required to confirm the hypothesis.

  14. 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.

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

    Science.gov (United States)

    Habila, Nathan; Agbaji, Abel S; Ladan, Zakari; Bello, Isaac A; Haruna, Emmanuel; Dakare, Monday A; Atolagbe, Taofiq O

    2010-01-01

    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. PMID:20700425

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

    Science.gov (United States)

    Habila, Nathan; Agbaji, Abel S.; Ladan, Zakari; Bello, Isaac A.; Haruna, Emmanuel; Dakare, Monday A.; Atolagbe, Taofiq O.

    2010-01-01

    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. PMID:20700425

  17. Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei.

    Science.gov (United States)

    Allmann, Stefan; Mazet, Muriel; Ziebart, Nicole; Bouyssou, Guillaume; Fouillen, Laetitia; Dupuy, Jean-William; Bonneu, Marc; Moreau, Patrick; Bringaud, Frédéric; Boshart, Michael

    2014-01-01

    Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4-5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4-5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 null mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse. PMID:25493940

  18. Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Stefan Allmann

    Full Text Available Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4-5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG content also increased 4-5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 null mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse.

  19. Simultaneous depletion of Atm and Mdl rebalances cytosolic Fe-S cluster assembly but not heme import into the mitochondrion of Trypanosoma brucei.

    Science.gov (United States)

    Horáková, Eva; Changmai, Piya; Paris, Zdeněk; Salmon, Didier; Lukeš, Julius

    2015-11-01

    ABC transporter mitochondrial 1 (Atm1) and multidrug resistance-like 1 (Mdl) are mitochondrial ABC transporters. Although Atm1 was recently suggested to transport different forms of glutathione from the mitochondrion, which are used for iron-sulfur (Fe-S) cluster maturation in the cytosol, the function of Mdl remains elusive. In Trypanosoma brucei, we identified one homolog of each of these genes, TbAtm and TbMdl, which were downregulated either separately or simultaneously using RNA interference. Individual depletion of TbAtm and TbMdl led to limited growth defects. In cells downregulated for TbAtm, the enzymatic activities of the Fe-S cluster proteins aconitase and fumarase significantly decreased in the cytosol but not in the mitochondrion. Downregulation of TbMdl did not cause any change in activities of the Fe-S proteins. Unexpectedly, the simultaneous downregulation of TbAtm and TbMdl did not result in any growth defect, nor were the Fe-S cluster protein activities altered in either the cytosolic or mitochondrial compartments. Additionally, TbAtm and TbMdl were able to partially restore the growth of the Saccharomyces cerevisiae Δatm1 and Δmdl2 null mutants, respectively. Because T. brucei completely lost the heme b biosynthesis pathway, this cofactor has to be obtained from the host. Based on our results, TbMdl is a candidate for mitochondrial import of heme b, which was markedly decreased in both TbMdl and TbAtm + TbMdl knockdowns. Moreover, the levels of heme a were strongly decreased in the same knockdowns, suggesting that TbMdl plays a key role in heme a biosynthesis, thus affecting the overall heme homeostasis in T. brucei. PMID:26277108

  20. 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

    2016-01-01

    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. PMID:27322225

  1. Phenethyl isothiocyanate-induced apoptosis in PC-3 human prostate cancer cells is mediated by reactive oxygen species-dependent disruption of the mitochondrial membrane potential.

    Science.gov (United States)

    Xiao, Dong; Lew, Karen L; Zeng, Yan; Xiao, Hui; Marynowski, Stanley W; Dhir, Rajiv; Singh, Shivendra V

    2006-11-01

    The present study was undertaken to gain insights into the molecular mechanism of apoptosis induction by phenethyl isothiocyanate (PEITC), which is a cancer chemopreventive constituent of cruciferous vegetables, using PC-3 human prostate cancer cells as a model. The PEITC-induced cell death in PC-3 cells was associated with disruption of the mitochondrial membrane potential, release of apoptogenic molecules (cytochrome c and Smac/DIABLO) from mitochondria to the cytosol and generation of reactive oxygen species (ROS), which were blocked in the presence of a combined mimetic of superoxide dismutase and catalase (Euk134). Ectopic expression of Bcl-xL, whose protein level is reduced markedly on treatment of PC-3 cells with PEITC, conferred partial protection against PEITC-induced apoptosis only at higher drug concentrations (>10 microM). Administration of 12 micromol PEITC/day (Monday through Friday) by oral gavage significantly retarded growth of PC-3 xenografts in athymic mice. For instance, 31 days after the initiation of PEITC administration, the average tumor volume in control mice (721 +/- 153 mm3) was approximately 2-fold higher compared with mice receiving 12 micromol PEITC/day. The PEITC-mediated inhibition of PC-3 xenograft growth was associated with induction of Bax and Bid proteins. In conclusion, the present study indicates that the PEITC-induced apoptosis in PC-3 cells is mediated by ROS-dependent disruption of the mitochondrial membrane potential and regulated by Bax and Bid. PMID:16774948

  2. Kaempferol ameliorates aflatoxin B1 (AFB1) induced hepatocellular carcinoma through modifying metabolizing enzymes, membrane bound ATPases and mitochondrial TCA cycle enzymes

    Institute of Scientific and Technical Information of China (English)

    Kulanthaivel Langeswaran; Rajendran Revathy; Subbaraj Gowtham Kumar; Shanmugam Vijayaprakash

    2012-01-01

    Objective: The present study was aimed to scrutinize the anticancer consequence of kaempferol against aflatoxin B1 induced hepatocarcinogenesis. Epidemiological studies of the incidence of liver cancer in the population, where dietary aflatoxin exposure is high, have provided much circumstantial evidence for the development of aflatoxin B1 induced primary liver cancer in humans. Methods:In the present investigation, aflatoxin B1 (2 mg/kg body weight i.p) was used as a hepatocarcinogen to induce hepatocellular carcinoma in experimental animals. Results: In the present analysis, on treatment with bioflavonoid kaempferol (100 mg/kg body weight p.o) the nucleic acids levels were brought back to normal and also the altered levels of biological enzymes such as membrane bound ATPase, carbohydrate metabolizing enzymes and mitochondrial TCA cycle enzymes levels (P<0.01).Conclusions:Membrane bound ATPase, carbohydrate metabolizing enzymes and mitochondrial TCA cycle enzymes were modulated by kaempferol evaluated on aflatoxin B1 induced primary liver carcinogenesis.

  3. 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.

  4. 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...

  5. Testicular pathology, gonadal and epididymal sperm reserves of Yankasa rams infected with experimental Trypanosoma brucei brucei and Trypanosoma evansi

    Science.gov (United States)

    Wada, Yunusa A.; Oniye, Sonnie J.; Rekwot, Peter I.; Okubanjo, Oluyinka O.

    2016-01-01

    Aim: The study was conducted to evaluate the pathological effects of trypanosomosis on the testes, gonadal, and epididymal sperm reserves of Yankasa rams for 98 days. Materials and Methods: A total of 16 Yankasa rams, aged between 24 and 30 months and weighed between 22 and 25 kg, were acclimatized for a period of 2-months in a clean fly proof house and were adequately fed and given water ad-libitum. Of the 16 rams, 12 that were clinically fit for the experiment at the end of the acclimatization period were randomly divided into four groups: Groups I, II, III, and IV, each having 3 rams. Groups I and II were each challenged singly with experimental Trypanosoma brucei brucei (Federer strain) and Trypanosoma evansi (Sokoto strain), respectively, while Group III was challenged with mixed T. brucei brucei and T. evansi parasites (50% of each species in the infective inoculum) and Group IV was left as an uninfected control. Each infected ram received 2 mL of the infected blood containing 2×106 trypomastigotes via the jugular vein, while the control group received 2 mL each, normal saline. Results: All the infected rams developed clinical signs typical of trypanosomosis at varying pre-patent periods. The gross lesions observed in the infected rams in Group II were moderate and more severe in those of Groups I and III. Histological sections of the testes of infected rams (Groups I, II, and III) showed moderate (T. evansi-infected group) to severe (mixed and T. brucei brucei-infected groups) testicular degenerations with reduction in number of spermatogenic cell layers, degenerated seminiferous tubules, congested interlobular spaces, loss of tissue architecture with significant (p<0.01) depletion, and loss of gonadal and epididymal sperm reserves in Groups I and III in comparison to Group II and the control Group IV. No observable clinical signs and histopathological lesions were found in those rams of the control Group IV. Conclusion: The study concluded that

  6. Testicular pathology, gonadal and epididymal sperm reserves of Yankasa rams infected with experimental Trypanosoma brucei brucei and Trypanosoma evansi

    Directory of Open Access Journals (Sweden)

    Yunusa A. Wada

    2016-07-01

    Full Text Available Aim: The study was conducted to evaluate the pathological effects of trypanosomosis on the testes, gonadal, and epididymal sperm reserves of Yankasa rams for 98 days. Materials and Methods: A total of 16 Yankasa rams, aged between 24 and 30 months and weighed between 22 and 25 kg, were acclimatized for a period of 2-months in a clean fly proof house and were adequately fed and given water ad-libitum. Of the 16 rams, 12 that were clinically fit for the experiment at the end of the acclimatization period were randomly divided into four groups: Groups I, II, III, and IV, each having 3 rams. Groups I and II were each challenged singly with experimental Trypanosoma brucei brucei (Federer strain and Trypanosoma evansi (Sokoto strain, respectively, while Group III was challenged with mixed T. brucei brucei and T. evansi parasites (50% of each species in the infective inoculum and Group IV was left as an uninfected control. Each infected ram received 2 mL of the infected blood containing 2×106 trypomastigotes via the jugular vein, while the control group received 2 mL each, normal saline. Results: All the infected rams developed clinical signs typical of trypanosomosis at varying pre-patent periods. The gross lesions observed in the infected rams in Group II were moderate and more severe in those of Groups I and III. Histological sections of the testes of infected rams (Groups I, II, and III showed moderate (T. evansi-infected group to severe (mixed and T. brucei brucei-infected groups testicular degenerations with reduction in number of spermatogenic cell layers, degenerated seminiferous tubules, congested interlobular spaces, loss of tissue architecture with significant (p<0.01 depletion, and loss of gonadal and epididymal sperm reserves in Groups I and III in comparison to Group II and the control Group IV. No observable clinical signs and histopathological lesions were found in those rams of the control Group IV. Conclusion: The study concluded

  7. Nanomolar Inhibitors of Trypanosoma brucei RNA Triphosphatase

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    Paul Smith

    2016-02-01

    Full Text Available Eukaryal taxa differ with respect to the structure and mechanism of the RNA triphosphatase (RTPase component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi knockdown methods to show that Trypanosoma brucei RTPase Cet1 (TbCet1 is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals—including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics—that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC50s. We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae. Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition.

  8. The Spectrum of Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy.

    Science.gov (United States)

    Vincent, Amy E; Ng, Yi Shiau; White, Kathryn; Davey, Tracey; Mannella, Carmen; Falkous, Gavin; Feeney, Catherine; Schaefer, Andrew M; McFarland, Robert; Gorman, Grainne S; Taylor, Robert W; Turnbull, Doug M; Picard, Martin

    2016-01-01

    Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases. Following a systematic literature review on ultrastructural defects in mitochondrial myopathy, we investigated skeletal muscle biopsies from seven subjects with genetically defined mtDNA mutations. Mitochondrial ultrastructure and morphology were characterized using two complimentary approaches: transmission electron microscopy (TEM) and serial block face scanning EM (SBF-SEM) with 3D reconstruction. Six ultrastructural abnormalities were identified including i) paracrystalline inclusions, ii) linearization of cristae and abnormal angular features, iii) concentric layering of cristae membranes, iv) matrix compartmentalization, v) nanotunelling, and vi) donut-shaped mitochondria. In light of recent molecular advances in mitochondrial biology, these findings reveal novel aspects of mitochondrial ultrastructure and morphology in human tissues with implications for understanding the mechanisms linking mitochondrial dysfunction to disease. PMID:27506553

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

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    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

  10. Alteration of mitochondrial membrane potential (DELTA_PSI_m and phosphatidylserine translocation as early indicators of heavy metal-induced apoptosis in the earthworm Eisenia hortensis

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    FM Bearoff

    2011-06-01

    Full Text Available The effects of the heavy metals cadmium and copper (50-500 ìM on the apoptotic events involving changes in mitochondrial membrane potential (ÄØm and phosphatidylserine (PS translocation were investigated in the immune cells (celomocytes of the earthworm Eisenia hortensis. Using the fluorescent probe JC-1, loss of membrane potential due to depolarization was detected in a greater proportion of cases when induced by cadmium compared to copper (58.7 % vs. 37 % and at a lower concentration (50 ìM vs. 125ìM. With the use of the general caspase inhibitor Z-VAD-fmk, PS translocation detected by annexin V-FITC was found to be caspase-dependent when induced by cadmium at 125-250 ìM but not at 50 ìM or 500 ìM; a high proportion of earthworms (60 % exhibited inhibitory effects. Additionally, the collapse in membrane potential and PS translocation were found to strongly correlate (r > 0.5 in 89 % of cases when induced by cadmium and copper. Thus, heavy metals appear to induce death in celomocytes of E. hortensis through apoptosis by means of caspase dependent pathways

  11. The role of nitric oxide signaling in food intake; insights from the inner mitochondrial membrane peptidase 2 mutant mice

    OpenAIRE

    Changjie Han; Qingguo Zhao; Baisong Lu

    2013-01-01

    Reactive oxygen species have been implicated in feeding control through involvement in brain lipid sensing, and regulating NPY/AgRP and pro-opiomelanocortin (POMC) neurons, although the underlying mechanisms are unclear. Nitric oxide is a signaling molecule in neurons and it stimulates feeding in many species. Whether reactive oxygen species affect feeding through interaction with nitric oxide is unclear. We previously reported that Immp2l mutation in mice causes excessive mitochondrial super...

  12. Fluctuating vs. continuous exposure to H₂O₂: the effects on mitochondrial membrane potential, intracellular calcium, and NF-κB in astroglia.

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    Aleksandar Bajić

    Full Text Available The effects of H2O2 are widely studied in cell cultures and other in vitro systems. However, such investigations are performed with the assumption that H2O2 concentration is constant, which may not properly reflect in vivo settings, particularly in redox-turbulent microenvironments such as mitochondria. Here we introduced and tested a novel concept of fluctuating oxidative stress. We treated C6 astroglial cells and primary astrocytes with H2O2, using three regimes of exposure - continuous, as well as fluctuating at low or high rate, and evaluated mitochondrial membrane potential and other parameters of mitochondrial activity - respiration, reducing capacity, and superoxide production, as well as intracellular ATP, intracellular calcium, and NF-κB activation. When compared to continuous exposure, fluctuating H2O2 induced a pronounced hyperpolarization in mitochondria, whereas the activity of electron transport chain appears not to be significantly affected. H2O2 provoked a decrease of ATP level and an increase of intracellular calcium concentration, independently of the regime of treatment. However, fluctuating H2O2 induced a specific pattern of large-amplitude fluctuations of calcium concentration. An impact on NF-κB activation was observed for high rate fluctuations, whereas continuous and low rate fluctuating oxidative stress did not provoke significant effects. Presented results outline the (pathophysiological relevance of redox fluctuations.

  13. Adverse Effect of H2O2 Change on Morphology, Mitochondrial Membrane Permeability and Antioxidant Enzyme in Root of Dianthus Chinensis L. under Salt Stress

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    Xue-qin He

    2013-04-01

    Full Text Available Dianthus Chinensis L. is a salt-tolerant ornamental plant. Root is the first and critical part of plant to encounter soli salinity. In order to elucidate H2O2 impact on root morphology and mitochondrial permeability transition as well as activities of antioxidant enzymes in root ofDianthus Chinensis L., we treated seedling with H2O2 and DMTU under NaCl. The results revealed that change of H2O2 level under NaCl would negatively influence the root growth, as well as lower the value of mitochodrial membrane absorbance at 540 nm and the ratio of Cyt c/a. Meanwhile, SOD and POD under NaCl plus H2O2 and NaCl plus DMTU were far lower than those under NaCl alone.

  14. 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.

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

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    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

  16. Independent analysis of the flagellum surface and matrix proteomes provides insight into flagellum signaling in mammalian-infectious Trypanosoma brucei.

    Science.gov (United States)

    Oberholzer, Michael; Langousis, Gerasimos; Nguyen, HoangKim T; Saada, Edwin A; Shimogawa, Michelle M; Jonsson, Zophonias O; Nguyen, Steven M; Wohlschlegel, James A; Hill, Kent L

    2011-10-01

    The flagellum of African trypanosomes is an essential and multifunctional organelle that functions in motility, cell morphogenesis, and host-parasite interaction. Previous studies of the trypanosome flagellum have been limited by the inability to purify flagella without first removing the flagellar membrane. This limitation is particularly relevant in the context of studying flagellum signaling, as signaling requires surface-exposed proteins in the flagellar membrane and soluble signaling proteins in the flagellar matrix. Here we employ a combination of genetic and mechanical approaches to purify intact flagella from the African trypanosome, Trypanosoma brucei, in its mammalian-infectious stage. We combined flagellum purification with affinity-purification of surface-exposed proteins to conduct independent proteomic analyses of the flagellum surface and matrix fractions. The proteins identified encompass a broad range of molecular functionalities, including many predicted to function in signaling. Immunofluorescence and RNA interference studies demonstrate flagellum localization and function for proteins identified and provide insight into mechanisms of flagellum attachment and motility. The flagellum surface proteome includes many T. brucei-specific proteins and is enriched for proteins up-regulated in the mammalian-infectious stage of the parasite life-cycle. The combined results indicate that the flagellum surface presents a diverse and dynamic host-parasite interface that is well-suited for host-parasite signaling. PMID:21685506

  17. A cyclopalladated complex interacts with mitochondrial membrane thiol-groups and induces the apoptotic intrinsic pathway in murine and cisplatin-resistant human tumor cells

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    Martins Rafael M

    2011-07-01

    Full Text Available Abstract Background Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd2 [S(-C2, N-dmpa]2 (μ-dppeCl2} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies. Methods B16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a. Results Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human

  18. A cyclopalladated complex interacts with mitochondrial membrane thiol-groups and induces the apoptotic intrinsic pathway in murine and cisplatin-resistant human tumor cells

    International Nuclear Information System (INIS)

    Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd2 [S(-)C2, N-dmpa]2 (μ-dppe)Cl2} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies. B16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a. Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells. The cyclopalladated C7a complex is an

  19. Functional Diversity of Human Mitochondrial J-proteins Is Independent of Their Association with the Inner Membrane Presequence Translocase.

    Science.gov (United States)

    Sinha, Devanjan; Srivastava, Shubhi; D'Silva, Patrick

    2016-08-12

    Mitochondrial J-proteins play a critical role in governing Hsp70 activity and, hence, are essential for organellar protein translocation and folding. In contrast to yeast, which has a single J-protein Pam18, humans involve two J-proteins, DnaJC15 and DnaJC19, associated with contrasting cellular phenotype, to transport proteins into the mitochondria. Mutation in DnaJC19 results in dilated cardiomyopathy and ataxia syndrome, whereas expression of DnaJC15 regulates the response of cancer cells to chemotherapy. In the present study we have comparatively assessed the biochemical properties of the J-protein paralogs in relation to their association with the import channel. Both DnaJC15 and DnaJC19 formed two distinct subcomplexes with Magmas at the import channel. Knockdown analysis suggested an essential role for Magmas and DnaJC19 in organellar protein translocation and mitochondria biogenesis, whereas DnaJC15 had dispensable supportive function. The J-proteins were found to have equal affinity for Magmas and could stimulate mitochondrial Hsp70 ATPase activity by equivalent levels. Interestingly, we observed that DnaJC15 exhibits bifunctional properties. At the translocation channel, it involves conserved interactions and mechanism to translocate the precursors into mitochondria. In addition to protein transport, DnaJC15 also showed a dual role in yeast where its expression elicited enhanced sensitivity of cells to cisplatin that required the presence of a functional J-domain. The amount of DnaJC15 expressed in the cell was directly proportional to the sensitivity of cells. Our analysis indicates that the differential cellular phenotype displayed by human mitochondrial J-proteins is independent of their activity and association with Magmas at the translocation channel.

  20. Functional Diversity of Human Mitochondrial J-proteins Is Independent of Their Association with the Inner Membrane Presequence Translocase.

    Science.gov (United States)

    Sinha, Devanjan; Srivastava, Shubhi; D'Silva, Patrick

    2016-08-12

    Mitochondrial J-proteins play a critical role in governing Hsp70 activity and, hence, are essential for organellar protein translocation and folding. In contrast to yeast, which has a single J-protein Pam18, humans involve two J-proteins, DnaJC15 and DnaJC19, associated with contrasting cellular phenotype, to transport proteins into the mitochondria. Mutation in DnaJC19 results in dilated cardiomyopathy and ataxia syndrome, whereas expression of DnaJC15 regulates the response of cancer cells to chemotherapy. In the present study we have comparatively assessed the biochemical properties of the J-protein paralogs in relation to their association with the import channel. Both DnaJC15 and DnaJC19 formed two distinct subcomplexes with Magmas at the import channel. Knockdown analysis suggested an essential role for Magmas and DnaJC19 in organellar protein translocation and mitochondria biogenesis, whereas DnaJC15 had dispensable supportive function. The J-proteins were found to have equal affinity for Magmas and could stimulate mitochondrial Hsp70 ATPase activity by equivalent levels. Interestingly, we observed that DnaJC15 exhibits bifunctional properties. At the translocation channel, it involves conserved interactions and mechanism to translocate the precursors into mitochondria. In addition to protein transport, DnaJC15 also showed a dual role in yeast where its expression elicited enhanced sensitivity of cells to cisplatin that required the presence of a functional J-domain. The amount of DnaJC15 expressed in the cell was directly proportional to the sensitivity of cells. Our analysis indicates that the differential cellular phenotype displayed by human mitochondrial J-proteins is independent of their activity and association with Magmas at the translocation channel. PMID:27330077

  1. Inhibition of N-Methyl-D-aspartate-induced Retinal Neuronal Death by Polyarginine Peptides Is Linked to the Attenuation of Stress-induced Hyperpolarization of the Inner Mitochondrial Membrane Potential.

    Science.gov (United States)

    Marshall, John; Wong, Kwoon Y; Rupasinghe, Chamila N; Tiwari, Rakesh; Zhao, Xiwu; Berberoglu, Eren D; Sinkler, Christopher; Liu, Jenney; Lee, Icksoo; Parang, Keykavous; Spaller, Mark R; Hüttemann, Maik; Goebel, Dennis J

    2015-09-01

    It is widely accepted that overactivation of NMDA receptors, resulting in calcium overload and consequent mitochondrial dysfunction in retinal ganglion neurons, plays a significant role in promoting neurodegenerative disorders such as glaucoma. Calcium has been shown to initiate a transient hyperpolarization of the mitochondrial membrane potential triggering a burst of reactive oxygen species leading to apoptosis. Strategies that enhance cell survival signaling pathways aimed at preventing this adverse hyperpolarization of the mitochondrial membrane potential may provide a novel therapeutic intervention in retinal disease. In the retina, brain-derived neurotrophic factor has been shown to be neuroprotective, and our group previously reported a PSD-95/PDZ-binding cyclic peptide (CN2097) that augments brain-derived neurotrophic factor-induced pro-survival signaling. Here, we examined the neuroprotective properties of CN2097 using an established retinal in vivo NMDA toxicity model. CN2097 completely attenuated NMDA-induced caspase 3-dependent and -independent cell death and PARP-1 activation pathways, blocked necrosis, and fully prevented the loss of long term ganglion cell viability. Although neuroprotection was partially dependent upon CN2097 binding to the PDZ domain of PSD-95, our results show that the polyarginine-rich transport moiety C-R(7), linked to the PDZ-PSD-95-binding cyclic peptide, was sufficient to mediate short and long term protection via a mitochondrial targeting mechanism. C-R(7) localized to mitochondria and was found to reduce mitochondrial respiration, mitochondrial membrane hyperpolarization, and the generation of reactive oxygen species, promoting survival of retinal neurons.

  2. Comparative genomics of drug resistance in Trypanosoma brucei rhodesiense.

    Science.gov (United States)

    Graf, Fabrice E; Ludin, Philipp; Arquint, Christian; Schmidt, Remo S; Schaub, Nadia; Kunz Renggli, Christina; Munday, Jane C; Krezdorn, Jessica; Baker, Nicola; Horn, David; Balmer, Oliver; Caccone, Adalgisa; de Koning, Harry P; Mäser, Pascal

    2016-09-01

    Trypanosoma brucei rhodesiense is one of the causative agents of human sleeping sickness, a fatal disease that is transmitted by tsetse flies and restricted to Sub-Saharan Africa. Here we investigate two independent lines of T. b. rhodesiense that have been selected with the drugs melarsoprol and pentamidine over the course of 2 years, until they exhibited stable cross-resistance to an unprecedented degree. We apply comparative genomics and transcriptomics to identify the underlying mutations. Only few mutations have become fixed during selection. Three genes were affected by mutations in both lines: the aminopurine transporter AT1, the aquaporin AQP2, and the RNA-binding protein UBP1. The melarsoprol-selected line carried a large deletion including the adenosine transporter gene AT1, whereas the pentamidine-selected line carried a heterozygous point mutation in AT1, G430R, which rendered the transporter non-functional. Both resistant lines had lost AQP2, and both lines carried the same point mutation, R131L, in the RNA-binding motif of UBP1. The finding that concomitant deletion of the known resistance genes AT1 and AQP2 in T. b. brucei failed to phenocopy the high levels of resistance of the T. b. rhodesiense mutants indicated a possible role of UBP1 in melarsoprol-pentamidine cross-resistance. However, homozygous in situ expression of UBP1-Leu(131) in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine. PMID:26973180

  3. Changes in blood sugar levels of rats experimentally infected with Trypanosoma brucei and treated with imidocarb dipropionate and diminazene aceturate

    OpenAIRE

    Nwoha Rosemary Ijeoma Ogechi; Omamegbe Joseph Omalathebu

    2016-01-01

    Objective: To determine the effect of Trypanosoma brucei (T. brucei) on blood sugar level of infected rats. Methods: The experiment was done with 42 albino rats grouped into 3 groups of 14 members each. Group A was uninfected (control group), Group B was infected with T. brucei and treated with diminazene aceturate, and Group C was infected with T. brucei and treated with imidocarb dipropionate. Blood samples were collected from the media canthus of the experimental rats on ...

  4. The role of nitric oxide signaling in food intake; insights from the inner mitochondrial membrane peptidase 2 mutant mice

    Directory of Open Access Journals (Sweden)

    Changjie Han

    2013-01-01

    Full Text Available Reactive oxygen species have been implicated in feeding control through involvement in brain lipid sensing, and regulating NPY/AgRP and pro-opiomelanocortin (POMC neurons, although the underlying mechanisms are unclear. Nitric oxide is a signaling molecule in neurons and it stimulates feeding in many species. Whether reactive oxygen species affect feeding through interaction with nitric oxide is unclear. We previously reported that Immp2l mutation in mice causes excessive mitochondrial superoxide generation, which causes infertility and early signs of aging. In our present study, reduced food intake in mutant mice resulted in significantly reduced body weight and fat composition while energy expenditure remained unchanged. Lysate from mutant brain showed a significant decrease in cGMP levels, suggesting insufficient nitric oxide signaling. Thus, our data suggests that reactive oxygen species may regulate food intake through modulating the bioavailability of nitric oxide.

  5. Cytotoxic effects induced by interferon-ω gene lipofection through ROS generation and mitochondrial membrane potential disruption in feline mammary carcinoma cells.

    Science.gov (United States)

    Villaverde, Marcela Solange; Targovnik, Alexandra Marisa; Miranda, María Victoria; Finocchiaro, Liliana María Elena; Glikin, Gerardo Claudio

    2016-08-01

    Progress in comparative oncology promises advances in clinical cancer treatments for both companion animals and humans. In this context, feline mammary carcinoma (FMC) cells have been proposed as a suitable model to study human breast cancer. Based on our previous data about the advantages of using type I interferon gene therapy over the respective recombinant DNA derived protein, the present work explored the effects of feline interferon-ω gene (fIFNω) transfer on FMC cells. Three different cell variants derived from a single spontaneous highly aggressive FMC tumor were successfully established and characterized. Lipofection of the fIFNω gene displayed a significant cytotoxic effect on the three cell variants. The extent of the response was proportional to ROS generation, mitochondrial membrane potential disruption and calcium uptake. Moreover, a lower sensitivity to the treatment correlated with a higher malignant phenotype. Our results suggest that fIFNω lipofection could offer an alternative approach in veterinary oncology with equal or superior outcome and with less adverse effects than recombinant fIFNω therapy. PMID:27236354

  6. T cells and macrophages in Trypanosoma brucei-related glomerulopathy.

    Science.gov (United States)

    van Velthuysen, M L; Mayen, A E; van Rooijen, N; Fleuren, G J; de Heer, E; Bruijn, J A

    1994-08-01

    In a previous study, susceptibility for Trypanosoma brucei-related glomerulopathy in mice was shown to be dependent on non-major histocompatibility complex genes. Glomerular disease in this model could not be explained by the production of autoantibodies alone. In order to analyze which part of the defense system, in addition to the B-cell compartment, is involved in the development of this infection-related glomerular disease, groups of athymic (BALB/c rnu/rnu), splenectomized, or macrophage-depleted BALB/c mice were inoculated with T. brucei parasites. Polyclonal B-cell activation, invariably observed in infected BALB/c mice, was absent in BALB/c rnu/rnu mice. Glomerular disease in athymic mice, however, as defined by albuminuria and deposition of immune complexes, was not different from that seen in euthymic infected BALB/c mice. Splenectomy prior to inoculation of parasites led to a decreased incidence of albuminuria in 40% of the animals, whereas splenectomy 21 days after inoculation reduced albuminuria significantly, suggesting a role for spleen cells in the induction of glomerular disease. After macrophage depletion with liposome-encapsulated dichlorodimethylene-diphosphonate, infected BALB/c mice developed significantly higher albuminuria levels for a period up to 2 weeks after depletion. Therefore, it was concluded that the development of T. brucei-related glomerular disease is independent of thymus-matured T cells, while the involvement of macrophages in the development of proteinuria is inhibitory rather than disease inducing. Spleen cells other than thymus-dependent T cells, B cells, and macrophages should be investigated for their role in the pathogenesis of this glomerulopathy. PMID:7913696

  7. VSG gene expression site control in insect form Trypanosoma brucei.

    OpenAIRE

    Rudenko, G; Blundell, P A; Taylor, M. C.; Kieft, R.; Borst, P

    1994-01-01

    When the African trypanosome Trypanosoma brucei is taken up from mammals by a tse-tse fly, it replaces its variant surface glycoprotein (VSG) coat by a procyclin coat. Transcription of VSG genes stops in the fly, but transcription of sequences derived from the promoter area of the VSG expression site(s) remains high. Whether this is due to continuing high activity of one promoter or to low activity of many promoters was unclear. We have used the small differences between the sequences of diff...

  8. Apolipoprotein L-I Promotes Trypanosome Lysis by Forming Pores in Lysosomal Membranes

    Science.gov (United States)

    Pérez-Morga, David; Vanhollebeke, Benoit; Paturiaux-Hanocq, Françoise; Nolan, Derek P.; Lins, Laurence; Homblé, Fabrice; Vanhamme, Luc; Tebabi, Patricia; Pays, Annette; Poelvoorde, Philippe; Jacquet, Alain; Brasseur, Robert; Pays, Etienne

    2005-07-01

    Apolipoprotein L-I is the trypanolytic factor of human serum. Here we show that this protein contains a membrane pore-forming domain functionally similar to that of bacterial colicins, flanked by a membrane-addressing domain. In lipid bilayer membranes, apolipoprotein L-I formed anion channels. In Trypanosoma brucei, apolipoprotein L-I was targeted to the lysosomal membrane and triggered depolarization of this membrane, continuous influx of chloride, and subsequent osmotic swelling of the lysosome until the trypanosome lysed.

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

    KAUST Repository

    Wang, Jingyan

    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.

  10. Mitochondrial Ion Channels

    Science.gov (United States)

    O’Rourke, Brian

    2009-01-01

    In work spanning more than a century, mitochondria have been recognized for their multifunctional roles in metabolism, energy transduction, ion transport, inheritance, signaling, and cell death. Foremost among these tasks is the continuous production of ATP through oxidative phosphorylation, which requires a large electrochemical driving force for protons across the mitochondrial inner membrane. This process requires a membrane with relatively low permeability to ions to minimize energy dissipation. However, a wealth of evidence now indicates that both selective and nonselective ion channels are present in the mitochondrial inner membrane, along with several known channels on the outer membrane. Some of these channels are active under physiological conditions, and others may be activated under pathophysiological conditions to act as the major determinants of cell life and death. This review summarizes research on mitochondrial ion channels and efforts to identify their molecular correlates. Except in a few cases, our understanding of the structure of mitochondrial ion channels is limited, indicating the need for focused discovery in this area. PMID:17059356

  11. Effects of DMSO on Diminazene Efficacy in Experimental Murine T. brucei Infection

    OpenAIRE

    K.I. Eghianruwa; Anika, S.M.

    2012-01-01

    This study evaluated the influence of dimethyl sulfoxide (DMSO) daily supplementation on diminazene treatment of trypanosomosis. Four groups of Trypanosoma brucei brucei infected rats received 7.0 mg/kg diminazene aceturate on day 7 post infection. Three of the four groups received different doses of DMSO (0.5, 1.0 and 2.0 g/kg, respectively) in addition to diminazene treatment. The changes in hematological parameters and the weights of liver, spleen and heart caused by T. brucei infection we...

  12. 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.

  13. Legionella pneumophila Secretes a Mitochondrial Carrier Protein during Infection

    OpenAIRE

    Pavel Dolezal; Margareta Aili; Janette Tong; Jhih-Hang Jiang; Marobbio, Carlo M.T.; Sau Fung Lee; Ralf Schuelein; Simon Belluzzo; Eva Binova; Aurelie Mousnier; Gad Frankel; Giulia Giannuzzi; Ferdinando Palmieri; Kipros Gabriel; Thomas Naderer

    2012-01-01

    Author Summary Mitochondrial carrier proteins evolved during endosymbiosis to transport substrates across the mitochondrial inner membrane. As such the proteins are associated exclusively with eukaryotic organisms. Despite this, we identified putative mitochondrial carrier proteins in the genomes of different intracellular bacterial pathogens, including Legionella pneumophila, the causative agent of Legionnaire's disease. We named the mitochondrial carrier protein from L. pneumophila LncP and...

  14. Sulforaphene-Carboplatin Combination Synergistically Enhances Apoptosis by Disruption of Mitochondrial Membrane Potential and Cell Cycle Arrest in Human Non-Small Cell Lung Carcinoma.

    Science.gov (United States)

    Chatterjee, Saswata; Rhee, Yun-Hee; Ahn, Jin-Chul

    2016-09-01

    Worldwide non-small cell lung cancer (NSCLC) causes substantial morbidity and mortality among human populations. Due to the severe side effects and low survival rate of patients with the conventional drugs, implementation of new combination therapies is much needed. The aim of this study was to evaluate the efficacy of a combination therapy with a conventional drug and a natural medicine. We compared the combination of chemotherapy drug carboplatin and the radish-derived isothiocyanate compound sulforaphene, which synergistically induces higher apoptosis and growth inhibition in A549, to the drug alone in human NSCLC cells. We found that this combination group significantly induced higher depolarization of mitochondrial membrane potential (MMP) and intracellular reactive oxygen species generation than the single drug dose, followed by cell cycle arrest at the G0/G1 phase after 24 h of incubation. In addition to that, the Western blot assays showed that combination treatment inhibited the expression of Bcl-2 and successively upregulated the expression of Bax, cytochrome C, apoptosis-inducing factor, caspase-9 and -3, and cleaved poly ADP ribose polymerase. It also modulated the expression of PI3K, p-extracellular signal-regulated kinase (1/2), and p-c-Jun N-terminal kinase indicating the involvement of antiproliferative properties. Further pretreatment with pan-caspase inhibitor Z-VAD-fmk was carried out to confirm the effect of caspases in the combination therapy-induced apoptosis. To summarize, this is the first report that sulforaphene-carboplatin combination treatment synergistically promotes enhanced apoptosis and antiproliferative effect over single drug treatment against A549, human NSCLC cells through caspase activation, MMP disruption, and cell cycle arrest. This study demonstrates that the duel character of this combination therapy may be an effective replacement for conventional therapy alone against NSCLC. PMID:27467015

  15. Removal of spermatozoa with externalized phosphatidylserine from sperm preparation in human assisted medical procreation: effects on viability, motility and mitochondrial membrane potential

    Directory of Open Access Journals (Sweden)

    de Agostini Ariane

    2009-01-01

    Full Text Available Abstract Background Externalization of phosphatidylserine (EPS occurs in apoptotic-like spermatozoa and could be used to remove them from sperm preparations to enhance sperm quality for assisted medical procreation. We first characterized EPS in sperms from infertile patients in terms of frequency of EPS spermatozoa as well as localization of phosphatidylserine (PS on spermatozoa. Subsequently, we determined the impact of depleting EPS spermatozoa on sperm quality. Methods EPS were visualized by fluorescently-labeled annexin V binding assay. Double staining with annexin V and Hoechst differentiates apoptotic from necrotic spermatozoa. We used magnetic-activated cell sorting using annexin V-conjugated microbeads (MACS-ANMB technique to remove EPS spermatozoa from sperm prepared by density gradient centrifugation (DGC. The impact of this technique on sperm quality was evaluated by measuring progressive motility, viability, and the integrity of the mitochondrial membrane potential (MMP by Rhodamine 123. Results Mean percentages of EPS spermatozoa were 14% in DGC sperm. Four subpopulations of spermatozoa were identified: 70% alive, 3% early apoptotic, 16% necrotic and 11% late apoptotic or necrotic. PS were localized on head and/or midpiece or on the whole spermatozoa. MACS efficiently eliminates EPS spermatozoa. MACS combined with DGC allows a mean reduction of 70% in EPS and of 60% in MMP-disrupted spermatozoa with a mean increase of 50% in sperm survival at 24 h. Conclusion Human ejaculates contain EPS spermatozoa which can mostly be eliminated by DGC plus MACS resulting in improved sperm long term viability, motility and MMP integrity. EPS may be used as an indicator of sperm quality and removal of EPS spermatozoa may enhance fertility potential in assisted medical procreation.

  16. Sclareol, a plant diterpene, exhibits potent antiproliferative effects via the induction of apoptosis and mitochondrial membrane potential loss in osteosarcoma cancer cells.

    Science.gov (United States)

    Wang, Lin; He, Hong-Sheng; Yu, Hua-Long; Zeng, Yun; Han, Heng; He, Ning; Liu, Zhi-Gang; Wang, Zhi-Yong; Xu, Shou-Jia; Xiong, Min

    2015-06-01

    The objective of the current study was to evaluate the antiproliferative activity of sclareol against MG63 osteosarcoma cells. A 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay was used to evaluate the cell viability of cells following treatment with sclareol. The extent of cell death induced by sclareol was evaluated using a lactate dehydrogenase (LDH) assay. The effect of sclareol on cell cycle progression and mitochondrial membrane potential (ΛΨm) was evaluated with flow cytometry using the DNA‑binding fluorescent dyes propidium iodide and rhodamine‑123, respectively. Fluorescence microscopy was used to detect the morphological changes in the MG63 osteosarcoma cancer cells and the appearance of apoptotic bodies following sclareol treatment. The results revealed that sclareol induced dose‑ and time‑dependent growth inhibition of MG63 cancer cells with an IC50 value of 65.2 µM following a 12‑h incubation. Furthermore, sclareol induced a significant increase in the release of LDH from MG63 cell cultures, which was much more pronounced at higher doses. Fluorescence microscopy revealed that sclareol induced characteristic morphological features of apoptosis and the appearance of apoptotic bodies. Flow cytometry revealed that sclareol induced G1‑phase cell cycle arrest, which showed significant dose‑dependence. Additionally, sclareol induced a progressive and dose‑dependent reduction in the ΛΨm. In summary, sclareol inhibits the growth of osteosarcoma cancer cells via the induction of apoptosis, which is accompanied by G1‑phase cell cycle arrest and loss of ΛΨm.

  17. 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.

  18. Cofactor-independent phosphoglycerate mutase is an essential gene in procyclic form Trypanosoma brucei.

    Science.gov (United States)

    Djikeng, Appolinaire; Raverdy, Sylvine; Foster, Jeremy; Bartholomeu, Daniella; Zhang, Yinhua; El-Sayed, Najib M; Carlow, Clotilde

    2007-03-01

    Glycolysis and gluconeogenesis are, in part, driven by the interconversion of 3- and 2-phosphoglycerate (3-PG and 2-PG) which is performed by phosphoglycerate mutases (PGAMs) which can be cofactor dependant (dPGAM) or cofactor independent (iPGAM). The African trypanosome, Trypanosoma brucei, possesses the iPGAM form which is thought to play an important role in glycolysis. Here, we report on the use of RNA interference to down-regulate the T. brucei iPGAM in procyclic form T. brucei and evaluation of the resulting phenotype. We first demonstrated biochemically that depletion of the steady state levels of iPGM mRNA correlates with a marked reduction of enzyme activity. We further show that iPGAM is required for cell growth in procyclic T. brucei.

  19. Hidden Markov Models for Gene Sequence Classification: Classifying the VSG genes in the Trypanosoma brucei Genome

    OpenAIRE

    Mesa, Andrea; Basterrech, Sebastián; Guerberoff, Gustavo; Alvarez-Valin, Fernando

    2015-01-01

    The article presents an application of Hidden Markov Models (HMMs) for pattern recognition on genome sequences. We apply HMM for identifying genes encoding the Variant Surface Glycoprotein (VSG) in the genomes of Trypanosoma brucei (T. brucei) and other African trypanosomes. These are parasitic protozoa causative agents of sleeping sickness and several diseases in domestic and wild animals. These parasites have a peculiar strategy to evade the host's immune system that consists in periodicall...

  20. Mechanism of Trypanosoma brucei gambiense (group 1) resistance to human trypanosome lytic factor.

    Science.gov (United States)

    Kieft, Rudo; Capewell, Paul; Turner, C Michael R; Veitch, Nicola J; MacLeod, Annette; Hajduk, Stephen

    2010-09-14

    Human innate immunity against most African trypanosomes, including Trypanosoma brucei brucei, is mediated by a minor subclass of toxic serum HDL, called trypanosome lytic factor-1 (TLF-1). This HDL contains two primate specific proteins, apolipoprotein L-1 and haptoglobin (Hp)-related protein, as well as apolipoprotein A-1. These assembled proteins provide a powerful defense against trypanosome infection. Trypanosoma brucei rhodesiense causes human African sleeping sickness because it has evolved an inhibitor of TLF-1, serum resistance-associated (SRA) protein. Trypanosoma brucei gambiense lacks the SRA gene, yet it infects humans. As transfection of T. b. gambiense (group 1) is not possible, we initially used in vitro-selected TLF-1-resistant T. b. brucei to examine SRA-independent mechanisms of TLF-1 resistance. Here we show that TLF-1 resistance in T. b. brucei is caused by reduced expression of the Hp/Hb receptor gene (TbbHpHbR). Importantly, T. b. gambiense (group 1) also showed a marked reduction in uptake of TLF-1 and a corresponding decrease in expression of T. b. gambiense Hp/Hb receptor (TbgHpHbR). Ectopic expression of TbbHpHbR in TLF-1-resistant T. b. brucei rescued TLF-1 uptake, demonstrating that decreased TbbHpHbR expression conferred TLF-1 resistance. Ectopic expression of TbgHpHbR in TLF-1-resistant T. b. brucei failed to rescue TLF-1 killing, suggesting that coding sequence changes altered Hp/Hb receptor binding affinity for TLF-1. We propose that the combination of coding sequence mutations and decreased expression of TbgHpHbR directly contribute to parasite evasion of human innate immunity and infectivity of group 1 T. b. gambiense. PMID:20805508

  1. 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...

  2. Mitochondrial Diseases

    Science.gov (United States)

    ... disorder, something goes wrong with this process. Mitochondrial diseases are a group of metabolic disorders. Mitochondria are ... cells and cause damage. The symptoms of mitochondrial disease can vary. It depends on how many mitochondria ...

  3. Mosaic VSGs and the scale of Trypanosoma brucei antigenic variation.

    Directory of Open Access Journals (Sweden)

    James P J Hall

    Full Text Available A main determinant of prolonged Trypanosoma brucei infection and transmission and success of the parasite is the interplay between host acquired immunity and antigenic variation of the parasite variant surface glycoprotein (VSG coat. About 0.1% of trypanosome divisions produce a switch to a different VSG through differential expression of an archive of hundreds of silent VSG genes and pseudogenes, but the patterns and extent of the trypanosome diversity phenotype, particularly in chronic infection, are unclear. We applied longitudinal VSG cDNA sequencing to estimate variant richness and test whether pseudogenes contribute to antigenic variation. We show that individual growth peaks can contain at least 15 distinct variants, are estimated computationally to comprise many more, and that antigenically distinct 'mosaic' VSGs arise from segmental gene conversion between donor VSG genes or pseudogenes. The potential for trypanosome antigenic variation is probably much greater than VSG archive size; mosaic VSGs are core to antigenic variation and chronic infection.

  4. Hsp90 inhibition decreases mitochondrial protein turnover.

    Directory of Open Access Journals (Sweden)

    Daciana H Margineantu

    Full Text Available BACKGROUND: Cells treated with hsp90 inhibitors exhibit pleiotropic changes, including an expansion of the mitochondrial compartment, accompanied by mitochondrial fragmentation and condensed mitochondrial morphology, with ultimate compromise of mitochondrial integrity and apoptosis. FINDINGS: We identified several mitochondrial oxidative phosphorylation complex subunits, including several encoded by mtDNA, that are upregulated by hsp90 inhibitors, without corresponding changes in mRNA abundance. Post-transcriptional accumulation of mitochondrial proteins observed with hsp90 inhibitors is also seen in cells treated with proteasome inhibitors. Detailed studies of the OSCP subunit of mitochondrial F1F0-ATPase revealed the presence of mono- and polyubiquitinated OSCP in mitochondrial fractions. We demonstrate that processed OSCP undergoes retrotranslocation to a trypsin-sensitive form associated with the outer mitochondrial membrane. Inhibition of proteasome or hsp90 function results in accumulation of both correctly targeted and retrotranslocated mitochondrial OSCP. CONCLUSIONS: Cytosolic turnover of mitochondrial proteins demonstrates a novel connection between mitochondrial and cytosolic compartments through the ubiquitin-proteasome system. Analogous to defective protein folding in the endoplasmic reticulum, a mitochondrial unfolded protein response may play a role in the apoptotic effects of hsp90 and proteasome inhibitors.

  5. Effects of nicorandil on cardiac plasma membrane and cardiac mitochondrial membrane potential of guinea-pig%尼可地尔对豚鼠心肌细胞膜及线粒体膜电位的影响

    Institute of Scientific and Technical Information of China (English)

    冯力; 刘伊丽; 刘杰; 金春华

    2001-01-01

    研究KATP通道开放剂尼可地尔(Nic)对豚鼠心肌细胞膜和线粒体膜电位的影响.用激光共聚焦显微镜和特异性荧光探针,观察不同剂量的Nic及KATP通道阻滞剂格列本脲(Gli)引起急性分离的豚鼠心肌细胞膜电位,线粒体膜电位荧光值的变化.Nic1mmol.L-1引起细胞膜电位在1min内迅速超极化〔膜电位荧光值减少(75±12)%〕,Gli3μmol.L-1可阻断其变化;0.1和1mmol.L-1Nic可使线粒体膜电位去极化和膜电位荧光值在1,2,5min分别增加(12±3)%和(32±8)%,(25±6)%和(39±9)%,(34±6)%和(45±12)%;3μmol.L-1Gli可抑制其变化.结果说明低浓度Nic只引起线粒体膜电位去极化,高浓度Nic还可使细胞膜电位发生超极化,引起KATP通道开放.%With digital imaging techniques of advanced laser confocalmicroscope, effects of KATP channel opener nicorandil(Nic) on cardiac plasma membrane(CPM) and cardiac mitochondrial membrane(CMM) potential of guinea-pig were studied. It was found that Nic 1 mmol.L-1 caused the potential of CPM more negative (hyperpolarization), fluorescence intensity(FI) decreased by (75±12)% of baseline within 1 min, but no effect at 0.1 mmol.L-1. CMM was depolarized by 0.1 mmol.L-1 Nic〔FI increased by (12±3)%, (25±6)%, (34±6)% of baseline within 1, 2, 5 min〕, and by 1 mmol.L-1 Nic〔FI remarkably increased by (32±8)%, (39±9)%, (45±12)% of baseline〕. KATP channel blocker glibenclamide 3 μmol.L-1 itself caused no effect on potential of CPM and CMM, but blocked the above effect on potential of CPM and CMM induced by Nic. The results suggest that KATP channel of CMM is activated by low dose of Nic, and the high dose of Nic activate both KATP channels of CPM and CMM.

  6. MIF Contributes to Trypanosoma brucei Associated Immunopathogenicity Development

    Science.gov (United States)

    Stijlemans, Benoît; Leng, Lin; Brys, Lea; Sparkes, Amanda; Vansintjan, Liese; Caljon, Guy; Raes, Geert; Van Den Abbeele, Jan; Van Ginderachter, Jo A.; Beschin, Alain

    2014-01-01

    African trypanosomiasis is a chronic debilitating disease affecting the health and economic well-being of many people in developing countries. The pathogenicity associated with this disease involves a persistent inflammatory response, whereby M1-type myeloid cells, including Ly6Chigh inflammatory monocytes, are centrally implicated. A comparative gene analysis between trypanosusceptible and trypanotolerant animals identified MIF (macrophage migrating inhibitory factor) as an important pathogenic candidate molecule. Using MIF-deficient mice and anti-MIF antibody treated mice, we show that MIF mediates the pathogenic inflammatory immune response and increases the recruitment of inflammatory monocytes and neutrophils to contribute to liver injury in Trypanosoma brucei infected mice. Moreover, neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to increased pathogenic liver TNF production and liver injury during trypanosome infection. MIF deficient animals also featured limited anemia, coinciding with increased iron bio-availability, improved erythropoiesis and reduced RBC clearance during the chronic phase of infection. Our data suggest that MIF promotes the most prominent pathological features of experimental trypanosome infections (i.e. anemia and liver injury), and prompt considering MIF as a novel target for treatment of trypanosomiasis-associated immunopathogenicity. PMID:25255103

  7. MIF contributes to Trypanosoma brucei associated immunopathogenicity development.

    Directory of Open Access Journals (Sweden)

    Benoît Stijlemans

    2014-09-01

    Full Text Available African trypanosomiasis is a chronic debilitating disease affecting the health and economic well-being of many people in developing countries. The pathogenicity associated with this disease involves a persistent inflammatory response, whereby M1-type myeloid cells, including Ly6C(high inflammatory monocytes, are centrally implicated. A comparative gene analysis between trypanosusceptible and trypanotolerant animals identified MIF (macrophage migrating inhibitory factor as an important pathogenic candidate molecule. Using MIF-deficient mice and anti-MIF antibody treated mice, we show that MIF mediates the pathogenic inflammatory immune response and increases the recruitment of inflammatory monocytes and neutrophils to contribute to liver injury in Trypanosoma brucei infected mice. Moreover, neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to increased pathogenic liver TNF production and liver injury during trypanosome infection. MIF deficient animals also featured limited anemia, coinciding with increased iron bio-availability, improved erythropoiesis and reduced RBC clearance during the chronic phase of infection. Our data suggest that MIF promotes the most prominent pathological features of experimental trypanosome infections (i.e. anemia and liver injury, and prompt considering MIF as a novel target for treatment of trypanosomiasis-associated immunopathogenicity.

  8. Mitochondrial Myopathy

    Science.gov (United States)

    ... NINDS supports research focused on effective treatments and cures for mitochondrial myopathies and other mitochondrial diseases. Scientists are investigating the possible benefits of exercise programs and nutritional supplements, primarily natural and synthetic versions of CoQ10. While CoQ10 has ...

  9. 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.

  10. Mitochondrial cytopathies.

    Science.gov (United States)

    El-Hattab, Ayman W; Scaglia, Fernando

    2016-09-01

    Mitochondria are found in all nucleated human cells and perform a variety of essential functions, including the generation of cellular energy. Most of mitochondrial proteins are encoded by the nuclear DNA (nDNA) whereas a very small fraction is encoded by the mitochondrial DNA (mtDNA). Mutations in mtDNA or mitochondria-related nDNA genes can result in mitochondrial dysfunction which leads to a wide range of cellular perturbations including aberrant calcium homeostasis, excessive reactive oxygen species production, dysregulated apoptosis, and insufficient energy generation to meet the needs of various organs, particularly those with high energy demand. Impaired mitochondrial function in various tissues and organs results in the multi-organ manifestations of mitochondrial diseases including epilepsy, intellectual disability, skeletal and cardiac myopathies, hepatopathies, endocrinopathies, and nephropathies. Defects in nDNA genes can be inherited in an autosomal or X-linked manners, whereas, mtDNA is maternally inherited. Mitochondrial diseases can result from mutations of nDNA genes encoding subunits of the electron transport chain complexes or their assembly factors, proteins associated with the mitochondrial import or networking, mitochondrial translation factors, or proteins involved in mtDNA maintenance. MtDNA defects can be either point mutations or rearrangements. The diagnosis of mitochondrial disorders can be challenging in many cases and is based on clinical recognition, biochemical screening, histopathological studies, functional studies, and molecular genetic testing. Currently, there are no satisfactory therapies available for mitochondrial disorders that significantly alter the course of the disease. Therapeutic options include symptomatic treatment, cofactor supplementation, and exercise. PMID:26996063

  11. 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

  12. Cancer: Mitochondrial Origins.

    Science.gov (United States)

    Stefano, George B; Kream, Richard M

    2015-12-01

    The primacy of glucose derived from photosynthesis as an existential source of chemical energy across plant and animal phyla is universally accepted as a core principle in the biological sciences. In mammalian cells, initial processing of glucose to triose phosphate intermediates takes place within the cytosolic glycolytic pathway and terminates with temporal transport of reducing equivalents derived from pyruvate metabolism by membrane-associated respiratory complexes in the mitochondrial matrix. The intra-mitochondrial availability of molecular oxygen as the ultimate electron acceptor drives the evolutionary fashioned chemiosmotic production of ATP as a high-efficiency biological process. The mechanistic bases of carcinogenesis have demonstrated profound alteration of normative mitochondrial function, notably dysregulated respiratory processes. Accordingly, the classic Warburg effect functionally links aerobic glycolysis, aberrant production and release of lactate, and metabolic down-regulation of mitochondrial oxidative processes with the carcinogenetic phenotype. We surmise, however, that aerobic fermentation by cancer cells may also represent a developmental re-emergence of an evolutionarily conserved early phenotype, which was "sidelined" with the emergence of mitochondrial oxidative phosphorylation as a primary mechanism for ATP production in normal cells. Regardless of state-dependent physiological status in mixed populations of cancer cells, it has been established that mitochondria are functionally linked to the initiation of cancer and its progression. Biochemical, molecular, and physiological differences in cancer cell mitochondria, notably mtDNA heteroplasmy and allele-specific expression of selected nuclear genes, may represent major focal points for novel targeting and elimination of cancer cells in metastatic disease afflicting human populations. To date, and despite considerable research efforts, the practical realization of advanced mitochondrial

  13. ERp57 modulates mitochondrial calcium uptake through the MCU.

    Science.gov (United States)

    He, Jingquan; Shi, Weikang; Guo, Yu; Chai, Zhen

    2014-06-01

    ERp57 participates in the regulation of calcium homeostasis. Although ERp57 modulates calcium flux across the plasma membrane and the endoplasmic reticulum membrane, its functions on mitochondria are largely unknown. Here, we found that ERp57 can regulate the expression of the mitochondrial calcium uniporter (MCU) and modulate mitochondrial calcium uptake. In ERp57-silenced HeLa cells, MCU was downregulated, and the mitochondrial calcium uptake was inhibited, consistent with the effect of MCU knockdown. When MCU was re-expressed in the ERp57 knockdown cells, mitochondrial calcium uptake was restored. Thus, ERp57 is a potent regulator of mitochondrial calcium homeostasis.

  14. Mitochondrial Cristae: Where Beauty Meets Functionality.

    Science.gov (United States)

    Cogliati, Sara; Enriquez, Jose A; Scorrano, Luca

    2016-03-01

    Mitochondrial cristae are dynamic bioenergetic compartments whose shape changes under different physiological conditions. Recent discoveries have unveiled the relation between cristae shape and oxidative phosphorylation (OXPHOS) function, suggesting that membrane morphology modulates the organization and function of the OXPHOS system, with a direct impact on cellular metabolism. As a corollary, cristae-shaping proteins have emerged as potential modulators of mitochondrial bioenergetics, a concept confirmed by genetic experiments in mouse models of respiratory chain deficiency. Here, we review our knowledge of mitochondrial ultrastructural organization and how it impacts mitochondrial metabolism.

  15. Mitochondrial Cristae: Where Beauty Meets Functionality.

    Science.gov (United States)

    Cogliati, Sara; Enriquez, Jose A; Scorrano, Luca

    2016-03-01

    Mitochondrial cristae are dynamic bioenergetic compartments whose shape changes under different physiological conditions. Recent discoveries have unveiled the relation between cristae shape and oxidative phosphorylation (OXPHOS) function, suggesting that membrane morphology modulates the organization and function of the OXPHOS system, with a direct impact on cellular metabolism. As a corollary, cristae-shaping proteins have emerged as potential modulators of mitochondrial bioenergetics, a concept confirmed by genetic experiments in mouse models of respiratory chain deficiency. Here, we review our knowledge of mitochondrial ultrastructural organization and how it impacts mitochondrial metabolism. PMID:26857402

  16. Novel super-resolution capable mitochondrial probe, MitoRed AIE, enables assessment of real-time molecular mitochondrial dynamics

    Science.gov (United States)

    Lo, Camden Yeung-Wah; Chen, Sijie; Creed, Sarah Jayne; Kang, Miaomiao; Zhao, Na; Tang, Ben Zhong; Elgass, Kirstin Diana

    2016-01-01

    Mitochondria and mitochondrial dynamics play vital roles in health and disease. With the intricate nanometer-scale structure and rapid dynamics of mitochondria, super-resolution microscopy techniques possess great un-tapped potential to significantly contribute to understanding mitochondrial biology and kinetics. Here we present a novel mitochondrial probe (MitoRed AIE) suitable for live mitochondrial dynamics imaging and single particle tracking (SPT), together with a multi-dimensional data analysis approach to assess local mitochondrial (membrane) fluidity. The MitoRed AIE probe localizes primarily to mitochondrial membranes, with 95 ms fluorophore on-time delivering 106 photons/ms, characteristics which we exploit to demonstrate live cell 100 fps 3D time-lapse tracking of mitochondria. Combining our experimental and analytical approaches, we uncover mitochondrial dynamics at unprecedented time scales. This approach opens up a new regime into high spatio-temporal resolution dynamics in many areas of mitochondrial biology. PMID:27492961

  17. 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 cod

  18. Involvement of lysosomes in the uptake of macromolecular material by bloodstream forms of Trypanosoma brucei.

    Science.gov (United States)

    Opperdoes, F R; Van Roy, J

    1982-09-01

    To investigate whether the lysosomes of Trypanosoma brucei are capable of uptake of macromolecules after internalization by the cell, we used Triton WR-1339, a non-digestible macromolecular compound, which is known to cause a marked decrease in the density of hepatic lysosomes due to massive intralysosomal storage. Intraperitoneal administration of 0.4 g/kg Triton WR-1339 to rats infected with T. brucei led to the development of a large vacuole in the trypanosomes between nucleus and kinetoplast within 22 h. Higher doses (2 g/kg) led to the disappearance of the trypanosomes from the blood and resulted in permanent cures (greater than 100 days). Lysosomes isolated from the trypanosomes of animals treated with a sub-curative dose showed a decrease in equilibrium density of 0.03 g/cm3 in sucrose gradients. These lysosomes were partly damaged as evidenced by a reduction in latency and an increase in the non-sedimentable part of lysosomal enzymes. We conclude that acid proteinase and alpha-mannosidase-containing organelles of T. brucei take up exogenous macromolecules and must therefore be considered as true lysosomes and that Triton WR-1339 acts in T. brucei as a true lysosomotropic drug. Its trypanocidal action probably results from an interference with lysosomal function.

  19. Dynamic Modelling under Uncertainty : The Case of Trypanosoma brucei Energy Metabolism

    NARCIS (Netherlands)

    Achcar, Fiona; Kerkhoven, Eduard J.; Bakker, Barbara M.; Barrett, Michael P.; Breitling, Rainer; Papin, Jason A.

    2012-01-01

    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 metabol

  20. Handling Uncertainty in Dynamic Models : The Pentose Phosphate Pathway in Trypanosoma brucei

    NARCIS (Netherlands)

    Kerkhoven, Eduard J.; Achcar, Fiona; Alibu, Vincent P.; Burchmore, Richard J.; Gilbert, Ian H.; Trybilo, Maciej; Driessen, Nicole N.; Gilbert, David; Breitling, Rainer; Bakker, Barbara M.; Barrett, Michael P.

    2013-01-01

    Dynamic models of metabolism can be useful in identifying potential drug targets, especially in unicellular organisms. A model of glycolysis in the causative agent of human African trypanosomiasis, Trypanosoma brucei, has already shown the utility of this approach. Here we add the pentose phosphate

  1. Telomere length affects the frequency and mechanism of antigenic variation in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Galadriel A Hovel-Miner

    Full Text Available Trypanosoma brucei is a master of antigenic variation and immune response evasion. Utilizing a genomic repertoire of more than 1000 Variant Surface Glycoprotein-encoding genes (VSGs, T. brucei can change its protein coat by "switching" from the expression of one VSG to another. Each active VSG is monoallelically expressed from only one of approximately 15 subtelomeric sites. Switching VSG expression occurs by three predominant mechanisms, arguably the most significant of which is the non-reciprocal exchange of VSG containing DNA by duplicative gene conversion (GC. How T. brucei orchestrates its complex switching mechanisms remains to be elucidated. Recent work has demonstrated that an exogenous DNA break in the active site could initiate a GC based switch, yet the source of the switch-initiating DNA lesion under natural conditions is still unknown. Here we investigated the hypothesis that telomere length directly affects VSG switching. We demonstrate that telomerase deficient strains with short telomeres switch more frequently than genetically identical strains with long telomeres and that, when the telomere is short, switching preferentially occurs by GC. Our data supports the hypothesis that a short telomere at the active VSG expression site results in an increase in subtelomeric DNA breaks, which can initiate GC based switching. In addition to their significance for T. brucei and telomere biology, the findings presented here have implications for the many diverse pathogens that organize their antigenic genes in subtelomeric regions.

  2. Nucleic acid sequence-based amplification with oligochromatography for detection of Trypanosoma brucei in clinical samples

    NARCIS (Netherlands)

    C.M. Mugasa; T. Laurent; G.J. Schoone; P.A. Kager; G.W. Lubega; H.D.F.H. Schallig

    2009-01-01

    Molecular tools, such as real-time nucleic acid sequence-based amplification (NASBA) and PCR, have been developed to detect Trypanosoma brucei parasites in blood for the diagnosis of human African trypanosomiasis (HAT). Despite good sensitivity, these techniques are not implemented in HAT control pr

  3. 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

  4. A core MRB1 complex component is indispensable for RNA editing in insect and human infective stages of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Michelle L Ammerman

    Full Text Available Uridine insertion/deletion RNA editing is a unique and vital process in kinetoplastids, required for creation of translatable open reading frames in most mitochondrially-encoded RNAs. Emerging as a key player in this process is the mitochondrial RNA binding 1 (MRB1 complex. MRB1 comprises an RNA-independent core complex of at least six proteins, including the GAP1/2 guide RNA (gRNA binding proteins. The core interacts in an RNA-enhanced or -dependent manner with imprecisely defined TbRGG2 subcomplexes, Armadillo protein MRB10130, and additional factors that comprise the dynamic MRB1 complex. Towards understanding MRB1 complex function in RNA editing, we present here functional characterization of the pentein domain-containing MRB1 core protein, MRB11870. Inducible RNAi studies demonstrate that MRB11870 is essential for proliferation of both insect vector and human infective stage T. brucei. MRB11870 ablation causes a massive defect in RNA editing, affecting both pan-edited and minimally edited mRNAs, but does not substantially affect mitochondrial RNA stability or processing of precursor transcripts. The editing defect in MRB1-depleted cells occurs at the initiation stage of editing, as pre-edited mRNAs accumulate. However, the gRNAs that direct editing remain abundant in the knockdown cells. To examine the contribution of MRB11870 to MRB1 macromolecular interactions, we tagged core complexes and analyzed their composition and associated proteins in the presence and absence of MRB11870. These studies demonstrated that MRB11870 is essential for association of GAP1/2 with the core, as well as for interaction of the core with other proteins and subcomplexes. Together, these data support a model in which the MRB1 core mediates functional interaction of gRNAs with the editing machinery, having GAP1/2 as its gRNA binding constituents. MRB11870 is a critical component of the core, essential for its structure and function.

  5. 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; 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 sequ

  6. Oxidative stress, mitochondrial damage and neurodegenerative diseases****

    Institute of Scientific and Technical Information of China (English)

    Chunyan Guo; Li Sun; Xueping Chen; Danshen Zhang

    2013-01-01

    Oxidative stress and mitochondrial damage have been implicated in the pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Oxidative stress is characterized by the overproduction of reactive oxygen species, which can induce mitochondrial DNA mutations, damage the mitochondrial respiratory chain, alter membrane permeability, and influence Ca2+ homeostasis and mitochondrial defense systems. Al these changes are implicated in the development of these neurodegenerative diseases, mediating or amplifying neuronal dysfunction and triggering neurodegeneration. This paper summarizes the contribution of oxidative stress and mitochondrial damage to the onset of neurodegenerative eases and discusses strategies to modify mitochondrial dysfunction that may be attractive thera-peutic interventions for the treatment of various neurodegenerative diseases.

  7. Flagellar membrane proteins in kinetoplastid parasites.

    Science.gov (United States)

    Landfear, Scott M; Tran, Khoa D; Sanchez, Marco A

    2015-09-01

    All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. Although flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites, including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted.

  8. Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry.

    Science.gov (United States)

    Guthrie, H D; Welch, G R

    2006-08-01

    The use of frozen semen in the swine industry is limited by problems with viability and fertility compared with liquid semen. Part of the reduction in sperm motility and fertility associated with cryopreservation may be due to oxidative damage from excessive or inappropriate formation of reactive oxygen species (ROS). Chemiluminescence measurements of ROS are not possible in live cells and are problematic because of poor specificity. An alternative approach, flow cytometry, was developed to identify viable boar sperm containing ROS utilizing the dyes hydroethidine and 2', 7'-dichlorodihydrofluorescein diacetate as oxidizable substrates and impermeant DNA dyes to exclude dead sperm. The percentage of sperm with high mitochondrial transmembrane potential was determined by flow cytometry using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide with propidium iodide staining to exclude nonviable cells. Sperm were incubated with and without ROS generators and free radical scavengers. Basal ROS formation was low (less than 4%) and did not differ (P = 0.26) between viable fresh and frozen-thawed boar sperm. In addition, fresh and frozen-thawed viable sperm were equally susceptible (P = 0.20) to intracellular formation of ROS produced by xanthine/xanthine oxidase (94.4 and 87.9% of sperm, respectively). Menadione increased (P boar sperm, both were quite susceptible to external sources of hydrogen peroxide. PMID:16864869

  9. Oxygen Glucose Deprivation in Rat Hippocampal Slice Cultures Results in Alterations in Carnitine Homeostasis and Mitochondrial Dysfunction

    OpenAIRE

    Thomas F. Rau; Qing Lu; Shruti Sharma; Xutong Sun; Gregory Leary; Beckman, Matthew L.; Yali Hou; Wainwright, Mark S; Michael Kavanaugh; Poulsen, David J.; Black, Stephen M.

    2012-01-01

    Mitochondrial dysfunction characterized by depolarization of mitochondrial membranes and the initiation of mitochondrial-mediated apoptosis are pathological responses to hypoxia-ischemia (HI) in the neonatal brain. Carnitine metabolism directly supports mitochondrial metabolism by shuttling long chain fatty acids across the inner mitochondrial membrane for beta-oxidation. Our previous studies have shown that HI disrupts carnitine homeostasis in neonatal rats and that L-carnitine can be neurop...

  10. The lysosomotropic drug LeuLeu-OMe induces lysosome disruption and autophagy-independent cell death in Trypanosoma brucei

    OpenAIRE

    Hazel Xinyu Koh; Htay Mon Aye; Tan, Kevin S W; He, Cynthia Y.

    2015-01-01

    Background: Trypanosoma brucei is a blood-borne, protozoan parasite that causes African sleeping sickness in humans and nagana in animals. The current chemotherapy relies on only a handful of drugs that display undesirable toxicity, poor efficacy and drug-resistance. In this study, we explored the use of lysosomotropic drugs to induce bloodstream form T. brucei cell death via lysosome destabilization. Methods: We measured drug concentrations that inhibit cell proliferation by 50% (...

  11. Experimental study on mitochondrial membrane potential and ultrastructure injuries of kidney in septic rats%脓毒症大鼠肾脏线粒体膜电位变化和超微结构损伤的研究

    Institute of Scientific and Technical Information of China (English)

    陈飞燕; 曾其毅; 赵明奇; 杨文敏; 连广琬

    2008-01-01

    Objective To study the mitochondrial membrane potential and ultrastructure injuries of kidney in septic rats. Methods Thirty SD rats were randomly divided into three groups: Saline control group, 6 h LPS group and 24 h LPS group. The rats in LPS groups received 10 mg/kg lipopolysaccharide by intraperitonesl injection. The levels of serum Cr and BUN were detected. The swelling (FSC/SSC) and the membrane potential (FL2/FL1) of isolated renal mitochondrion were tested by flow cytometry. The morphologic change of mitochondria in kidney was observed by electronic microscopy. Results Compared with control group, serum Cr and BUN increased in septic rats. Compared with control group, swelling of the mirochondrion significantly increased in 24 h LPS group ( Control: 0.55 ± 0.10; 6 h LPS: 0.58 ± 0.10; 24 h LPS: 0.66 ± 0.12, P<0.05). Renal mitochondrial membrane potential significantly decreased in 6 h LPS group and 24 h LPS group (Control:0.77 ± 0.26; 6 h LPS: 0.32 ± 0.19;24 h LPS: 0.30 ± 0.17, P < 0.01). The mitochondrial membrane potential in kidney were inversely correlated with serum Cr level( r = -0.510, P = 0.004). The injuries of mitochondrial ultrastructure increased in 24 h LPS group. Conclusion The injury of the renal mitochondrial membrane potential has occurred in early stage of sepsis before obvious ultrastructure damage of the renal mitochondrion in septic rats.%目的 探讨脓毒症时大鼠肾脏线粒体的损伤情况.方法 采用腹腔注射内毒素(LPS)建立脓毒症大鼠模型.30只SD大鼠随机分为对照组、6 h脓毒症组和24 h脓毒症组.分别检测各组大鼠血清肌酐(Cr)、尿素氮(BUN)水平;电镜观察肾脏线粒体形态学变化;运用流式细胞术检测分离肾脏线粒体的肿胀程度和线粒体膜电位,用前向角与侧向角的平均荧光强度比值(FSC/SSC)反映线粒体肿胀程度,用二通道和一通道平均荧光强度比值(FL2/FL1)确定线粒体膜电位.结果 脓毒症大鼠的血清Cr、BUN

  12. Mitochondrial dysfunction in Parkinson's disease.

    Science.gov (United States)

    Hu, Qingsong; Wang, Guanghui

    2016-01-01

    Parkinson's disease (PD) is the second most common neurodegenerative disease, which is characterized by loss of dopaminergic (DA) neurons in the substantia nigra pars compacta and the formation of Lewy bodies and Lewy neurites in surviving DA neurons in most cases. Although the cause of PD is still unclear, the remarkable advances have been made in understanding the possible causative mechanisms of PD pathogenesis. Numerous studies showed that dysfunction of mitochondria may play key roles in DA neuronal loss. Both genetic and environmental factors that are associated with PD contribute to mitochondrial dysfunction and PD pathogenesis. The induction of PD by neurotoxins that inhibit mitochondrial complex I provides direct evidence linking mitochondrial dysfunction to PD. Decrease of mitochondrial complex I activity is present in PD brain and in neurotoxin- or genetic factor-induced PD cellular and animal models. Moreover, PINK1 and parkin, two autosomal recessive PD gene products, have important roles in mitophagy, a cellular process to clear damaged mitochondria. PINK1 activates parkin to ubiquitinate outer mitochondrial membrane proteins to induce a selective degradation of damaged mitochondria by autophagy. In this review, we summarize the factors associated with PD and recent advances in understanding mitochondrial dysfunction in PD. PMID:27453777

  13. A mechanistic view of mitochondrial death decision pores

    OpenAIRE

    Belizário, J E; Alves, J.; J.M. Occhiucci; M. Garay-Malpartida; Sesso, A.

    2007-01-01

    Mitochondria increase their outer and inner membrane permeability to solutes, protons and metabolites in response to a variety of extrinsic and intrinsic signaling events. The maintenance of cellular and intraorganelle ionic homeostasis, particularly for Ca2+, can determine cell survival or death. Mitochondrial death decision is centered on two processes: inner membrane permeabilization, such as that promoted by the mitochondrial permeability transition pore, formed across inner membranes whe...

  14. Pyr3, a TRPC3 channel blocker, potentiates dexamethasone sensitivity and apoptosis in acute lymphoblastic leukemia cells by disturbing Ca(2+) signaling, mitochondrial membrane potential changes and reactive oxygen species production.

    Science.gov (United States)

    Abdoul-Azize, Souleymane; Buquet, Catherine; Vannier, Jean-Pierre; Dubus, Isabelle

    2016-08-01

    Dexamethasone (Dex) is used as a chemotherapeutic drug in the treatment of acute lymphoblastic leukemia (ALL) because of its capacity to induce apoptosis. However, some ALL patients acquire resistance to glucocorticoids (GC). Thus, it is important to explore new agents to overcome GC resistance. The aim of the present work was to assess the ability of Pyr3, a selective inhibitor of transient receptor potential canonical 3 (TRPC3), to sensitize human ALL cells to Dex. We show here, for the first time, that Pyr3 enhances Dex sensitivity through the distraction of Dex-mediated Ca(2+) signaling in ALL cells (in vitro) and primary blasts (ex vivo) associated with mitochondrial-mediated reactive oxygen species production in ALL cells. Pyr3 alone induced Ca(2+) signaling via only endoplasmic reticulum-released Ca(2+) and exerted inhibitory effect on store-operated Ca(2+) entry in dose-dependent manner in ALL cell lines. Pre-incubation of cells with Pyr3 significantly curtailed the thapsigargin- and Dex-evoked Ca(2+) signaling in ALL cell lines. Pyr3 synergistically potentiated Dex lethality, as shown by the induction of cell mortality, G2/M cell cycle arrest and apoptosis in ALL cell lines. Moreover, Pyr3 disrupted Dex-mediated Ca(2+) signaling and increased the sensitivity of Dex-induced cell death in primary blasts from ALL patients. Additional analysis showed that co-treatment with Dex and Pyr3 results in mitochondrial membrane potential depolarization and reactive oxygen species production in ALL cells. Together, Pyr3 exhibited potential therapeutic benefit in combination with Dex to inverse glucocorticoid resistance in human ALL and probably in other lymphoid malignancies. PMID:27179991

  15. Identification of a Wee1-like kinase gene essential for procyclic Trypanosoma brucei survival.

    Directory of Open Access Journals (Sweden)

    Natalia Y Boynak

    Full Text Available Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs. Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.

  16. 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.

  17. 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.

  18. 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.

  19. Mitochondrial dysfunction and organophosphorus compounds

    International Nuclear Information System (INIS)

    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

  20. 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

  1. Mitochondrial Fusion in Yeast Requires the Transmembrane GTPase Fzo1p

    OpenAIRE

    Hermann, Greg J.; Thatcher, John W.; Mills, John P.; Hales, Karen G.; Fuller, Margaret T.; Nunnari, Jodi; Shaw, Janet M.

    1998-01-01

    Membrane fusion is required to establish the morphology and cellular distribution of the mitochondrial compartment. In Drosophila, mutations in the fuzzy onions (fzo) GTPase block a developmentally regulated mitochondrial fusion event during spermatogenesis. Here we report that the yeast orthologue of fuzzy onions, Fzo1p, plays a direct and conserved role in mitochondrial fusion. A conditional fzo1 mutation causes the mitochondrial reticulum to fragment and blocks mitochondrial fusion during ...

  2. Trypanosoma brucei Infection in asymptomatic greater Kudus (Tragelaphus strepsiceros) on a game ranch in Zambia.

    Science.gov (United States)

    Munang'andu, Hetron Mweemba; Siamudaala, Victor; Munyeme, Musso; Nambota, Andrew; Mutoloki, Stephen; Matandiko, Wigganson

    2010-03-01

    Trypomastogotes of Trypanosoma brucei were detected from 4 asymptomatic kudus (Tragelaphus strepsiceros) on a game ranch located approximately 45 km north east of Lusaka, Zambia. Blood smears examined from 14 wildlife species comprising of the impala (Aepyceros melampus), Kafue lechwe (kobus leche kafuensis), sable antelope (Hippotragus niger), tsessebe (Damaliscus lunatus), warthog (Phacochoerus aethiopicus), puku (Kobus vardoni), zebra (Equus burchelli), waterbuck (Kobus ellipsiprymnus), bushbuck (Tragelaphus scriptus), reedbuck (Redunca arundinum), wilderbeest (Connochaetes taurinus), hartebeest (Alcephelus lichtensteini), African buffalo (Syncerus caffer), and kudu (Tragelaphus strepsiceros) showed that only the kudu had T. brucei. Although game ranching has emerged to be a successful ex-situ conservation strategy aimed at saving the declining wildlife population in the National Parks, our findings suggest that it has the potential of aiding the re-distribution of animal diseases. Hence, there is a need for augmenting wildlife conservation with disease control strategies aimed at reducing the risk of disease transmission between wildlife and domestic animals. PMID:20333288

  3. Identification of Paralogous Life-Cycle Stage Specific Cytoskeletal Proteins in the Parasite Trypanosoma brucei

    OpenAIRE

    Neil Portman; Keith Gull

    2014-01-01

    The life cycle of the African trypanosome Trypanosoma brucei, is characterised by a transition between insect and mammalian hosts representing very different environments that present the parasite with very different challenges. These challenges are met by the expression of life-cycle stage-specific cohorts of proteins, which function in systems such as metabolism and immune evasion. These life-cycle transitions are also accompanied by morphological rearrangements orchestrated by microtubule ...

  4. Ribose 5-Phosphate Isomerase B Knockdown Compromises Trypanosoma brucei Bloodstream Form Infectivity

    OpenAIRE

    Inês Loureiro; Joana Faria; Christine Clayton; Sandra Macedo-Ribeiro; Nuno Santarém; Nilanjan Roy; Anabela Cordeiro-da-Siva; Joana Tavares

    2015-01-01

    Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive dru...

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

    OpenAIRE

    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 specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5' ends of the major transcripts coming from the initiation region m...

  6. Reconstitution of a surface transferrin binding complex in insect form Trypanosoma brucei.

    OpenAIRE

    Ligtenberg, M.J.; Bitter, W.; Kieft, R.; Steverding, D; Janssen, H.; Calafat, J.; Borst, P

    1994-01-01

    In the bloodstream of the mammalian host, Trypanosoma brucei takes up host transferrin by means of a high-affinity uptake system, presumably a transferrin receptor. Transferrin-binding activity is seen in the flagellar pocket and is absent in insect form trypanosomes. By transfection we have reconstituted a transferrin-binding complex in insect form trypanosomes. Formation of this complex requires the products of two genes that are part of a variant surface glycoprotein expression site, expre...

  7. Molecular variation of Trypanosoma brucei subspecies as revealed by AFLP fingerprinting

    OpenAIRE

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

    2002-01-01

    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 pri...

  8. Genetic validation of aminoacyl-tRNA synthetases as drug targets in Trypanosoma brucei.

    Science.gov (United States)

    Kalidas, Savitha; Cestari, Igor; Monnerat, Severine; Li, Qiong; Regmi, Sandesh; Hasle, Nicholas; Labaied, Mehdi; Parsons, Marilyn; Stuart, Kenneth; Phillips, Margaret A

    2014-04-01

    Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts. PMID:24562907

  9. A Pre-clinical Animal Model of Trypanosoma brucei Infection Demonstrating Cardiac Dysfunction

    OpenAIRE

    McCarroll, Charlotte S; Rossor, Charlotte L.; Linda R Morrison; Morrison, Liam J.; Loughrey, Christopher M.

    2015-01-01

    Author Summary African trypanosomiasis (AT) is a disease caused by the single-celled protozoan parasite Trypanosoma brucei. In humans, AT causes neurological problems including sleep disturbances, which give the disease its colloquial name of “sleeping sickness”. Much of the focus of AT research has been on the neurological deficits, but other major organs are also affected, including the heart. Previous studies in humans and animals with AT have identified heart abnormalities such as contrac...

  10. An essential signal peptide peptidase identified in an RNAi screen of serine peptidases of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Catherine X Moss

    Full Text Available The serine peptidases of Trypanosoma brucei have been viewed as potential drug targets. In particular, the S9 prolyl oligopeptidase subfamily is thought to be a good avenue for drug discovery. This is based on the finding that some S9 peptidases are secreted and active in the mammalian bloodstream, and that they are a class of enzyme against which drugs have successfully been developed. We collated a list of all serine peptidases in T. brucei, identifying 20 serine peptidase genes, of which nine are S9 peptidases. We screened all 20 serine peptidases by RNAi to determine which, if any, are essential for bloodstream form T. brucei survival. All S9 serine peptidases were dispensable for parasite survival in vitro, even when pairs of similar genes, coding for oligopeptidase B or prolyl oligopeptidase, were targeted simultaneously. We also found no effect on parasite survival in an animal host when the S9 peptidases oligopeptidase B, prolyl oligopeptidase or dipeptidyl peptidase 8 were targeted. The only serine peptidase to emerge from the RNAi screen as essential was a putative type-I signal peptide peptidase (SPP1. This gene was essential for parasite survival both in vitro and in vivo. The growth defect conferred by RNAi depletion of SPP1 was rescued by expression of a functional peptidase from an RNAi resistant SPP1 gene. However, expression of catalytically inactive SPP1 was unable to rescue cells from the SPP1 depleted phenotype, demonstrating that SPP1 serine peptidase activity is necessary for T. brucei survival.

  11. Wild fauna as a probable animal reservoir for Trypanosoma brucei gambiense in Cameroon

    OpenAIRE

    Njiokou, F.; Laveissière, Claude; Simo, G.; Nkinin, S.; Grébaut, Pascal; Cuny, Gérard; Herder, Stéphane

    2006-01-01

    In order to Study the existence of a wild animal reservoir for Trypanosoma brucei gambiense in South Cameroon, blood was collected from wild animals in three human African trypanosomiasis foci and from a nonendemic control area. The 1142 wild animals sampled belonged to 36 different species pertaining to eight orders (407 primates, 347 artiodactyls, 265 rodents, 54 pangolins, 53 carnivores, 11 Saurians and crocodilians, and five hyraxes). QBC (R) and KIVI tests detected trypanosomes on 1.7% (...

  12. Experimental Trypanosoma brucei infection at immediate post partum period:effects on dam and the offspring

    Institute of Scientific and Technical Information of China (English)

    Izuchukwu S Ochiogu; Chukwuka N Uchendu; John I Ihedioha

    2010-01-01

    Objective:To investigate the effects of immediate post-partum infection with Trypanosoma brucei (T. brucei) on dam and offspring. Methods:Sixty female Albino rats (Rattus norvegicus) weighing between 130-170 g were used as animal model. The animals were divided as follows:25 infected between 1-5 days post partum; 10 infected unbred as positive controls; and 25 uninfected as negative controls. The following parameters were evaluated:packed cell volume (PCV), level of parasitaemia, survival time, litter size and litter weight at birth and on days 7, 14 and 21 post delivery, using conventional methods. Possible trans-mammary transmission of infection to litter through milk was also assessed. Results:The results showed a comparatively (P<0.05) higher mean PCV value for the uninfected negative control on the 8th day post infection compared with the infected groups which corresponded with the increasing level of parasitaemia in the two infected groups. Mean litter size and litter weights were higher (P< 0.05) in the uninfected controls on the 21st day. Survival time in the infected groups were similar. No evidence of trans-mammary transfer of infection was recorded. Conclusion:T. brucei infection during immediate post partum period is detrimental to the dam and impairs growth of the offspring.

  13. Advancing Trypanosoma brucei genome annotation through ribosome profiling and spliced leader mapping.

    Science.gov (United States)

    Parsons, Marilyn; Ramasamy, Gowthaman; Vasconcelos, Elton J R; Jensen, Bryan C; Myler, Peter J

    2015-08-01

    Since the initial publication of the trypanosomatid genomes, curation has been ongoing. Here we make use of existing Trypanosoma brucei ribosome profiling data to provide evidence of ribosome occupancy (and likely translation) of mRNAs from 225 currently unannotated coding sequences (CDSs). A small number of these putative genes correspond to extra copies of previously annotated genes, but 85% are novel. The median size of these novels CDSs is small (81 aa), indicating that past annotation work has excelled at detecting large CDSs. Of the unique CDSs confirmed here, over half have candidate orthologues in other trypanosomatid genomes, most of which were not yet annotated as protein-coding genes. Nonetheless, approximately one-third of the new CDSs were found only in T. brucei subspecies. Using ribosome footprints, RNA-Seq and spliced leader mapping data, we updated previous work to definitively revise the start sites for 414 CDSs as compared to the current gene models. The data pointed to several regions of the genome that had sequence errors that altered coding region boundaries. Finally, we consolidated this data with our previous work to propose elimination of 683 putative genes as protein-coding and arrive at a view of the translatome of slender bloodstream and procyclic culture form T. brucei.

  14. Discovery of Inhibitors of Trypanosoma brucei by Phenotypic Screening of a Focused Protein Kinase Library.

    Science.gov (United States)

    Woodland, Andrew; Thompson, Stephen; Cleghorn, Laura A T; Norcross, Neil; De Rycker, Manu; Grimaldi, Raffaella; Hallyburton, Irene; Rao, Bhavya; Norval, Suzanne; Stojanovski, Laste; Brun, Reto; Kaiser, Marcel; Frearson, Julie A; Gray, David W; Wyatt, Paul G; Read, Kevin D; Gilbert, Ian H

    2015-11-01

    A screen of a focused kinase inhibitor library against Trypanosoma brucei rhodesiense led to the identification of seven series, totaling 121 compounds, which showed >50 % inhibition at 5 μm. Screening of these hits in a T. b. brucei proliferation assay highlighted three compounds with a 1H-imidazo[4,5-b]pyrazin-2(3H)-one scaffold that showed sub-micromolar activity and excellent selectivity against the MRC5 cell line. Subsequent rounds of optimisation led to the identification of compounds that exhibited good in vitro drug metabolism and pharmacokinetics (DMPK) properties, although in general this series suffered from poor solubility. A scaffold-hopping exercise led to the identification of a 1H-pyrazolo[3,4-b]pyridine scaffold, which retained potency. A number of examples were assessed in a T. b. brucei growth assay, which could differentiate static and cidal action. Compounds from the 1H-imidazo[4,5-b]pyrazin-2(3H)-one series were found to be either static or growth-slowing and not cidal. Compounds with the 1H-pyrazolo[3,4-b]pyridine scaffold were found to be cidal and showed an unusual biphasic nature in this assay, suggesting they act by at least two mechanisms.

  15. Torilis japonica extract-generated intracellular ROS induces apoptosis by reducing the mitochondrial membrane potential via regulation of the AMPK-p38 MAPK signaling pathway in HCT116 colon cancer.

    Science.gov (United States)

    Kim, Guen Tae; Lee, Se Hee; Kim, Young Min

    2016-09-01

    Torilis japonica extract (TJE) has been reported to possess diverse medicinal properties including anti‑inflammatory and antibacterial activities. However, the precise mechanism of its anticancer effect is not understood. Thus, we evaluated the apoptotic effects of TJE and examined its underlying molecular mechanisms in HCT116 colorectal cancer cells. Our results show that TJE induces apoptosis through the generation of intracellular reactive oxygen species (ROS), and that it regulates the mitochondrial outer membrane potential via the AMPK/p38 MAPK signaling pathway. Importantly, ~50% of cancer cells have p53 mutations. Thus, the ability to induce apoptosis in a p53-independent manner would be of great value in cancer treatment. Our results show that not only does TJE regulate the AMPK/p38 signaling pathway, but it induces apoptosis in cells in which p53 has been knocked down using siRNA. Moreover, as in in vitro studies, TJE induced apoptosis and regulated apoptosis related-proteins in an HCT 116 xenograft model. Taken together, our results demonstrate that TJE, a natural compound that may provide a substitute for chemotherapeutic drugs, has potential as an anticancer agent. PMID:27314881

  16. L-Galactono-1,4-lactone dehydrogenase is an assembly factor of the membrane arm of mitochondrial complex I in Arabidopsis.

    Science.gov (United States)

    Schimmeyer, Joram; Bock, Ralph; Meyer, Etienne H

    2016-01-01

    L-Galactono-1,4-lactone dehydrogenase (GLDH) catalyses the last enzymatic step of the ascorbate biosynthetic pathway in plants. GLDH is localised to mitochondria and several reports have shown that GLDH is associated with complex I of the respiratory chain. In a gldh knock-out mutant, complex I is not detectable, suggesting that GLDH is essential for complex I assembly or stability. GLDH has not been identified as a genuine complex I subunit, instead, it is present in a smaller, lowly abundant version of complex I called complex I*. In addition, GLDH activity has also been detected in smaller protein complexes within mitochondria membranes. Here, we investigated the role of GLDH during complex I assembly. We identified GLDH in complexes co-localising with some complex I assembly intermediates. Using a mutant that accumulates complex I assembly intermediates, we confirmed that GLDH is associated with the complex I assembly intermediates of 400 and 450 kDa. In addition, we detected accumulation of the 200 kDa complex I assembly intermediate in the gldh mutant. Taken together, our data suggest that GLDH is an assembly factor of the membrane arm of complex I. This function appears to be independent of the role of GLDH in ascorbate synthesis, as evidenced by the ascorbate-deficient mutant vtc2-1 accumulating wild-type levels of complex I. Therefore, we propose that GLDH is a dual-function protein that has a second, non-enzymatic function in complex I assembly as a plant-specific assembly factor. We propose an updated model for complex I assembly that includes complex I* as an assembly intermediate.

  17. 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 ...

  18. Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization.

    Science.gov (United States)

    Zhou, Qinghua; Li, Haimin; Li, Hanzeng; Nakagawa, Akihisa; Lin, Jason L J; Lee, Eui-Seung; Harry, Brian L; Skeen-Gaar, Riley Robert; Suehiro, Yuji; William, Donna; Mitani, Shohei; Yuan, Hanna S; Kang, Byung-Ho; Xue, Ding

    2016-07-22

    Mitochondria are inherited maternally in most animals, but the mechanisms of selective paternal mitochondrial elimination (PME) are unknown. While examining fertilization in Caenorhabditis elegans, we observed that paternal mitochondria rapidly lose their inner membrane integrity. CPS-6, a mitochondrial endonuclease G, serves as a paternal mitochondrial factor that is critical for PME. We found that CPS-6 relocates from the intermembrane space of paternal mitochondria to the matrix after fertilization to degrade mitochondrial DNA. It acts with maternal autophagy and proteasome machineries to promote PME. Loss of cps-6 delays breakdown of mitochondrial inner membranes, autophagosome enclosure of paternal mitochondria, and PME. Delayed removal of paternal mitochondria causes increased embryonic lethality, demonstrating that PME is important for normal animal development. Thus, CPS-6 functions as a paternal mitochondrial degradation factor during animal development.

  19. Mitochondrial Reactive Oxygen Species Modulate Mosquito Susceptibility to Plasmodium Infection

    OpenAIRE

    Gonçalves, Renata L. S.; Oliveira, Jose Henrique M.; Oliveira, Giselle A.; Andersen, John F.; Oliveira, Marcus F.; Pedro L Oliveira; Barillas-Mury, Carolina

    2012-01-01

    Background Mitochondria perform multiple roles in cell biology, acting as the site of aerobic energy-transducing pathways and as an important source of reactive oxygen species (ROS) that modulate redox metabolism. Methodology/Principal Findings We demonstrate that a novel member of the mitochondrial transporter protein family, Anopheles gambiae mitochondrial carrier 1 (AgMC1), is required to maintain mitochondrial membrane potential in mosquito midgut cells and modulates epithelial responses ...

  20. A mitochondrial import receptor for the ADP/ATP carrier

    OpenAIRE

    Söllner, Thomas; Griffiths, Gareth; Pfanner, Nikolaus; Neupert, Walter

    1990-01-01

    We have identified a mitochondrial outer membrane protein of 72 kd (MOM72) that exhibits the properties of an import receptor for the ADP/ATP carrier (AAC), the most abundant mitochondrial protein. Monospecific antibodies and Fab fragments against MOM72 selectively inhibit import of AAC at the level of specific binding to the mitochondria. AAC bound to the mitochondrial surface is coprecipitated with antibodies against MOM72 after lysis of mitochondria with detergent. MOM72 thus has a complem...

  1. Parkin suppresses Drp1-independent mitochondrial division.

    Science.gov (United States)

    Roy, Madhuparna; Itoh, Kie; Iijima, Miho; Sesaki, Hiromi

    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.

  2. Parkin suppresses Drp1-independent mitochondrial division.

    Science.gov (United States)

    Roy, Madhuparna; Itoh, Kie; Iijima, Miho; Sesaki, Hiromi

    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. PMID:27181353

  3. The plant mitochondrial carrier family: functional and evolutionary aspects

    OpenAIRE

    Ilka eHaferkamp; Stephan eSchmitz-Esser

    2012-01-01

    Mitochondria play a key role in respiration and energy production and are involved in multiple eukaryotic but also in several plant specific metabolic pathways. Solute carriers in the inner mitochondrial membrane connect the internal metabolism with that of the surrounding cell. Because of their common basic structure, these transport proteins affiliate to the mitochondrial carrier family (MCF). Generally, MCF proteins consist of six membrane-spanning helices, exhibit typical conserved domain...

  4. Berberine inhibits growth, induces G1 arrest and apoptosis in human epidermoid carcinoma A431 cells by regulating Cdki-Cdk-cyclin cascade, disruption of mitochondrial membrane potential and cleavage of caspase 3 and PARP.

    Science.gov (United States)

    Mantena, Sudheer K; Sharma, Som D; Katiyar, Santosh K

    2006-10-01

    Chemotherapeutic approach using non-toxic botanicals may be one of the strategies for the management of the skin cancers. Here we report that in vitro treatment of human epidermoid carcinoma A431 cells with berberine, a naturally occurring isoquinoline alkaloid, decreased cell viability (3-77%, P berberine-induced G(1) cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), a simultaneous decrease in Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and enhanced binding of Cdki-Cdk. In additional studies, treatment of A431 cells with berberine (15-75 microM) for 72 h resulted in a significant dose-dependent increase in apoptosis (31-60%, P berberine-treated control (11.7%), which was associated with an increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase. Pretreatment of A431 cells with the pan-caspase inhibitor (z-VAD-fmk) significantly blocked the berberine-induced apoptosis in A431 cells confirmed that berberine-induced apoptosis is mediated through activation of caspase 3-dependent pathway. Together, this study for the first time identified berberine as a chemotherapeutic agent against human epidermoid carcinoma A431 cells in vitro, further in vivo studies are required to determine whether berberine could be an effective chemotherapeutic agent for the management of non-melanoma skin cancers.

  5. Grape seed proanthocyanidins promote apoptosis in human epidermoid carcinoma A431 cells through alterations in Cdki-Cdk-cyclin cascade, and caspase-3 activation via loss of mitochondrial membrane potential.

    Science.gov (United States)

    Meeran, Syed M; Katiyar, Santosh K

    2007-05-01

    Dietary grape seed proanthocyanidins (GSPs) prevent photocarcinogenesis in mice. Here, we report that in vitro treatment of human epidermoid carcinoma A431 cells with GSPs inhibited cellular proliferation (13-89%) and induced cell death (1-48%) in a dose (5-100 mug/ml)- and time (24, 48 and 72 h)-dependent manner. GSP-induced inhibition of cell proliferation was associated with an increase in G1-phase arrest at 24 h, which was mediated through the inhibition of cyclin-dependent kinases (Cdk) Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and simultaneous increase in protein expression of cyclin-dependent kinase inhibitors (Cdki), Cip1/p21 and Kip1/p27, and enhanced binding of Cdki-Cdk. The treatment of A431 cells with GSPs (20-80 mug/ml) resulted in a dose-dependent increase in apoptotic cell death (26-58%), which was associated with an increased protein expression of proapoptotic Bax, decreased expression of antiapoptotic Bcl-2 and Bcl-xl, loss of mitochondrial membrane potential, and cleavage of caspase-9, caspase-3 and PARP. Pretreatment with the pan-caspase inhibitor (z-VAD-fmk) blocked the GSP-induced apoptosis in A431 cells suggesting that GSP-induced apoptosis is associated primarily with the caspase-3-dependent pathway. Together, our study suggests that GSPs possess chemotherapeutic potential against human epidermoid carcinoma cells in vitro, further in vivo mechanistic studies are required to verify the chemotherapeutic effect of GSPs in skin cancers. PMID:17437483

  6. Ethanolamine phosphoglycerol attachment to eEF1A is not essential for normal growth of Trypanosoma brucei

    OpenAIRE

    Eva Greganova; Peter Bütikofer

    2012-01-01

    Eukaryotic elongation factor 1A (eEF1A) is the only protein modified by ethanolamine phosphoglycerol (EPG). In mammals and plants, EPG is attached to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote, Trypanosoma brucei, a single EPG moiety is attached to domain III. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but the role of this unique protein modification in cellul...

  7. Central Nervous System Parasitosis and Neuroinflammation Ameliorated by Systemic IL-10 Administration in Trypanosoma brucei-Infected Mice.

    Science.gov (United States)

    Rodgers, Jean; Bradley, Barbara; Kennedy, Peter G E; Sternberg, Jeremy M

    2015-01-01

    Invasion of the central nervous system (CNS) by African trypanosomes represents a critical step in the development of human African trypanosomiasis. In both clinical cases and experimental mouse infections it has been demonstrated that predisposition to CNS invasion is associated with a type 1 systemic inflammatory response. Using the Trypanosoma brucei brucei GVR35 experimental infection model, we demonstrate that systemic delivery of the counter-inflammatory cytokine IL-10 lowers plasma IFN-γ and TNF-α concentrations, CNS parasitosis and ameliorates neuro-inflammatory pathology and clinical symptoms of disease. The results provide evidence that CNS invasion may be susceptible to immunological attenuation.

  8. Trypanosoma brucei modifies the tsetse salivary composition, altering the fly feeding behavior that favors parasite transmission.

    Directory of Open Access Journals (Sweden)

    Jan Van Den Abbeele

    Full Text Available Tsetse flies are the notorious transmitters of African trypanosomiasis, a disease caused by the Trypanosoma parasite that affects humans and livestock on the African continent. Metacyclic infection rates in natural tsetse populations with Trypanosoma brucei, including the two human-pathogenic subspecies, are very low, even in epidemic situations. Therefore, the infected fly/host contact frequency is a key determinant of the transmission dynamics. As an obligate blood feeder, tsetse flies rely on their complex salivary potion to inhibit host haemostatic reactions ensuring an efficient feeding. The results of this experimental study suggest that the parasite might promote its transmission through manipulation of the tsetse feeding behavior by modifying the saliva composition. Indeed, salivary gland Trypanosoma brucei-infected flies display a significantly prolonged feeding time, thereby enhancing the likelihood of infecting multiple hosts during the process of a single blood meal cycle. Comparison of the two major anti-haemostatic activities i.e. anti-platelet aggregation and anti-coagulation activity in these flies versus non-infected tsetse flies demonstrates a significant suppression of these activities as a result of the trypanosome-infection status. This effect was mainly related to the parasite-induced reduction in salivary gland gene transcription, resulting in a strong decrease in protein content and related biological activities. Additionally, the anti-thrombin activity and inhibition of thrombin-induced coagulation was even more severely hampered as a result of the trypanosome infection. Indeed, while naive tsetse saliva strongly inhibited human thrombin activity and thrombin-induced blood coagulation, saliva from T. brucei-infected flies showed a significantly enhanced thrombinase activity resulting in a far less potent anti-coagulation activity. These data clearly provide evidence for a trypanosome-mediated modification of the tsetse

  9. Mitochondrial Dynamics and Mitochondrial Dysfunction in Diabetes.

    Science.gov (United States)

    Wada, Jun; Nakatsuka, Atsuko

    2016-06-01

    The mitochondria are involved in active and dynamic processes, such as mitochondrial biogenesis, fission, fusion and mitophagy to maintain mitochondrial and cellular functions. In obesity and type 2 diabetes, impaired oxidation, reduced mitochondrial contents, lowered rates of oxidative phosphorylation and excessive reactive oxygen species (ROS) production have been reported. Mitochondrial biogenesis is regulated by various transcription factors such as peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), peroxisome proliferator-activated receptors (PPARs), estrogen-related receptors (ERRs), and nuclear respiratory factors (NRFs). Mitochondrial fusion is promoted by mitofusin 1 (MFN1), mitofusin 2 (MFN2) and optic atrophy 1 (OPA1), while fission is governed by the recruitment of dynamin-related protein 1 (DRP1) by adaptor proteins such as mitochondrial fission factor (MFF), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51), and fission 1 (FIS1). Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARKIN promote DRP1-dependent mitochondrial fission, and the outer mitochondrial adaptor MiD51 is required in DRP1 recruitment and PARKIN-dependent mitophagy. This review describes the molecular mechanism of mitochondrial dynamics, its abnormality in diabetes and obesity, and pharmaceuticals targeting mitochondrial biogenesis, fission, fusion and mitophagy. PMID:27339203

  10. Increased intrinsic mitochondrial function in humans with mitochondrial haplogroup H

    DEFF Research Database (Denmark)

    Larsen, Steen; Díez-Sánchez, Carmen; Rabøl, Rasmus;

    2014-01-01

    and determined their mitochondrial haplogroup, mitochondrial oxidative phosphorylation capacity (OXPHOS), mitochondrial content (citrate synthase (CS)) and VO2max. Intrinsic mitochondrial function is calculated as mitochondrial OXPHOS capacity divided by mitochondrial content (CS). Haplogroup H showed a 30......% higher intrinsic mitochondrial function compared with the other haplo group U. There was no relationship between haplogroups and VO2max. In skeletal muscle from men with mitochondrial haplogroup H, an increased intrinsic mitochondrial function is present....

  11. A Gateway® compatible vector for gene silencing in bloodstream form Trypanosoma brucei

    OpenAIRE

    Kalidas, Savitha; Li, Qiong; Margaret A Phillips

    2011-01-01

    RNA interference is the most rapid method for generation of conditional knockdown mutants in Trypanosoma brucei. The dual T7 promoter (pZJM) and the stem-loop vectors have been widely used to generate stable inducible RNAi cell lines with the latter providing tighter regulatory control. However, the steps for cloning stem-loop constructs are cumbersome requiring either multiple cloning steps or multi-fragment ligation reactions. We report the development of a vector (pTrypRNAiGate) derived fr...

  12. Development of Simplified Heterocyclic Acetogenin Analogues as Potent and Selective Trypanosoma brucei Inhibitors.

    Science.gov (United States)

    Florence, Gordon J; Fraser, Andrew L; Gould, Eoin R; King, Elizabeth F; Menzies, Stefanie K; Morris, Joanne C; Thomson, Marie I; Tulloch, Lindsay B; Zacharova, Marija K; Smith, Terry K

    2016-07-19

    Neglected tropical diseases caused by parasitic infections are an ongoing and increasing concern. They are a burden to human and animal health, having the most devastating effect on the world's poorest countries. Building upon our previously reported triazole analogues, in this study we describe the synthesis and biological testing of other novel heterocyclic acetogenin-inspired derivatives, namely 3,5-isoxazoles, furoxans, and furazans. Several of these compounds maintain low-micromolar levels of inhibition against Trypanosoma brucei, whilst having no observable inhibitory effect on mammalian cells, leading to the possibility of novel lead compounds for selective treatment. PMID:27283448

  13. cAMP signalling meets mitochondrial compartments.

    Science.gov (United States)

    Lefkimmiatis, Konstantinos

    2014-04-01

    Mitochondria are highly dynamic organelles comprising at least three distinct areas, the OMM (outer mitochondrial membrane), the IMS (intermembrane space) and the mitochondrial matrix. Physical compartmentalization allows these organelles to host different functional domains and therefore participate in a variety of important cellular actions such as ATP synthesis and programmed cell death. In a surprising homology, it is now widely accepted that the ubiquitous second messenger cAMP uses the same stratagem, compartmentalization, in order to achieve the characteristic functional pleiotropy of its pathway. Accumulating evidence suggests that all the main mitochondrial compartments contain segregated cAMP cascades; however, the regulatory properties and functional significance of such domains are not fully understood and often remain controversial issues. The present mini-review discusses our current knowledge of how the marriage between mitochondrial and cAMP compartmentalization is achieved and its effects on the biology of the cell. PMID:24646228

  14. Evidence of mitochondrial dysfunction in obese adolescents

    DEFF Research Database (Denmark)

    Wilms, L; Larsen, J; Pedersen, P L;

    2010-01-01

    Abstract Aim: Although obesity and weight gain generally are anticipated to be caused by an imbalance between energy intake and energy expenditure, the significance of thyroid hormones (TH) remains unclear. Examination of mitochondrial function may reflect intracellular thyroid hormone effect...... and mitochondrial function in peripheral blood monocytes was determined by flow cytometry. Results: Significant increase in TSH (3.06 +/- 1.56 mU/L vs. 2.33 +/- 0.91 mU/L, p ... compared with lean adolescents. Flow cytometry analysis demonstrated a lower mitochondrial mass (6385 +/- 1962 a.u. vs. 7608 +/- 2328 a.u., p mitochondrial membrane potential (11426 +/- 3861 a.u. vs. 14017 +/- 5536 a.u., p

  15. 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.

  16. NEW EMBO MEMBER’S REVIEW: Viral and bacterial proteins regulating apoptosis at the mitochondrial level

    OpenAIRE

    Boya, Patricia; Roques, Bernard,; Kroemer, Guido

    2001-01-01

    Mitochondrial membrane permeabilization (MMP) is a critical step of several apoptotic pathways. Some infectious intracellular pathogens can regulate (induce or inhibit) apoptosis of their host cells at the mitochondrial level, by targeting proteins to mitochondrial membranes that either induce or inhibit MMP. Pathogen-encoded mitochondrion-targeted proteins may or may not show amino acid sequence homology to Bcl-2-like proteins. Among the Bcl-2-unrelated, mitochondrion-targeted proteins, seve...

  17. 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.

  18. Tissue-Specific Remodeling of the Mitochondrial Proteome in Type 1 Diabetic Akita Mice

    OpenAIRE

    Bugger, Heiko; Dong CHEN; Riehle, Christian; Soto, Jamie; Theobald, Heather A.; Hu, Xiao X; Ganesan, Balasubramanian; Bart C Weimer; Abel, E. Dale

    2009-01-01

    OBJECTIVE To elucidate the molecular basis for mitochondrial dysfunction, which has been implicated in the pathogenesis of diabetes complications. RESEARCH DESIGN AND METHODS Mitochondrial matrix and membrane fractions were generated from liver, brain, heart, and kidney of wild-type and type 1 diabetic Akita mice. Comparative proteomics was performed using label-free proteome expression analysis. Mitochondrial state 3 respirations and ATP synthesis were measured, and mitochondrial morphology ...

  19. A mechanistic view of mitochondrial death decision pores

    Directory of Open Access Journals (Sweden)

    J.E. Belizário

    2007-08-01

    Full Text Available Mitochondria increase their outer and inner membrane permeability to solutes, protons and metabolites in response to a variety of extrinsic and intrinsic signaling events. The maintenance of cellular and intraorganelle ionic homeostasis, particularly for Ca2+, can determine cell survival or death. Mitochondrial death decision is centered on two processes: inner membrane permeabilization, such as that promoted by the mitochondrial permeability transition pore, formed across inner membranes when Ca2+ reaches a critical threshold, and mitochondrial outer membrane permeabilization, in which the pro-apoptotic proteins BID, BAX, and BAK play active roles. Membrane permeabilization leads to the release of apoptogenic proteins: cytochrome c, apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G. Cytochrome c initiates the proteolytic activation of caspases, which in turn cleave hundreds of proteins to produce the morphological and biochemical changes of apoptosis. Voltage-dependent anion channel, cyclophilin D, adenine nucleotide translocase, and the pro-apoptotic proteins BID, BAX, and BAK may be part of the molecular composition of membrane pores leading to mitochondrial permeabilization, but this remains a central question to be resolved. Other transporting pores and channels, including the ceramide channel, the mitochondrial apoptosis-induced channel, as well as a non-specific outer membrane rupture may also be potential release pathways for these apoptogenic factors. In this review, we discuss the mechanistic models by which reactive oxygen species and caspases, via structural and conformational changes of membrane lipids and proteins, promote conditions for inner/outer membrane permeabilization, which may be followed by either opening of pores or a rupture of the outer mitochondrial membrane.

  20. The TgsGP gene is essential for resistance to human serum in Trypanosoma brucei gambiense.

    Science.gov (United States)

    Capewell, Paul; Clucas, Caroline; DeJesus, Eric; Kieft, Rudo; Hajduk, Stephen; Veitch, Nicola; Steketee, Pieter C; Cooper, Anneli; Weir, William; MacLeod, Annette

    2013-01-01

    Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1) delivered via two trypanosome lytic factors (TLF-1 and TLF-2). Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense. PMID:24098129

  1. The TgsGP gene is essential for resistance to human serum in Trypanosoma brucei gambiense.

    Directory of Open Access Journals (Sweden)

    Paul Capewell

    Full Text Available Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1 delivered via two trypanosome lytic factors (TLF-1 and TLF-2. Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense.

  2. Trypanosoma Brucei Aquaglyceroporins Facilitate the Uptake of Arsenite and Antimonite in a pH Dependent Way

    Directory of Open Access Journals (Sweden)

    Néstor L. Uzcátegui

    2013-09-01

    Full Text Available Background: Trypanosoma brucei is a primitive parasitic protozoan that thrives in diverse environments such as the midgut of the tsetse fly and the blood of a mammalian host. For an adequate adaptation to these environments, the parasite´s aquaglyceroporins play an important role. Methods and Results: In order to test their ability to transport trivalent arsenic and antimony, we expressed the three known Trypanosoma brucei aquaglyceroporins (TbAQPs in the heterologous systems of yeast null aquaporin mutant and Xenopus laevis oocytes. For both expression systems, we found a pH dependent intracellular accumulation of As(III or Sb(III mediated by all of the three TbAQPs, with the exception of TbAQP1-As(III uptake. Additionally, we observed that Trypanosoma brucei aquaglyceroporins allow the passage of As(III in both directions. Conclusion: Taken together, these results demonstrated that T. brucei aquaglyceroporins can serve as entry routes for As(III and Sb(III into the parasitic cell, and that this uptake is pH sensitive. Therefore, aquaporins of protozoan parasites may be considered useful as a vehicle for drug delivery.

  3. 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

  4. 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

  5. Gemini surfactants mediate efficient mitochondrial gene delivery and expression.

    Science.gov (United States)

    Cardoso, Ana M; Morais, Catarina M; Cruz, A Rita; Cardoso, Ana L; Silva, Sandra G; do Vale, M Luísa; Marques, Eduardo F; Pedroso de Lima, Maria C; Jurado, Amália S

    2015-03-01

    Gene delivery targeting mitochondria has the potential to transform the therapeutic landscape of mitochondrial genetic diseases. Taking advantage of the nonuniversal genetic code used by mitochondria, a plasmid DNA construct able to be specifically expressed in these organelles was designed by including a codon, which codes for an amino acid only if read by the mitochondrial ribosomes. In the present work, gemini surfactants were shown to successfully deliver plasmid DNA to mitochondria. Gemini surfactant-based DNA complexes were taken up by cells through a variety of routes, including endocytic pathways, and showed propensity for inducing membrane destabilization under acidic conditions, thus facilitating cytoplasmic release of DNA. Furthermore, the complexes interacted extensively with lipid membrane models mimicking the composition of the mitochondrial membrane, which predicts a favored interaction of the complexes with mitochondria in the intracellular environment. This work unravels new possibilities for gene therapy toward mitochondrial diseases. PMID:25634573

  6. 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

  7. Pivotal role of AKAP121 in mitochondrial physiology.

    Science.gov (United States)

    Czachor, Alexander; Failla, Athena; Lockey, Richard; Kolliputi, Narasaiah

    2016-04-15

    In this Perspective, we discuss some recent developments in the study of the mitochondrial scaffolding protein AKAP121 (also known as AKAP1, or AKAP149 as the human homolog), with an emphasis on its role in mitochondrial physiology. AKAP121 has been identified to function as a key regulatory molecule in several mitochondrial events including oxidative phosphorylation, the control of membrane potential, fission-induced apoptosis, maintenance of mitochondrial Ca(2+)homeostasis, and the phosphorylation of various mitochondrial respiratory chain substrate molecules. Furthermore, we discuss the role of hypoxia in prompting cellular stress and damage, which has been demonstrated to mediate the proteosomal degradation of AKAP121, leading to an increase in reactive oxgyen species production, mitochondrial dysfunction, and ultimately cell death. PMID:26825124

  8. The Plant Mitochondrial Transportome: Balancing Metabolic Demands with Energetic Constraints.

    Science.gov (United States)

    Lee, Chun Pong; Millar, A Harvey

    2016-08-01

    In plants, mitochondrial function is associated with hundreds of metabolic reactions. To facilitate these reactions, charged substrates and cofactors move across the charge-impermeable inner mitochondrial membrane via specialized transporters and must work cooperatively with the electrochemical gradient which is essential for mitochondrial function. The regulatory framework for mitochondrial metabolite transport is expected to be more complex in plants than in mammals owing to the close metabolic association between mitochondrial, plastids, and peroxisome metabolism, as well as to the major diurnal fluctuations in plant metabolic function. We propose here how recent advances can be integrated towards defining the mitochondrial transportome in plants. We also discuss what this reveals about sustaining cooperativity between bioenergetics, metabolism, and transport in typical and challenging environments. PMID:27162080

  9. Control mechanisms in mitochondrial oxidative phosphorylation

    Institute of Scientific and Technical Information of China (English)

    Jana Hroudová; Zdeněk Fi(s)ar

    2013-01-01

    Distribution and activity of mitochondria are key factors in neuronal development, synaptic plasticity and axogenesis. The majority of energy sources, necessary for cellular functions, originate from oxidative phosphorylation located in the inner mitochondrial membrane. The adenosine-5'- triphosphate production is regulated by many control mechanism–firstly by oxygen, substrate level, adenosine-5'-diphosphate level, mitochondrial membrane potential, and rate of coupling and proton leak. Recently, these mechanisms have been implemented by "second control mechanisms," such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes, allosteric inhibition of cytochrome c oxidase, thyroid hormones, effects of fatty acids and uncoupling proteins. Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia. Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5'-triphosphate in response to energy demands. Large amounts of reactive oxygen species are released by defective mitochondria, similarly, decline of antioxidative enzyme activities (e.g. in the elderly) enhances reactive oxygen species production. We reviewed data concerning neuroplasticity, physiology, and control of mitochondrial oxidative phosphorylation and reactive oxygen species production.

  10. Almacenamiento en frío de espermatozoides de trucha arcoiris (Oncorhynchus mykiss: Efectos en la motilidad, superóxido intracelular, integridad de la membrana plasmática y potencial de membrana mitocondrial Cold storage of sperm of rainbow trout (oncorhynchus mykiss: Effect on motility, intracellular superoxide, plasma membrane integrity and mitochondrial membrane potential

    Directory of Open Access Journals (Sweden)

    O Berríos

    2010-01-01

    Full Text Available A diferencia de lo que ocurre en mamíferos, en teleósteos la mayoría de los estudios que evalúan la calidad del semen almacenado están orientados a la exposición de los espermatozoides a algunas especies reactivas de oxígeno (ROS, a la incorporación de antioxidantes en la dieta o a la aplicación de éstos en el plasma seminal. No se encuentran trabajos disponibles que traten la presencia del radical superóxido (O2._, ni la función que éste cumple al interior del espermatozoide cuando se encuentran almacenados. En la presente investigación se evaluó el efecto del almacenamiento en el O2._, motilidad, integridad de la membrana plasmática y potencial de membrana mitocondrial (ΔΨMit en espermatozoides de trucha arcoiris (oncorhynchus mykiss. Para ello se extrajo el semen, el cual fue almacenado durante 12 días a 4 ºC. Cada 4 días se evaluó motilidad, ΔΨMit, integridad de la membrana plasmática y se detectó O2._ intracelular en los espermatozoides. Se encontró un 82,59% de células con tinción positiva para O2._ el día de extracción de la muestra, mientras que la motilidad, ΔΨMit y la integridad de la membrana plasmática, solo mostraron deterioro después del octavo día de almacenamiento. Únicamente el ΔΨMit se correlaciona negativamente con O2._ a partir del octavo día de almacenamiento (r = -0,56 P In teleostei, as opposed to what happens in mammals, most of the studies that evaluate the quality storages of semen are oriented toward the exposure of spermatozoa to some reactive oxygen species (ROS, the utilization of antioxidants in the diet, or the incorporation of these in seminal plasma. There is no available the literature covering the presence of superoxide ions (O2._, or the function of these on the interior of the spermatozoa that have been stored. In this study, we evaluated the effect of storage on intracellular O2._, motility, plasmatic membrane integrity, and mitochondrial membrane potential (

  11. 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.

  12. Mitochondrial involvement in drug-induced liver injury.

    Science.gov (United States)

    Pessayre, Dominique; Mansouri, Abdellah; Berson, Alain; Fromenty, Bernard

    2010-01-01

    Mitochondrial dysfunction is a major mechanism of liver injury. A parent drug or its reactive metabolite can trigger outer mitochondrial membrane permeabilization or rupture due to mitochondrial permeability transition. The latter can severely deplete ATP and cause liver cell necrosis, or it can instead lead to apoptosis by releasing cytochrome c, which activates caspases in the cytosol. Necrosis and apoptosis can trigger cytolytic hepatitis resulting in lethal fulminant hepatitis in some patients. Other drugs severely inhibit mitochondrial function and trigger extensive microvesicular steatosis, hypoglycaemia, coma, and death. Milder and more prolonged forms of drug-induced mitochondrial dysfunction can also cause macrovacuolar steatosis. Although this is a benign liver lesion in the short-term, it can progress to steatohepatitis and then to cirrhosis. Patient susceptibility to drug-induced mitochondrial dysfunction and liver injury can sometimes be explained by genetic or acquired variations in drug metabolism and/or elimination that increase the concentration of the toxic species (parent drug or metabolite). Susceptibility may also be increased by the presence of another condition, which also impairs mitochondrial function, such as an inborn mitochondrial cytopathy, beta-oxidation defect, certain viral infections, pregnancy, or the obesity-associated metabolic syndrome. Liver injury due to mitochondrial dysfunction can have important consequences for pharmaceutical companies. It has led to the interruption of clinical trials, the recall of several drugs after marketing, or the introduction of severe black box warnings by drug agencies. Pharmaceutical companies should systematically investigate mitochondrial effects during lead selection or preclinical safety studies. PMID:20020267

  13. Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression.

    Science.gov (United States)

    Jourdain, Alexis A; Boehm, Erik; Maundrell, Kinsey; Martinou, Jean-Claude

    2016-03-14

    In mitochondria, DNA replication, gene expression, and RNA degradation machineries coexist within a common nondelimited space, raising the question of how functional compartmentalization of gene expression is achieved. Here, we discuss the recently characterized "mitochondrial RNA granules," mitochondrial subdomains with an emerging role in the regulation of gene expression. PMID:26953349

  14. 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.

  15. Altered Mitochondrial Respiration and Other Features of Mitochondrial Function in Parkin-Mutant Fibroblasts from Parkinson’s Disease Patients

    Directory of Open Access Journals (Sweden)

    William Haylett

    2016-01-01

    Full Text Available Mutations in the parkin gene are the most common cause of early-onset Parkinson’s disease (PD. Parkin, an E3 ubiquitin ligase, is involved in respiratory chain function, mitophagy, and mitochondrial dynamics. Human cellular models with parkin null mutations are particularly valuable for investigating the mitochondrial functions of parkin. However, published results reporting on patient-derived parkin-mutant fibroblasts have been inconsistent. This study aimed to functionally compare parkin-mutant fibroblasts from PD patients with wild-type control fibroblasts using a variety of assays to gain a better understanding of the role of mitochondrial dysfunction in PD. To this end, dermal fibroblasts were obtained from three PD patients with homozygous whole exon deletions in parkin and three unaffected controls. Assays of mitochondrial respiration, mitochondrial network integrity, mitochondrial membrane potential, and cell growth were performed as informative markers of mitochondrial function. Surprisingly, it was found that mitochondrial respiratory rates were markedly higher in the parkin-mutant fibroblasts compared to control fibroblasts (p = 0.0093, while exhibiting more fragmented mitochondrial networks (p=0.0304. Moreover, cell growth of the parkin-mutant fibroblasts was significantly higher than that of controls (p=0.0001. These unanticipated findings are suggestive of a compensatory mechanism to preserve mitochondrial function and quality control in the absence of parkin in fibroblasts, which warrants further investigation.

  16. Altered Mitochondrial Respiration and Other Features of Mitochondrial Function in Parkin-Mutant Fibroblasts from Parkinson's Disease Patients.

    Science.gov (United States)

    Haylett, William; Swart, Chrisna; van der Westhuizen, Francois; van Dyk, Hayley; van der Merwe, Lize; van der Merwe, Celia; Loos, Ben; Carr, Jonathan; Kinnear, Craig; Bardien, Soraya

    2016-01-01

    Mutations in the parkin gene are the most common cause of early-onset Parkinson's disease (PD). Parkin, an E3 ubiquitin ligase, is involved in respiratory chain function, mitophagy, and mitochondrial dynamics. Human cellular models with parkin null mutations are particularly valuable for investigating the mitochondrial functions of parkin. However, published results reporting on patient-derived parkin-mutant fibroblasts have been inconsistent. This study aimed to functionally compare parkin-mutant fibroblasts from PD patients with wild-type control fibroblasts using a variety of assays to gain a better understanding of the role of mitochondrial dysfunction in PD. To this end, dermal fibroblasts were obtained from three PD patients with homozygous whole exon deletions in parkin and three unaffected controls. Assays of mitochondrial respiration, mitochondrial network integrity, mitochondrial membrane potential, and cell growth were performed as informative markers of mitochondrial function. Surprisingly, it was found that mitochondrial respiratory rates were markedly higher in the parkin-mutant fibroblasts compared to control fibroblasts (p = 0.0093), while exhibiting more fragmented mitochondrial networks (p = 0.0304). Moreover, cell growth of the parkin-mutant fibroblasts was significantly higher than that of controls (p = 0.0001). These unanticipated findings are suggestive of a compensatory mechanism to preserve mitochondrial function and quality control in the absence of parkin in fibroblasts, which warrants further investigation. PMID:27034887

  17. Altered Mitochondrial Respiration and Other Features of Mitochondrial Function in Parkin-Mutant Fibroblasts from Parkinson's Disease Patients

    Science.gov (United States)

    Swart, Chrisna; van der Westhuizen, Francois; van Dyk, Hayley; van der Merwe, Lize; van der Merwe, Celia; Loos, Ben; Carr, Jonathan; Kinnear, Craig; Bardien, Soraya

    2016-01-01

    Mutations in the parkin gene are the most common cause of early-onset Parkinson's disease (PD). Parkin, an E3 ubiquitin ligase, is involved in respiratory chain function, mitophagy, and mitochondrial dynamics. Human cellular models with parkin null mutations are particularly valuable for investigating the mitochondrial functions of parkin. However, published results reporting on patient-derived parkin-mutant fibroblasts have been inconsistent. This study aimed to functionally compare parkin-mutant fibroblasts from PD patients with wild-type control fibroblasts using a variety of assays to gain a better understanding of the role of mitochondrial dysfunction in PD. To this end, dermal fibroblasts were obtained from three PD patients with homozygous whole exon deletions in parkin and three unaffected controls. Assays of mitochondrial respiration, mitochondrial network integrity, mitochondrial membrane potential, and cell growth were performed as informative markers of mitochondrial function. Surprisingly, it was found that mitochondrial respiratory rates were markedly higher in the parkin-mutant fibroblasts compared to control fibroblasts (p = 0.0093), while exhibiting more fragmented mitochondrial networks (p = 0.0304). Moreover, cell growth of the parkin-mutant fibroblasts was significantly higher than that of controls (p = 0.0001). These unanticipated findings are suggestive of a compensatory mechanism to preserve mitochondrial function and quality control in the absence of parkin in fibroblasts, which warrants further investigation. PMID:27034887

  18. Altered Mitochondrial Respiration and Other Features of Mitochondrial Function in Parkin-Mutant Fibroblasts from Parkinson's Disease Patients.

    Science.gov (United States)

    Haylett, William; Swart, Chrisna; van der Westhuizen, Francois; van Dyk, Hayley; van der Merwe, Lize; van der Merwe, Celia; Loos, Ben; Carr, Jonathan; Kinnear, Craig; Bardien, Soraya

    2016-01-01

    Mutations in the parkin gene are the most common cause of early-onset Parkinson's disease (PD). Parkin, an E3 ubiquitin ligase, is involved in respiratory chain function, mitophagy, and mitochondrial dynamics. Human cellular models with parkin null mutations are particularly valuable for investigating the mitochondrial functions of parkin. However, published results reporting on patient-derived parkin-mutant fibroblasts have been inconsistent. This study aimed to functionally compare parkin-mutant fibroblasts from PD patients with wild-type control fibroblasts using a variety of assays to gain a better understanding of the role of mitochondrial dysfunction in PD. To this end, dermal fibroblasts were obtained from three PD patients with homozygous whole exon deletions in parkin and three unaffected controls. Assays of mitochondrial respiration, mitochondrial network integrity, mitochondrial membrane potential, and cell growth were performed as informative markers of mitochondrial function. Surprisingly, it was found that mitochondrial respiratory rates were markedly higher in the parkin-mutant fibroblasts compared to control fibroblasts (p = 0.0093), while exhibiting more fragmented mitochondrial networks (p = 0.0304). Moreover, cell growth of the parkin-mutant fibroblasts was significantly higher than that of controls (p = 0.0001). These unanticipated findings are suggestive of a compensatory mechanism to preserve mitochondrial function and quality control in the absence of parkin in fibroblasts, which warrants further investigation.

  19. Exercise alters liver mitochondria phospholipidomic profile and mitochondrial activity in non-alcoholic steatohepatitis

    OpenAIRE

    Gonçalves, Inês O; Maciel, Elisabete; Passos, Emanuel; Torrella, Joan R.; Rizo, David; Viscor, Ginés; Rocha-Rodrigues, Silvia; Santos-Alves, Estela; Domingues, Maria R.; Oliveira, Paulo J; Ascensão, António; Magalhães, José

    2014-01-01

    Mitochondrial membrane lipid composition is a critical factor in non-alcoholic steatohepatitis (NASH). Exercise is the most prescribed therapeutic strategy against NASH and a potential modulator of lipid membrane. Thus, we aimed to analyze whether physical exercise exerted preventive (voluntary physical activity – VPA) and therapeutic (endurance training – ET) effect on NASH-induced mitochondrial membrane changes. Sprague-Dawley rats (n = 36) were divided into standard-diet sedentary (SS, n =...

  20. Evaluation of the In Vitro Efficacy of Artemisia annua, Rumex abyssinicus, and Catha edulis Forsk Extracts in Cancer and Trypanosoma brucei Cells.

    Science.gov (United States)

    Worku, Netsanet; Mossie, Andualem; Stich, August; Daugschies, Arwid; Trettner, Susanne; Hemdan, Nasr Y A; Birkenmeier, Gerd

    2013-01-01

    The current drugs against sleeping sickness are derived from cancer chemotherapeutic approaches. Herein, we aimed at evaluating the in vitro effect of alcoholic extracts of Artemisia annua (AMR), Rumex abyssinicus (RMA), and Catha edulis Forsk (CEF) on proliferation/viability of 1321N1 astrocytoma, MCF-7 breast cancer, THP-1 leukemia, and LNCaP, Du-145, and PC-3 prostate cancer cells and on Trypanosoma brucei cells. Proliferation of tumor cells was evaluated by WST-1 assay and viability/behaviour of T. brucei by cell counting and light microscopy. CEF was the most efficient growth inhibitor in comparison to AMR and RMA. Nevertheless, in LNCaP and THP-1 cells, all extracts significantly inhibited tumor growth at 3 μg/mL. All extracts inhibited proliferation of T. brucei cells in a concentration-dependent manner. Microscopic analysis revealed that 95% of the T. brucei cells died when exposed to 33 μg/mL CEF for 3 hrs. Similar results were obtained using 33 μg/mL AMR for 6 hrs. In case of RMA, however, higher concentrations were necessary to obtain similar effects on T. brucei. This demonstrates the antitumor efficacy of these extracts as well as their ability to dampen viability and proliferation of T. brucei, suggesting a common mechanism of action on highly proliferative cells, most probably by targeting cell metabolism. PMID:25937964

  1. Uncoupling Mitochondrial Respiration for Diabesity.

    Science.gov (United States)

    Larrick, James W; Larrick, Jasmine W; Mendelsohn, Andrew R

    2016-08-01

    Until recently, the mechanism of adaptive thermogenesis was ascribed to the expression of uncoupling protein 1 (UCP1) in brown and beige adipocytes. UCP1 is known to catalyze a proton leak of the inner mitochondrial membrane, resulting in uncoupled oxidative metabolism with no production of adenosine triphosphate and increased energy expenditure. Thus increasing brown and beige adipose tissue with augmented UCP1 expression is a viable target for obesity-related disorders. Recent work demonstrates an UCP1-independent pathway to uncouple mitochondrial respiration. A secreted enzyme, PM20D1, enriched in UCP1+ adipocytes, exhibits catalytic and hydrolytic activity to reversibly form N-acyl amino acids. N-acyl amino acids act as endogenous uncouplers of mitochondrial respiration at physiological concentrations. Administration of PM20D1 or its products, N-acyl amino acids, to diet-induced obese mice improves glucose tolerance by increasing energy expenditure. In short-term studies, treated animals exhibit no toxicity while experiencing 10% weight loss primarily of adipose tissue. Further study of this metabolic pathway may identify novel therapies for diabesity, the disease state associated with diabetes and obesity. PMID:27378359

  2. 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.

  3. The acylphloroglucinols hyperforin and myrtucommulone A cause mitochondrial dysfunctions in leukemic cells by direct interference with mitochondria.

    Science.gov (United States)

    Wiechmann, Katja; Müller, Hans; Fischer, Dagmar; Jauch, Johann; Werz, Oliver

    2015-11-01

    The acylphloroglucinols hyperforin (Hypf) and myrtucommulone A (MC A) induce death of cancer cells by triggering the intrinsic/mitochondrial pathway of apoptosis, accompanied by a loss of the mitochondrial membrane potential and release of cytochrome c. However, the upstream targets and mechanisms leading to these mitochondrial events in cancer cells remain elusive. Here we show that Hypf and MC A directly act on mitochondria derived from human leukemic HL-60 cells and thus, disrupt mitochondrial functions. In isolated mitochondria, Hypf and MC A efficiently impaired mitochondrial viability (EC50 = 0.2 and 0.9 µM, respectively), caused loss of the mitochondrial membrane potential (at 0.03 and 0.1 µM, respectively), and suppressed mitochondrial ATP synthesis (IC50 = 0.2 and 0.5 µM, respectively). Consequently, the compounds activated the adenosine monophosphate-activated protein kinase (AMPK) in HL-60 cells, a cellular energy sensor involved in apoptosis of cancer cells. Side by side comparison with the protonophore CCCP and the ATP synthase inhibitor oligomycin suggest that Hypf and MC A act as protonophores that primarily dissipate the mitochondrial membrane potential by direct interaction with the mitochondrial membrane. Together, Hypf and MC A abolish the mitochondrial proton motive force that on one hand impairs mitochondrial viability and on the other cause activation of AMPK due to lowered ATP levels which may further facilitate the intrinsic mitochondrial pathway of apoptosis.

  4. Ionizing radiation accelerates Drp1-dependent mitochondrial fission, which involves delayed mitochondrial reactive oxygen species production in normal human fibroblast-like cells

    International Nuclear Information System (INIS)

    Highlights: ► We report first time that ionizing radiation induces mitochondrial dynamic changes. ► Radiation-induced mitochondrial fission was caused by Drp1 localization. ► We found that radiation causes delayed ROS from mitochondria. ► Down regulation of Drp1 rescued mitochondrial dysfunction after radiation exposure. -- Abstract: Ionizing radiation is known to increase intracellular level of reactive oxygen species (ROS) through mitochondrial dysfunction. Although it has been as a basis of radiation-induced genetic instability, the mechanism involving mitochondrial dysfunction remains unclear. Here we studied the dynamics of mitochondrial structure in normal human fibroblast like cells exposed to ionizing radiation. Delayed mitochondrial O2·- production was peaked 3 days after irradiation, which was coupled with accelerated mitochondrial fission. We found that radiation exposure accumulated dynamin-related protein 1 (Drp1) to mitochondria. Knocking down of Drp1 expression prevented radiation induced acceleration of mitochondrial fission. Furthermore, knockdown of Drp1 significantly suppressed delayed production of mitochondrial O2·-. Since the loss of mitochondrial membrane potential, which was induced by radiation was prevented in cells knocking down of Drp1 expression, indicating that the excessive mitochondrial fission was involved in delayed mitochondrial dysfunction after irradiation.

  5. 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.

  6. Trypanosoma brucei: Enrichment by UV of intergenic transcripts from the variable surface glycoprotein gene expression site

    International Nuclear Information System (INIS)

    The expression site for the variable surface glycoprotein (VSG) gene AnTat 1.3A of Trypanosoma brucei is 45 kilobases long and encompasses seven expression site-associated genes (ESAGs). After UV irradiation, several large transcripts from the putative promoter region were strongly enriched. We report that one such major transcript starts near the poly(A) addition site of the first gene (ESAG 7), spans the intergenic region, and extends to the poly(A) addition site of the second gene (ESAG 6), thus bypassing the normal 3' splice site of the ESAG 6 mRNA. Since this transcript is spliced, we conclude that UV irradiation does not inhibit splicing but stabilizes unstable processing products. This demonstrates that at least some intergenic regions of the VSG gene expression site are continuously transcribed in accordance with a polycistronic transcription model

  7. Strokes in mitochondrial diseases

    Directory of Open Access Journals (Sweden)

    N V Pizova

    2012-01-01

    Full Text Available It is suggested that mitochondrial diseases might be identified in 22—33% of cryptogenic stroke cases in young subjects. The incidence of mitochondrial disorders in patients with stroke is unknown; it is 0.8 to 7.2% according to the data of some authors. The paper gives data on the prevalence, pathogenesis, and clinical manifestations of mitochondrial diseases, such as mitochondrial encephalopathy, lactic acidosis, and stroke-like syndrome (MELAS and insulin-like episodes; myoclonic epilepsy and ragged-red fibers (MERRF syndrome, and Kearns-Sayre syndrome (sporadic multisystem mitochondrial pathology.

  8. A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence.

    Directory of Open Access Journals (Sweden)

    Bidyottam Mittra

    2016-01-01

    Full Text Available Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast and mitoferrin-1 (human that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1 showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes.

  9. Altered Mitochondrial Dynamics and TBI Pathophysiology

    Directory of Open Access Journals (Sweden)

    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

  10. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Gualdrón-López, Melisa [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium); Michels, Paul A.M., E-mail: paul.michels@uclouvain.be [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium)

    2013-02-01

    Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M{sub r} of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M{sub r} of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and {sup 35}S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed.

  11. Knockdown of Inner Arm Protein IC138 in Trypanosoma brucei Causes Defective Motility and Flagellar Detachment.

    Directory of Open Access Journals (Sweden)

    Corinne S Wilson

    Full Text Available Motility in the protozoan parasite Trypanosoma brucei is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged In situ-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in T. brucei.

  12. 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.

  13. 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.

  14. 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.

  15. 信息动态%Evaluation of Mitochondrial Damage of lsletβCells by Mitochondrial Permeability Transition Pore

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Objective To evaluate the mitochondrial damage of islet β cells under glucolipotoxicity by investigating the mitochondrial permeability transition pore (mPTP). Methods Pancreatic β cell lines INS-1 cells were treated with 0. 4 mmol/L palmitic acid and different concentrations of glucose (5.6 mmol/L or 25 mmol/L). The mitochondrial membrane potential, mPTP and reactive oxygen species (ROS) were measured by flow cytometry and fluorescence staining technique to assess the mitochon drial damage. Cell proliferation was measured by 5-bromodeoxyuridine incorporation and cell apoptosis was detected by Annexin V method. Results Compared with the low glucose concentration, the high glucose concentration resulted in decreased mPTP activity (P<0.05), increased mitochondrial membrane potential (P<0.05) and increased cell proliferation rate (P<0.05). There was no significant change in ROS generation. When cells were exposed to high glucose concentration and palmitic acid, both mPTP activity and mitochonhdrial membrane potential reduced (P<0.05), with increased cell apoptosis rate (P <0.05) and increased ROS generation. Conclusion The high glucose concentration decreases mPTP and increases mitochondrial membrane potential, suggesting that cells may remain in an unstable high metabolic state. Evaluation of mPTP may contribute to a more comprehensive understanding of mitochondrial dysfunction under glucotoxictiy.

  16. Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU.

    Science.gov (United States)

    Shanmughapriya, Santhanam; Rajan, Sudarsan; Hoffman, Nicholas E; Zhang, Xueqian; Guo, Shuchi; Kolesar, Jill E; Hines, Kevin J; Ragheb, Jonathan; Jog, Neelakshi R; Caricchio, Roberto; Baba, Yoshihiro; Zhou, Yandong; Kaufman, Brett A; Cheung, Joseph Y; Kurosaki, Tomohiro; Gill, Donald L; Madesh, Muniswamy

    2015-03-03

    Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. Mitochondrial Ca2+ is important for mitochondrial function, and when cytosolic Ca2+ concentration becomes too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+ -permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+ -regulated transcription factor CREB (cyclic adenosine monophosphate response element-binding protein) directly bound the MCU promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulates mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+ -dependent activation of CREB controls the Ca2+ uptake capability of mitochondria and hence regulates mitochondrial metabolism.

  17. Disorders of phospholipid metabolism: an emerging class of mitochondrial disease due to defects in nuclear genes

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    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.

  18. 肥胖男性不育患者精子线粒体膜电位、游离脂肪酸、活性氧的关系%Relationship of Free Fatty Acid,Reactive Oxygen Species and Sperm Mitochondrial Membrane Potential in Obese Male Infertility Patients

    Institute of Scientific and Technical Information of China (English)

    白双勇; 王剑松; 赵庆华

    2015-01-01

    Objective To investigate the change of sperm mitochondrial membrane potential in obese male infertility patients,and to explore the re⁃lationship of sperm mitochondrial membrane potential with the reactive oxygen species and free fatty acids in seminal plasma. Methods According to the research conditions,samples were randomly selected by cluster sampling from outpatient,and divided into normal body mass index fertile men as control group(n=51),normal body mass index infertile group(n=36),overweight infertile group(n=44),and obesity infertile group(n=45). Semen routine analysis was performed. Free fatty acid and reactive oxygen species in the seminal plasma was determined by ELSA method,sperm mitochondrial membrane potential was determined by flow cytometry. Results The rate of normal sperm mitochondrial membrane potential in nor⁃mal weight infertility(27.34%±13.38%),overweight infertility(28.26%±9.76%),obesity infertility group(25.27%±7.51%)were lower than the control group(35.12%±15.90%),the difference was statistically significant(P<0.05). Although obesity infertility group normal rate of mitochon⁃drial membrane potential were lower than normal weight infertility group and the overweight infertility group,but there was no statistically significant difference. The rate of sperm normal mitochondrial membrane potential was positively correlated with the rate of sperm progressive motility(r=0.29, P<0.01). Free fatty acid was positively correlated with reactive oxygen species in seminal plasma(r=0.30,P<0.01),reactive oxygen species in seminal plasma was negatively correlated with sperm normal mitochondrial membrane potential(r=-0.24,P<0.01). Conclusion Free fatty acid was elevated in the seminal plasma of overweight and obese patients with male infertility,which causes increased reactive oxygen species,reduced mitochondrial membrane potential,and eventually lead to the decline of sperm movement ability. Patients undergoing the treatment should be ad

  19. 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 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...... 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....

  20. 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.

  1. Mitochondrial reactive oxygen species modulate mosquito susceptibility to Plasmodium infection.

    Directory of Open Access Journals (Sweden)

    Renata L S Gonçalves

    Full Text Available BACKGROUND: Mitochondria perform multiple roles in cell biology, acting as the site of aerobic energy-transducing pathways and as an important source of reactive oxygen species (ROS that modulate redox metabolism. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that a novel member of the mitochondrial transporter protein family, Anopheles gambiae mitochondrial carrier 1 (AgMC1, is required to maintain mitochondrial membrane potential in mosquito midgut cells and modulates epithelial responses to Plasmodium infection. AgMC1 silencing reduces mitochondrial membrane potential, resulting in increased proton-leak and uncoupling of oxidative phosphorylation. These metabolic changes reduce midgut ROS generation and increase A. gambiae susceptibility to Plasmodium infection. CONCLUSION: We provide direct experimental evidence indicating that ROS derived from mitochondria can modulate mosquito epithelial responses to Plasmodium infection.

  2. Mitochondrial Reactive Oxygen Species Modulate Mosquito Susceptibility to Plasmodium Infection

    Science.gov (United States)

    Oliveira, Giselle A.; Andersen, John F.; Oliveira, Marcus F.; Oliveira, Pedro L.; Barillas-Mury, Carolina

    2012-01-01

    Background Mitochondria perform multiple roles in cell biology, acting as the site of aerobic energy-transducing pathways and as an important source of reactive oxygen species (ROS) that modulate redox metabolism. Methodology/Principal Findings We demonstrate that a novel member of the mitochondrial transporter protein family, Anopheles gambiae mitochondrial carrier 1 (AgMC1), is required to maintain mitochondrial membrane potential in mosquito midgut cells and modulates epithelial responses to Plasmodium infection. AgMC1 silencing reduces mitochondrial membrane potential, resulting in increased proton-leak and uncoupling of oxidative phosphorylation. These metabolic changes reduce midgut ROS generation and increase A. gambiae susceptibility to Plasmodium infection. Conclusion We provide direct experimental evidence indicating that ROS derived from mitochondria can modulate mosquito epithelial responses to Plasmodium infection. PMID:22815925

  3. In vitro investigation of Brazilian Cerrado plant extract activity against Plasmodium falciparum, Trypanosoma cruzi and T. brucei gambiense.

    Science.gov (United States)

    Charneau, Sébastien; de Mesquita, Mariana Laundry; Bastos, Izabela Marques Dourado; Santana, Jaime Martins; de Paula, José Elias; Grellier, Philippe; Espindola, Laila Salmen

    2016-06-01

    The threatened Brazilian Cerrado biome is an important biodiversity hotspot but still few explored that constitutes a potential reservoir of molecules to treat infectious diseases. We selected eight Cerrado plant species for screening against the erythrocytic stages of Plasmodium falciparum, human intracellular stages of Trypanosoma cruzi and bloodstream forms of T. brucei gambiense, and for their cytotoxicity upon the rat L6-myoblast cell line. Bioassays were performed with 37 hexane, ethyl acetate and ethanol extracts prepared from different plant organs. Activities against parasites were observed for 24 extracts: 9 with anti-P. falciparum, 4 with anti-T. cruzi and 11 with anti-T. brucei gambiense activities. High anti-protozoal activity (IC50 values knowledge essential for Cerrado conservation and sustainable development. PMID:26222897

  4. 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. PMID:26681804

  5. Connexin 43 impacts on mitochondrial potassium uptake

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    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.

  6. Functional dissection of T. brucei Protein Tyrosine Phosphatase 1 and investigation of its development as a therapeutic target

    OpenAIRE

    Ruberto, Irene

    2011-01-01

    Trypanosoma brucei undergoes developmentally regulated morphological and biochemical changes during its life cycle, being transmitted between the mammalian host and the tsetse fly. It is generally recognized that cellular responses to environmental changes are mediated through signalling pathways, but our understanding of trypanosome signal transduction during differentiation is limited. Protein Tyrosine Phosphatase 1 (TbPTP1) is the one of the few factors identified to b...

  7. A structural domain mediates attachment of ethanolamine phosphoglycerol to eukaryotic elongation factor 1A in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Eva Greganova

    Full Text Available Ethanolamine phosphoglycerol (EPG represents a protein modification that so far has only been found in eukaryotic elongation factor 1A (eEF1A. In mammals and plants, EPG is covalently attached to two conserved glutamate residues located in domains II and III of eEF1A. In contrast, Trypanosoma brucei eEF1A contains a single EPG attached to Glu362 in domain III. The sequence and/or structural requirements for covalent linkage of EPG to eEF1A have not been determined for any organism. Using a combination of biosynthetic labelling of parasites with tritiated ethanolamine and mass spectrometry analyses, we demonstrate that replacement of Glu362 in T. brucei eEF1A by site-directed mutagenesis prevents EPG attachment, whereas single or multiple amino acid substitutions around the attachment site are not critical. In addition, by expressing a series of eEF1A deletion mutants in T. brucei procyclic forms, we demonstrate that a peptide consisting of 80 amino acids of domain III of eEF1A is sufficient for EPG attachment to occur. Furthermore, EPG addition also occurs if domain III of eEF1A is fused to a soluble reporter protein. To our knowledge, this is the first report addressing amino acid sequence, or structure, requirements for EPG modification of eEF1A in any organism. Using T. brucei as a model organism, we show that amino acid substitutions around the modification site are not critical for EPG attachment and that a truncated version of domain III of eEF1A is sufficient to mediate EPG addition.

  8. Comparative genomics reveals two novel RNAi factors in Trypanosoma brucei and provides insight into the core machinery.

    Directory of Open Access Journals (Sweden)

    Rebecca L Barnes

    Full Text Available The introduction ten years ago of RNA interference (RNAi as a tool for molecular exploration in Trypanosoma brucei has led to a surge in our understanding of the pathogenesis and biology of this human parasite. In particular, a genome-wide RNAi screen has recently been combined with next-generation Illumina sequencing to expose catalogues of genes associated with loss of fitness in distinct developmental stages. At present, this technology is restricted to RNAi-positive protozoan parasites, which excludes T. cruzi, Leishmania major, and Plasmodium falciparum. Therefore, elucidating the mechanism of RNAi and identifying the essential components of the pathway is fundamental for improving RNAi efficiency in T. brucei and for transferring the RNAi tool to RNAi-deficient pathogens. Here we used comparative genomics of RNAi-positive and -negative trypanosomatid protozoans to identify the repertoire of factors in T. brucei. In addition to the previously characterized Argonaute 1 (AGO1 protein and the cytoplasmic and nuclear Dicers, TbDCL1 and TbDCL2, respectively, we identified the RNA Interference Factors 4 and 5 (TbRIF4 and TbRIF5. TbRIF4 is a 3'-5' exonuclease of the DnaQ superfamily and plays a critical role in the conversion of duplex siRNAs to the single-stranded form, thus generating a TbAGO1-siRNA complex required for target-specific cleavage. TbRIF5 is essential for cytoplasmic RNAi and appears to act as a TbDCL1 cofactor. The availability of the core RNAi machinery in T. brucei provides a platform to gain mechanistic insights in this ancient eukaryote and to identify the minimal set of components required to reconstitute RNAi in RNAi-deficient parasites.

  9. High-resolution complex of papain with remnants of a cysteine protease inhibitor derived from Trypanosoma brucei

    OpenAIRE

    Alphey, Magnus S.; Hunter, William N.

    2006-01-01

    Attempts to cocrystallize the cysteine protease papain derived from the latex of Carica papaya with an inhibitor of cysteine proteases (ICP) from Trypanosoma brucei were unsuccessful. However, crystals of papain that diffracted to higher resolution, 1.5 Å, than other crystals of this archetypal cysteine protease were obtained, so the analysis was continued. Surprisingly, the substrate-binding cleft was occupied by two short peptide fragments which have been assigned as remnants of ICP. Compar...

  10. 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

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome.

  11. Evolutionary consequences of a large duplication event in Trypanosoma brucei: Chromosomes 4 and 8 are partial duplicons

    Directory of Open Access Journals (Sweden)

    Jackson Andrew P

    2007-11-01

    Full Text Available Abstract Background Gene order along the genome sequence of the human parasite Trypanosoma brucei provides evidence for a 0.5 Mb duplication, comprising the 3' regions of chromosomes 4 and 8. Here, the principal aim was to examine the contribution made by this duplication event to the T. brucei genome sequence, emphasising the consequences for gene content and the evolutionary change subsequently experienced by paralogous gene copies. The duplicated region may be browsed online at http://www.genedb.org/genedb/tryp/48dup_image.jsp Results Comparisons of trypanosomatid genomes demonstrated widespread gene loss from each duplicon, but also showed that 47% of duplicated genes were retained on both chromosomes as paralogous loci. Secreted and surface-expressed genes were over-represented among retained paralogs, reflecting a bias towards important factors at the host-parasite interface, and consistent with a dosage-balance hypothesis. Genetic divergence in both coding and regulatory regions of retained paralogs was bimodal, with a deficit in moderately divergent paralogs; in particular, non-coding sequences were either conserved or entirely remodelled. The conserved paralogs included examples of remarkable sequence conservation, but also considerable divergence of both coding and regulatory regions. Sequence divergence typically displayed strong negative selection; but several features, such as asymmetric evolutionary rates, positively-selected codons and other non-neutral substitutions, suggested that divergence of some paralogs was driven by functional change. The absence of orthologs to retained paralogs in T. congolense indicated that the duplication event was specific to T. brucei. Conclusion The duplication of this chromosomal region doubled the dosage of many genes. Rather than creating 'more of the same', these results show that paralogs were structurally modified according to various evolutionary trajectories. The retention of paralogs, and

  12. Mitochondrial Dysfunction and Chronic Disease: Treatment With Natural Supplements

    OpenAIRE

    Nicolson, Garth L.

    2014-01-01

    Loss of function in mitochondria, the key organelle responsible for cellular energy production, can result in the excess fatigue and other symptoms that are common complaints in almost every chronic disease. At the molecular level, a reduction in mitochondrial function occurs as a result of the following changes: (1) a loss of maintenance of the electrical and chemical transmembrane potential of the inner mitochondrial membrane, (2) alterations in the function of the electron transport chain,...

  13. Yeast PPR proteins, watchdogs of mitochondrial gene expression

    OpenAIRE

    Herbert, Christopher J.; Golik, Pawel; Bonnefoy, Nathalie

    2013-01-01

    PPR proteins are a family of ubiquitous RNA-binding factors, found in all the Eukaryotic lineages, and are particularly numerous in higher plants. According to recent bioinformatic analyses, yeast genomes encode from 10 (in S. pombe) to 15 (in S. cerevisiae) PPR proteins. All of these proteins are mitochondrial and very often interact with the mitochondrial membrane. Apart from the general factors, RNA polymerase and RNase P, most yeast PPR proteins are involved in the stability and/or transl...

  14. Kinetics and specificity of paternal mitochondrial elimination in Caenorhabditis elegans

    Science.gov (United States)

    Wang, Yang; Zhang, Yi; Chen, Lianwan; Liang, Qian; Yin, Xiao-Ming; Miao, Long; Kang, Byung-Ho; Xue, Ding

    2016-01-01

    In most eukaryotes, mitochondria are inherited maternally. The autophagy process is critical for paternal mitochondrial elimination (PME) in Caenorhabditis elegans, but how paternal mitochondria, but not maternal mitochondria, are selectively targeted for degradation is poorly understood. Here we report that mitochondrial dynamics have a profound effect on PME. A defect in fission of paternal mitochondria delays PME, whereas a defect in fusion of paternal mitochondria accelerates PME. Surprisingly, a defect in maternal mitochondrial fusion delays PME, which is reversed by a fission defect in maternal mitochondria or by increasing maternal mitochondrial membrane potential using oligomycin. Electron microscopy and tomography analyses reveal that a proportion of maternal mitochondria are compromised when they fail to fuse normally, leading to their competition for the autophagy machinery with damaged paternal mitochondria and delayed PME. Our study indicates that mitochondrial dynamics play a critical role in regulating both the kinetics and the specificity of PME. PMID:27581092

  15. Kinetics and specificity of paternal mitochondrial elimination in Caenorhabditis elegans.

    Science.gov (United States)

    Wang, Yang; Zhang, Yi; Chen, Lianwan; Liang, Qian; Yin, Xiao-Ming; Miao, Long; Kang, Byung-Ho; Xue, Ding

    2016-09-01

    In most eukaryotes, mitochondria are inherited maternally. The autophagy process is critical for paternal mitochondrial elimination (PME) in Caenorhabditis elegans, but how paternal mitochondria, but not maternal mitochondria, are selectively targeted for degradation is poorly understood. Here we report that mitochondrial dynamics have a profound effect on PME. A defect in fission of paternal mitochondria delays PME, whereas a defect in fusion of paternal mitochondria accelerates PME. Surprisingly, a defect in maternal mitochondrial fusion delays PME, which is reversed by a fission defect in maternal mitochondria or by increasing maternal mitochondrial membrane potential using oligomycin. Electron microscopy and tomography analyses reveal that a proportion of maternal mitochondria are compromised when they fail to fuse normally, leading to their competition for the autophagy machinery with damaged paternal mitochondria and delayed PME. Our study indicates that mitochondrial dynamics play a critical role in regulating both the kinetics and the specificity of PME.

  16. Kinetics and specificity of paternal mitochondrial elimination in Caenorhabditis elegans.

    Science.gov (United States)

    Wang, Yang; Zhang, Yi; Chen, Lianwan; Liang, Qian; Yin, Xiao-Ming; Miao, Long; Kang, Byung-Ho; Xue, Ding

    2016-01-01

    In most eukaryotes, mitochondria are inherited maternally. The autophagy process is critical for paternal mitochondrial elimination (PME) in Caenorhabditis elegans, but how paternal mitochondria, but not maternal mitochondria, are selectively targeted for degradation is poorly understood. Here we report that mitochondrial dynamics have a profound effect on PME. A defect in fission of paternal mitochondria delays PME, whereas a defect in fusion of paternal mitochondria accelerates PME. Surprisingly, a defect in maternal mitochondrial fusion delays PME, which is reversed by a fission defect in maternal mitochondria or by increasing maternal mitochondrial membrane potential using oligomycin. Electron microscopy and tomography analyses reveal that a proportion of maternal mitochondria are compromised when they fail to fuse normally, leading to their competition for the autophagy machinery with damaged paternal mitochondria and delayed PME. Our study indicates that mitochondrial dynamics play a critical role in regulating both the kinetics and the specificity of PME. PMID:27581092

  17. Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

    International Nuclear Information System (INIS)

    Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H2O2 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 H2O2 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 2O2 release, assessed by Amplex Red, was reduced by about 45% (p 2O2 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 H2O2 release and increases ROS. ► Aquaporin-8 knockdown causes ROS-induced mitochondrial depolarization and cell death. ► Mitochondrial permeability transition blockage prevents depolarization and cell death.

  18. Impaired expression of the mitochondrial calcium uniporter suppresses mast cell degranulation.

    Science.gov (United States)

    Furuno, Tadahide; Shinkai, Narumi; Inoh, Yoshikazu; Nakanishi, Mamoru

    2015-12-01

    Calcium ion (Ca(2+)) uptake into the mitochondrial matrix influences ATP production, Ca(2+) homeostasis, and apoptosis regulation. Ca(2+) uptake across the ion-impermeable inner mitochondrial membrane is mediated by the mitochondrial Ca(2+) uniporter (MCU) complex. The MCU complex forms a pore structure composed of several proteins. MCU is a Ca(2+)-selective channel in the inner-mitochondrial membrane that allows electrophoretic Ca(2+) entry into the matrix. Mitochondrial Ca(2+) uptake 1 (MICU1) functions as a Ca(2+)-sensing regulator of the MCU complex. Previously, by microscopic analysis at the single-cell level, we found that during mast cell activation, mitochondria capture cytosolic Ca(2+) in two steps. Consequently, mitochondrial Ca(2+) uptake likely plays a role in cellular function through cytosolic Ca(2+) buffering. Here, we investigate the role of MCU and MICU1 in mitochondrial Ca(2+) uptake and mast cell degranulation using MCU- and MICU1-knockdown (KD) mast cells. Whereas MCU- and MICU1-KD mast cells show normal proliferation rates and mitochondrial membrane potential, they exhibit slow and reduced cytosolic and mitochondrial Ca(2+) elevation after antigen stimulation. Moreover, β-hexosaminidase release induced by antigen was significantly suppressed in MCU-KD cells but not MICU1-KD cells. This suggests that both MCU and MICU1 are involved in mitochondrial Ca(2+) uptake in mast cells, while MCU plays a role in mast cell degranulation.

  19. Chemopreventive effect of methanolic extract of Azadirachta indica on experimental Trypanosoma brucei induced oxidative stress in dogs

    Directory of Open Access Journals (Sweden)

    Temidayo O Omobowale

    2015-01-01

    Full Text Available Introduction: The medicinal properties of Azadirachta indica have been harnessed for many years in the treatment of many diseases in both humans and animals. Materials and Methods: 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. Results: 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 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. Conclusion: From this study, MEAI showed significant ability to attenuate oxidative stress and inflammation during experimental T. brucei infection.

  20. Ab initio identification of novel regulatory elements in the genome of Trypanosoma brucei by Bayesian inference on sequence segmentation.

    Directory of Open Access Journals (Sweden)

    Steven Kelly

    Full Text Available BACKGROUND: The rapid increase in the availability of genome information has created considerable demand for both comparative and ab initio predictive bioinformatic analyses. The biology laid bare in the genomes of many organisms is often novel, presenting new challenges for bioinformatic interrogation. A paradigm for this is the collected genomes of the kinetoplastid parasites, a group which includes Trypanosoma brucei the causative agent of human African trypanosomiasis. These genomes, though outwardly simple in organisation and gene content, have historically challenged many theories for gene expression regulation in eukaryotes. METHODOLOGY/PRINCIPLE FINDINGS: Here we utilise a Bayesian approach to identify local changes in nucleotide composition in the genome of T. brucei. We show that there are several elements which are found at the starts and ends of multicopy gene arrays and that there are compositional elements that are common to all intergenic regions. We also show that there is a composition-inversion element that occurs at the position of the trans-splice site. CONCLUSIONS/SIGNIFICANCE: The nature of the elements discovered reinforces the hypothesis that context dependant RNA secondary structure has an important influence on gene expression regulation in Trypanosoma brucei.

  1. Effects of the Czech Propolis on Sperm Mitochondrial Function

    Science.gov (United States)

    Cedikova, Miroslava; Miklikova, Michaela; Stachova, Lenka; Grundmanova, Martina; Tuma, Zdenek; Vetvicka, Vaclav; Zech, Nicolas; Kralickova, Milena; Kuncova, Jitka

    2014-01-01

    Propolis is a natural product that honeybees collect from various plants. It is known for its beneficial pharmacological effects. The aim of our study was to evaluate the impact of propolis on human sperm motility, mitochondrial respiratory activity, and membrane potential. Semen samples from 10 normozoospermic donors were processed according to the World Health Organization criteria. Propolis effects on the sperm motility and mitochondrial activity parameters were tested in the fresh ejaculate and purified spermatozoa. Propolis preserved progressive motility of spermatozoa in the native semen samples. Oxygen consumption determined in purified permeabilized spermatozoa by high-resolution respirometry in the presence of adenosine diphosphate and substrates of complex I and complex II (state OXPHOSI+II) was significantly increased in the propolis-treated samples. Propolis also increased uncoupled respiration in the presence of rotenone (state ETSII) and complex IV activity, but it did not influence state LEAK induced by oligomycin. Mitochondrial membrane potential was not affected by propolis. This study demonstrates that propolis maintains sperm motility in the native ejaculates and increases activities of mitochondrial respiratory complexes II and IV without affecting mitochondrial membrane potential. The data suggest that propolis improves the total mitochondrial respiratory efficiency in the human spermatozoa in vitro thereby having potential to improve sperm motility. PMID:25104965

  2. Mitochondrial DNA repair and association with aging--an update

    DEFF Research Database (Denmark)

    Diaz, Ricardo Gredilla; Bohr, Vilhelm A; Stevnsner, Tinna V.

    2010-01-01

    Mitochondrial DNA is constantly exposed to oxidative injury. Due to its location close to the main site of reactive oxygen species, the inner mitochondrial membrane, mtDNA is more susceptible than nuclear DNA to oxidative damage. The accumulation of DNA damage is thought to play a critical role...... proteins and novel DNA repair pathways, thought to be exclusively present in the nucleus, have recently been described also to be present in mitochondria. Here we review the main mitochondrial DNA repair pathways and their association with the aging process....

  3. The mitochondrial calcium uniporter (MCU): molecular identity and physiological roles.

    Science.gov (United States)

    Patron, Maria; Raffaello, Anna; Granatiero, Veronica; Tosatto, Anna; Merli, Giulia; De Stefani, Diego; Wright, Lauren; Pallafacchina, Giorgia; Terrin, Anna; Mammucari, Cristina; Rizzuto, Rosario

    2013-04-12

    The direct measurement of mitochondrial [Ca(2+)] with highly specific probes demonstrated that major swings in organellar [Ca(2+)] parallel the changes occurring in the cytosol and regulate processes as diverse as aerobic metabolism and cell death by necrosis and apoptosis. Despite great biological relevance, insight was limited by the complete lack of molecular understanding. The situation has changed, and new perspectives have emerged following the very recent identification of the mitochondrial Ca(2+) uniporter, the channel allowing rapid Ca(2+) accumulation across the inner mitochondrial membrane.

  4. Deceleration of fusion-fission cycles improves mitochondrial quality control during aging.

    Directory of Open Access Journals (Sweden)

    Marc Thilo Figge

    Full Text Available Mitochondrial dynamics and mitophagy play a key role in ensuring mitochondrial quality control. Impairment thereof was proposed to be causative to neurodegenerative diseases, diabetes, and cancer. Accumulation of mitochondrial dysfunction was further linked to aging. Here we applied a probabilistic modeling approach integrating our current knowledge on mitochondrial biology allowing us to simulate mitochondrial function and quality control during aging in silico. We demonstrate that cycles of fusion and fission and mitophagy indeed are essential for ensuring a high average quality of mitochondria, even under conditions in which random molecular damage is present. Prompted by earlier observations that mitochondrial fission itself can cause a partial drop in mitochondrial membrane potential, we tested the consequences of mitochondrial dynamics being harmful on its own. Next to directly impairing mitochondrial function, pre-existing molecular damage may be propagated and enhanced across the mitochondrial population by content mixing. In this situation, such an infection-like phenomenon impairs mitochondrial quality control progressively. However, when imposing an age-dependent deceleration of cycles of fusion and fission, we observe a delay in the loss of average quality of mitochondria. This provides a rational why fusion and fission rates are reduced during aging and why loss of a mitochondrial fission factor can extend life span in fungi. We propose the 'mitochondrial infectious damage adaptation' (MIDA model according to which a deceleration of fusion-fission cycles reflects a systemic adaptation increasing life span.

  5. MAVS maintains mitochondrial homeostasis via autophagy

    Science.gov (United States)

    Sun, Xiaofeng; Sun, Liwei; Zhao, Yuanyuan; Li, Ying; Lin, Wei; Chen, Dahua; Sun, Qinmiao

    2016-01-01

    Mitochondrial antiviral signalling protein (MAVS) acts as a critical adaptor protein to transduce antiviral signalling by physically interacting with activated RIG-I and MDA5 receptors. MAVS executes its functions at the outer membrane of mitochondria to regulate downstream antiviral signalling, indicating that the mitochondria provides a functional platform for innate antiviral signalling transduction. However, little is known about whether and how MAVS-mediated antiviral signalling contributes to mitochondrial homeostasis. Here we show that the activation of MAVS is sufficient to induce autophagic signalling, which may mediate the turnover of the damaged mitochondria. Importantly, we find MAVS directly interacts with LC3 through its LC3-binding motif ‘YxxI’, suggesting that MAVS might act as an autophagy receptor to mediate mitochondrial turnover upon excessive activation of RLR signalling. Furthermore, we provide evidence that both MAVS self-aggregation and its interaction with TRAF2/6 proteins are important for MAVS-mediated mitochondrial turnover. Collectively, our findings suggest that MAVS acts as a potential receptor for mitochondria-associated autophagic signalling to maintain mitochondrial homeostasis. PMID:27551434

  6. Characteristics of Mitochondrial Transformation into Human Cells

    Science.gov (United States)

    Kesner, E. E.; Saada-Reich, A.; Lorberboum-Galski, H.

    2016-01-01

    Mitochondria can be incorporated into mammalian cells by simple co-incubation of isolated mitochondria with cells, without the need of transfection reagents or any other type of intervention. This phenomenon was termed mitochondrial transformation, and although it was discovered in 1982, currently little is known regarding its mechanism(s). Here we demonstrate that mitochondria can be transformed into recipient cells very quickly, and co-localize with endogenous mitochondria. The isolated mitochondria interact directly with cells, which engulf the mitochondria with cellular extensions in a way, which may suggest the involvement of macropinocytosis or macropinocytosis-like mechanisms in mitochondrial transformation. Indeed, macropinocytosis inhibitors but not clathrin-mediated endocytosis inhibition-treatments, blocks mitochondria transformation. The integrity of the mitochondrial outer membrane and its proteins is essential for the transformation of the mitochondria into cells; cells can distinguish mitochondria from similar particles and transform only intact mitochondria. Mitochondrial transformation is blocked in the presence of the heparan sulfate molecules pentosan polysulfate and heparin, which indicate crucial involvement of cellular heparan sulfate proteoglycans in the mitochondrial transformation process. PMID:27184109

  7. Mitochondrial functions on oocytes and preimplantation embryos

    Institute of Scientific and Technical Information of China (English)

    Li-ya WANG; Da-hui WANG; Xiang-yang ZOU; Chen-ming XU

    2009-01-01

    Oocyte quality has long been considered as a main limiting factor for in vitro fertilization (IVF). In the past decade,extensive observations demonstrated that the mitochondrion plays a vital role in the oocyte cytoplasm, for it can provide adenosine triphosphate (ATP) for fertilization and preimplantation embryo development and also act as stores of intracellular calcium and proapoptotic factors. During the oocyte maturation, mitochondria are characterized by distinct changes of their distribution pattern from being homogeneous to heterogeneous, which is correlated with the cumulus apoptosis. Oocyte quality decreases with the increasing maternal age. Recent studies have shown that low quality oocytes have some age-related dysfunctions, which include the decrease in mitochondrial membrane potential, increase of mitochondrial DNA (mtDNA) damages, chromosomal aneuploidies,the incidence of apoptosis, and changes in mitochondrial gene expression. All these dysfunctions may cause a high level of developmental retardation and arrest of preimplantation embryos. It has been suggested that these mitochondrial changes may arise from excessive reactive oxygen species (ROS) that is closely associated with the oxidative energy production or calcium overload,which may trigger permeability transition pore opening and subsequent apoptosis. Therefore, mitochondria can be seen as signs for oocyte quality evaluation, and it is possible that the oocyte quality can be improved by enhancing the physical function of mitochondria. Here we reviewed recent advances in mitochondrial functions on oocytes.

  8. Characteristics of Mitochondrial Transformation into Human Cells.

    Science.gov (United States)

    Kesner, E E; Saada-Reich, A; Lorberboum-Galski, H

    2016-01-01

    Mitochondria can be incorporated into mammalian cells by simple co-incubation of isolated mitochondria with cells, without the need of transfection reagents or any other type of intervention. This phenomenon was termed mitochondrial transformation, and although it was discovered in 1982, currently little is known regarding its mechanism(s). Here we demonstrate that mitochondria can be transformed into recipient cells very quickly, and co-localize with endogenous mitochondria. The isolated mitochondria interact directly with cells, which engulf the mitochondria with cellular extensions in a way, which may suggest the involvement of macropinocytosis or macropinocytosis-like mechanisms in mitochondrial transformation. Indeed, macropinocytosis inhibitors but not clathrin-mediated endocytosis inhibition-treatments, blocks mitochondria transformation. The integrity of the mitochondrial outer membrane and its proteins is essential for the transformation of the mitochondria into cells; cells can distinguish mitochondria from similar particles and transform only intact mitochondria. Mitochondrial transformation is blocked in the presence of the heparan sulfate molecules pentosan polysulfate and heparin, which indicate crucial involvement of cellular heparan sulfate proteoglycans in the mitochondrial transformation process.

  9. MAVS maintains mitochondrial homeostasis via autophagy.

    Science.gov (United States)

    Sun, Xiaofeng; Sun, Liwei; Zhao, Yuanyuan; Li, Ying; Lin, Wei; Chen, Dahua; Sun, Qinmiao

    2016-01-01

    Mitochondrial antiviral signalling protein (MAVS) acts as a critical adaptor protein to transduce antiviral signalling by physically interacting with activated RIG-I and MDA5 receptors. MAVS executes its functions at the outer membrane of mitochondria to regulate downstream antiviral signalling, indicating that the mitochondria provides a functional platform for innate antiviral signalling transduction. However, little is known about whether and how MAVS-mediated antiviral signalling contributes to mitochondrial homeostasis. Here we show that the activation of MAVS is sufficient to induce autophagic signalling, which may mediate the turnover of the damaged mitochondria. Importantly, we find MAVS directly interacts with LC3 through its LC3-binding motif 'YxxI', suggesting that MAVS might act as an autophagy receptor to mediate mitochondrial turnover upon excessive activation of RLR signalling. Furthermore, we provide evidence that both MAVS self-aggregation and its interaction with TRAF2/6 proteins are important for MAVS-mediated mitochondrial turnover. Collectively, our findings suggest that MAVS acts as a potential receptor for mitochondria-associated autophagic signalling to maintain mitochondrial homeostasis. PMID:27551434

  10. Scoliosis in Mitochondrial Myopathy

    OpenAIRE

    Li, Zheng; Shen, Jianxiong; Liang, Jinqian

    2015-01-01

    Abstract The mitochondrial myopathies include a diverse group of disorders characterized by morphological abnormalities of muscle mitochondria. Little is reported about spinal deformity associated with this syndrome. This study presents a case of scoliosis occurring in the setting of mitochondrial myopathies and explores the possible mechanisms between the 2 diseases. A previously unreported scoliosis in mitochondrial myopathies is described. The patient was a 16-year-old Chinese adolescent b...

  11. Keshan disease and mitochondrial cardiomyopathy

    Institute of Scientific and Technical Information of China (English)

    YANG; Fuyu

    2006-01-01

    Keshan disease (KD) is a potentially fatal form of cardiomyopathy (disease of the heart muscle) endemic in certain areas of China. From 1984 to 1986, a national comprehensive scientific investigation on KD in Chuxiong region of Yunnan Province in the southwest China was conducted. The investigation team was composed of epidemiologists, clinic doctors, pathologists, biochemists, biophysicists and specialists in ecological environment. Results of pathological, biochemical and biophysical as well as clinical studies showed: an obvious increase of enlarged and swollen mitochondria with distended crista membranes in myocardium from patients with KD; significant reductions in the activity of oxidative phosphorylation (succinate dehydrogenase, cytochrome oxidase, succinate oxidase, H+-ATPase) of affected mitochondria; decrease in CoQ, cardiolipin, Se and GSHPx activity, while obvious increase in the Ca2+ content. So, it was suggested that mitochondria are the predominant target of the pathogenic factors of KD. Before Chuxiong KD survey only a few cases of mitochondrial cardiomyopathy were studied. During the multidisciplinary scientific investigation on KD in Chuxiong a large amount of samples from KD cases and the positive controls were examined. On the basis of the results obtained it was suggested that KD might be classified as a "Mitochondrial Cardiomyopathy" endemic in China. This is one of the achievements in the three years' survey in Chuxiong and is valuable not only to the deeper understanding of pathogenic mechanism of KD but also to the study of mitochondrial cardiomyopathy in general.Keshan disease is not a genetic disease, but is closely related to the malnutrition (especially microelement Se deficiency). KD occurs along a low Se belt, and Se supplementation has been effective in prevention of such disease. The incidence of KD has sharply decreased along with the steady raise of living standard and realization of preventive measures. At present, patients of

  12. Mitochondrial morphology and cardiovascular disease

    OpenAIRE

    Ong, Sang-Bing; Hausenloy, Derek J

    2010-01-01

    Mitochondria are dynamic and are able to interchange their morphology between elongated interconnected mitochondrial networks and a fragmented disconnected arrangement by the processes of mitochondrial fusion and fission, respectively. Changes in mitochondrial morphology are regulated by the mitochondrial fusion proteins (mitofusins 1 and 2, and optic atrophy 1) and the mitochondrial fission proteins (dynamin-related peptide 1 and mitochondrial fission protein 1) and have been implicated in a...

  13. Transport proteins determine drug sensitivity and resistance in a protozoan parasite, Trypanosoma brucei

    Science.gov (United States)

    Munday, Jane C.; Settimo, Luca; de Koning, Harry P.

    2015-01-01

    Drug resistance in pathogenic protozoa is very often caused by changes to the ‘transportome’ of the parasites. In Trypanosoma brucei, several transporters have been implicated in uptake of the main classes of drugs, diamidines and melaminophenyl arsenicals. The resistance mechanism had been thought to be due to loss of a transporter known to carry both types of agents: the aminopurine transporter P2, encoded by the gene TbAT1. However, although loss of P2 activity is well-documented as the cause of resistance to the veterinary diamidine diminazene aceturate (DA; Berenil®), cross-resistance between the human-use arsenical melarsoprol and the diamidine pentamidine (melarsoprol/pentamidine cross resistance, MPXR) is the result of loss of a separate high affinity pentamidine transporter (HAPT1). A genome-wide RNAi library screen for resistance to pentamidine, published in 2012, gave the key to the genetic identity of HAPT1 by linking the phenomenon to a locus that contains the closely related T. brucei aquaglyceroporin genes TbAQP2 and TbAQP3. Further analysis determined that knockdown of only one pore, TbAQP2, produced the MPXR phenotype. TbAQP2 is an unconventional aquaglyceroporin with unique residues in the “selectivity region” of the pore, and it was found that in several MPXR lab strains the WT gene was either absent or replaced by a chimeric protein, recombined with parts of TbAQP3. Importantly, wild-type AQP2 was also absent in field isolates of T. b. gambiense, correlating with the outcome of melarsoprol treatment. Expression of a wild-type copy of TbAQP2 in even the most resistant strain completely reversed MPXR and re-introduced HAPT1 function and transport kinetics. Expression of TbAQP2 in Leishmania mexicana introduced a pentamidine transport activity indistinguishable from HAPT1. Although TbAQP2 has been shown to function as a classical aquaglyceroporin it is now clear that it is also a high affinity drug transporter, HAPT1. We discuss here a

  14. Changes in blood sugar levels of rats experimentally infected withTrypanosoma brucei and treated with imidocarb dipropionate and diminazene aceturate

    Institute of Scientific and Technical Information of China (English)

    Nwoha Rosemary Ijeoma Ogechi; Omamegbe Joseph Omalathebu

    2016-01-01

    Objective:To determine the effect ofTrypanosoma brucei (T. brucei) on blood sugar level of infected rats. Methods: The experiment was done with 42 albino rats grouped into 3 groups of 14 members each. Group A was uninfected (control group), Group B was infected withT. brucei and treated with diminazene aceturate, and Group C was infected withT. brucei and treated with imidocarb dipropionate. Blood samples were collected from the media canthus of the experimental rats on Days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 for the assessment of change in blood sugar levels. The blood sugar levels were determined with a glucometer (Accu-chek active serialNo.GN:10023338). Results: By 4 to 5 days post infection, there was a significant increase (P 0.05) was observed in the groups when compared with the control group till Day 12 of the experiment. Conclusions:T. brucei caused a significant increase in blood sugar of infected rats.

  15. Neurodegenerative and Fatiguing Illnesses, Infections and Mitochondrial Dysfunction: Use of Natural Supplements to Improve Mitochondrial Function

    Directory of Open Access Journals (Sweden)

    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. Phenyl-α-tert-Butyl Nitrone Reverses Mitochondrial Decay in Acute Chagas’ Disease

    OpenAIRE

    Wen, Jian-jun; Bhatia, Vandanajay; Popov, Vsevolod L.; Garg, Nisha Jain

    2006-01-01

    In this study, we investigated the mechanism(s) of mitochondrial functional decline in acute Chagas’ disease. Our data show a substantial decline in respiratory complex activities (39 to 58%) and ATP (38%) content in Trypanosoma cruzi-infected murine hearts compared with normal controls. These metabolic alterations were associated with an approximately fivefold increase in mitochondrial reactive oxygen species production rate, substantial oxidative insult of mitochondrial membranes and respir...

  17. MICU1 motifs define mitochondrial calcium uniporter binding and activity.

    Science.gov (United States)

    Hoffman, Nicholas E; Chandramoorthy, Harish C; Shamugapriya, Santhanam; Zhang, Xueqian; Rajan, Sudarsan; Mallilankaraman, Karthik; Gandhirajan, Rajesh Kumar; Vagnozzi, Ronald J; Ferrer, Lucas M; Sreekrishnanilayam, Krishnalatha; Natarajaseenivasan, Kalimuthusamy; Vallem, Sandhya; Force, Thomas; Choi, Eric T; Cheung, Joseph Y; Madesh, Muniswamy

    2013-12-26

    Resting mitochondrial matrix Ca(2+) is maintained through a mitochondrial calcium uptake 1 (MICU1)-established threshold inhibition of mitochondrial calcium uniporter (MCU) activity. It is not known how MICU1 interacts with MCU to establish this Ca(2+) threshold for mitochondrial Ca(2+) uptake and MCU activity. Here, we show that MICU1 localizes to the mitochondrial matrix side of the inner mitochondrial membrane and MICU1/MCU binding is determined by a MICU1 N-terminal polybasic domain and two interacting coiled-coil domains of MCU. Further investigation reveals that MICU1 forms homo-oligomers, and this oligomerization is independent of the polybasic region. However, the polybasic region confers MICU1 oligomeric binding to MCU and controls mitochondrial Ca(2+) current (IMCU). Moreover, MICU1 EF hands regulate MCU channel activity, but do not determine MCU binding. Loss of MICU1 promotes MCU activation leading to oxidative burden and a halt to cell migration. These studies establish a molecular mechanism for MICU1 control of MCU-mediated mitochondrial Ca(2+) accumulation, and dysregulation of this mechanism probably enhances vascular dysfunction.

  18. Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

    Energy Technology Data Exchange (ETDEWEB)

    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

  19. Mitochondrial Dynamics in Diabetes

    OpenAIRE

    Yoon, Yisang; Galloway, Chad A.; Jhun, Bong Sook; Yu, Tianzheng

    2011-01-01

    Mitochondria are at the center of cellular energy metabolism and regulate cell life and death. The cell biological aspect of mitochondria, especially mitochondrial dynamics, has drawn much attention through implications in human pathology, including neurological disorders and metabolic diseases. Mitochondrial fission and fusion are the main processes governing the morphological plasticity and are controlled by multiple factors, including mechanochemical enzymes and accessory proteins. Emergin...

  20. Defects of mitochondrial DNA replication.

    Science.gov (United States)

    Copeland, William C

    2014-09-01

    Mitochondrial DNA is replicated by DNA polymerase γ in concert with accessory proteins such as the mitochondrial DNA helicase, single-stranded DNA binding protein, topoisomerase, and initiating factors. Defects in mitochondrial DNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mitochondrial DNA deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mitochondrial DNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mitochondrial DNA deletion disorders, such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. This review focuses on our current knowledge of genetic defects of mitochondrial DNA replication (POLG, POLG2, C10orf2, and MGME1) that cause instability of mitochondrial DNA and mitochondrial disease.

  1. Avocado Oil Improves Mitochondrial Function and Decreases Oxidative Stress in Brain of Diabetic Rats

    Directory of Open Access Journals (Sweden)

    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.

  2. 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.

  3. Is cell aging caused by respiration-dependent injury to the mitochondrial genome

    Science.gov (United States)

    Fleming, J. E.; Yengoyan, L. S.; Miquel, J.; Cottrell, S. F.; Economos, A. C.

    1982-01-01

    Though intrinsic mitochondrial aging has been considered before as a possible cause of cellular senescence, the mechanisms of such mitochondrial aging have remained obscure. In this article, the hypothesis of free-radical-induced inhibition of mitochondrial replenishment in fixed postmitotic cells is expanded. It is maintained that the respiration-dependent production of superoxide and hydroxyl radicals may not be fully counteracted, leading to a continuous production of lipoperoxides and malonaldehyde in actively respiring mitochondria. These compounds, in turn, can easily react with the mitochondrial DNA which is in close spatial relationship with the inner mitochondrial membrane, producing an injury that the mitochondria may be unable to counteract because of their apparent lack of adequate repair mechanisms. Mitochondrial division may thus be inhibited leading to age-related reduction of mitochondrial numbers, a deficit in energy production with a concomitant decrease in protein synthesis, deterioration of physiological performance, and, therefore, of organismic performance.

  4. 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. PMID:26180820

  5. Flux Analysis of the Trypanosoma brucei Glycolysis Based on a Multiobjective-Criteria Bioinformatic Approach

    Directory of Open Access Journals (Sweden)

    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.

  6. A new generation of T7 RNA polymerase-independent inducible expression plasmids for Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Jack Sunter

    Full Text Available Expression of transgenes is central to forward and reverse genetic analysis in Trypanosoma brucei. The inducible expression of transgenes in trypanosomes is based on the tetracycline repressor binding to a tetracycline operator to prevent transcription in the absence of tetracycline. The same inducible system is used to produce double-stranded RNA for RNAi knockdown of target genes. This study describes a new plasmid pSPR2.1 that drives consistent high-level expression of tetracycline repressor in procyclic form trypanosomes. A complementary expression plasmid, p3227, was constructed. The major difference between this and current plasmids is the separation of the inducible transgene and selectable marker promoters by the plasmid backbone. The plasmid p3227 was able to support inducible expression in cell lines containing pSPR2.1 as well as the established Lister 427 29-13 cell line. p3666, a derivative of p3227, was made for inducible expression of stem loop RNAi constructs and was effective for knockdown of DRBD3, which had proved problematic using existing RNAi plasmids with head-to-head promoters. The plasmid system was also able to support inducible transgene expression and DRBD3 RNAi knockdown in bloodstream form cells expressing tetracycline repressor from an integrated copy of the plasmid pHD1313.

  7. Immune Evasion Strategies of Trypanosoma brucei within the Mammalian Host: Progression to Pathogenicity

    Science.gov (United States)

    Stijlemans, Benoît; Caljon, Guy; Van Den Abbeele, Jan; Van Ginderachter, Jo A.; Magez, Stefan; De Trez, Carl

    2016-01-01

    The diseases caused by African trypanosomes (AT) are of both medical and veterinary importance and have adversely influenced the economic development of sub-Saharan Africa. Moreover, so far not a single field applicable vaccine exists, and chemotherapy is the only strategy available to treat the disease. These strictly extracellular protozoan parasites are confronted with different arms of the host’s immune response (cellular as well as humoral) and via an elaborate and efficient (vector)–parasite–host interplay they have evolved efficient immune escape mechanisms to evade/manipulate the entire host immune response. This is of importance, since these parasites need to survive sufficiently long in their mammalian/vector host in order to complete their life cycle/transmission. Here, we will give an overview of the different mechanisms AT (i.e. T. brucei as a model organism) employ, comprising both tsetse fly saliva and parasite-derived components to modulate host innate immune responses thereby sculpturing an environment that allows survival and development within the mammalian host.

  8. Latent Trypanosoma brucei gambiense foci in Uganda: a silent epidemic in children and adults?

    Science.gov (United States)

    Wastling, S L; Picozzi, K; Wamboga, C; VON Wissmann, B; Amongi-Accup, C; Wardrop, N A; Stothard, J R; Kakembo, A; Welburn, S C

    2011-10-01

    Trypanosoma brucei gambiense sleeping sickness follows a long asymptomatic phase and persists in ancient foci from which epidemic clinical disease arises. A putative focus of T. b. gambiense infections has been identified, initially in mothers and young children, on the Lake Albert shoreline of Western Uganda leading to mass screening of 6207 individuals in September 2008. T. b. gambiense infections were identified by Card Agglutination Test for Trypanosomiasis (CATT) and sub-species-specific PCR although parasitological methods failed to confirm any patent trypanosome infections. In April 2009, CATT positives were re-visited; diagnosis of individuals by CATT and PCR was unstable over the two time points and parasites remained undetected, even using mini Anion Exchange Centrifugation Technique (mAECT). These observations suggest the possibility of a silent focus of disease, where all infected individuals are in a latent stage, and highlight our limited understanding of the local natural history and disease progression of T. b. gambiense in children and adults. PMID:21554841

  9. Synchronous expression of individual metacyclic variant surface glycoprotein genes in Trypanosoma brucei.

    Science.gov (United States)

    Ramey-Butler, Kiantra; Ullu, Elisabetta; Kolev, Nikolay G; Tschudi, Christian

    2015-01-01

    One distinctive feature of the Trypanosoma brucei life cycle is the presence of two discrete populations that are based on differential expression of variant surface glycoproteins (VSGs). Both are adapted to the environmental pressures they face and more importantly, both contribute directly to transmission. Metacyclics in the tsetse fly enable transmission to a new mammalian host, whereas bloodstream trypanosomes must avoid immune destruction to the extent that sufficient numbers are available for transmission, when the insect vector takes a blood meal. At present, there are few investigations on the molecular aspects of parasite biology in the tsetse vector and specifically about the activation of metacyclic VSG gene expression. Here we used an established in vitro differentiation system based on the overexpression of the RNA-binding protein 6 (RBP6), to monitor two metacyclic VSGs (VSG 397 and VSG 653) during development from procyclics to infectious metacyclic forms. We observed that activation of these two mVSGs was simultaneous both at the transcript and protein level, and manifested by the appearance of only one of the mVSGs in individual cells. PMID:25896436

  10. Identification and characterization of an unusual class I myosin involved in vesicle traffic in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Diana Spitznagel

    Full Text Available Myosins are a multimember family of motor proteins with diverse functions in eukaryotic cells. African trypanosomes possess only two candidate myosins and thus represent a useful system for functional analysis of these motors. One of these candidates is an unusual class I myosin (TbMyo1 that is expressed at similar levels but organized differently during the life cycle of Trypanosoma brucei. This myosin localizes to the polarized endocytic pathway in bloodstream forms of the parasite. This organization is actin dependent. Knock down of TbMyo1 results in a significant reduction in endocytic activity, a cessation in cell division and eventually cell death. A striking morphological feature in these cells is an enlargement of the flagellar pocket, which is consistent with an imbalance in traffic to and from the surface. In contrast TbMyo1 is distributed throughout procyclic forms of the tsetse vector and a loss of approximately 90% of the protein has no obvious effects on growth or morphology. These results reveal a life cycle stage specific requirement for this myosin in essential endocytic traffic and represent the first description of the involvement of a motor protein in vesicle traffic in these parasites.

  11. Identification and characterization of an unusual class I myosin involved in vesicle traffic in Trypanosoma brucei.

    Science.gov (United States)

    Spitznagel, Diana; O'Rourke, John F; Leddy, Neal; Hanrahan, Orla; Nolan, Derek P

    2010-01-01

    Myosins are a multimember family of motor proteins with diverse functions in eukaryotic cells. African trypanosomes possess only two candidate myosins and thus represent a useful system for functional analysis of these motors. One of these candidates is an unusual class I myosin (TbMyo1) that is expressed at similar levels but organized differently during the life cycle of Trypanosoma brucei. This myosin localizes to the polarized endocytic pathway in bloodstream forms of the parasite. This organization is actin dependent. Knock down of TbMyo1 results in a significant reduction in endocytic activity, a cessation in cell division and eventually cell death. A striking morphological feature in these cells is an enlargement of the flagellar pocket, which is consistent with an imbalance in traffic to and from the surface. In contrast TbMyo1 is distributed throughout procyclic forms of the tsetse vector and a loss of approximately 90% of the protein has no obvious effects on growth or morphology. These results reveal a life cycle stage specific requirement for this myosin in essential endocytic traffic and represent the first description of the involvement of a motor protein in vesicle traffic in these parasites.

  12. Isothermal microcalorimetry, a new tool to monitor drug action against Trypanosoma brucei and Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    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.

  13. Identification of paralogous life-cycle stage specific cytoskeletal proteins in the parasite Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Neil Portman

    Full Text Available The life cycle of the African trypanosome Trypanosoma brucei, is characterised by a transition between insect and mammalian hosts representing very different environments that present the parasite with very different challenges. These challenges are met by the expression of life-cycle stage-specific cohorts of proteins, which function in systems such as metabolism and immune evasion. These life-cycle transitions are also accompanied by morphological rearrangements orchestrated by microtubule dynamics and associated proteins of the subpellicular microtubule array. Here we employed a gel-based comparative proteomic technique, Difference Gel Electrophoresis, to identify cytoskeletal proteins that are expressed differentially in mammalian infective and insect form trypanosomes. From this analysis we identified a pair of novel, paralogous proteins, one of which is expressed in the procyclic form and the other in the bloodstream form. We show that these proteins, CAP51 and CAP51V, localise to the subpellicular corset of microtubules and are essential for correct organisation of the cytoskeleton and successful cytokinesis in their respective life cycle stages. We demonstrate for the first time redundancy of function between life-cycle stage specific paralogous sets in the cytoskeleton and reveal modification of cytoskeletal components in situ prior to their removal during differentiation from the bloodstream form to the insect form. These specific results emphasise a more generic concept that the trypanosome genome encodes a cohort of cytoskeletal components that are present in at least two forms with life-cycle stage-specific expression.

  14. Dynamic modelling under uncertainty: the case of Trypanosoma brucei energy metabolism.

    Directory of Open Access Journals (Sweden)

    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.

  15. Fluorinated Sterols Are Suicide Inhibitors of Ergosterol Biosynthesis and Growth in Trypanosoma brucei.

    Science.gov (United States)

    Leaver, David J; Patkar, Presheet; Singha, Ujjal K; Miller, Matthew B; Haubrich, Brad A; Chaudhuri, Minu; Nes, W David

    2015-10-22

    Trypanosoma brucei, the causal agent for sleeping sickness, depends on ergosterol for growth. Here, we describe the effects of a mechanism-based inhibitor, 26-fluorolanosterol (26FL), which converts in vivo to a fluorinated substrate of the sterol C24-methyltransferase essential for sterol methylation and function of ergosterol, and missing from the human host. 26FL showed potent inhibition of ergosterol biosynthesis and growth of procyclic and bloodstream forms while having no effect on cholesterol biosynthesis or growth of human epithelial kidney cells. During exposure of cloned TbSMT to 26-fluorocholesta-5,7,24-trienol, the enzyme is gradually killed as a consequence of the covalent binding of the intermediate C25 cation to the active site (kcat/kinact = 0.26 min(-1)/0.24 min(-1); partition ratio of 1.08), whereas 26FL is non-productively bound. These results demonstrate that poisoning of ergosterol biosynthesis by a 26-fluorinated Δ(24)-sterol is a promising strategy for developing a new treatment for trypanosomiasis.

  16. Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Daniel Nilsson

    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/.

  17. Dual Effect of Phosphate Transport on Mitochondrial Ca2+ Dynamics.

    Science.gov (United States)

    Wei, An-Chi; Liu, Ting; O'Rourke, Brian

    2015-06-26

    The large inner membrane electrochemical driving force and restricted volume of the matrix confer unique constraints on mitochondrial ion transport. Cation uptake along with anion and water movement induces swelling if not compensated by other processes. For mitochondrial Ca(2+) uptake, these include activation of countertransporters (Na(+)/Ca(2+) exchanger and Na(+)/H(+) exchanger) coupled to the proton gradient, ultimately maintained by the proton pumps of the respiratory chain, and Ca(2+) binding to matrix buffers. Inorganic phosphate (Pi) is known to affect both the Ca(2+) uptake rate and the buffering reaction, but the role of anion transport in determining mitochondrial Ca(2+) dynamics is poorly understood. Here we simultaneously monitor extra- and intra-mitochondrial Ca(2+) and mitochondrial membrane potential (ΔΨm) to examine the effects of anion transport on mitochondrial Ca(2+) flux and buffering in Pi-depleted guinea pig cardiac mitochondria. Mitochondrial Ca(2+) uptake proceeded slowly in the absence of Pi but matrix free Ca(2+) ([Ca(2+)]mito) still rose to ~50 μm. Pi (0.001-1 mm) accelerated Ca(2+) uptake but decreased [Ca(2+)]mito by almost 50% while restoring ΔΨm. Pi-dependent effects on Ca(2+) were blocked by inhibiting the phosphate carrier. Mitochondrial Ca(2+) uptake rate was also increased by vanadate (Vi), acetate, ATP, or a non-hydrolyzable ATP analog (AMP-PNP), with differential effects on matrix Ca(2+) buffering and ΔΨm recovery. Interestingly, ATP or AMP-PNP prevented the effects of Pi on Ca(2+) uptake. The results show that anion transport imposes an upper limit on mitochondrial Ca(2+) uptake and modifies the [Ca(2+)]mito response in a complex manner. PMID:25963147

  18. A crucial role of the mitochondrial protein import receptor MOM19 for the biogenesis of mitochondria

    OpenAIRE

    Harkness, Troy A. A.; Nargang, Frank E.; van der Klei, Ida; Neupert, Walter; Lill, Roland

    1994-01-01

    The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activit...

  19. MOM22 is a receptor for mitochondrial targeting sequences and cooperates with MOM19.

    OpenAIRE

    Mayer, A.; Nargang, F E; Neupert, W; Lill, R

    1995-01-01

    Recognition of targeting signals is a crucial step in protein sorting within the cell. So far, only a few components capable of deciphering targeting signals have been identified, and insights into the chemical nature of the interaction between the signals and their receptors are scarce. Using highly purified mitochondrial outer membrane vesicles, we demonstrate that MOM22 and MOM19, components of the protein import complex of the outer membrane, bind preproteins at the mitochondrial surface ...

  20. Effect of Anthocyanin Extract of Mulberry Fruit on the Apoptosis and the Mitochondrial Membrane Potential of Breast Cancer Cells%桑葚花色苷提取物对乳腺癌细胞凋亡及线粒体膜电位的影响

    Institute of Scientific and Technical Information of China (English)

    常徽; 王湛; 袁丽佳; 付钰洁; 糜漫天

    2012-01-01

    目的:观察桑葚花色苷提取物对人乳腺癌细胞株MDA-MB-453、MDA-MB-231和MCF-7细胞凋亡及线粒体膜电位的影响.方法:利用超声辅助乙醇萃取法提取桑葚花色苷,pH示差法测定提取物花色苷总含量,以50、100和150 mg/mL桑葚花色苷提取物作用三种乳腺癌细胞MDA-MB-231、MDA-MB-453和MCF-7 24h,采用Annexin V/PI双染流式细胞分析法检测细胞凋亡水平变化,JC-1探针染色激光共聚焦扫描显微镜观察MDA-MB-453细胞线粒体膜电位水平变化.结果:凋亡分析结果表明,桑葚花色苷提取物作用后三种乳腺癌细胞凋亡率均升高,显示出促凋亡效应,且具有剂量-效应关系,100和150 mg/mL组凋亡率显著升高(P<0.05).激光共聚焦扫描显微镜检测结果显示,桑葚花色苷提取物作用24h,可使MDA-MB-453细胞线粒体膜电位显著下降,表现为红色/绿色荧光的比值显著降低(P<0.05).结论:桑葚花色苷提取物可显著降低乳腺癌细胞线粒体膜电位,并促发细胞凋亡.%Objective: To study the effect of anthocyanin extract of mulberry fruit on the apoptosis and the mitochondrial membrane potential of breast cancer cells MDA-MB-453, MDA-MB-231 and MCF-7. Methods: Preparation of anthocyanin-rich extract from mulberry fruits was carried out by ultrasonic extraction with acidified-ethanol. The breast cancer cells MDA-MB-453, MDA-MB-231 and MCF-7 were reated with 50, 100 or 150 mg/mL of the anthocyanin-rich extract for 24h; the cells apoptosis were analyzed by Annexin V/PI dyeing and flow cytometric assay. To characterize the upstream factors involved in the intrinsic apoptosis pathway, the mitochondrial permeability of MDA-MB-453 cells were measured by JC-1 staining and the laser confocal scanning microscopy. Results: The apoptosis analysis results indicated that the anthocyanin-rich mulberry fruit extract treated for 24 h, the apoptosis rate of these three breast cells in 100 and 150 mg/mL treated groups markedly

  1. Mitochondrial toxicity of depleted uranium: protection by Beta-glucan.

    Science.gov (United States)

    Shaki, Fatemeh; Pourahmad, Jalal

    2013-01-01

    Considerable evidence suggests that mitochondrial dysfunction contributes to the toxicity of uranyl acetate (UA), a soluble salt of depleted uranium (DU). We examined the ability of the two antioxidants, beta-glucan and butylated hydroxyl toluene (BHT), to prevent UA-induced mitochondrial dysfunction using rat-isolated kidney mitochondria. Beta-glucan (150 nM) and BHT (20 nM) attenuated UA-induced mitochondrial reactive oxygen species (ROS) formation, lipid peroxidation and glutathione oxidation. Beta-glucan and BHT also prevented the loss of mitochondrial membrane potential (MMP) and mitochondrial swelling following the UA treatment in isolated mitochondria. Our results show that beta-glucan and BHT prevented UA-induced mitochondrial outer membrane damage as well as release of cytochrome c from mitochondria. UA also decreased the ATP production in isolated mitochondria significantly inhibited with beta-glucan and BHT pre-treatment. Our results showed that beta-glucan may be mitochondria-targeted antioxidant and suggested this compound as a possible drug candidate for prophylaxis and treatment against DU-induced nephrotoxicity. PMID:24250581

  2. 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.

  3. Mitochondrial benzodiazepine receptors regulate steroid biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Mitochondrial benzodiazepine receptors regulate steroid biosynthesis

    International Nuclear Information System (INIS)

    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'-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 3H-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

  5. Probing glycolytic and membrane potential oscillations in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

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

    2008-01-01

    We have investigated glycolytic oscillations under semi-anaerobic conditions in Saccharomyces cerevisiae by means of NADH fluorescence, measurements of intracellular glucose concentration, and mitochondrial membrane potential. The glucose concentration was measured using an optical nanosensor, wh...

  6. Mitochondrial biogenesis: pharmacological approaches.

    Science.gov (United States)

    Valero, Teresa

    2014-01-01

    Organelle biogenesis is concomitant to organelle inheritance during cell division. It is necessary that organelles double their size and divide to give rise to two identical daughter cells. Mitochondrial biogenesis occurs by growth and division of pre-existing organelles and is temporally coordinated with cell cycle events [1]. However, mitochondrial biogenesis is not only produced in association with cell division. It can be produced in response to an oxidative stimulus, to an increase in the energy requirements of the cells, to exercise training, to electrical stimulation, to hormones, during development, in certain mitochondrial diseases, etc. [2]. Mitochondrial biogenesis is therefore defined as the process via which cells increase their individual mitochondrial mass [3]. Recent discoveries have raised attention to mitochondrial biogenesis as a potential target to treat diseases which up to date do not have an efficient cure. Mitochondria, as the major ROS producer and the major antioxidant producer exert a crucial role within the cell mediating processes such as apoptosis, detoxification, Ca2+ buffering, etc. This pivotal role makes mitochondria a potential target to treat a great variety of diseases. Mitochondrial biogenesis can be pharmacologically manipulated. This issue tries to cover a number of approaches to treat several diseases through triggering mitochondrial biogenesis. It contains recent discoveries in this novel field, focusing on advanced mitochondrial therapies to chronic and degenerative diseases, mitochondrial diseases, lifespan extension, mitohormesis, intracellular signaling, new pharmacological targets and natural therapies. It contributes to the field by covering and gathering the scarcely reported pharmacological approaches in the novel and promising field of mitochondrial biogenesis. There are several diseases that have a mitochondrial origin such as chronic progressive external ophthalmoplegia (CPEO) and the Kearns- Sayre syndrome (KSS

  7. THE MULTIPLE ROLES OF CYCLIN E1 IN CONTROLLING CELL CYCLE PROGRESSION AND CELLULAR MORPHOLOGY OF TRYPANOSOMA BRUCEI

    OpenAIRE

    Gourguechon, Stéphane; Savich, Jason M.; Ching C Wang

    2007-01-01

    Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases. Previous RNA interference (RNAi) experiments in Trypanosoma brucei indicated that cyclin E1, cdc2-related kinase (CRK)1 and CRK2 are involved in regulating G1/S transition, whereas cyclin B2 and CRK3 play a pivotal role in controlling the G2/M checkpoint. To search for potential interactions between the other cyclins and CRKs that may not have been revealed by the RNAi ...

  8. Mitochondrial Ca2+ uptake in skeletal muscle health and disease

    CERN Document Server

    Zhou, Jingsong; Yi, Jianxun

    2016-01-01

    Muscle uses Ca2+ as a messenger to control contraction and relies on ATP to maintain the intracellular Ca2+ homeostasis. Mitochondria are the major sub-cellular organelle of ATP production. With a negative inner membrane potential, mitochondria take up Ca2+ from their surroundings, a process called mitochondrial Ca2+ uptake. Under physiological conditions, Ca2+ uptake into mitochondria promotes ATP production. Excessive uptake causes mitochondrial Ca2+ overload, which activates downstream adverse responses leading to cell dysfunction. Moreover, mitochondrial Ca2+ uptake could shape spatio-temporal patterns of intracellular Ca2+ signaling. Malfunction of mitochondrial Ca2+ uptake is implicated in muscle degeneration. Unlike non-excitable cells, mitochondria in muscle cells experience dramatic changes of intracellular Ca2+ levels. Besides the sudden elevation of Ca2+ level induced by action potentials, Ca2+ transients in muscle cells can be as short as a few milliseconds during a single twitch or as long as min...

  9. United Mitochondrial Disease Foundation

    Science.gov (United States)

    ... to Mitochondrial Disease FAQ's MitoFirst Handbook More Information Mito 101 Symposium Archives Get Connected Find an Event Adult Advisory Council Team Ask The Mito Doc Grand Rounds Kids & Teens Medical Child Abuse ...

  10. Mitochondrial dysfunction induced by different concentrations of gadolinium ion.

    Science.gov (United States)

    Zhao, Jie; Zhou, Zhi-Qiang; Jin, Jian-Cheng; Yuan, Lian; He, Huan; Jiang, Feng-Lei; Yang, Xiao-Gang; Dai, Jie; Liu, Yi

    2014-04-01

    Gadolinium-based compounds are the most widely used paramagnetic contrast agents in magnetic resonance imaging on the world. But the tricationic gadolinium ion (Gd(3+)) could induce cell apoptosis probably because of its effects on mitochondria. Until now, the mechanism about how Gd(3+) interacts with mitochondria is not well elucidated. In this work, mitochondrial swelling, collapsed transmembrane potential and decreased membrane fluidity were observed to be important factors for mitochondrial permeability transition pore (mtPTP) opening induced by Gd(3+). The protection effect of CsA (Cyclosporin A) could confirm high concentration of Gd(3+) (500 μM) would trigger mtPTP opening. Moreover, mitochondrial outer membrane breakdown and volume expansion observed clearly by transmission electron microscopy and the release of Cyt c (Cytochrome c) could explain the mtPTP opening from another aspect. In addition, MBM(+) (monobromobimane(+)) and DTT (dithiothreitol) could protect thiol (-SH) groups from oxidation so that the toxicity of Gd(3+) might be resulted from the chelation of -SH of membrane proteins by free Gd(3+). Gd(3+) could inhibit the initiation of mitochondrial membrane lipid peroxidation, so it might interact with anionic lipids too. These findings will highly contribute to the safe applications of Gd-based agents.

  11. Mechanistic insight from the crystal structure of mitochondrial complex I

    NARCIS (Netherlands)

    Zickermann, V.; Wirth, C.; Nasiri, H.; Siegmund, K.; Schwalbe, H.; Hunte, C.; Brandt, U.

    2015-01-01

    Proton-pumping complex I of the mitochondrial respiratory chain is among the largest and most complicated membrane protein complexes. The enzyme contributes substantially to oxidative energy conversion in eukaryotic cells. Its malfunctions are implicated in many hereditary and degenerative disorders

  12. The causes and functions of mitochondrial proton leak.

    Science.gov (United States)

    Brand, M D; Chien, L F; Ainscow, E K; Rolfe, D F; Porter, R K

    1994-08-30

    The non-linear relationship between respiration rate and protonmotive force in isolated mitochondria is explained entirely by delta p-dependent changes in the proton conductance of the mitochondrial inner membrane and is not caused by redox slip in the proton pumps. Mitochondrial proton leak occurs in intact cells and tissues: the futile cycle of proton pumping and proton leak accounts for 26% +/- 7% of the total oxygen consumption rate or 33% +/- 7% of the mitochondrial respiration rate of isolated hepatocytes (mean +/- S.D. for 43 rats); 52% of the oxygen consumption rate of resting perfused muscle and up to 38% of the basal metabolic rate of a rat, suggesting that heat production may be an important function in the proton leak in homeotherms. Together with non-mitochondrial oxygen consumption, it lowers the effective P/O ratio in cells from maximum possible values of 2.33 (palmitate oxidation) or 2.58 (glucose oxidation) to as low as 1.1 in liver or 0.8 in muscle. The effective P/O ratio increases in response to ATP demand; the ability to allow rapid switching of flux from leak to ATP turnover may be an even more important function of the leak reaction than heat production. The mitochondrial proton conductance in isolated mitochondria and in hepatocytes is greatly modulated by thyroid hormones, by phylogeny and by body mass. Usually the reactions of ATP turnover change in parallel so that the coupling ratio is not greatly affected. Changes in proton leak in tissues are brought about in the short term by changes in mitochondrial protonmotive force and in the longer term by changes in the surface area and proton permeability of the mitochondrial inner membrane. Permeability changes are probably caused by changes in the fatty acid composition of the membrane phospholipids.

  13. Multiple Targets for Drug-Induced Mitochondrial Toxicity.

    Science.gov (United States)

    Wallace, Kendall B

    2015-01-01

    Mitochondrial toxicity is rapidly gaining the interest of researchers and practitioners as a prominent liability in drug discovery and development, accounting for a growing proportion of preclinical drug attrition and post-market withdrawals or black box warnings by the U.S. FDA. To date, the focus of registries of drugs that elicit mitochondrial toxicity has been largely restricted to those that either inhibit the mitochondrial electron transport chain (ETC) or uncouple mitochondrial oxidative phosphorylation. Less appreciated are the toxicities that are secondary to the drug affecting either the molecular regulation, assembly or incorporation of the ETC into the inner mitochondrial membrane or those that limit substrate availability. The current article describes the complexities of molecular events and biochemical pathways required to sustain mitochondrial fidelity and substrate homeostasis with examples of drugs that interfere which the various pathways. The principal objective of this review is to shed light on the broader scope of drug-induced mitochondrial toxicities and how these secondary targets may account for a large portion of drug failures. PMID:25973981

  14. Analysis of Mitochondrial haemoglobin in Parkinson's disease brain.

    Science.gov (United States)

    Shephard, Freya; Greville-Heygate, Oliver; Liddell, Susan; Emes, Richard; Chakrabarti, Lisa

    2016-07-01

    Mitochondrial dysfunction is an early feature of neurodegeneration. We have shown there are mitochondrial haemoglobin changes with age and neurodegeneration. We hypothesised that altered physiological processes are associated with recruitment and localisation of haemoglobin to these organelles. To confirm a dynamic localisation of haemoglobin we exposed Drosophila melanogaster to cyclical hypoxia with recovery. With a single cycle of hypoxia and recovery we found a relative accumulation of haemoglobin in the mitochondria compared with the cytosol. An additional cycle of hypoxia and recovery led to a significant increase of mitochondrial haemoglobin (pbrains. Relative mitochondrial/cytosolic quantities of haemoglobin were obtained for the cortical region, substantia nigra and cerebellum. In age matched post-mortem brain mitochondrial haemoglobin ratios change, decreasing with disease duration in female cerebellum samples (n=7). The change is less discernible in male cerebellum (n=18). In cerebellar mitochondria, haemoglobin localisation in males with long disease duration shifts from the intermembrane space to the outer membrane of the organelle. These new data illustrate dynamic localisation of mitochondrial haemoglobin within the cell. Mitochondrial haemoglobin should be considered in the context of gender differences characterised in Parkinson's disease. It has been postulated that cerebellar circuitry may be activated to play a protective role in individuals with Parkinson's. The changing localisation of intracellular haemoglobin in response to hypoxia presents a novel pathway to delineate the role of the cerebellum in Parkinson's disease. PMID:27181046

  15. [Effects of exogenous spermidine on mitochondrial function of tomato seedling roots under salinity-alkalinity stress].

    Science.gov (United States)

    Pan, Xiong-bo; Xiang, Li-xia; Hu, Xiao-hui; Ren, Wen-qi; Zhang, Li; Ni, Xin-xin

    2016-02-01

    Two cultivars of tomato (Solanum lycopersicum, cvs. 'Jinpengchaoguan' and 'Zhongza No. 9', with the former being more tolerant to saline-alkaline stress) seedlings grown hydroponically were subjected to salinity-alkalinity stress condition (NaCl: Na2SO4:NaHCO3:Na2CO3 = 1:9:9:1) without or with foliar application of 0.25 mmol . L-1 spermidine (Spd), and the root morphology and physiological characteristics of mitochondrial membrane were analyzed 8 days after treatment, to explore the protective effects of exogenous Spd on mitochondrial function in tomato roots under salinity-alkalinity stress. The results showed that the salinity-alkalinity stress increased the concentrations of both mitochondrial H2O2 and MDA as well as the mitochondrial membrane permeability in the roots of the two cultivars, while it decreased the mitochondrial membrane fluidity, membrane potential, Cyt c/a and H+-ATPase activity, which impaired the mitochondria and therefore inhibited the root growth; and these effects were more obvious in 'Zhongza No. 9' than in 'Jinpengechaoguan'. Under the salinity-alkalinity stress, foliar application Spd could effectively decrease the concentrations of mitochondrial H2O2 and MDA and mitochondrial membrane permeability, while increased the mitochondrial membrane fluidity, membrane potential, Cyt c/a and H+-ATPase activity. These results suggested that exogenous Spd could effectively mitigate the damage on mitochondria induced by salinity-alkalinity stress, and the alleviation effect was more obvious in 'Zhongza No. 9' than in 'Jinpengchaoguan'. PMID:27396122

  16. 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...

  17. The plant mitochondrial proteome

    DEFF Research Database (Denmark)

    Millar, A.H.; Heazlewood, J.L.; Kristensen, B.K.;

    2005-01-01

    The plant mitochondrial proteome might contain as many as 2000-3000 different gene products, each of which might undergo post-translational modification. Recent studies using analytical methods, such as one-, two- and three-dimensional gel electrophoresis and one- and two-dimensional liquid...... context to be defined for them. There are indications that some of these proteins add novel activities to mitochondrial protein complexes in plants....

  18. Mitochondrial metabolism and diabetes

    OpenAIRE

    Kwak, Soo Heon; Park, Kyong Soo; Lee, Ki‐Up; Lee, Hong Kyu

    2010-01-01

    Abstract The oversupply of calories and sedentary lifestyle has resulted in a rapid increase of diabetes prevalence worldwide. During the past two decades, lines of evidence suggest that mitochondrial dysfunction plays a key role in the pathophysiology of diabetes. Mitochondria are vital to most of the eukaryotic cells as they provide energy in the form of adenosine triphosphate by oxidative phosphorylation. In addition, mitochondrial function is an integral part of glucose‐stimulated insulin...

  19. Mitochondrial dynamics and apoptosis

    OpenAIRE

    Suen, Der-Fen; Norris, Kristi L.; Youle, Richard J.

    2008-01-01

    In healthy cells, mitochondria continually divide and fuse to form a dynamic interconnecting network. The molecular machinery that mediates this organelle fission and fusion is necessary to maintain mitochondrial integrity, perhaps by facilitating DNA or protein quality control. This network disintegrates during apoptosis at the time of cytochrome c release and prior to caspase activation, yielding more numerous and smaller mitochondria. Recent work shows that proteins involved in mitochondri...

  20. Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using yeast models of OXPHOS deficiencies.

    Science.gov (United States)

    Fontanesi, Flavia; Diaz, Francisca; Barrientos, Antoni

    2009-10-01

    The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. Several approaches for the assessment of the OXPHOS system enzymes have been developed. Based on the methods described elsewhere, this unit describes the creation and study of yeast models of mitochondrial OXPHOS deficiencies.

  1. Loop-mediated isothermal amplification (LAMP method for rapid detection of Trypanosoma brucei rhodesiense.

    Directory of Open Access Journals (Sweden)

    Zablon Kithinji Njiru

    Full Text Available Loop-mediated isothermal amplification (LAMP of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62 degrees C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml and 0.1 pg (1 trypanosome/ml using heat-treated buffy coat, while the detection limit for conventional SRA PCR was approximately 1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions.

  2. Genome-wide dissection of the quorum sensing signalling pathway in Trypanosoma brucei.

    Science.gov (United States)

    Mony, Binny M; MacGregor, Paula; Ivens, Alasdair; Rojas, Federico; Cowton, Andrew; Young, Julie; Horn, David; Matthews, Keith

    2014-01-30

    The protozoan parasites Trypanosoma brucei spp. cause important human and livestock diseases in sub-Saharan Africa. In mammalian blood, two developmental forms of the parasite exist: proliferative 'slender' forms and arrested 'stumpy' forms that are responsible for transmission to tsetse flies. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission. This response is triggered by an elusive 'stumpy induction factor' (SIF) whose intracellular signalling pathway is also uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains respond inefficiently to SIF but can generate forms with stumpy characteristics when exposed to cell-permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNA interference library screen to identify the signalling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) or 8-pCPT-2'-O-methyl-5'-AMP to select cells that were unresponsive to these signals and hence remained proliferative. Genome-wide Ion Torrent based RNAi target sequencing identified cohorts of genes implicated in each step of the signalling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. Genes at each step were independently validated in cells naturally capable of stumpy formation, confirming their role in density sensing in vivo. The putative RNA-binding protein, RBP7, was required for normal quorum sensing and promoted cell-cycle arrest and transmission competence when overexpressed. This study reveals that quorum sensing signalling in trypanosomes shares similarities to fundamental quiescence pathways in eukaryotic cells, its components providing targets for quorum-sensing interference-based therapeutics.

  3. Enhanced succinic acid production in Aspergillus saccharolyticus by heterologous expression of fumarate reductase from Trypanosoma brucei.

    Science.gov (United States)

    Yang, Lei; Lübeck, Mette; Ahring, Birgitte K; Lübeck, Peter S

    2016-02-01

    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 of the enzymes, pyruvate carboxylase (PYC), malate dehydrogenase (MDH), and fumarase (FUM), involved in the reductive tricarboxylic acid (rTCA) branch, were examined and compared in cells harvested from the acid production medium and a complete medium. The results showed that ambient pH had a significant impact 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 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 to an enhanced production of succinic acid in frd transformants compared with the wild-type in both pH buffered and pH non-buffered conditions with highest amount produced in the pH buffered condition (16.2 ± 0.5 g/L). This study demonstrates the feasibility of increasing succinic acid production through the cytosolic reductive pathway by genetic engineering in A. saccharolyticus.

  4. Trypanosoma brucei gambiense Adaptation to Different Mammalian Sera Is Associated with VSG Expression Site Plasticity

    Science.gov (United States)

    Cordon-Obras, Carlos; Cano, Jorge; González-Pacanowska, Dolores; Benito, Agustin; Navarro, Miguel; Bart, Jean-Mathieu

    2013-01-01

    Trypanosoma brucei gambiense infection is widely considered an anthroponosis, although it has also been found in wild and domestic animals. Thus, fauna could act as reservoir, constraining the elimination of the parasite in hypo-endemic foci. To better understand the possible maintenance of T. b. gambiense in local fauna and investigate the molecular mechanisms underlying adaptation, we generated adapted cells lines (ACLs) by in vitro culture of the parasites in different mammalian sera. Using specific antibodies against the Variant Surface Glycoproteins (VSGs) we found that serum ACLs exhibited different VSG variants when maintained in pig, goat or human sera. Although newly detected VSGs were independent of the sera used, the consistent appearance of different VSGs suggested remodelling of the co-transcribed genes at the telomeric Expression Site (VSG-ES). Thus, Expression Site Associated Genes (ESAGs) sequences were analysed to investigate possible polymorphism selection. ESAGs 6 and 7 genotypes, encoding the transferrin receptor (TfR), expressed in different ACLs were characterised. In addition, we quantified the ESAG6/7 mRNA levels and analysed transferrin (Tf) uptake. Interestingly, the best growth occurred in pig and human serum ACLs, which consistently exhibited a predominant ESAG7 genotype and higher Tf uptake than those obtained in calf and goat sera. We also detected an apparent selection of specific ESAG3 genotypes in the pig and human serum ACLs, suggesting that other ESAGs could be involved in the host adaptation processes. Altogether, these results suggest a model whereby VSG-ES remodelling allows the parasite to express a specific set of ESAGs to provide selective advantages in different hosts. Finally, pig serum ACLs display phenotypic adaptation parameters closely related to human serum ACLs but distinct to parasites grown in calf and goat sera. These results suggest a better suitability of swine to maintain T. b. gambiense infection supporting

  5. Untreated human infections by Trypanosoma brucei gambiense are not 100% fatal.

    Directory of Open Access Journals (Sweden)

    Vincent Jamonneau

    Full Text Available The final outcome of infection by Trypanosoma brucei gambiense, the main agent of sleeping sickness, has always been considered as invariably fatal. While scarce and old reports have mentioned cases of self-cure in untreated patients, these studies suffered from the lack of accurate diagnostic tools available at that time. Here, using the most specific and sensitive tools available to date, we report on a long-term follow-up (15 years of a cohort of 50 human African trypanosomiasis (HAT patients from the Ivory Coast among whom 11 refused treatment after their initial diagnosis. In 10 out of 11 subjects who continued to refuse treatment despite repeated visits, parasite clearance was observed using both microscopy and polymerase chain reaction (PCR. Most of these subjects (7/10 also displayed decreasing serological responses, becoming progressively negative to trypanosome variable antigens (LiTat 1.3, 1.5 and 1.6. Hence, in addition to the "classic" lethal outcome of HAT, we show that alternative natural progressions of HAT may occur: progression to an apparently aparasitaemic and asymptomatic infection associated with strong long-lasting serological responses and progression to an apparently spontaneous resolution of infection (with negative results in parasitological tests and PCR associated with a progressive drop in antibody titres as observed in treated cases. While this study does not precisely estimate the frequency of the alternative courses for this infection, it is noteworthy that in the field national control programs encounter a significant proportion of subjects displaying positive serologic test results but negative results in parasitological testing. These findings demonstrate that a number of these subjects display such infection courses. From our point of view, recognising that trypanotolerance exists in humans, as is now widely accepted for animals, is a major step forward for future research in the field of HAT.

  6. Simulating the complex cell design of Trypanosoma brucei and its motility.

    Directory of Open Access Journals (Sweden)

    Davod Alizadehrad

    2015-01-01

    Full Text Available The flagellate Trypanosoma brucei, which causes the sleeping sickness when infecting a mammalian host, goes through an intricate life cycle. It has a rather complex propulsion mechanism and swims in diverse microenvironments. These continuously exert selective pressure, to which the trypanosome adjusts with its architecture and behavior. As a result, the trypanosome assumes a diversity of complex morphotypes during its life cycle. However, although cell biology has detailed form and function of most of them, experimental data on the dynamic behavior and development of most morphotypes is lacking. Here we show that simulation science can predict intermediate cell designs by conducting specific and controlled modifications of an accurate, nature-inspired cell model, which we developed using information from live cell analyses. The cell models account for several important characteristics of the real trypanosomal morphotypes, such as the geometry and elastic properties of the cell body, and their swimming mechanism using an eukaryotic flagellum. We introduce an elastic network model for the cell body, including bending rigidity and simulate swimming in a fluid environment, using the mesoscale simulation technique called multi-particle collision dynamics. The in silico trypanosome of the bloodstream form displays the characteristic in vivo rotational and translational motility pattern that is crucial for survival and virulence in the vertebrate host. Moreover, our model accurately simulates the trypanosome's tumbling and backward motion. We show that the distinctive course of the attached flagellum around the cell body is one important aspect to produce the observed swimming behavior in a viscous fluid, and also required to reach the maximal swimming velocity. Changing details of the flagellar attachment generates less efficient swimmers. We also simulate different morphotypes that occur during the parasite's development in the tsetse fly, and

  7. 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction.

    Directory of Open Access Journals (Sweden)

    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.

  8. Time representation of mitochondrial morphology and function after acute spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Zhi-qiang Jia; Gang Li; Zhen-yu Zhang; Hao-tian Li; Ji-quan Wang; Zhong-kai Fan; Gang Lv

    2016-01-01

    Changes in mitochondrial morphology and function play an important role in secondary damage after acute spinal cord injury. We re-corded the time representation of mitochondrial morphology and function in rats with acute spinal cord injury. Results showed that mitochondria had an irregular shape, and increased in size. Mitochondrial cristae were disordered and mitochondrial membrane rupture was visible at 2–24 hours after injury. Fusion protein mitofusin 1 expression gradually increased, peaked at 8 hours after injury, and then decreased to its lowest level at 24 hours. Expression of dynamin-related protein 1, amitochondrial ifssion protein, showed the opposite kinetics. At 2–24 hours after acute spinal cord injury, malondialdehyde content, cytochrome c levels and caspase-3 expression were in-creased, but glutathione content, adenosine triphosphate content, Na+-K+-ATPase activity and mitochondrial membrane potential were gradually reduced. Furthermore, mitochondrial morphology altered during the acute stage of spinal cord injury. Fusion was important within the ifrst 8 hours, but ifssion played a key role at 24 hours. Oxidative stress was inhibited, biological productivity was diminished, and mitochondrial membrane potential and permeability were reduced in the acute stage of injury. In summary, mitochondrial apoptosis is activated when the time of spinal cord injury is prolonged.

  9. Phosphodiesterase-3 inhibitor (cilostazol) attenuates oxidative stress-induced mitochondrial dysfunction in the heart

    Institute of Scientific and Technical Information of China (English)

    Siriporn C.Chattipakorn; Savitree Thummasorn; Jantira Sanit; Nipon Chattipakorn

    2014-01-01

    Background Cilostazol is a type 3 phosphodiesterase inhibitor which has been previously demonstrated to prevent the occurrence of tachyarrhythmia and improve defibrillation efficacy. However, the mechanism for this beneficial effect is still unclear. Since cardiac mito-chondria have been shown to play a crucial role in fatal cardiac arrhythmias and that oxidative stress is one of the main contributors to arr-hythmia generation, we tested the effects of cilostazol on cardiac mitochondria under severe oxidative stress. Methods Mitochondria were isolated from rat hearts and treated with H2O2 to induce oxidative stress. Cilostazol, at various concentrations, was used to study its protective effects. Pharmacological interventions, including a mitochondrial permeability transition pore (mPTP) blocker, cyclosporine A (CsA), and an inner membrane anion channel (IMAC) blocker, 4’-chlorodiazepam (CDP), were used to investigate the mechanistic role of cilostazol on cardiac mitochondria. Cardiac mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential change and mi-tochondrial swelling were determined as indicators of cardiac mitochondrial function. Results Cilostazol preserved cardiac mitochondrial function when exposed to oxidative stress by preventing mitochondrial depolarization, mitochondrial swelling, and decreasing ROS produc-tion. Conclusions Our findings suggest that cardioprotective effects of cilostazol reported previously could be due to its prevention of car-diac mitochondrial dysfunction caused by severe oxidative stress.

  10. The role of myeloid differentiation factor 88 on mitochondrial dysfunction of peritoneal leukocytes during polymicrobial sepsis

    Science.gov (United States)

    Zou, Lin; Chen, Dunjin; Chao, Wei

    2016-01-01

    Objective To investigate the role of myeloid differentiation factor 88 (MyD88) on mitochondrial dysfunction of peritoneal leukocytes during polymicrobial sepsis. Material and methods Polymicrobial peritonitis, a clinically relevant mouse model of sepsis, was generated by cecum ligation and puncture (CLP) in both male C57BL/6J wild-type (WT) and MyD88 knockout (MyD88–/–) mice. Twenty-four hours after surgeries, peritoneal leukocytes were collected and four parameters of mitochondrial function, including total intracellular and mitochondrial ROS burst, mitochondrial membrane depolarization and ATP depletion, were measured by flow cytometry or ATP assay, and then compared. Results Polymicrobial sepsis led to a marked mitochondrial dysfunction of peritoneal leukocytes with total intracellular and mitochondrial ROS overproduction, decreased mitochondrial membrane potential and reduced intracellular ATP production. In comparison, there was no significant difference in the extent of mitochondrial dysfunction of peritoneal leukocytes between WT and MyD88–/– septic mice. Conclusions MyD88 may be not sufficient to regulate mitochondrial dysfunction of peritoneal leukocytes during polymicrobial sepsis. PMID:27536200

  11. Mitochondria-associated membranes as hubs for neurodegeneration

    OpenAIRE

    Krols, Michiel; Van Isterdael, Gert; Asselbergh, Bob; Kremer, Anna; Lippens, Saskia; Timmerman, Vincent; Janssens, Sophie

    2016-01-01

    There is a growing appreciation that membrane-bound organelles in eukaryotic cells communicate directly with one another through direct membrane contact sites. Mitochondria-associated membranes are specialized subdomains of the endoplasmic reticulum that function as membrane contact sites between the endoplasmic reticulum and mitochondria. These sites have emerged as major players in lipid metabolism and calcium signaling. More recently also autophagy and mitochondrial dynamics have been foun...

  12. Mitochondrial-targeted DNA delivery using a DF-MITO-Porter, an innovative nano carrier with cytoplasmic and mitochondrial fusogenic envelopes

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Yuma; Kawamura, Eriko; Harashima, Hideyoshi, E-mail: harasima@pharm.hokudai.ac.jp [Hokkaido University, Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences (Japan)

    2012-08-15

    Mitochondrial gene therapy has the potential for curing a variety of diseases that are associated with mitochondrial DNA mutations and/or defects. To achieve this, it will be necessary to deliver therapeutic agents into the mitochondria in diseased cells. A number of mitochondrial drug delivery systems have been reported to date. However, reports of mitochondrial-targeted DNA delivery are limited. To achieve this, the therapeutic agent must be taken up by the cell (1), after which, the multi-processes associated with intracellular trafficking must be sophisticatedly regulated so as to release the agent from the endosome and deliver it to the cytosol (2) and to pass through the mitochondrial membrane (3). We report herein on the mitochondrial delivery of oligo DNA as a model therapeutic using a Dual Function (DF)-MITO-Porter, an innovative nano carrier designed for mitochondrial delivery. The critical structural elements of the DF-MITO-Porter include mitochondria-fusogenic inner envelopes and endosome-fusogenic outer envelopes, modified with octaarginine which greatly assists in cellular uptake. Inside the cell, the carrier passes through the endosomal and mitochondrial membranes via step-wise membrane fusion. When the oligo DNA was packaged in the DF-MITO-Porter, cellular uptake efficiency was strongly enhanced. Intracellular observation using confocal laser scanning microscopy showed that the DF-MITO-Porter was effectively released from endosomes. Moreover, the findings confirmed that the mitochondrial targeting activity of the DF-MITO-Porter was significantly higher than that of a carrier without outer endosome-fusogenic envelopes. These results support the conclusion that mitochondrial-targeted DNA delivery using a DF-MITO-Porter can be achieved when intracellular trafficking is optimally regulated.

  13. Mitochondria-targeted antioxidant mitotempo protects mitochondrial function against amyloid beta toxicity in primary cultured mouse neurons.

    Science.gov (United States)

    Hu, Hongtao; Li, Mo

    2016-09-01

    Mitochondrial defects including excess reactive oxygen species (ROS) production and compromised ATP generation are featured pathology in Alzheimer's disease (AD). Amyloid beta (Aβ)-mediated mitochondrial ROS overproduction disrupts intra-neuronal Redox balance, in turn exacerbating mitochondrial dysfunction leading to neuronal injury. Previous studies have found the beneficial effects of mitochondria-targeted antioxidants in preventing mitochondrial dysfunction and neuronal injury in AD animal and cell models, suggesting that mitochondrial ROS scavengers hold promise for the treatment of this neurological disorder. In this study, we have determined that mitotempo, a novel mitochondria-targeted antioxidant protects mitochondrial function from the toxicity of Aβ in primary cultured neurons. Our results showed that Aβ-promoted mitochondrial superoxide production and neuronal lipid oxidation were significantly suppressed by the application of mitotempo. Moreover, mitotempo also demonstrated protective effects on mitochondrial bioenergetics evidenced by preserved mitochondrial membrane potential, cytochrome c oxidase activity as well as ATP production. In addition, the Aβ-induced mitochondrial DNA (mtDNA) depletion and decreased expression levels of mtDNA replication-related DNA polymerase gamma (DNA pol γ) and Twinkle were substantially mitigated by mitotempo. Therefore, our study suggests that elimination of excess mitochondrial ROS rescues mitochondrial function in Aβ-insulted neruons; and mitotempo has the potential to be a promising therapeutic agent to protect mitochondrial and neuronal function in AD. PMID:27444386

  14. Mitochondria-targeted antioxidant mitotempo protects mitochondrial function against amyloid beta toxicity in primary cultured mouse neurons.

    Science.gov (United States)

    Hu, Hongtao; Li, Mo

    2016-09-01

    Mitochondrial defects including excess reactive oxygen species (ROS) production and compromised ATP generation are featured pathology in Alzheimer's disease (AD). Amyloid beta (Aβ)-mediated mitochondrial ROS overproduction disrupts intra-neuronal Redox balance, in turn exacerbating mitochondrial dysfunction leading to neuronal injury. Previous studies have found the beneficial effects of mitochondria-targeted antioxidants in preventing mitochondrial dysfunction and neuronal injury in AD animal and cell models, suggesting that mitochondrial ROS scavengers hold promise for the treatment of this neurological disorder. In this study, we have determined that mitotempo, a novel mitochondria-targeted antioxidant protects mitochondrial function from the toxicity of Aβ in primary cultured neurons. Our results showed that Aβ-promoted mitochondrial superoxide production and neuronal lipid oxidation were significantly suppressed by the application of mitotempo. Moreover, mitotempo also demonstrated protective effects on mitochondrial bioenergetics evidenced by preserved mitochondrial membrane potential, cytochrome c oxidase activity as well as ATP production. In addition, the Aβ-induced mitochondrial DNA (mtDNA) depletion and decreased expression levels of mtDNA replication-related DNA polymerase gamma (DNA pol γ) and Twinkle were substantially mitigated by mitotempo. Therefore, our study suggests that elimination of excess mitochondrial ROS rescues mitochondrial function in Aβ-insulted neruons; and mitotempo has the potential to be a promising therapeutic agent to protect mitochondrial and neuronal function in AD.

  15. In Silico Identification and in Vitro Activity of Novel Natural Inhibitors of Trypanosoma brucei Glyceraldehyde-3-phosphate-dehydrogenase

    Directory of Open Access Journals (Sweden)

    Fabian C. Herrmann

    2015-09-01

    Full Text Available As part of our ongoing efforts to identify natural products with activity against pathogens causing neglected tropical diseases, we are currently performing an extensive screening of natural product (NP databases against a multitude of protozoan parasite proteins. Within this project, we screened a database of NPs from a commercial supplier, AnalytiCon Discovery (Potsdam, Germany, against Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH, a glycolytic enzyme whose inhibition deprives the parasite of energy supply. NPs acting as potential inhibitors of the mentioned enzyme were identified using a pharmacophore-based virtual screening and subsequent docking of the identified hits into the active site of interest. In a set of 700 structures chosen for the screening, 13 (1.9% were predicted to possess significant affinity towards the enzyme and were therefore tested in an in vitro enzyme assay using recombinant TbGAPDH. Nine of these in silico hits (69% showed significant inhibitory activity at 50 µM, of which two geranylated benzophenone derivatives proved to be particularly active with IC50 values below 10 µM. These compounds also showed moderate in vitro activity against T. brucei rhodesiense and may thus represent interesting starting points for further optimization.

  16. Tracking the Biogenesis and Inheritance of Subpellicular Microtubule in Trypanosoma brucei with Inducible YFP-α-Tubulin

    Directory of Open Access Journals (Sweden)

    Omar Sheriff

    2014-01-01

    Full Text Available The microtubule cytoskeleton forms the most prominent structural system in Trypanosoma brucei, undergoing extensive modifications during the cell cycle. Visualization of tyrosinated microtubules leads to a semiconservative mode of inheritance, whereas recent studies employing microtubule plus end tracking proteins have hinted at an asymmetric pattern of cytoskeletal inheritance. To further the knowledge of microtubule synthesis and inheritance during T. brucei cell cycle, the dynamics of the microtubule cytoskeleton was visualized by inducible YFP-α-tubulin expression. During new flagellum/flagellum attachment zone (FAZ biogenesis and cell growth, YFP-α-tubulin was incorporated mainly between the old and new flagellum/FAZ complexes. Cytoskeletal modifications at the posterior end of the cells were observed with EB1, a microtubule plus end binding protein, particularly during mitosis. Additionally, the newly formed microtubules segregated asymmetrically, with the daughter cell inheriting the new flagellum/FAZ complex retaining most of the new microtubules. Together, our results suggest an intimate connection between new microtubule formation and new FAZ assembly, consequently leading to asymmetric microtubule inheritance and cell division.

  17. Genome-wide Analysis Reveals Extensive Functional Interaction between DNA Replication Initiation and Transcription in the Genome of Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Calvin Tiengwe

    2012-07-01

    Full Text Available Identification of replication initiation sites, termed origins, is a crucial step in understanding genome transmission in any organism. Transcription of the Trypanosoma brucei genome is highly unusual, with each chromosome comprising a few discrete transcription units. To understand how DNA replication occurs in the context of such organization, we have performed genome-wide mapping of the binding sites of the replication initiator ORC1/CDC6 and have identified replication origins, revealing that both localize to the boundaries of the transcription units. A remarkably small number of active origins is seen, whose spacing is greater than in any other eukaryote. We show that replication and transcription in T. brucei have a profound functional overlap, as reducing ORC1/CDC6 levels leads to genome-wide increases in mRNA levels arising from the boundaries of the transcription units. In addition, ORC1/CDC6 loss causes derepression of silent Variant Surface Glycoprotein genes, which are critical for host immune evasion.

  18. Deviating the level of proliferating cell nuclear antigen in Trypanosoma brucei elicits distinct mechanisms for inhibiting proliferation and cell cycle progression.

    Science.gov (United States)

    Valenciano, Ana L; Ramsey, Aaron C; Mackey, Zachary B

    2015-01-01

    The DNA replication machinery is spatially and temporally coordinated in all cells to reproduce a single exact copy of the genome per division, but its regulation in the protozoan parasite Trypanosoma brucei is not well characterized. We characterized the effects of altering the levels of proliferating cell nuclear antigen, a key component of the DNA replication machinery, in bloodstream form T. brucei. This study demonstrated that tight regulation of TbPCNA levels was critical for normal proliferation and DNA replication in the parasite. Depleting TbPCNA mRNA reduced proliferation, severely diminished DNA replication, arrested the synthesis of new DNA and caused the parasites to accumulated in G2/M. Attenuating the parasite by downregulating TbPCNA caused it to become hypersensitive to hydroxyurea. Overexpressing TbPCNA in T. brucei arrested proliferation, inhibited DNA replication and prevented the parasite from exiting G2/M. These results indicate that distinct mechanisms of cell cycle arrest are associated with upregulating or downregulating TbPCNA. The findings of this study validate deregulating intra-parasite levels of TbPCNA as a potential strategy for therapeutically exploiting this target in bloodstream form T. brucei.

  19. Identification and Characterization of Hundreds of Potent and Selective Inhibitors of Trypanosoma brucei Growth from a Kinase-Targeted Library Screening Campaign

    Science.gov (United States)

    Diaz, Rosario; Luengo-Arratta, Sandra A.; Seixas, João D.; Amata, Emanuele; Devine, William; Cordon-Obras, Carlos; Rojas-Barros, Domingo I.; Jimenez, Elena; Ortega, Fatima; Crouch, Sabrinia; Colmenarejo, Gonzalo; Fiandor, Jose Maria; Martin, Jose Julio; Berlanga, Manuela; Gonzalez, Silvia; Manzano, Pilar; Navarro, Miguel; Pollastri, Michael P.

    2014-01-01

    In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2) cells. In this way, we have identified 797 sub-micromolar inhibitors of T. brucei growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of in silico and in vitro physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of T. brucei rhodesiense with one of these compounds (NEU-1053). This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for T. brucei, and new HAT therapeutics discovery programs. PMID:25340575

  20. A proteomics approach reveals molecular manipulators of distinct cellular processes in the salivary glands of Glossina m. morsitans in response to Trypanosoma b. brucei infections

    NARCIS (Netherlands)

    Kariithi, Henry M.; Boeren, Sjef; Murungi, Edwin K.; Vlak, Just M.; Abd-Alla, Adly M.M.

    2016-01-01

    Background: Glossina m. morsitans is the primary vector of the Trypanosoma brucei group, one of the causative agents of African trypanosomoses. The parasites undergo metacyclogenesis, i.e. transformation into the mammalian-infective metacyclic trypomastigote (MT) parasites, in the salivary glands

  1. [Cyclosporin A causes oxidative stress and mitochondrial dysfunction in renal tubular cells].

    Science.gov (United States)

    Pérez de Hornedo, J; de Arriba, G; Calvino, M; Benito, S; Parra, T

    2007-01-01

    Reactive oxygen species (ROS) have been implicated in cyclosporin A (CsA) nephrotoxicity. As mitochondria are one of the main sources of ROS in cells, we evaluated the role of CsA in mitochondrial structure and function in LLC-PK1 cells. We incubated cells with CsA 1 microM for 24 hours and studies were performed with flow citometry and confocal microscopy. We studied mitochondrial NAD(P)H content, superoxide anion (O2.-) production (MitoSOX Red), oxidation of cardiolipin of inner mitochondrial membrane (NAO) and mitochondrial membrane potential (DIOC2(3)). Also we analyzed the intracellular ROS synthesis (H2DCF-DA) and reduced glutation (GSH) of cells. Our results showed that CsA decreased NAD(P)H and membrane potential, and increased O2.- in mitochondria. CsA also provoked oxidation of cardiolipin. Furthermore, CsA increased intracellular ROS production and decreased GSH content. These results suggest that CsA has crucial effects in mitochondria. CsA modified mitochondrial physiology through the decrease of antioxidant mitochondrial compounds as NAD(P)H and the dissipation of mitochondrial membrane potential and increase of oxidants as O2.-. Also, CsA alters lipidic structure of inner mitochondrial membrane through the oxidation of cardiolipin. These effects trigger a chain of events that favour intracellular synthesis of ROS and depletion of GSH that can compromise cellular viability. Nephrotoxic cellular effects of CsA can be explained, at least in part, through its influence on mitochondrial functionalism.

  2. Cultured senescent myoblasts derived from human vastus lateralis exhibit normal mitochondrial ATP synthesis capacities with correlating concomitant ROS production while whole cell ATP production is decreased

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2012-01-01

    satellite cells at early and late passage numbers. We show that cultured muscle satellite cells undergoing senescence express a reduced mitochondrial mass, decreased whole cell ATP level, normal to increased mitochondrial ATP production under ATP utilization, increased mitochondrial membrane potential......The free radical theory of aging says that increased oxidative stress and mitochondrial dysfunction are associated with old age. In the present study we have investigated the effects of cellular senescence on muscle energetic by comparing mitochondrial content and function in cultured muscle...... and increased superoxide/mitochondrial mass and hydrogen peroxide/mitochondrial mass ratios. Moreover, the increased ROS production correlates with the corresponding mitochondrial ATP production. Thus, myotubes differentiated from human myoblasts undergoing senescence have a reduced mitochondrial content...

  3. Calpastatin overexpression reduces oxidative stress-induced mitochondrial impairment and cell death in human neuroblastoma SH-SY5Y cells by decreasing calpain and calcineurin activation, induction of mitochondrial fission and destruction of mitochondrial fusion.

    Science.gov (United States)

    Tangmansakulchai, Kulvadee; Abubakar, Zuroida; Kitiyanant, Narisorn; Suwanjang, Wilasinee; Leepiyasakulchai, Chaniya; Govitrapong, Piyarat; Chetsawang, Banthit

    2016-09-01

    Calpain is an intracellular Ca(2+)-dependent protease, and the activation of calpain has been implicated in neurodegenerative diseases. Calpain activity can be regulated by calpastatin, an endogenous specific calpain inhibitor. Several lines of evidence have demonstrated a potential role of calpastatin in preventing calpain-mediated pathogenesis. Additionally, several studies have revealed that calpain activation and mitochondrial damage are involved in the cell death process; however, recent evidence has not clearly indicated a neuroprotective mechanism of calpastatin against calpain-dependent mitochondrial impairment in the process of neuronal cell death. Therefore, the purpose of this study was to investigate the potential ability of calpastatin to inhibit calpain activation and mitochondrial impairment in oxidative stress-induced neuron degeneration. Calpastatin was stably overexpressed in human neuroblastoma SH-SY5Y cells. In non-calpastatin overexpressing SH-SY5Y cells, hydrogen peroxide significantly decreased cell viability, superoxide dismutase activity, mitochondrial membrane potential, ATP production and mitochondrial fusion protein (Opa1) levels in the mitochondrial fraction but increased reactive oxygen species formation, calpain and calcineurin activation, mitochondrial fission protein (Fis1 and Drp1) levels in the mitochondrial fraction and apoptotic cells. Nevertheless, these toxic effects were abolished in hydrogen peroxide-treated calpastatin-overexpressing SH-SY5Y cells. The results of the present study demonstrate the potential ability of calpastatin to diminish calpain and calcineurin activation and mitochondrial impairment in neurons that are affected by oxidative damage. PMID:27453331

  4. The morphological changes of cardiomyocytes and mitochondrial dysfunction in spontaneous hypertensive rats with experimental diabetes mellitus

    Directory of Open Access Journals (Sweden)

    Kolesnyk M.Yu.

    2013-01-01

    Full Text Available The conception of energetic deficiency in the pathogenesis of arterial hypertension and diabetes mellitus presents new perspectives in the understanding of molecular and biochemical mechanisms of these diseases. It was performed the comparison between morphological changes and mitochondrial dysfunction in spontaneous hypertensive rats with experimental diabetes mellitus. The mitochondrial state was assessed by investigation of the permeability of the giant mitochondrial pore. It was found that the permeability of mitochondrial pore is increased in spontaneous hypertensive rats. It was registrated the significant increasing of mitochondrial membrane permeability in case of diabetes. It was observed the increased area of cardiomyocytes’ nuclei and decreased nuclear cytoplasmic ratio in diabetic animals. It was demonstrated that nucleic and cytoplasmic RNA concentration is decreased in comparison with the intact spontaneous hypertensive rats. The RNA biosynthesis abnormalities are associated with the degree of mitochondrial dysfunction in the myocardium of spontaneous hypertensive rats with experimental diabetes mellitus.

  5. Assessment of cardiac function in mice lacking the mitochondrial calcium uniporter.

    Science.gov (United States)

    Holmström, Kira M; Pan, Xin; Liu, Julia C; Menazza, Sara; Liu, Jie; Nguyen, Tiffany T; Pan, Haihui; Parks, Randi J; Anderson, Stasia; Noguchi, Audrey; Springer, Danielle; Murphy, Elizabeth; Finkel, Toren

    2015-08-01

    Mitochondrial calcium is thought to play an important role in the regulation of cardiac bioenergetics and function. The entry of calcium into the mitochondrial matrix requires that the divalent cation pass through the inner mitochondrial membrane via a specialized pore known as the mitochondrial calcium uniporter (MCU). Here, we use mice deficient of MCU expression to rigorously assess the role of mitochondrial calcium in cardiac function. Mitochondria isolated from MCU(-/-) mice have reduced matrix calcium levels, impaired calcium uptake and a defect in calcium-stimulated respiration. Nonetheless, we find that the absence of MCU expression does not affect basal cardiac function at either 12 or 20months of age. Moreover, the physiological response of MCU(-/-) mice to isoproterenol challenge or transverse aortic constriction appears similar to control mice. Thus, while mitochondria derived from MCU(-/-) mice have markedly impaired mitochondrial calcium handling, the hearts of these animals surprisingly appear to function relatively normally under basal conditions and during stress.

  6. Progress in surface and membrane science

    CERN Document Server

    Cadenhead, D A; Rosenberg, M D

    1974-01-01

    Progress in Surface and Membrane Science, Volume 8 covers the developments in the study of surface and membrane science. The book discusses the applications of statistical mechanics to physical adsorption; the impact of electron spectroscopy and cognate techniques on the study of solid surfaces; and the ellipsometric studies of thin films. The text also describes the interfacial photochemistry of bilayer lipid membranes; cell junctions and their development; and the composition and function of the inner mitochondrial membrane. The role of the cell surface in contact inhibition of cell division

  7. Neurological mitochondrial cytopathies.

    Directory of Open Access Journals (Sweden)

    Mehndiratta M

    2002-04-01

    Full Text Available The mitochondrial cytopathies are genetically and phenotypically heterogeneous group of disorders caused by structural and functional abnormalities in mitochondria. To the best of our knowledge, there are very few studies published from India till date. Selected and confirmed fourteen cases of neurological mitochondrial cytopathies with different clinical syndromes admitted between 1997 and 2000 are being reported. There were 8 male and 6 female patients. The mean age was 24.42+/-11.18 years (range 4-40 years. Twelve patients could be categorized into well-defined syndromes, while two belonged to undefined group. In the defined syndrome categories, three patients had MELAS (mitochondrial encephalopathy, lactic acidosis and stroke like episodes, three had MERRF (myoclonic epilepsy and ragged red fibre myopathy, three cases had KSS (Kearns-Sayre Syndrome and three were diagnosed to be suffering from mitochondrial myopathy. In the uncategorized group, one case presented with paroxysmal kinesogenic dystonia and the other manifested with generalized chorea alone. Serum lactic acid level was significantly increased in all the patients (fasting 28.96+/-4.59 mg%, post exercise 41.02+/-4.93 mg%. Muscle biopsy was done in all cases. Succinic dehydrogenase staining of muscle tissue showed subsarcolemmal accumulation of mitochondria in 12 cases. Mitochondrial DNA study could be performed in one case only and it did not reveal any mutation at nucleotides 3243 and 8344. MRI brain showed multiple infarcts in MELAS, hyperintensities in putaminal areas in chorea and bilateral cerebellar atrophy in MERRF.

  8. Altered mitochondrial function and oxidative stress in leukocytes of anorexia nervosa patients.

    Directory of Open Access Journals (Sweden)

    Victor M Victor

    Full Text Available CONTEXT: Anorexia nervosa is a common illness among adolescents and is characterised by oxidative stress. OBJECTIVE: The effects of anorexia on mitochondrial function and redox state in leukocytes from anorexic subjects were evaluated. DESIGN AND SETTING: A multi-centre, cross-sectional case-control study was performed. PATIENTS: Our study population consisted of 20 anorexic patients and 20 age-matched controls, all of which were Caucasian women. MAIN OUTCOME MEASURES: Anthropometric and metabolic parameters were evaluated in the study population. To assess whether anorexia nervosa affects mitochondrial function and redox state in leukocytes of anorexic patients, we measured mitochondrial oxygen consumption, membrane potential, reactive oxygen species production, glutathione levels, mitochondrial mass, and complex I and III activity in polymorphonuclear cells. RESULTS: Mitochondrial function was impaired in the leukocytes of the anorexic patients. This was evident in a decrease in mitochondrial O2 consumption (P<0.05, mitochondrial membrane potential (P<0.01 and GSH levels (P<0.05, and an increase in ROS production (P<0.05 with respect to control subjects. Furthermore, a reduction of mitochondrial mass was detected in leukocytes of the anorexic patients (P<0.05, while the activity of mitochondrial complex I (P<0.001, but not that of complex III, was found to be inhibited in the same population. CONCLUSIONS: Oxidative stress is produced in the leukocytes of anorexic patients and is closely related to mitochondrial dysfunction. Our results lead us to propose that the oxidative stress that occurs in anorexia takes place at mitochondrial complex I. Future research concerning mitochondrial dysfunction and oxidative stress should aim to determine the physiological mechanism involved in this effect and the physiological impact of anorexia.

  9. Mitochondrial fusion and inheritance of the mitochondrial genome.

    Science.gov (United States)

    Takano, Hiroyoshi; Onoue, Kenta; Kawano, Shigeyuki

    2010-03-01

    Although maternal or uniparental inheritance of mitochondrial genomes is a general rule, biparental inheritance is sometimes observed in protists and fungi,including yeasts. In yeast, recombination occurs between the mitochondrial genomes inherited from both parents.Mitochondrial fusion observed in yeast zygotes is thought to set up a space for DNA recombination. In the last decade,a universal mitochondrial fusion mechanism has been uncovered, using yeast as a model. On the other hand, an alternative mitochondrial fusion mechanism has been identified in the true slime mold Physarum polycephalum.A specific mitochondrial plasmid, mF, has been detected as the genetic material that causes mitochondrial fusion in P. polycephalum. Without mF, fusion of the mitochondria is not observed throughout the life cycle, suggesting that Physarum has no constitutive mitochondrial fusion mechanism.Conversely, mitochondria fuse in zygotes and during sporulation with mF. The complete mF sequence suggests that one gene, ORF640, encodes a fusogen for Physarum mitochondria. Although in general, mitochondria are inherited uniparentally, biparental inheritance occurs with specific sexual crossing in P. polycephalum.An analysis of the transmission of mitochondrial genomes has shown that recombinations between two parental mitochondrial genomes require mitochondrial fusion,mediated by mF. Physarum is a unique organism for studying mitochondrial fusion. PMID:20196232

  10. Impaired Transport of Mitochondrial Transcription Factor and the Metabolic Memory Phenomenon Associated with the Progression of Diabetic Retinopathy

    OpenAIRE

    Santos, Julia M.; Kowluru, Renu A.

    2013-01-01

    Diabetes damages retinal mitochondrial DNA (mtDNA), and compromises the mtDNA transcription. In the transcription and replication of mtDNA, nuclear-encoded transcription factor A (TFAM) is considered as a key activator, and we have shown that in diabetes while retinal TFAM gene expression is increased, its mitochondrial levels are decreased. This study investigates the role of mitochondrial outer and inner membrane transport systems in the transfer of TFAM into the mitochondria in diabetes, a...

  11. Fine-Tuning of Drp1/Fis1 Availability by AKAP121/Siah2 Regulates Mitochondrial Adaptation to Hypoxia

    OpenAIRE

    Kim, Hyungsoo; Scimia, Maria C.; Wilkinson, Deepti; Trelles, Ramon D.; Wood, Malcolm R.; Bowtell, David; Dillin, Andrew; Mercola, Mark; Ronai, Ze’ev A.

    2011-01-01

    Defining the mechanisms underlying the control of mitochondrial fusion and fission is critical to understanding cellular adaptation to diverse physiological conditions. Here we demonstrate that hypoxia induces fission of mitochondrial membranes, dependent on availability of the mitochondrial scaffolding protein AKAP121. AKAP121 controls mitochondria dynamics through PKA-dependent inhibitory phosphorylation of Drp1 and PKA-independent inhibition of Drp1-Fis1 interaction. Reduced availability o...

  12. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

  13. The neurogenic basic helix-loop-helix transcription factor NeuroD6 enhances mitochondrial biogenesis and bioenergetics to confer tolerance of neuronal PC12-NeuroD6 cells to the mitochondrial stressor rotenone

    Energy Technology Data Exchange (ETDEWEB)

    Baxter, Kristin Kathleen; Uittenbogaard, Martine [Department of Anatomy and Regenerative Biology, George Washington University Medical Center, Washington, DC (United States); Chiaramello, Anne, E-mail: achiaram@gwu.edu [Department of Anatomy and Regenerative Biology, George Washington University Medical Center, Washington, DC (United States)

    2012-10-15

    The fundamental question of how and which neuronal specific transcription factors tailor mitochondrial biogenesis and bioenergetics to the need of developing neuronal cells has remained largely unexplored. In this study, we report that the neurogenic basic helix-loop-helix transcription factor NeuroD6 possesses mitochondrial biogenic properties by amplifying the mitochondrial DNA content and TFAM expression levels, a key regulator for mitochondrial biogenesis. NeuroD6-mediated increase in mitochondrial biogenesis in the neuronal progenitor-like PC12-NEUROD6 cells is concomitant with enhanced mitochondrial bioenergetic functions, including increased expression levels of specific subunits of respiratory complexes of the electron transport chain, elevated mitochondrial membrane potential and ATP levels produced by oxidative phosphorylation. Thus, NeuroD6 augments the bioenergetic capacity of PC12-NEUROD6 cells to generate an energetic reserve, which confers tolerance to the mitochondrial stressor, rotenone. We found that NeuroD6 induces an adaptive bioenergetic response throughout rotenone treatment involving maintenance of the mitochondrial membrane potential and ATP levels in conjunction with preservation of the actin network. In conclusion, our results support the concept that NeuroD6 plays an integrative role in regulating and coordinating the onset of neuronal differentiation with acquisition of adequate mitochondrial mass and energetic capacity to ensure energy demanding events, such as cytoskeletal remodeling, plasmalemmal expansion, and growth cone formation. -- Highlights: Black-Right-Pointing-Pointer NeuroD6 induces mitochondrial biogenesis in neuroprogenitor-like cells. Black-Right-Pointing-Pointer NeuroD6 augments the bioenergetic reserve of the neuronal PC12-NeuroD6 cells. Black-Right-Pointing-Pointer NeuroD6 increases the mitochondrial membrane potential and ATP levels. Black-Right-Pointing-Pointer NeuroD6 confers tolerance to rotenone via an adaptive

  14. Adult-onset mitochondrial myopathy.

    Science.gov (United States)

    Fernandez-Sola, J.; Casademont, J.; Grau, J. M.; Graus, F.; Cardellach, F.; Pedrol, E.; Urbano-Marquez, A.

    1992-01-01

    Mitochondrial diseases are polymorphic entities which may affect many organs and systems. Skeletal muscle involvement is frequent in the context of systemic mitochondrial disease, but adult-onset pure mitochondrial myopathy appears to be rare. We report 3 patients with progressive skeletal mitochondrial myopathy starting in adult age. In all cases, the proximal myopathy was the only clinical feature. Mitochondrial pathology was confirmed by evidence of ragged-red fibres in muscle histochemistry, an abnormal mitochondrial morphology in electron microscopy and by exclusion of other underlying diseases. No deletions of mitochondrial DNA were found. We emphasize the need to look for a mitochondrial disorder in some non-specific myopathies starting in adult life. Images Figure 1 Figure 2 PMID:1589382

  15. Pig Brain Mitochondria as a Biological Model for Study of Mitochondrial Respiration.

    Science.gov (United States)

    Fišar, Z; Hroudová, J

    2016-01-01

    Oxidative phosphorylation is a key process of intracellular energy transfer by which mitochondria produce ATP. Isolated mitochondria serve as a biological model for understanding the mitochondrial respiration control, effects of various biologically active substances, and pathophysiology of mitochondrial diseases. The aim of our study was to evaluate pig brain mitochondria as a proper biological model for investigation of activity of the mitochondrial electron transport chain. Oxygen consumption rates of isolated pig brain mitochondria were measured using high-resolution respirometry. Mitochondrial respiration of crude mitochondrial fraction, mitochondria purified in sucrose gradient, and mitochondria purified in Percoll gradient were assayed as a function of storage time. Oxygen flux and various mitochondrial respiratory control ratios were not changed within two days of mitochondria storage on ice. Leak respiration was found higher and Complex I-linked respiration lower in purified mitochondria compared to the crude mitochondrial fraction. Damage to both outer and inner mitochondrial membrane caused by the isolation procedure was the greatest after purification in a sucrose gradient. We confirmed that pig brain mitochondria can serve as a biological model for investigation of mitochondrial respiration. The advantage of this biological model is the stability of respiratory parameters for more than 48 h and the possibility to isolate large amounts of mitochondria from specific brain areas without the need to kill laboratory animals. We suggest the use of high-resolution respirometry of pig brain mitochondria for research of the neuroprotective effects and/or mitochondrial toxicity of new medical drugs.

  16. Reduced calcium-dependent mitochondrial damage underlies the reduced vulnerability of excitotoxicity-tolerant hippocampal neurons.

    Science.gov (United States)

    Pivovarova, Natalia B; Stanika, Ruslan I; Watts, Charlotte A; Brantner, Christine A; Smith, Carolyn L; Andrews, S Brian

    2008-03-01

    In central neurons, over-stimulation of NMDA receptors leads to excessive mitochondrial calcium accumulation and damage, which is a critical step in excitotoxic death. This raises the possibility that low susceptibility to calcium overload-induced mitochondrial damage might characterize excitotoxicity-resistant neurons. In this study, we have exploited two complementary models of preconditioning-induced excitotoxicity resistance to demonstrate reduced calcium-dependent mitochondrial damage in NMDA-tolerant hippocampal neurons. We have further identified adaptations in mitochondrial calcium handling that account for enhanced mitochondrial integrity. In both models, enhanced tolerance was associated with improved preservation of mitochondrial membrane potential and structure. In the first model, which exhibited modest neuroprotection, mitochondria-dependent calcium deregulation was delayed, even though cytosolic and mitochondrial calcium loads were quantitatively unchanged, indicating that enhanced mitochondrial calcium capacity accounts for reduced injury. In contrast, the second model, which exhibited strong neuroprotection, displayed further delayed calcium deregulation and reduced mitochondrial damage because downregulation of NMDA receptor surface expression depressed calcium loading. Reducing calcium entry also modified the chemical composition of the calcium-buffering precipitates that form in calcium-loaded mitochondria. It thus appears that reduced mitochondrial calcium loading is a major factor underlying the robust neuroprotection seen in highly tolerant cells. PMID:18036152

  17. Mitochondrial calcium uptake.

    Science.gov (United States)

    Williams, George S B; Boyman, Liron; Chikando, Aristide C; Khairallah, Ramzi J; Lederer, W J

    2013-06-25

    Calcium (Ca(2+)) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca(2+) levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca(2+) ([Ca(2+)]i) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca(2+) uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca(2+) uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca(2+) uniporter (MCU) candidate. This quantitative analysis yields four clear results: (i) under physiological conditions, Ca(2+) influx into the mitochondria via the MCU is small relative to other cytosolic Ca(2+) extrusion pathways; (ii) single MCU conductance is ∼6-7 pS (105 mM [Ca(2+)]), and MCU flux appears to be modulated by [Ca(2+)]i, suggesting Ca(2+) regulation of MCU open probability (P(O)); (iii) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and (iv) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca(2+) critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca(2+) under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca(2+)]i, mitochondrial Ca(2+) uptake can increase 10- to 1,000-fold and begin to shape [Ca(2+)]i dynamics.

  18. FUNDC1 regulates mitochondrial dynamics at the ER-mitochondrial contact site under hypoxic conditions.

    Science.gov (United States)

    Wu, Wenxian; Lin, Chunxia; Wu, Keng; Jiang, Lei; Wang, Xiaojing; Li, Wen; Zhuang, Haixia; Zhang, Xingliang; Chen, Hao; Li, Shupeng; Yang, Yue; Lu, Yue; Wang, Jingjing; Zhu, Runzhi; Zhang, Liangqing; Sui, Senfang; Tan, Ning; Zhao, Bin; Zhang, Jingjing; Li, Longxuan; Feng, Du

    2016-07-01

    In hypoxic cells, dysfunctional mitochondria are selectively removed by a specialized autophagic process called mitophagy. The ER-mitochondrial contact site (MAM) is essential for fission of mitochondria prior to engulfment, and the outer mitochondrial membrane protein FUNDC1 interacts with LC3 to recruit autophagosomes, but the mechanisms integrating these processes are poorly understood. Here, we describe a new pathway mediating mitochondrial fission and subsequent mitophagy under hypoxic conditions. FUNDC1 accumulates at the MAM by associating with the ER membrane protein calnexin. As mitophagy proceeds, FUNDC1/calnexin association attenuates and the exposed cytosolic loop of FUNDC1 interacts with DRP1 instead. DRP1 is thereby recruited to the MAM, and mitochondrial fission then occurs. Knockdown of FUNDC1, DRP1, or calnexin prevents fission and mitophagy under hypoxic conditions. Thus, FUNDC1 integrates mitochondrial fission and mitophagy at the interface of the MAM by working in concert with DRP1 and calnexin under hypoxic conditions in mammalian cells. PMID:27145933

  19. Mitochondrial Dysfunction and Psychiatric Disorders

    OpenAIRE

    Shaw-Hwa Jou; Nan-Yin Chiu; Chin-San Liu

    2009-01-01

    Mitochondria are intracellular organelles crucial in the production of cellular energy.Mitochondrial diseases may result from malfunctions in this biochemical cascade. Severalinvestigators have proposed that mitochondrial dysfunction is related to the pathophysiologyof bipolar disorder (BD), major depressive disorder (MDD) and schizophrenia (SZ). Theauthors reviewed recent study findings and tried to delineate the current understanding of thecorrelation between mitochondrial dysfunction and p...

  20. Pore dynamics in lipid membranes

    Science.gov (United States)

    Gozen, I.; Dommersnes, P.

    2014-09-01

    Transient circular pores can open in plasma membrane of cells due to mechanical stress, and failure to repair such pores lead to cell death. Similar pores in the form of defects also exist among smectic membranes, such as in myelin sheaths or mitochondrial membranes. The formation and growth of membrane defects are associated with diseases, for example multiple sclerosis. A deeper understanding of membrane pore dynamics can provide a more refined picture of membrane integrity-related disease development, and possibly also treatment options and strategies. Pore dynamics is also of great importance regarding healthcare applications such as drug delivery, gene or as recently been implied, cancer therapy. The dynamics of pores significantly differ in stacks which are confined in 2D compared to those in cells or vesicles. In this short review, we will summarize the dynamics of different types of pores that can be observed in biological membranes, which include circular transient, fusion and hemi-fusion pores. We will dedicate a section to floral and fractal pores which were discovered a few years ago and have highly peculiar characteristics. Finally, we will discuss the repair mechanisms of large area pores in conjunction with the current cell membrane repair hypotheses.

  1. Effect of Dietary Bioactive Compounds on Mitochondrial and Metabolic Flexibility

    Directory of Open Access Journals (Sweden)

    Jose C. E. Serrano

    2016-03-01

    Full Text Available Metabolic flexibility is the capacity of an organism to adequately respond to changes in the environment, such as nutritional input, energetic demand, etc. An important player in the capacity of adaptation through different stages of metabolic demands is the mitochondrion. In this context, mitochondrial dysfunction has been attributed to be the onset and center of many chronic diseases, which are denoted by an inability to adapt fuel preferences and induce mitochondrial morphological changes to respond to metabolic demands, such as mitochondrial number, structure and function. Several nutritional interventions have shown the capacity to induce changes in mitochondrial biogenesis/degradation, oxidative phosphorylation efficiency, mitochondrial membrane composition, electron transfer chain capacity, etc., in metabolic inflexibility states that may open new target options and mechanisms of action of bioactive compounds for the treatment of metabolic diseases. This review is focused in three well-recognized food bioactive compounds that modulate insulin sensitivity, polyphenols, ω-3 fatty acids and dietary fiber, by several mechanism of action, like caloric restriction properties and inflammatory environment modulation, both closely related to mitochondrial function and dynamics.

  2. Mitochondrial cereblon functions as a Lon-type protease

    Science.gov (United States)

    Kataoka, Kosuke; Nakamura, China; Asahi, Toru; Sawamura, Naoya

    2016-01-01

    Lon protease plays a major role in the protein quality control system in mammalian cell mitochondria. It is present in the mitochondrial matrix, and degrades oxidized and misfolded proteins, thereby protecting the cell from various extracellular stresses, including oxidative stress. The intellectual disability-associated and thalidomide-binding protein cereblon (CRBN) contains a large, highly conserved Lon domain. However, whether CRBN has Lon protease-like function remains unknown. Here, we determined if CRBN has a protective function against oxidative stress, similar to Lon protease. We report that CRBN partially distributes in mitochondria, suggesting it has a mitochondrial function. To specify the mitochondrial role of CRBN, we mitochondrially expressed CRBN in human neuroblastoma SH-SY5Y cells. The resulting stable SH-SY5Y cell line showed no apparent effect on the mitochondrial functions of fusion, fission, and membrane potential. However, mitochondrially expressed CRBN exhibited protease activity, and was induced by oxidative stress. In addition, stably expressed cells exhibited suppressed neuronal cell death induced by hydrogen peroxide. These results suggest that CRBN functions specifically as a Lon-type protease in mitochondria. PMID:27417535

  3. Mitochondrial Ca(2+) uptake in skeletal muscle health and disease.

    Science.gov (United States)

    Zhou, Jingsong; Dhakal, Kamal; Yi, Jianxun

    2016-08-01

    Muscle uses Ca(2+) as a messenger to control contraction and relies on ATP to maintain the intracellular Ca(2+) homeostasis. Mitochondria are the major sub-cellular organelle of ATP production. With a negative inner membrane potential, mitochondria take up Ca(2+) from their surroundings, a process called mitochondrial Ca(2+) uptake. Under physiological conditions, Ca(2+) uptake into mitochondria promotes ATP production. Excessive uptake causes mitochondrial Ca(2+) overload, which activates downstream adverse responses leading to cell dysfunction. Moreover, mitochondrial Ca(2+) uptake could shape spatio-temporal patterns of intracellular Ca(2+) signaling. Malfunction of mitochondrial Ca(2+) uptake is implicated in muscle degeneration. Unlike non-excitable cells, mitochondria in muscle cells experience dramatic changes of intracellular Ca(2+) levels. Besides the sudden elevation of Ca(2+) level induced by action potentials, Ca(2+) transients in muscle cells can be as short as a few milliseconds during a single twitch or as long as minutes during tetanic contraction, which raises the question whether mitochondrial Ca(2+) uptake is fast and big enough to shape intracellular Ca(2+) signaling during excitation-contraction coupling and creates technical challenges for quantification of the dynamic changes of Ca(2+) inside mitochondria. This review focuses on characterization of mitochondrial Ca(2+) uptake in skeletal muscle and its role in muscle physiology and diseases. PMID:27430885

  4. Implications of mitochondrial DNA mutations and mitochondrial dysfunction in tumorigenesis

    Institute of Scientific and Technical Information of China (English)

    Jianxin Lu; Lokendra Kumar Sharma; Yidong Bai

    2009-01-01

    Alterations in oxidative phosphorylation resulting from mitochondrial dysfunction have long been hypothesized to be involved in tumorigenesis. Mitochondria have recently been shown to play an important role in regulating both programmed cell death and cell proliferation. Furthermore, mitochondrial DNA (mtDNA) mutations have been found in various cancer cells. However, the role of these mtDNA mutations in tumorigenesis remains largely unknown. This review focuses on basic mitochondrial genetics, mtDNA mutations and consequential mitochondrial dysfunction associated with cancer. The potential molecular mechanisms, mediating the pathogenesis from mtDNA mutations and mitochondrial dysfunction to tumorigenesis are also discussed.

  5. Regulation of Mitochondrial Function by Voltage Dependent Anion Channels in Ethanol Metabolism and the Warburg Effect

    Science.gov (United States)

    Lemasters, John J.; Holmuhamedov, Ekhson L.; Czerny, Christoph; Zhong, Zhi; Maldonado, Eduardo N.

    2012-01-01

    Voltage dependent anion channels (VDAC) are highly conserved proteins that are responsible for permeability of the mitochondrial outer membrane to hydrophilic metabolites like ATP, ADP and respiratory substrates. Although previously assumed to remain open, VDAC closure is emerging as an important mechanism for regulation of global mitochondrial metabolism in apoptotic cells and also in cells that are not dying. During hepatic ethanol oxidation to acetaldehyde, VDAC closure suppresses exchange of mitochondrial metabolites, resulting in inhibition of ureagenesis. In vivo, VDAC closure after ethanol occurs coordinately with mitochondrial uncoupling. Since acetaldehyde passes through membranes independently of channels and transporters, VDAC closure and uncoupling together foster selective and more rapid oxidative metabolism of toxic acetaldehyde to nontoxic acetate by mitochondrial aldehyde dehydrogenase. In single reconstituted VDAC, tubulin decreases VDAC conductance, and in HepG2 hepatoma cells, free tubulin negatively modulates mitochondrial membrane potential, an effect enhanced by protein kinase A. Tubulin-dependent closure of VDAC in cancer cells contributes to suppression of mitochondrial metabolism and may underlie the Warburg phenomenon of aerobic glycolysis. PMID:22172804

  6. Condurango (Gonolobus condurango Extract Activates Fas Receptor and Depolarizes Mitochondrial Membrane Potential to Induce ROS-dependent Apoptosis in Cancer Cells in vitro CE-treatment on HeLa: a ROS-dependent mechanism

    Directory of Open Access Journals (Sweden)

    Kausik Bishayee

    2015-09-01

    Full Text Available Objectives: Condurango (Gonolobus condurango extract is used by complementary and alternative medicine (CAM practitioners as a traditional medicine, including homeopathy, mainly for the treatment of syphilis. Condurango bark extract is also known to reduce tumor volume, but the underlying molecular mechanisms still remain unclear. Methods: Using a cervical cancer cell line (HeLa as our model, the molecular events behind condurango extract’s (CE’s anticancer effect were investigated by using flow cytometry, immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR. Other included cell types were prostate cancer cells (PC3, transformed liver cells (WRL-68, and peripheral blood mononuclear cells (PBMCs. Results: Condurango extract (CE was found to be cytotoxic against target cells, and this was significantly deactivated in the presence of N-acetyl cysteine (NAC, a scavenger of reactive oxygen species (ROS, suggesting that its action could be mediated through ROS generation. CE caused an increase in the HeLa cell population containing deoxyribonucleic acid (DNA damage at the G zero/Growth 1 (G0/G1 stage. Further, CE increased the tumor necrosis factor alpha (TNF-α and the fas receptor (FasR levels both at the ribonucleic acid (RNA and the protein levels, indicating that CE might have a cytotoxic mechanism of action. CE also triggered a sharp decrease in the expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB both at the RNA and the protein levels, a possible route to attenuation of B-cell lymphoma 2 (Bcl-2, and caused an opening of the mitochondrial membrane’s permeability transition (MPT pores, thus enhancing caspase activities. Conclusion: Overall, our results suggest possible pathways for CE mediated cytotoxicity in model cancer cells.

  7. Cardiolipin linoleic acid content and mitochondrial cytochrome c oxidase activity are associated in rat skeletal muscle.

    Science.gov (United States)

    Fajardo, Val Andrew; McMeekin, Lauren; Saint, Caitlin; LeBlanc, Paul J

    2015-04-01

    Cardiolipin (CL) is an inner-mitochondrial membrane phospholipid that is important for optimal mitochondrial function. Specifically, CL and CL linoleic (18:2ω6) content are known to be positively associated with cytochrome c oxidase (COX) activity. However, this association has not been examined in skeletal muscle. In this study, rats were fed high-fat diets with a naturally occurring gradient in linoleic acid (coconut oil [CO], 5.8%; flaxseed oil [FO], 13.2%; safflower oil [SO], 75.1%) in an attempt to alter both mitochondrial CL fatty acyl composition and COX activity in rat mixed hind-limb muscle. In general, mitochondrial membrane lipid composition was fairly resistant to dietary treatments as only modest changes in fatty acyl composition were detected in CL and other major mitochondrial phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). As a result of this resistance, CL 18:2ω6 content was not different between the dietary groups. Consistent with the lack of changes in CL 18:2ω6 content, mitochondrial COX activity was also not different between the dietary groups. However, correlational analysis using data obtained from rats across the dietary groups showed a significant relationship (p = 0.009, R(2) = 0.21). Specifically, our results suggest that CL 18:2ω6 content may positively influence mitochondrial COX activity thereby making this lipid molecule a potential factor related to mitochondrial health and function in skeletal muscle.

  8. Dietary ω-3 Fatty Acids Alter Cardiac Mitochondrial Phospholipid Composition and Delay Ca2+-Induced Permeability Transition

    OpenAIRE

    O’Shea, Karen M.; Khairallah, Ramzi J.; Sparagna, Genevieve C.; Xu, Wenhong; Hecker, Peter A; Robillard-Frayne, Isabelle; Des Rosiers, Christine; Kristian, Tibor; Robert C. Murphy; Fiskum, Gary; Stanley, William C.

    2009-01-01

    Consumption of ω-3 fatty acids from fish oil, specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), decreases risk for heart failure and attenuates pathologic cardiac remodeling in response to pressure overload. Dietary supplementation with EPA+DHA may also impact cardiac mitochondrial function and energetics through alteration of membrane phospholipids. We assessed the role of EPA+DHA supplementation on left ventricular (LV) function, cardiac mitochondrial membrane phospho...

  9. Mitochondrial Dysfunction in Cancer

    Directory of Open Access Journals (Sweden)

    Michelle L Boland

    2013-12-01

    Full Text Available A mechanistic understanding of how mitochondrial dysfunction contributes to cell growth and tumorigenesis is emerging beyond Warburg as an area of research that is under-explored in terms of its significance for clinical management of cancer. Work discussed in this review focuses less on the Warburg effect and more on mitochondria and how dysfunctional mitochondria modulate cell cycle, gene expression, metabolism, cell viability and other more conventional aspects of cell growth and stress responses. There is increasing evidence that key oncogenes and tumor suppressors modulate mitochondrial dynamics through important signaling pathways and that mitochondrial mass and function vary between tumors and individuals but the sigificance of these events for cancer are not fully appreciated. We explore the interplay between key molecules involved in mitochondrial fission and fusion and in apoptosis, as well as in mitophagy, biogenesis and spatial dynamics and consider how these distinct mechanisms are coordinated in response to physiological stresses such as hypoxia and nutrient deprivation. Importantly, we examine how deregulation of these processes in cancer has knockon effects for cell proliferation and growth. Scientifically, there is also scope for defining what mitochondria dysfunction is and here we address the extent to which the functional consequences of such dysfunction can be determined and exploited for cancer diagnosis and treatment.

  10. The effect of ethidium bromide and chloramphenicol on mitochondrial biogenesis in primary human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Kao, Li-Pin; Ovchinnikov, Dmitry; Wolvetang, Ernst, E-mail: e.wolvetang@uq.edu.au

    2012-05-15

    The expression of mitochondrial components is controlled by an intricate interplay between nuclear transcription factors and retrograde signaling from mitochondria. The role of mitochondrial DNA (mtDNA) and mtDNA-encoded proteins in mitochondrial biogenesis is, however, poorly understood and thus far has mainly been studied in transformed cell lines. We treated primary human fibroblasts with ethidium bromide (EtBr) or chloramphenicol for six weeks to inhibit mtDNA replication or mitochondrial protein synthesis, respectively, and investigated how the cells recovered from these insults two weeks after removal of the drugs. Although cellular growth and mitochondrial gene expression were severely impaired after both inhibitor treatments we observed marked differences in mitochondrial structure, membrane potential, glycolysis, gene expression, and redox status between fibroblasts treated with EtBr and chloramphenicol. Following removal of the drugs we further detected clear differences in expression of both mtDNA-encoded genes and nuclear transcription factors that control mitochondrial biogenesis, suggesting that the cells possess different compensatory mechanisms to recover from drug-induced mitochondrial dysfunction. Our data reveal new aspects of the interplay between mitochondrial retrograde signaling and the expression of nuclear regulators of mitochondrial biogenesis, a process with direct relevance to mitochondrial diseases and chloramphenicol toxicity in humans. -- Highlights: ► Cells respond to certain environmental toxins by increasing mitochondrial biogenesis. ► We investigated the effect of Chloramphenicol and EtBr in primary human fibroblasts. ► Inhibiting mitochondrial protein synthesis or DNA replication elicit different effects. ► We provide novel insights into the cellular responses toxins and antibiotics.

  11. Legionella pneumophila secretes a mitochondrial carrier protein during infection.

    Directory of Open Access Journals (Sweden)

    Pavel Dolezal

    2012-01-01

    Full Text Available The Mitochondrial Carrier Family (MCF is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP, encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

  12. Synthesis of novel guttiferone A derivatives: in-vitro evaluation toward Plasmodium falciparum, Trypanosoma brucei and Leishmania donovani.

    Science.gov (United States)

    Fromentin, Yann; Gaboriaud-Kolar, Nicolas; Lenta, Bruno Ndjakou; Wansi, Jean Duplex; Buisson, Didier; Mouray, Elisabeth; Grellier, Philippe; Loiseau, Philippe M; Lallemand, Marie-Christine; Michel, Sylvie

    2013-07-01

    The catechol pharmacomodulation of the natural product guttiferone A, isolated from the Symphonia globulifera tree, led to the semisynthesis of a collection of twenty derivatives. The ester and ether derivatives of guttiferone A were evaluated for their anti-plasmodial, trypanocidal and anti-leishmanial activities. Some compounds described below have shown potent antiparasitic activity against Plasmodium falciparum, Trypanosoma brucei and Leishmania donovani in a range from 1 to 5 μM. The evaluation of guttiferone A derivatives against VERO cells highlighted catechol modulations as an interesting tool to decrease the toxicity and keep the activity of this natural compound. The current study revealed new molecules as promising new antiparasitic drug candidates. PMID:23727538

  13. JBP2, a SWI2/SNF2-like protein, regulates de novo telomeric DNA glycosylation in bloodstream form Trypanosoma brucei.

    Science.gov (United States)

    Kieft, Rudo; Brand, Verena; Ekanayake, Dilrukshi K; Sweeney, Kate; DiPaolo, Courtney; Reznikoff, William S; Sabatini, Robert

    2007-11-01

    Synthesis of the modified thymine base, beta-d-glucosyl-hydroxymethyluracil or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream form specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. In order to analyze the function of base J in the regulation of antigenic variation, we are characterizing the regulatory mechanism of J biosynthesis. We have recently proposed a model in which chromatin remodeling by a SWI2/SNF2-like protein (JBP2) regulates the developmental and de novo site-specific localization of J synthesis within bloodstream form trypanosome DNA. Consistent with this model, we now show that JBP2 (-/-) bloodstream form trypanosomes contain five-fold less base J and are unable to stimulate de novo J synthesis in newly generated telomeric arrays. PMID:17706299

  14. Crystal structures of Trypanosoma brucei oligopeptidase B broaden the paradigm of catalytic regulation in prolyl oligopeptidase family enzymes.

    Directory of Open Access Journals (Sweden)

    Peter Canning

    Full Text Available Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life.

  15. Immunospecific immunoglobulins and IL-10 as markers for Trypanosoma brucei rhodesiense late stage disease in experimentally infected vervet monkeys

    DEFF Research Database (Denmark)

    Ngotho, Maina; Kagira, J.M.; Jensen, Henrik Michael Elvang;

    2009-01-01

    OBJECTIVE: To determine the usefulness of IL-10 and immunoglobulin M (IgM) as biomarkers for staging HAT in vervet monkeys, a useful pathogenesis model for humans. METHODS: Vervet monkeys were infected with Trypanosoma brucei rhodesiense and subsequently given sub-curative and curative treatment 28...... and 140 days post-infection (dpi) respectively. Matched serum and CSF samples were obtained at regular intervals and immunospecific IgM, immunoglobulin G (IgG) and IL-10 were quantified by ELISA. RESULTS: There was no detectable immunospecific IgM and IgG in the CSF before 49 dpi. CSF IgM and Ig...... followed a pattern that mimics the progression of the disease and may present reliable and useful biomarkers of the disease stage. Due to rapid decline, serum IgM and IL-10 are, additionally, potential biomarkers of the success of chemotherapy....

  16. Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

    Institute of Scientific and Technical Information of China (English)

    Min Chen; Yanru Wang; Tingting Hou; Huiliang Zhang; Aijuan Qu; Xianhua Wang

    2011-01-01

    Mitochondrial calcium plays a crucial role in mitochondriai metabolism,cell calcium handling,and cell death.However,some mechanisms concerning mitochondrial calcium regulation are still unknown,especially how mitochondrial calcium couples with cytosolic calcium.In this work,we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation.Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester,a mitochondrial membrane potential indicator.The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2.The apparent Kd of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes.Furthermore,we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria.In HeLa cells,we found that mitochondrial calcium ([Ca2+]mito)responded to the changes of cytosolic calcium ([Ca2+]cyto)induced by histamine or thapasigargin.Moreover,external Ca2+ (100 μmol/L) directly induced an increase of [Ca2+]mito in permeabilized HeLa cells.However,in rat cardiomyocytes [Ca2+]mito did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine.In permeabilized cardiomyocytes,600 nmol/L free Ca2+ repeatedly increased the fluorescent signals of mito-GCaMP2,which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria.These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.

  17. Mitochondrial remodeling following fission inhibition by 15d-PGJ2 involves molecular changes in mitochondrial fusion protein OPA1

    International Nuclear Information System (INIS)

    Research highlights: → Chemical inhibition of fission protein Drp1 leads to mitochondrial fusion. → Increased fusion stimulates molecular changes in mitochondrial fusion protein OPA1. → Proteolysis of larger isoforms, new synthesis and ubiquitination of OPA1 occur. → Loss of mitochondrial tubular rigidity and disorganization of cristae. → Generation of large swollen dysfunctional mitochondria. -- Abstract: We showed earlier that 15 deoxy Δ12,14 prostaglandin J2 (15d-PGJ2) inactivates Drp1 and induces mitochondrial fusion . However, prolonged incubation of cells with 15d-PGJ2 resulted in remodeling of fused mitochondria into large swollen mitochondria with irregular cristae structure. While initial fusion of mitochondria by 15d-PGJ2 required the presence of both outer (Mfn1 and Mfn2) and inner (OPA1) mitochondrial membrane fusion proteins, later mitochondrial changes involved increased degradation of the fusion protein OPA1 and ubiquitination of newly synthesized OPA1 along with decreased expression of Mfn1 and Mfn2, which likely contributed to the loss of tubular rigidity, disorganization of cristae, and formation of large swollen degenerated dysfunctional mitochondria. Similar to inhibition of Drp1 by 15d-PGJ2, decreased expression of fission protein Drp1 by siRNA also resulted in the loss of fusion proteins. Prevention of 15d-PGJ2 induced mitochondrial elongation by thiol antioxidants prevented not only loss of OPA1 isoforms but also its ubiquitination. These findings provide novel insights into unforeseen complexity of molecular events that modulate mitochondrial plasticity.

  18. Crystal Structures of TbCatB and rhodesain, potential chemotherapeutic targets and major cysteine proteases of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Iain D Kerr

    Full Text Available BACKGROUND: Trypanosoma brucei is the etiological agent of Human African Trypanosomiasis, an endemic parasitic disease of sub-Saharan Africa. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei. METHODS AND FINDINGS: We have determined, by X-ray crystallography, the first reported structure of TbCatB in complex with the cathepsin B selective inhibitor CA074. In addition we report the structure of rhodesain in complex with the vinyl-sulfone K11002. CONCLUSIONS: The mature domain of our TbCat*CA074 structure contains unique features for a cathepsin B-like enzyme including an elongated N-terminus extending 16 residues past the predicted maturation cleavage site. N-terminal Edman sequencing reveals an even longer extension than is observed amongst the ordered portions of the crystal structure. The TbCat*CA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures. Our data further highlight enhanced flexibility in the occluding loop main chain and structural deviations from mammalian cathepsin B enzymes that may affect activity and inhibitor design. Comparisons with the rhodesain*K11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs.

  19. The multiple roles of cyclin E1 in controlling cell cycle progression and cellular morphology of Trypanosoma brucei.

    Science.gov (United States)

    Gourguechon, Stéphane; Savich, Jason M; Wang, Ching C

    2007-05-11

    Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases. Previous RNA interference (RNAi) experiments in Trypanosoma brucei indicated that cyclin E1, cdc2-related kinase (CRK)1 and CRK2 are involved in regulating G1/S transition, whereas cyclin B2 and CRK3 play a pivotal role in controlling the G2/M checkpoint. To search for potential interactions between the other cyclins and CRKs that may not have been revealed by the RNAi assays, we used the yeast two-hybrid system and an in vitro glutathione-S-transferase pulldown assay and observed interactions between cyclin E1 and CRK1, CRK2 and CRK3. Cyclins E1-E4 are homologues of yeast Pho80 cyclin. But yeast complementation assays indicated that none of them possesses a Pho80-like function. Analysis of cyclin E1+CRK1 and cyclin E1+CRK2 double knockdowns in the procyclic form of T. brucei indicated that the cells were arrested more extensively in the G1 phase beyond the cumulative effect of individual knockdowns. But BrdU incorporation was impaired significantly only in cyclin E1+CRK1-depleted cells, whereas a higher percentage of cyclin E1+CRK2 knockdown cells assumed a grossly elongated posterior end morphology. A double knockdown of cyclin E1 and CRK3 arrested cells in G2/M much more efficiently than if only CRK3 was depleted. Taken together, these data suggest multiple functions of cyclin E1: it forms a complex with CRK1 in promoting G1/S phase transition; it forms a complex with CRK2 in controlling the posterior morphogenesis during G1/S transition; and it forms a complex with CRK3 in promoting passage across the G2/M checkpoint in the trypanosome. PMID:17376478

  20. The orthologue of Sjogren's syndrome nuclear autoantigen 1 (SSNA1 in Trypanosoma brucei is an immunogenic self-assembling molecule.

    Directory of Open Access Journals (Sweden)

    Helen P Price

    Full Text Available Primary Sjögren's Syndrome (PSS is a highly prevalent autoimmune disease, typically manifesting as lymphocytic infiltration of the exocrine glands leading to chronically impaired lacrimal and salivary secretion. Sjögren's Syndrome nuclear autoantigen 1 (SSNA1 or NA14 is a major specific target for autoantibodies in PSS but the precise function and clinical relevance of this protein are largely unknown. Orthologues of the gene are absent from many of the commonly used model organisms but are present in Chlamyodomonas reinhardtii (in which it has been termed DIP13 and most protozoa. We report the functional characterisation of the orthologue of SSNA1 in the kinetoplastid parasite, Trypanosoma brucei. Both TbDIP13 and human SSNA1 are small coiled-coil proteins which are predicted to be remote homologues of the actin-binding protein tropomyosin. We use comparative proteomic methods to identify potential interacting partners of TbDIP13. We also show evidence that TbDIP13 is able to self-assemble into fibril-like structures both in vitro and in vivo, a property which may contribute to its immunogenicity. Endogenous TbDIP13 partially co-localises with acetylated α-tubulin in the insect procyclic stage of the parasite. However, deletion of the DIP13 gene in cultured bloodstream and procyclic stages of T. brucei has little effect on parasite growth or morphology, indicating either a degree of functional redundancy or a function in an alternative stage of the parasite life cycle.

  1. The spliceosomal snRNP core complex of Trypanosoma brucei: Cloning and functional analysis reveals seven Sm protein constituents

    Science.gov (United States)

    Palfi, Zsofia; Lücke, Stephan; Lahm, Hans-Werner; Lane, William S.; Kruft, Volker; Bragado-Nilsson, Elisabeth; Séraphin, Bertrand; Bindereif, Albrecht

    2000-01-01

    Each of the trypanosome small nuclear ribonucleoproteins (snRNPs) U2, U4/U6, and U5, as well as the spliced leader (SL) RNP, contains a core of common proteins, which we have previously identified. This core is unusual because it is not recognized by anti-Sm Abs and it associates with an Sm-related sequence in the trypanosome small nuclear RNAs (snRNAs). Using peptide sequences derived from affinity-purified U2 snRNP proteins, we have cloned cDNAs for five common proteins of 8.5, 10, 12.5, 14, and 15 kDa of Trypanosoma brucei and identified them as Sm proteins SmF (8.5 kDa), -E (10 kDa), -D1 (12.5 kDa), -G (14 kDa), and -D2 (15 kDa), respectively. Furthermore, we found the trypanosome SmB (T. brucei) and SmD3 (Trypanosoma cruzi) homologues through database searches, thus completing a set of seven canonical Sm proteins. Sequence comparisons of the trypanosome proteins revealed several deviations in highly conserved positions from the Sm consensus motif. We have identified a network of specific heterodimeric and -trimeric Sm protein interactions in vitro. These results are summarized in a model of the trypanosome Sm core, which argues for a strong conservation of the Sm particle structure. The conservation extends also to the functional level, because at least one trypanosome Sm protein, SmG, was able to specifically complement a corresponding mutation in yeast. PMID:10900267

  2. Structural characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei bound to the antifungal drugs posaconazole and fluconazole.

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    Chiung-Kuang Chen

    Full Text Available BACKGROUND: Chagas Disease is the leading cause of heart failure in Latin America. Current drug therapy is limited by issues of both efficacy and severe side effects. Trypansoma cruzi, the protozoan agent of Chagas Disease, is closely related to two other major global pathogens, Leishmania spp., responsible for leishmaniasis, and Trypansoma brucei, the causative agent of African Sleeping Sickness. Both T. cruzi and Leishmania parasites have an essential requirement for ergosterol, and are thus vulnerable to inhibitors of sterol 14alpha-demethylase (CYP51, which catalyzes the conversion of lanosterol to ergosterol. Clinically employed anti-fungal azoles inhibit ergosterol biosynthesis in fungi, and specific azoles are also effective against both Trypanosoma and Leishmania parasites. However, modification of azoles to enhance efficacy and circumvent potential drug resistance has been problematic for both parasitic and fungal infections due to the lack of structural insights into drug binding. METHODOLOGY/PRINCIPAL FINDINGS: We have determined the crystal structures for CYP51 from T. cruzi (resolutions of 2.35 A and 2.27 A, and from the related pathogen T. brucei (resolutions of 2.7 A and 2.6 A, co-crystallized with the antifungal drugs fluconazole and posaconazole. Remarkably, both drugs adopt multiple conformations when binding the target. The fluconazole 2,4-difluorophenyl ring flips 180 degrees depending on the H-bonding interactions with the BC-loop. The terminus of the long functional tail group of posaconazole is bound loosely in the mouth of the hydrophobic substrate binding tunnel, suggesting that the major contribution of the tail to drug efficacy is for pharmacokinetics rather than in interactions with the target. CONCLUSIONS/SIGNIFICANCE: The structures provide new insights into binding of azoles to CYP51 and mechanisms of potential drug resistance. Our studies define in structural detail the CYP51 therapeutic target in T. cruzi, and

  3. Revealing various coupling of electron transfer and proton pumping in mitochondrial respiratory chain.

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    Sun, Fei; Zhou, Qiangjun; Pang, Xiaoyun; Xu, Yingzhi; Rao, Zihe

    2013-08-01

    Cellular respiration is the process that releases energy from food and supplies energy for life processes. The mitochondrial respiratory chain is the final and most important step for cellular respiration and is located on the inner membrane of mitochondrion and comprises four large trans-membrane protein complexes (respiratory chain Complexes I, II, III and IV) as well as ubiquinone between Complexes I/II and III and cytochrome c between Complexes III and IV. The function of mitochondrial respiratory chain is biological oxidation by transferring electrons from NADH and succinate to oxygen and then generating proton gradient across the inner membrane. Such proton gradient is utilized by ATP synthase (ATPase, also called as Complex V) to produce energy molecules ATP. Structural studies of mitochondrial respiratory membrane protein complexes are important to understand the mechanism of electron transfer and the redox-coupled proton translocation across the inner membrane. Here, according to the time line, we reviewed the great achievements on structural studies of mitochondrial respiratory complexes in the past twenty years as well as the recent research progresses on the structures of mitochondrial respiratory supra-complexes.

  4. Preventing mitochondrial fission impairs mitochondrial function and leads to loss of mitochondrial DNA.

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    Philippe A Parone

    Full Text Available Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS. At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.

  5. Oxidative phosphorylation differences between mitochondrial DNA haplogroups modify the risk of Leber's hereditary optic neuropathy.

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    Gómez-Durán, Aurora; Pacheu-Grau, David; Martínez-Romero, Iñigo; López-Gallardo, Ester; López-Pérez, Manuel J; Montoya, Julio; Ruiz-Pesini, Eduardo

    2012-08-01

    Leber's hereditary optic neuropathy is a maternally inherited optic atrophy caused by mitochondrial DNA point mutations. Previous epidemiological studies have shown that individuals from mitochondrial genetic backgrounds (haplogroups) J/Uk and H have a higher and a lower risk, respectively, of suffering this disorder. To analyze the bases of these associations at cellular and molecular levels, functional studies with cybrids provide high quality evidence. Cybrids from haplogroup J contain less mitochondrial deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and synthesize a smaller amount of mitochondrial DNA-encoded polypeptides than those from haplogroup H. Haplogroup J cybrids also display lower oxygen consumption, mitochondrial inner membrane potential and total adenosine-5'-triphosphate (ATP) levels. Moreover, mitochondrial DNA levels correlate with many parameters of the oxidative phosphorylation system. These results suggest that the mitochondrial DNA amount determines oxidative phosphorylation capacity and, along with other recently published observations, support the possibility that mitochondrial DNA levels may be responsible for the bias of the disorder toward males, for the incomplete penetrance of mutations causing Leber's hereditary optic neuropathy and for the association of the disease with particular mitochondrial DNA haplogroups.

  6. The mitochondrial Ca2+ uniporter: regulation by auxiliary subunits and signal transduction pathways.

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    Jhun, Bong Sook; Mishra, Jyotsna; Monaco, Sarah; Fu, Deming; Jiang, Wenmin; Sheu, Shey-Shing; O-Uchi, Jin

    2016-07-01

    Mitochondrial Ca(2+) homeostasis, the Ca(2+) influx-efflux balance, is responsible for the control of numerous cellular functions, including energy metabolism, generation of reactive oxygen species, spatiotemporal dynamics of Ca(2+) signaling, and cell growth and death. Recent discovery of the molecular identity of the mitochondrial Ca(2+) uniporter (MCU) provides new possibilities for application of genetic approaches to study the mitochondrial Ca(2+) influx mechanism in various cell types and tissues. In addition, the subsequent discovery of various auxiliary subunits associated with MCU suggests that mitochondrial Ca(2+) uptake is not solely regulated by a single protein (MCU), but likely by a macromolecular protein complex, referred to as the MCU-protein complex (mtCUC). Moreover, recent reports have shown the potential role of MCU posttranslational modifications in the regulation of mitochondrial Ca(2+) uptake through mtCUC. These observations indicate that mtCUCs form a local signaling complex at the inner mitochondrial membrane that could significantly regulate mitochondrial Ca(2+) handling, as well as numerous mitochondrial and cellular functions. In this review we discuss the current literature on mitochondrial Ca(2+) uptake mechanisms, with a particular focus on the structure and function of mtCUC, as well as its regulation by signal transduction pathways, highlighting current controversies and discrepancies.

  7. Mitochondrial calcium uniporter Mcu controls excitotoxicity and is transcriptionally repressed by neuroprotective nuclear calcium signals.

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    Qiu, Jing; Tan, Yan-Wei; Hagenston, Anna M; Martel, Marc-Andre; Kneisel, Niclas; Skehel, Paul A; Wyllie, David J A; Bading, Hilmar; Hardingham, Giles E

    2013-01-01

    The recent identification of the mitochondrial Ca(2+) uniporter gene (Mcu/Ccdc109a) has enabled us to address its role, and that of mitochondrial Ca(2+) uptake, in neuronal excitotoxicity. Here we show that exogenously expressed Mcu is mitochondrially localized and increases mitochondrial Ca(2+) levels following NMDA receptor activation, leading to increased mitochondrial membrane depolarization and excitotoxic cell death. Knockdown of endogenous Mcu expression reduces NMDA-induced increases in mitochondrial Ca(2+), resulting in lower levels of mitochondrial depolarization and resistance to excitotoxicity. Mcu is subject to dynamic regulation as part of an activity-dependent adaptive mechanism that limits mitochondrial Ca(2+) overload when cytoplasmic Ca(2+) levels are high. Specifically, synaptic activity transcriptionally represses Mcu, via a mechanism involving the nuclear Ca(2+) and CaM kinase-mediated induction of Npas4, resulting in the inhibition of NMDA receptor-induced mitochondrial Ca(2+) uptake and preventing excitotoxic death. This establishes Mcu and the pathways regulating its expression as important determinants of excitotoxicity, which may represent therapeutic targets for excitotoxic disorders.

  8. Mechanism of mitochondrial respiratory control in caspase-3 induced positive feed back loop in apoptosis

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Caspase-3 plays a central role in the execution of apoptosis. Besides many substrates of caspase-3, mitochondria seem to be one of the candidate targets in the apoptotic process. We evaluated the effects of caspase-3 on the isolated mitochondria in detail, and especially focused on the mechanism involved in mitochondrial functions, which were not fully assessed till now. Our results showed that recombinant caspase-3 induced the increase of superoxide production, the dissipation of mitochondrial membrane potential and rate increasing of mitochondrial state 4 respiration. Caspases inhibitor, z-VAD-fmk can inhibit these effects of caspase-3 on mitochondria. Bcl-xL and cyclosporin A were also shown to be able to inhibit these changes. These results suggested a possible mechanism in caspase-3 induced disruption of mitochondrial membrane barrier which formed a positive feedback loop in apoptosis.

  9. The mitochondrial function was impaired in APP knockout mouse embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    SHENG BaiYang; NIU Ying; ZHOU Hui; YAN JiaXin; ZHAO NanMing; ZHANG XiuFang; GONG YanDao

    2009-01-01

    The amyloid precursor protein (APP) is recognized as the source of Aβ, which plays an important role in Alzheimer's disease. However, the biological function of APP is obscure. Previous studies showed that mitochondria could be a target of APP. In this work, APP knockout mouse embryo fibroblast (MEF) cells were used to test if APP plays any role in maintaining the mitochondrial function. As the result, APP knockout MEF cells (APP-/- cells) showed the abnormal mitochondrial function, including slower cell proliferation, lower mitochondrial membrane potential, lower intracellular ROS, higher mitochon-drial membrane fluidity and lower cytochrome c oxidase activity than their wild-type counterparts. However, no change was found in the amount of mitochondria in MEF APP-/- cells.

  10. Polybrominated diphenyl ether congener (BDE-100) induces mitochondrial impairment.

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    Pereira, Lílian Cristina; de Souza, Alecsandra Oliveira; Dorta, Daniel Junqueira

    2013-06-01

    Brominated flame retardants are used in various consumer products to increase their resistance to fire and/or high temperatures. Polybrominated diphenyl ethers (PBDEs) are representatives of this class and among the most widely used congeners, and BDE-100 is produced on a large scale. There is a lack of toxicological data about these compounds, which has recently become a matter of concern to the scientific community. The mitochondria are recognized as the main energy-producing organelles, as well as playing a vital role in the maintenance of many cell functions. Therefore, mitochondria were used in the present work as an experimental model to evaluate the effects of the BDE-100 congeners at concentrations ranging from 0.1 μM to 50 μM. The results showed that high concentrations of BDE-100 were able to induce mitochondrial alterations. It was observed that the substance had an affinity for the hydrophilic portion of the mitochondrial membrane, as monitored by ANS, inhibiting the glutamate + malate-stimulated mitochondrial respiration and also inducing dissipation of the mitochondrial membrane potential, deregulation of calcium homoeostasis and mitochondrial swelling, the latter being insensitive to cyclosporin A (CsA) but partially inhibited by Ruthenium Red and N-ethyl maleimide. In addition, a significant reduction in mitochondrial ATP content was found, but on the other hand, no oxidative stress was observed after exposure of the mitochondria to BDE-100. These results show the key role of mitochondria in the cytotoxicity induced by BDE-100. PMID:23302053

  11. Polybrominated diphenyl ether congener (BDE-100) induces mitochondrial impairment.

    Science.gov (United States)

    Pereira, Lílian Cristina; de Souza, Alecsandra Oliveira; Dorta, Daniel Junqueira

    2013-06-01

    Brominated flame retardants are used in various consumer products to increase their resistance to fire and/or high temperatures. Polybrominated diphenyl ethers (PBDEs) are representatives of this class and among the most widely used congeners, and BDE-100 is produced on a large scale. There is a lack of toxicological data about these compounds, which has recently become a matter of concern to the scientific community. The mitochondria are recognized as the main energy-producing organelles, as well as playing a vital role in the maintenance of many cell functions. Therefore, mitochondria were used in the present work as an experimental model to evaluate the effects of the BDE-100 congeners at concentrations ranging from 0.1 μM to 50 μM. The results showed that high concentrations of BDE-100 were able to induce mitochondrial alterations. It was observed that the substance had an affinity for the hydrophilic portion of the mitochondrial membrane, as monitored by ANS, inhibiting the glutamate + malate-stimulated mitochondrial respiration and also inducing dissipation of the mitochondrial membrane potential, deregulation of calcium homoeostasis and mitochondrial swelling, the latter being insensitive to cyclosporin A (CsA) but partially inhibited by Ruthenium Red and N-ethyl maleimide. In addition, a significant reduction in mitochondr